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Fiber based scaffolds in connective tissue engineering : using the architecture of woven scaffolds to influence and control the formation of organized tissues

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Fiber based scaffolds in connective tissue engineering : using the architecture of woven scaffolds to influence and control the formation of organized tissues
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Seegert, Charles Alan, 1971-
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English
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xiv, 150 leaves : ill. ; 29 cm.

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Bones ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Collagens ( jstor )
Cultured cells ( jstor )
Diameters ( jstor )
Osteoblasts ( jstor )
Scaffolds ( jstor )
Solar fibrils ( jstor )
Stainless steels ( jstor )
Biomedical Engineering thesis, Ph.D ( lcsh )
Dissertations, Academic -- Biomedical Engineering -- UF ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2002.
Bibliography:
Includes bibliographical references (leaves 138-149).
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Also available online.
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Printout.
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Vita.
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by Charles Alan Seegert.

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University of Florida
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FIBER BASED SCAFFOLDS IN CONNECTIVE
TISSUE ENGINEERING:
USING THE ARCHITECTURE OF WOVEN SCAFFOLDS
TO INFLUENCE AND CONTROL THE FORMATION
OF ORGANIZED TISSUES












By

CHARLES ALAN SEEGERT












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2002


























Copyright 2002

by

CHARLES ALAN SEEGERT





























To the Pinnacle














ACKNOWLEDGMENTS

I would like to thank everyone who made it possible for me to perform the research in this dissertation. In particular I would like to acknowledge Dr. Colin Sumners and the people in his lab who provided me with the tissues that I used to develop my cell cultures.

I would especially like to thank Dr. Brennan, my major adviser, who allowed me the time and provided me with the resources I needed to develop my ideas.

































iv














TABLE OF CONTENTS
pne

ACKN OW LED GM EN TS ................................................................................................. iv

LIST OF TABLES ........................................................................................................... viii

LIST O F FIGU RES ........................................................................................................... ix

ABSTRA CT ..................................................................................................................... xiii

CHAPTERS

I INTRODU CTION ............................................................................................................ I

Autogeneic Bone Grafting .............................................................................................. 2
Allogeneic Bone Grafts ................................................................................................... 3
Synthetic Bone Replacement Materials and Xenogeneic Bone Grafts ........................... 5
Problem to be Approached .............................................................................................. 6
Tissue Engineering .......................................................................................................... 8
The Case for Hierarchical Organization of Fiber Constructs ......................................... 9


2 BA CK GRO UN D ............................................................................................................ 13

Bone Form ation in Utero .............................................................................................. 13
M SC Differentiation ..................................................................................................... 15
Bone Developm ent and Bone A natom y ....................................................................... 21
Fracture Healing and Ectopic Bone Form ation ............................................................ 25
Porosity and Diffusion Properties of Implant Materials ............................................... 25
Rem odeling and Cellular Orientation ........................................................................... 28
Contact Guidance .......................................................................................................... 29
Architecture of Fiber Based Scaffolds: Development of Cell-Based Tension ............. 33
Autocrine/Paracrine Considerations in Scaffold Design .............................................. 37


3 SIN GLE FIBER STUD IES ............................................................................................. 42

Introduction ................................................................................................................... 42
M aterials ....................................................................................................................... 44
M ethods ......................................................................................................................... 45
Single Fiber Scaffolds ............................................................................................ 45


v








Cell Culture ............................................................................................................ 45
M SC Seeding ......................................................................................................... 46
SEM Imaging (Nylon sutures) ............................................................................... 46
Light M icroscopy (M axon Sutures) ....................................................................... 47
Results ........................................................................................................................... 47
SEM Studies ........................................................................................................... 47
4-0 nylon sutures .............................................................................................. 47
9-0 nylon sutures .............................................................................................. 49
10-0 nylon sutures ............................................................................................ 50
M axon sutures .................................................................................................. 51
Light M icroscopy Studies of M axon ..................................................................... 53
Nuclear form factor .......................................................................................... 55
Nuclear angle analysis ..................................................................................... 58
Discussion ..................................................................................................................... 61
Conclusions ................................................................................................................... 62


4 CELLULAR BRIDGING PHENOM ENA ..................................................................... 64

Introduction ................................................................................................................... 64
M aterials ....................................................................................................................... 66
M ethods ......................................................................................................................... 66
Culture M ethods ..................................................................................................... 66
SEM Imaging ......................................................................................................... 67
Light M icroscopy Studies ...................................................................................... 67
Von Kossa Staining ................................................................................................ 67
Proliferation Studies ............................................................................................... 68
Results ........................................................................................................................... 69
Light Microscopy of Bioactive Glass Fibers After 6 Days in Culture ................... 69
Scanning Electron M icroscopy .............................................................................. 71
Bioactive glass fibers after 6 days in culture ................................................... 71
Polym er fibers .................................................................................................. 72
Light microscopy of Bridge Development on Polystyrene Culture Flask ............. 73
BiTdge Development on Stainless Steel Screens (Light Microscopy and SEM) ... 77 Proliferation Studies of RM SCs on Bioactive Glass .............................................. 81
Discussion ..................................................................................................................... 83
Conclusions ................................................................................................................... 92


5 M ULTI-FIBER STUDIES .............................................................................................. 94

Introduction ................................................................................................................... 94
M aterials ....................................................................................................................... 97
M ethods ......................................................................................................................... 97
Cell Culture ............................................................................................................ 97
Construct Preparation ............................................................................................. 97
M axon Construct Sterilization ............................................................................... 99


vi








Stainless Steel Construct Preparation ................................................................... 100
Transmission Electron M icroscopy ...................................................................... 101
Statistical Analysis ............................................................................................... 101
Results ......................................................................................................................... 104
M axonTm Bridging ................................................................................................ 104
M axonTm bridging day 3 ................................................................................ 104
M axonTm bridging day 6 ................................................................................ 104
M axonTm bridging day 9 ................................................................................ 105
M axonTm bridging day 10 .............................................................................. 105
M axonTm bridging day I I .............................................................................. 105
M ulti-Layer Construct Bridging .......................................................................... 107
Cell Angle and Spacing Distance ......................................................................... 109
Stainless Steel Bridging ....................................................................................... 109
Stainless steel bridging day 3 ......................................................................... 109
Stainless steel bridging day 6 ......................................................................... 110
Stainless steel bridging day 8 ......................................................................... 110
Stainless steel bridging day 10 (mineralization analysis) .............................. 110
Transm ission Electron M icroscopy ...................................................................... III
Transmission electron microscopy of 7-0 single fiber ................................... III
Transmission electron microscopy of 7-0 25 jim spaced parallel array ........ 112
Transmission electron microscopy of contracted 7-0 25 PM parallel
array ......................................................................................................... 114
Discussion ................................................................................................................... 117
Conclusions ................................................................................................................. 123


6 CON CLUSION S AND FUTURE W ORK ................................................................... 125

APPENDIX QUANTITATIVE CONTACT GUIDANCE ANALYSIS METHODS .... 132

Conceptual and Illustrated Review of Nuclear Form Factor (NFF) ........................... 132
Extension of NFF Correction Concept to Nuclear Angle Measurements ................... 134
M athem atical Derivation ............................................................................................ 135


LIST OF REFERENCES ................................................................................................. 138

BIOGRAPHICAL SKETCH ........................................................................................... 150











vii















LIST OF TABLES

Table page

1-1. List of Design Requirements for More Ideal Bone Replacement Material .................. 6

5-1. Summary of Statistical Results from the Mineralization Study Performed on
Stainless Steel Screens ......................................................................................... III






































viii















LIST OF FIGURES

Figure page

1-1 An example of a femoral reconstruction using a segment of allograft bone ....... 5 1-2 Schematic representation of diaphyseal bone replacement...................... 7

1-3 A diagram of the general orientation of lamellae in a segment of bone............. 9

1-4 Schematic representation of collagen fibril orientation within a segment of bone ... 10 1-5 General concept of the hierarchical level of woven fiber based scaffolds ........ 11 2-1 Schematic of the cell cycle ............................................................. 14

2-2 MSC developmental sequence leading to bone producing cells ................... 17

2-3 Lines of force seen in proximal portions of the femur ............................... 21

2-4 Examples of lamellar orientation within a section of bone ......................... 23

2-5 Fibrillar orientation within bone ....................................................... 23

2-6 Rotated plywood model................................................................. 24

2-7 Schematic representation of ectopic bone formation around implanted,
demineralized bone chips............................................................... 26

2-8 Receptor mediated adhesion of osteoblasts via fibronectin ........................ 30

2-9 Diagram of autocrine signaling ........................................................ 38

2-10 Diagram of paracrine signaling ........................................................ 40

3-1 Examples of MaxonTm single fiber constructs........................................ 45

3-2 4-0 nylon fibers with RMSCs after being cultured for 5 days ..................... 48

3-3 Examples of MSC growth on the surface of 4-0 nylon fibers ...................... 49

3-4 Examples of RMSC growth on the surface of 9-0 nylon fibers .................... 50




ix








3-5 Examples of RMSC growth on the surface of 9-0 nylon fibers ............................. 51

3-6 SEM images of 10-0 nylon sutures at various magnifications ............................ 52

3-7 5-0 Maxon sutures exhibiting RMSC adhesion and growth ................................ 53

3-8 6-0 Maxon sutures exhibiting RMSC adhesion and growth ............................... 54

3-9 7-0 Maxon sutures exhibiting RMSC adhesion and growth ................................ 54

3-9 Continued. 7-0 Maxon sutures exhibiting RMSC adhesion and growth ............. 55

3-10 Example of nuclear form factor measurements ................................................... 55

3-11 Graph showing the independent influence of fiber diameter on NFF .................. 56

3-12 Graph showing the independent influence of time on NFF ................................... 57

3-13 Graph showing the effects of time on NFF for all diameters studied .................. 57

3-14 Example of nuclear angle measurements ............................................................... 59

3-15 Graph showing the effects of diameter independent of time ................................ 59

3-16 Graph showing the effect of time in culture on nuclear orientation as measured via
nuclear angle ....................................................................................................... 60

3-17 Graph showing the effects of time and diameter together .................................. 60

4-1 Multilayering on adjacent fibers leading to interactions due to their proximity .... 65

4-2 Example of bioactive glass fiber constructs used for the proliferation study ....... 68

4.3 Montage of light micrographs portraying the cellular interaction with bioactive
glass fibers placed in standard culture well ......................................................... 70

44 Light micrograph of area without fibers in the same culture well as that shown in
Figure 4-3 ............................................................................................................. 70

4-5 Examples of cellular growth on fiber placed in the RMSC system ..................... 71

4-6 Examples of unicellular bridging on bioactive glass over distances of -70 Pm. ..72

4-7 Examples of unicellular bridging between nylon fibers ...................................... 73

4-8 Bridging between Maxon fibers ............................................................................. 73

4-9 Initial RM SC elongation ...................................................................................... 74



x









4-9 continued. Initial RMSC unicellular bridging........................................ 75

4-10 Multicellular bridge at 7 days in the RMSC culture.................................. 76

4-11 Multicellular bridge at 8 days in the RMSC culture.................................. 76

4-12 Multicellular bridge at 10 days in the RMSC culture ................................ 77

4-13 Multicellular bridge at I11 days in the RMSC culture................................ 77

4-14 Development of bridging on stainless steel screens.................................. 79

4-15 SEM of stainless steel screens after 10 days in RMSC culture .................... 80

4-16 SEM of stainless steel screens after 12 days in RMSC culture..................... 80

4-17 SEM of stainless steel screens after 15 days in RMSC culture..................... 81

4-18 SEM of stainless steel screens after 18 days in RMSC culture..................... 81

4-19 SEM of stainless steel screens after 23 days in RMSC culture..................... 82

4-20 RMSC growth curves for each fiber density.......................................... 83

4-21 Schematic generalization of multicellular bridging .................................. 85

5-1 Eye shaped bridging phenomenon caused by the weave of the screen ............ 96

5-2 Pictures of mult-fiber micromanipulator.............................................. 98

5-3 Examples of multi-fiber constructs for RMSC culture .............................. 99

5-4 Diagram of stainless steel construct preparation.................................... 100

5-5 Diagram of stainless steel statistical test............................................. 102

5-6 Examples of single fiber RMSC growth ............................................. 106

5-7 Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 25 im. 107 5-8 Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 55 Pm. 108 5-9 Micrographs of multi-layer parallel arrays........................................... 108

5-10 Examples of bridging between fibers of 25 pm and 55 pm spaced parallel arrays. 109 5-12 Transmission electron micrograph montage of 7-0 single fiber construct in crosssection .................................................................................. 112




xi








5-13 Transmission electron micrograph montage of 7-0 single fiber construct in longsection ................................................................................................................ 113

5-14 Transmission electron micrograph montage of 7-0 multi-fiber 25 pm spaced
parallel array in cross-section ............................................................................ 114

5-15 Transmission electron micrograph montage of contracted 7-0 multi-fiber 25 Pm
spaced parallel array in cross-section ................................................................ 115

5-16 TEM images of longitudinal section of the contracted portion of 7-0 25 Wn
spaced parallel array ........................................................................................... 116

5-17 Characteristic banding pattern of collagen fibrils .............................................. 117

5-18 Schematic representation of flat surface multilayering versus bridging ............ 122

6-1 Surface of a 5-0 maxon fiber imaged with light microscope ............................. 127

6-2 Conceptual diagram of bone replacement .......................................................... 128

6-3 A possible sequence of growth factors for release in a bone replacement system. 131

A-I Diagram of NFF measurement ............................................................................. 132

A-2 Diagram of relationship between nuclear dimensions and fiber geometry .......... 133

A-3 Diagram of trigonometric approximations used to determine nuclear dimensions. 133

A-4 Diagrammatic comparison of NFF and nuclear angle ........................................ 134

A-5 Diagrammatic relationship between NFF measurements and nuclear angle
m easurem ents ....................................................................................................... 135

A-6 Diagrammatic representation of calculated rotational correction ........................ 136

















xii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

FIBER BASED SCAFFOLDS IN CONNECTIVE TISSUE ENGINEERING:
USING THE ARCHITECTURE OF WOVEN SCAFFOLDS
TO INFLUENCE AND CONTROL THE FORMATION OF ORGANIZED TISSUES

By

Charles Alan Seegert

August 2002



Chair: Anthony B. Brennan Ph.D. Major Department: Biomedical Engineering

The skeleton generates locomotion and provides mechanical support for the human body. It is essential in every aspect of normal living, which is most apparent when it is damaged or rendered useless by disease. The repair of the skeletal system by orthopedists is rather common, occurring millions of times each year. During the 1990s it was estimated that nearly a million cases per year were bone graft cases, which makes them one of the most common forms of transplantation in the United States today.

Orthopedic reconstruction restores ftmction, extending and increasing quality of life. Though progress has been made toward reaching this ideal, bone grafting is still fraught with shortcomings. Its weaknesses become most apparent when large skeletal defects like those seen in osteosarcomal resection are treated. Commonly these reconstructions are


xiii








performed using allografts combined with steel rods and other devices. Synthetic materials may also be used, but full skeletal incorporation never occurs and they remain inanimate. Because they are non-living, these materials accumulate fatigue and eventually fail.

Cell-based tissue-engineered replacements are a strong candidate to address these challenges. This treatment system would be composed of a synthetic material, like resorbable polymer fibers, combined with cells that are loaded onto the fibers. After implantation the system would foster development of a living and thus self-repairing replacement tissue.

This study focuses on synthetic scaffolds and how they interact with the cells loaded onto them. Established principles of contact guidance were applied to influence orientation and growth of cells on these scaffolds. Guidance of cells and their extracellular matrix (ECM) products is shown on the level of single fibers. More important, however, is the direction of cells and ECM when fibers were organized into regular 3-D structures. Cellular organization and direction far exceeded what has been seen on flat surfaces, or single fibers. The goal of this work was to use scaffold architectures to organize cells and ECM in a 3-D manner as is seen in normal tissues. The results of this study indicate the success of this concept and represent a large step toward the development of this technology.












xiv














CHAPTER 1
INTRODUCTION

In addition to providing mechanical support for the human body, the skeleton is integral in generating locomotion. Indeed the musculoskeletal system is an essential component in nearly every aspect of a person's life, a fact that is made most apparent when a portion of this system is damaged or rendered useless by disease. Unfortunately the repair of the skeletal system by orthopedists is rather common, occurring millions of times each year alone. During the 1990s it was estimated that nearly a million cases per year were bone graft cases [I], making bone grafting one of the most common forms of transplantation in the United States today.

The intent of orthopedic reconstruction is to restore fiction, thus extending and increasing the quality of life an individual experiences after injury. Clearly much progress has been made toward reaching this goal. However, despite this progress and the frequency with which bone grafting is used, this treatment method is fraught with difficulties and shortcomings. Implantation of a bone graft or a synthetic bone replacement material is performed when a void in the skeleton is created by trauma or resection for pathological treatment. Another major implementation is when injured bones fail to re-join as is seen in non-unions at a fracture site [2,3]. In these situations, emplacement of a bone graft, or a synthetic bone replacement material is then performed to bridge the gap, restoring skeletal integrity.


I







2

When reconstructing skeletal defects, three major graft materials are used: autogeneic bone, allogeneic bone and xenogeneic bone. Synthetic materials like porous hydroxyapatite and bioactive ceramics have been used in some cases, as well. A degree of success has been realized with each of these materials, but there are also significant characteristic limitations to all of them.

Autogeneic Bone Grafting

Autogeneic bone grafting is considered the gold standard and all other materials are compared to it when their efficacy is evaluated [4]. This type of bone graft is taken from a site within the patient's own body and thus it is recognized by the body when implanted elsewhere. Additionally, autograft bone is a living graft, which contains bone producing cells. Because of this feature, cells are deposited into the site where new bone growth is desired, thus stimulating a much more rapid recovery. This type of bone deposition and growth is "osteogenic," a classification that includes grafts containing phenotypically committed osteoblasts, or grafts that stimulate proliferation of committed osteoblasts [4]. Other graft materials depend on the infiltration of cells from the surrounding area; thus recovery takes longer if it occurs at all. This type of healing, which is characterized by materials providing a scaffold to direct bone growth, is called "osteoconductive" [4]. One further classification of bone graft materials that is particularly germane to this study is "osteoinductive" materials. Osteoinductive grafts lead to differentiation of mesenchymal stem cells, or osteoblastic precursors, thus causing them to become fully committed bone producing cells [4].

Though autogeneic bone is considered the best material, there are still significant drawbacks to its use. The quantity of this bone graft material available is very limited.







3

Clearly only so much bone can be removed from one part of the body in order to heal another part; anything more would only create a new defect in skeletal integrity. In addition to this, harvest sites often experience lengthy and painful recovery periods. In many cases the recovery of the harvest site takes longer than the recovery of the original injury the bone graft was used to treat [4].

Allogeneic Bone Grafts

Allogeneic bone grafts are widely used, as well. Unlike autograft bone, allograft bone is not as limited by supply. This material is taken from other human bodies, usually deceased, and then passed through a series of treatments ideally rendering it nonimmunogenic and free of pathogens. Allograft bone is used to repair small skeletal defects with much success. For this use, it is usually "morselized," or broken up into small fragments that are then packed into the fracture site. The volume of most defects treated this way is relatively small, which is related to the success of this application. Allograft bone is acellular and osteoconductive; therefore it must be infiltrated with bone producing cells in order to recover. Additionally, there are a number of growth factors that are utilized by these cells during recovery. Depths of cellular migration into the graft material, as well as the distance that growth factors can diffuse without degradation, are again quite limited [5]. Full integration of allograft bone does occur; however. These distance related factors seem to prevent integration with the body beyond relatively small distances on the order of a few centimeters [6].

In addition to treating smaller skeletal defects with morselized allograft material, large skeletal reconstructions are performed with intact, non-morselized segments of allograft bone. In situations like this the drawbacks of allograft bone become most apparent.







4

Integration by the body ensures the presence of cells and the formation of a living tissue. Living tissues are capable of self-repair and are regenerate in the presence of stresses associated with daily activity. Non-animate replacements, even though of a biological origin, do not repair themselves and normal fatigue processes are continuously at work.

Treatment of osteosarcomas often requires large resections. Historically, when this disease was encountered, a patient's limb was amputated leading to a life long handicap. In the early 1960s, however, limb salvage became the standard of care in these cases. Providing a mechanical means of support via implantation of allograft bone allows the patient to maintain the reconstructed limb and a much higher quality of life [7]. During limb reconstruction, allogeneic bone grafts are frequently augmented with metal fixation devices, like inter-medullary nails, or plates. Figure 1-1 shows an example of just such a construction using allograft bone.

Sometimes, in addition to the fixation devices, bone cements are also used, leading to large static composite structures. At best, these rigid conglomerates of organic and inorganic materials are fractionally incorporated into the patient's skeleton due to the distance limitations mentioned previously. This limited amount of repair occurs at the ends of the allograft, where it comes in contact with the living bone allowing cells to infiltrate [3]. The rest of the composite structure remains a rigid mass of dead bone and inorganic components and even the limited amount of incorporation described above only occurs about 70 % of the time [8].

This remedy is better than the alternative--no limb--but the allograft is never fully integrated and the accumulation of fatigue eventually culminates in failure of the device.







5


















Figure 1-1. An example of a femoral reconstruction using a segment of allograft bone. The white structure in the center of the image is a metal rod holding the composite in place.

This failure tends to occur in areas where a hole has been introduced into the allograft material, sometimes as soon as 1-2 years after implantation [9].

Synthetic Bone Replacement Materials and Xenogeneic Bone Grafts

Synthetic bone replacement materials and xenogeneic bone grafts, like allograft bone, are not limited by supply. Examples of this class of materials are porous hydroxyapatite, choraline hydroxyapatite and bioactive glass materials. Generally these materials are processed into bulk materials with a porous structure leaving relatively poor allowances for diffusion of nutrients. For example, porous hydroxyapatite only possesses a porous volume of approximately 30% [10]. Because of this, when it comes to integration these materials exhibit distance limitations in a way similar to allograft bone. Xenografts, or grafts derived from other species, are rarely used due to the high chances of rejection and the possibility of disease transmission between species [4].






6

Problem to be Approached

Bone grafting, particularly of large diaphyseal segments, is far from ideal. Synthetic materials can only be used to fill small defects, while large defects are repaired using allograft material. Allograft bone is useful in that it is of the correct dimensions and density and does possess some of its original mechanical strength; however, fatigue problems limit its viability over time. For smaller bone defects autograft bone is the material of choice, but it is limited in quantity and its removal often leads to a long painful recovery in the area from which it was taken. A number of synthetic materials have also been used with some limited success, but no truly suitable substitute yet exists, especially for large segmental bone defects [4].

After examining the currently used bone graft materials and methods, a number of deficiencies become apparent that must be overcome in order to develop more viable synthetic bone replacement systems. Opportunities for improvement are summarized in table 1-1.



Table I 1. List of Design Requirements for More Ideal Bone Replacement Material
-Unlimited Quantity

-No Pathogenic Transmission

-No Immunogenicity

-Osteoinductive and Osteoconductive to be Fully Incorporated over Large Volumes.
-Highly Porous to Allow Proper Diffusion and Infiltration of Cells.
-Mechanically Robust to Provide Support Until New Tissue Becomes Self Supporting.







7

Overall, a bone replacement system capable of filling large skeletal defects would be ideal. Using the replacement of a diaphyseal segment of long bone as a goal, or model system presents a somewhat simplified bone replacement problem. Figure 1-2 shows a schematic representation of this idea, which if successfully achieved would pave the way for more complex problems like joint reconstruction, or tendinous attachments. Additionally, many of the problems associated with replacement of bone in other areas of the body would be answered.





. .













XX








Figure 1-2. Schematic representation of diaphyseal bone replacement. A. Damaged, or diseased segment of a long bone. B. Removal of diseased area with margins of healthy bone, followed by emplacement of a graft/ engineered synthetic replacement. C. The reconstructed bone with natural and synthetic components. D. After healing and subsequent resorption of the synthetic replacement material a new segment of living tissue persists and the patient is healed.






8

Tissue Engineering

Tissue engineering is defined as: the application of engineering disciplines to either maintain existing tissue structures, or to enable tissue growth [11]. A tissue engineered construct would solve many of the problems associated with bone grafting. Indeed there are already many studies in the literature that implant scaffold materials loaded with cells taken from the patient, or subject [12-20]. This method, where autogeneic cells are taken from a patient, loaded onto a scaffold and then implanted, is referred to as a "cell based approach" to tissue engineering.

Using this approach requires a scaffold, or support matrix that cells can adhere to and proliferate on prior to implantation. Eventually this scaffold would resort and be excreted firorn the body, leaving behind a living segment of bone tissue that is completely biological. In this model the scaffold is only present to facilitate and direct the growth of cells and the deposition of cell products, while its eventual resorption allows the induced tissue to become completely integrated into a subjects anatomical and physiological systems. Resorbable polymers, particularly in the fiber form meet this requirement, as well as all the needs listed in table I 1. Using appropriate processing methods, individual fibers can be made with remarkably robust tensile strength [12-23]; more importantly, however, these fibers can be organized in a woven construct that is very rigid [11]. Once cells have been induced to adhere and grow on a scaffold they will require adequate nourishment and gas exchange, which presents another major advantage of fiber based constructs: difflusive properties [23,24].

As mentioned previously, other materials commonly used to replace, or reconstruct bone have relatively poor allowances for diffusion of nutrients and thus are limited.







9

Randomly packed fibers, on the other hand, exhibit porosities greater than 95%, allowing for much greater nutrient inflow and waste matter outflow [251.

The Case for Hierarchical Organization of Fiber Constructs

Bone, like many other tissues in the body, exhibits anisotropic mechanical properties. This directional difference in stiffness, which depends on orientation with respect to the bone's long axis, is a characteristic that is directly tied to its layered molecular and cellular organization. Bone's structure is hierarchical [26] with two levels of this hierarchy that are particularly relevant to its mechanical properties: its lamellae, or layered organization and the collagen fibril arrays within each lamella. Lamellar units are approximately 3 gm wide and are oriented in a direction parallel to the long axis of the bone itself (Figure 1-3).











Figure 1-3. A diagram of the general orientation of lamellae in a segment of bone. Red arrow points out the plane the lamellae are oriented in is in the direction of the long axis of the bone. Adapted from Liu et al. [63].

Each of these arrays, though rotated around an axis perpendicular to the bone's long axis, remains parallel to that axis in their other dimensions (Figure 1-4). Each layer represents an oriented collagen fibril array and as these parallel fibrils are stacked each layer is rotated approximately 30 degrees, as is the case in bone [27,28]. The orientations







10

of these collagen fibrils and the presence of many lamellae are responsible for the mechanical anisotropy of bone as will be discussed more thoroughly in the background.

Logically, when engineering a replacement for large segments of bone, it is desirable that these systems introduce a similar anisotropy. Fibers below a diameter of -100 gm have been shown to influence the orientation of cells grown on them, a phenomenon known as contact guidance [29]. Cells that have been oriented using principles of contact guidance have also been shown to deposit their extra-cellular matrix (ECM) products parallel to their orientation [30-32]. Most notably this has been seen in cases where fibers are used to replace tendons [30].














Figure 14. Schematic representation of collagen fibril orientation within a segment of bone. The cylinders that are fanning out with respect to each other (red arrows) represent the orientations of collagen fibrils as they are organized within a single lamellae. Therefore each lamella is composed of collagen fibril layers organized in this pattern. Adapted fi-om Weiner and Wagner [26].

By creating parallel arrays of fibers that are then organized into layers it stands to reason that cells oriented by each lamella would deposit ECM in a manner directed by that layer (Figure 1-5A). Many layers sandwiched together with angles of rotation between adjacent layers conceivably could produce an anisotropic engineered material (Figure 1-5B). This material would be a laminated composite similar to that of normal







11

bone. Weaving scaffolds with desired orientations would then make it possible to design large scale constructs with the architecture necessary for replacing large segments of bone (Figure 1-5Q.


C



B e "











Figure 1-5. General concept of the hierarchical level of woven fiber based scaffolds. A.) represents the effects of the first level, which is composed of the fibers themselves. B.) Shows the second level of the hierarchy, which is weaving of the individual fibers to form large woven sheets responsible for cellular orientation on a lamellar basis. C.) Finally combining these many lamellae together and putting them in to a 3-D structure provides the highest level of the hierarchy and even more mechanical integrity.

A first step toward developing this technology is understanding how mesenchymal stem cells, the osteoblastic precursors, interact and are oriented by fibers over time. Additionally, understanding how MSCs are influenced by the spacing and organization of fibers in their multi-layer configurations is a key requirement.

The specific aims of this work are designed to elucidate the effects on cellular activity resulting from some of the levels in the hierarchy described above. Studying and extending what is known about the effects of fiber diameter on cellular and ECM orientation will be first. Following the single fiber studies will be multi-fiber studies, which focus on the effects of fiber spacing within a construct composed of fibers







12

arranged in parallel array. Finally, multi-layered constructs with parallel arrays arranged as shown in Figure 1-513 will be examined.













CHAPTER 2
BACKGROUND

To engineer any synthetic tissue replacement material, it is essential to have an understanding of the normal physiology of that tissue. Once a familiarity with the systems involved and the interplay of these systems is achieved, likely sites of manipulation become evident. Controlling these critical sites and thus the development of the desired organ system is the goal of a true tissue engineer.

Bone Formation in Utero

Bone formation begins early on in fetal development and progresses rapidly

throughout normal gestation. The cartilaginous beginnings of the skeletal system are seen as early as the first month of fetal growth when mnesenchymal cells begin to lay down different forms of collagen in an organized manner. These cells separate into two layers, the outer layer, which is composed of differentiated fibroblasts, and the inner layer, which remains undifferentiated mesenchymal cells. These layers together are referred to as the perichondrium, a structure that later becomes the periosteum. [33].

Ossification, or the mineralization of the developing skeleton, begins in the second month of pregnancy and proceeds via two mechanisms. The first mechanism of mineralization to occur is known as intra-membranous ossification and it takes place in sites like the calvarium and the clavicles. Intra-membranous ossification is a direct mineralization in the connective tissues of the fetus, which contrasts with the other mechanism of bone formation: endochondral ossification.




13






14


Endochondral ossification occurs after a scaffold of cartilage is laid down. This

scaffold of cartilage is then mineralized and eventually replaced with bone that is guided by its presence. The only difference between intra-membranous ossification and endochondral ossification is the latter's requirement of a cartilaginous scaffold, which must be deposited first [33].

After the initial mineralization of the cartilaginous scaffold, the development of true bone begins. This formation of true bone begins following the infiltration of vasculature and subsequent supply of oxygen and nutrients [33-34]. MSCs are the developmental precursors to nearly all the connective tissues in the body as schematically shown in Figure 2- 1. The presence of oxygen as supplied by the vasculature plays a significant role in controlling the level of differentiation achieved by MSCs [33-35].








-Bone
-Cartilage
Interh~se-Ligament
-Tendon
-Marrow
-Connective Fibroblast
C, S G2 M-Dermal Fibroblast






Figure 2-1. Schematic of the cell cycle. Top left corresponds to the diagram of cell division below it; outlining the proliferative stage of MSC development. With the advent of correct environmental cues MSCs undergo differentiation and enter the Go phase. Depending on the cues sensed by MSCs they are capable of becoming many different tissue types as listed on the right.






15


MSC Differentiation

Transformation of MSCs into mature bone producing osteoblasts is a multi-step

process involving a number of environmental and cell based signals. This differentiation occurs in the Go phase of the cell cycle (Figure 2-1), which is also known as the quiescent phase since cells are no longer multiplying [3 6-391. The diagram also shows that differentiation at this point leads to different tissue types depending on the cues provided by the environment.

It is hypothesized that bone development along this pathway depends in part on one major environmental cue: the supply of oxygen by newly formed vasculature [33-35]. Indeed it appears that MSCs become fibroblasts if there is a relatively low supply of 02, but osteoblasts if 02 is readily available. Once neovascularization has occurred and this signal has been received, passage of MSCs to fully developed bone producing cells proceeds along an orderly and well-defined path, which is reviewed below. This development is governed by a cascade of cellular based signals, or cytokines, which act in close concert with the cellular events seen in the developing tissue.

The differentiation of MSCs that leads to bone occurs in the GO phase of the cell cycle and is schemnatized below in Figure 2-2. Osteogenic cells develop in a three stage process with two point in between called restriction points [36-39]. Differentiation and advancement through each point are restricted until certain conditions are met; then development may proceed [36,37].

It is generally held that this progression of cellular events is regulated by an intricate system of feedback mechanisms and chemical cues among the developing cells themselves and between the cells and their environment [40-421. The proteinaceous chemical cues, or cytokines, involved in osteogenic cell development are composed of a






16

family of growth factors called bone morphogenetic proteins (BMPs). This family of proteins is quite large and includes the TGF-P isoforms. Each protein is numbered; for example the protein involved in the first stages of MSC differentiation is labeled BMP-2.

The role of the BMPs in bone development has been examined from many different viewpoints. One type of study involves following bone formation, while examining the locations and temporal sequence of the individual components of the skeletal system that are produced [43]. Other types of studies follow the production of growth factors and BMPs spatially and temporally in vivo. The first type of study shows each cell product is characteristic of a particular stage of cellular development. The second type of study shows where and when the growth factors and BMPs are released, allowing the two to be inter-related by shared temporal and spatial relationships [40,411]

In addition, there are other studies that focus on the function of individual growth

factors and BMPs, which provide corroborating evidence for the conclusions drawn from the inter-related temporal and spatial studies [44]. Essentially knowledge of the BMP location in the temporal and spatial sequence can be ascertained by the cellular responses they have been seen to induce. The validity of correlating specific protein production with a level of bone cell differentiation, as is described above, has been examined in the past and found to be acceptable in analogous situations [44]. Using this method, the systemic influences that guide the development of the cells and the location of these BMPs in the cycle can be understood.

The development of osteogenic cells proceeds in a three stage process with two points in between called restriction points (Figure 2-2). Differentiation and advancement






17

through each point are prevented until certain conditions are met; then development may proceed.

Stage one is proliferation. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-P3 (TGF-P) are active during this first and earliest phase of cellular development. BMP-2 is responsible for the initial levels of differentiation [45]. In fact BMP-2 is specifically located to MSCs, which are considered its target cell [41]. TGF-P is largely responsible for proliferation, or expansion, and it also stimulates production of extra-cellular matrix (ECM) components [35,46]. BMP-2 and TGF-P3 each have an influence on MSC development when they are present individually, but when they are both present concurrently, they act in a synergistic manner [47].



1PROLIFE oN

NEGATIVE FEEDBACK COLLAGEN/
DOWN REGULATING FIBRONECTIN
PROLIFERATION SYNTHESIS


ECMVMATURA1O ,*,
NEGATIVE FEEDBACK
DOWN REGULATING
ECM MATURATION
ECM MINERLIZATION I-


Figure 2-2. MSC developmental sequence leading to bone producing cells. This sequence occurs in the Go phase of the cell cycle as seen in Figure 2-1. Adapted from Stein etal. [36].

In addition to proliferation, the production of ECM is also an important aspect of the first stage in bone development [48]. TGF-13 stimulates production of collagen I, but collagen II is also produced in significant portions during this time. Collagen II is integrally associated with endochondral ossification [44] and a cell product that has been






18

associated with BMP-4 [49,50]. BMP-4 appears to be responsible for the number of chondrocytes that are recruited into the bone producing pathway [51 ], and it has been linked to the production of alkaline phosphatase [52,53]. Alkaline phosphatase indicates that the cells have progressed past the first restriction point, which implicates the value of BMP-4 in stage one with its activity beginning after TGF-P3 and BMP-2 have started to produce their effects.

The first restriction point resides between stage 1 and stage 2 of MSC differentiation. Proliferation and production of ECM occur simultaneously. During stage one, cells continue to proliferate until they are closely associated with each other, forcing each other into a less flattened shape. This change in cell density [36] and shape [54] signals the cells to further differentiate, forming products that lead to ECM maturation, the next stage in cell development.

The ECM and developing osteogenic cells also interact enhancing differentiation and cessation of proliferation, as BMP-4 comes into play. This occurs in a feed forward mechanism where the ECM influences the cells, which in turn influences the ECM [55]. So cell shape (which depends on proliferation) and the ECM largely dictate passage into the second stage.

The second stage of differentiation is the maturation and modification of ECM, thus preparing it for mineralization. The Vgr-1 gene is produced at this time by osteogenic cells and localized into the ECM surrounding hypertrophic chondrocytes [43]. This specifically happens around cells that are implicated in mineralization later. Vgr-1 has been associated with vascularization as bone development continues [56], as well as the further differentiation of osteogenic cells. Interestingly, Vgr-1 must be integrated into the






19

ECM for it to be active, which suggests as conformational change, or cleavage of a portion of the molecule by the ECM.

The Vgr-1 gene leads to the production of BMP-6; that is BMP-6 is the gene product of Vgr-1. BMP-6 leads to the production of the LMP-1 protein, a cell product which is important and necessary for the final differentiation of MSCs. LMP-1 is not regulated by BMP-2, or BMP-4, only BMP-6 has influence on this protein [57], which makes it the main growth factor involved in the second stage of MSC differentiation.

Another aspect of the second developmental stage is the preparation of the ECM for mineralization. It is thought that mineralization of bone requires nucleation sites for the hydroxyapatite crystals that compose bone. Bone Sialoprotein, (BSP) a non-collagenous ECM protein, is expressed in high levels in areas of bone that first begin mineralization, showing its probable importance as a nucleation [58]. This evidence is further supported by in vitro studies, which show BSP specifically causes nucleation of hydroxyapatite crystals, where other non-collagenous ECM proteins do not [59].

The second restriction point is after the ECM maturation of the second stage. BMP-6 and the presence of BSP prepare the cellular environment for this transition, which occurs when that environment is adequately supplied with the necessary quantity of ECM (BSP, collagen, etc...) and level of differentiation [36,37].

Mineralization is the last stage in bone development and MSC differentiation. Mature osteoblasts lead to the production of a number of non-collagenous protein, which are integral for mineralization. This production largely occurs during the second stage of development with the activity of BMP-6 and BSP. There are proteins, however, that are






20

produced during this last stage of development. Osteocalcin is one of these proteins and is a calcium binding protein necessary for mineralization [46].

OP- I (BMP-7) has been purported to be responsible for mineralization and, in conjunction with the ECM changes, cause terminal stages of osteogenic cell differentiation [60]. OP-i also leads to the up-regulation of BMP-6 and the downregulation of BMP-2 and -4, which implies that its activity normally occurs in the later stages of cell development [61].

OP- I has been shown to induce differentiation of osteoblasts and production of bone in a number of studies. Its role, however, appears to be in this later stage of bone development. Using OP- I on cells that have varying levels of development, causes many of them to differentiate before they normally would without its stimulation [44]. Many more cells can be stimulated to begin mineralization, inducing bone formation. OP-i1, however, does not lead to ECM production [44]. Essentially, if OP- I is present too soon it forces cells to differentiate before they can produce the necessary ECM for vascular development and other features of filly formed bone.

Presence of OP- I can lead to fully developed bone when it is administered alone

[44,45], but important aspects of development that are dependent on ECM and the noncollagenous proteins are attenuated. This implicates OP- I as the signal protein that is most important later in development, whose action is to stimulate final differentiation and aid mineralization. After the ECM has been developed by the earlier stages of growth, cells that are growing exist in population with varied levels of differentiation depending on the specific local environment. Terminal differentiation stimulated by OP- I seems to influence all cells at their given stage of development, speeding them to final






21


differentiation and mineralization, bringing the whole tissue to a final differentiated whole.

Bone Development and Bone Anatomy

Growth occurs rapidly at the ends, or epiphyses of developing bones and more slowly toward the centers of the shafts, or the diaphyses. Rapid growth primarily leads to spongy bone, while the relatively slow build up of bone that occurs in the diaphysis is more compact and dense. Spongy bone is composed of trabeculae, a porous honeycomb of interconnected bony processes. Trabeculae initially develop with a random orientation, but with the application of the stresses associated with living the orientation of these processes assume a pattern (Figure 2-3). In a probabilistic manner, trabeculae oriented at


Force












Figure 2-3. Lines of force seen in proximal portions of the femur. Weight bearing pressures as represented by the arrow are responsible for development of trabecular organization Adapted from Sinclair [331.

odd angles with the lines of stress imposed on the bone are broken down, while those that are aligned are relatively unchanged [33].

Bone tissue has been optimized through evolution in ways that are very specific to its function. During development, as mentioned above, it is remodeled through the stresses associated with weight bearing and muscular tension, so that it exhibits properties of






22

mechanical anisotropy. The source of this anisotropy is traced to the macromolecular and cellular level of bone.

There are a many different types of bone to be found in nature, but one of the most important is larnellar bone. This bone type, named for its organizational structure, is the most common bone type found in humans and is primarily responsible for the load bearing fimction of the skeleton. Lamellar bone consists of many mineralized layers, a characteristic which was noticed as early as 1906 [27]. The elucidation of the lamellar structure has taken some time, but now it is generally held that a "rotated plywood" motif best describes the organization of this bone type [26-28, 62,63].

The structure of bone is hierarchical [27]. Two levels of this hierarchy that are

particularly relevant to its mechanical properties its the larnellae and the collagen fibril arrays within each lammela. Lamellar units are approximately 3 Pm wide and are oriented in a direction parallel to the long axis of the bone itself (Figure 2-4). The next step down the hierarchy is the collagen fibril. Each lamella is composed of a number of collagen fibril arrays rotated at angles of about 30 degrees with each other. That is, each subsequent collagen array is rotated with respect to the one before it as one imagines, passing from one lamellar boundary to the next. Each array, though rotated around an axis perpendicular to the bone's long axis, remains parallel to that axis in their other dimension (Figure 2-5). When the rotated plywood structures, which compose larnella, are viewed in cross section they produce microscopic patterns referred to as "nested arcs" (Figure 2-613) [28,62].





23













Figure 2-4. Examples of lamellar orientation within a section of bone Adapted firom. Liu et al. [63].

.... ..........











Figure 2-5. Fibrillar orientation within bone. Fibrils are oriented in one plane which parallels that of the bone's long axis, while rotating at 30 degree increments with respect to each other. Adapted from Weiner [27].

Figure 2-6A shows how the rotated layers, when stacked, produce this effect. Each layer represents an oriented collagen fibril layer and as they are stacked each layer is rotated approximately 30 degrees, as is the case in bone [27,281.

One can envision how the orientations of these collagen fibrils and the presence of many lamellae make bone so mechanically anisotropic. The anisotropic nature of bone has been studied for some time, particularly on the macroscopic level. Directionally oriented bone specimens, which are very large compared to the larnellar sub-units, have been subjected to stress-strain measurements. Tensile and compressive examinations of






24

P/2




. . 2 A[A]











Figure 2-6. Rotated plywood model. A.) Drawing of many parallel fibril arrays each rotated 30 degrees with respect to the adjacent layers. The blue arrows highlight the centers of the nested arcs. B.)Notice how the rotation leads to a nested arc motif as seen in this cross-section. Adapted from Giraud-Guille [28]. these samples have shown that lamellar bone possesses markedly higher modulus values when loaded parallel to its long axis than in any other direction [27,641. Extending these measurements down to the lamellar and fibrillar subunit scale has been difficult, however, some results have been forthcoming supporting the relationship between anisotropy and the rotated plywood model.

Using microhardness instruments, the presence of anisotropy on a very small scale was related to the orientation of the mineralized fibrils [65]. Similarly, stress-strain data using very small scale bending specimens (-160 Lm diameter) supports the relationship between the lamellar structure and its function [63].

Overall, the numerous layers present in lamellar bone confer its highest strength in the longitudinal direction. In addition to this quality, the multi-angular orientation of the rotated plywood model makes the bone very resistant to fracture with the application of lateral stresses [26]. These examples demonstrate a relationship similar to what has been






25


described in aortic leaflets and arterial tissue, namely: the lamellar nature of bone, which is derived on the macromolecular and cellular level, is a quality integral to its physiologic function.

Fracture Healing and Ectopic Bone Formation Skeletal repair after fracture follows a sequence of events that is virtually analogous to that seen during development [35]. MSCs gather at the fracture site and form a repair blastema, or fracture callus. If the fracture site is stable and not subject to micro-motion, the MSCs will directly differentiate and become bone producing cells. If, however, there is instability that allows small amounts of motion the MSCs will form a more cartilaginous callus that will stabilize the break. With stability the ability for vasculature to successfully infiltrate increases, a condition that leads to endochondral ossification [35].

Ectopic bone formation, or the formation of bone in sites well away from the skeleton proper, proceeds very similarly to that of fetal bone formation as well. Use of demineralized bone chips, or polymer carriers loaded with BMPs leads to the development of ectopic bone [ 15,3 5,66]. The initial inflammation associated with implantation of the bone chips, or carrier is responsible for delivering MSCs to the ectopic site, which can be subcutaneous, or within a muscle. Figure 2-7 schematically represents this process, which again is dependent on the infiltration of vasculature.

Porosity and Diffusion Properties of Implant Materials

Proper porosity is an integral quality in a scaffold material as it controls the influx of nutrients to the cells. Initially the nutrients must be able to diffuse into the scaffold until such time as new vasculature has established itself. In order for vascularization to occur






26



Encysted
Demineralized by MSCS Cartilage Differentiation
Bone Chips


00









Bone Formation I Vascular Cartilage Hypertrophy/
Marrowization Invasion First Bone Formation



Figure 2-7. Schematic representation of ectopic bone formation around implanted, demineralized bone chips. It is thought that demineralized bone chips lead to ectopic bone formation because they contain BMPs. Adapted from Caplan [35]. the pore structures must be on the order of 200-500 pm in diameter [14]. This requirement is easily manipulated by varying the weave, tightness and diameter of the fibers used in the fabric.

There are many studies in the literature that attempt to use osteoconductive scaffolds to act as a synthetic bone replacement material [4,6,10,67]. Osteoconduction is defined as a material that allows vascular ingress, cellular infiltration, cartilage formation and mineral deposition [4]. These properties are indeed required in a bone replacement material, though they are by no means inclusive. Loading scaffolds with MSCs has also been used with some success in studies examining bone replacement [ 10,16,67,681. Osteoblastic precursors, which differentiate and become fully functioning osteoblasts are encompassed in a phenomena defined as osteoinduction. Any method or material that






27


induces differentiation of precursor cells into adult osteoblasts is included in this definition.

Large mechanical supports like allografi material have been shown to be

osteoconductive, but only at the ends of the allograft where it contacts the living bone [3]. Allograft bone has been fully incorporated in some instances where it has been used to fill areas around a collapsed acetabulum [6]. This was limited to only a few centimeters of material that was surrounded by living bone on all sides. Tricalcium phosphate scaffolds with pores ranging from 100 gim to 300 gim and a 36% porous volume show similar results, with vascular and cellular invasion of only 0.75 cm into the synthetic [10]. Replacing large volumes of bone has never been successful in this respect. Scaffold materials when used alone; seem to limit the nutrient supply, which prevents continued ingrowth.

Fibers have also been effective as a substrate in fixed bed bioreactors. Fiber type beds, or substrates, possess porosity much higher than other types of fixed beds (> 90% porosity). This porosity facilitates nutrient exchange by increasing the volume of medium allowed in and out of the scaffolds structure [24,25]. Because of the fiber architecture versus beads, or some other substrate, the cells in bioreactors with fiber beds produced

0. 15 IU of interferon per cell greater than an order of magnitude higher than cells on other substrates. A lack of diffusive ability has been a major drawback of the porous scaffolds mentioned previously.

A fixed bed is basically a solid support matrix that is used in bioreactor technology to maintain contact dependent cells in the culture environment. Its 3-D nature increases the surface area for cell adhesion beyond that of a 2-D culture dish. Some systems use glass






28


beads to pack the fixed bed, but their geometry limits the void fraction available for cell growth and diffusion [24]. Using 3 mm glass beads the void fraction is as low as 35 %, but using fibers the void fractions increase to > 90%, providing much better flow medium flow and thus greater cell growth [25].

Remodeling and Cellular Orientation

During development, as well as fracture healing reorganization of cells and

remodeling of their ECM products occurs. This remodeling and organization is in response to mechanical stresses and is responsible for the development of anisotropic strength characteristic of bone [33]. This portion of the healing sequence can be quite lengthy and logically, it depends on how much the cells are organized when remodeling begins. It stands to reason that cells that start the remodeling process in a more organized state will have to undergo less change than randomly oriented cells and ECM.

A number of resorbable polymer scaffolds have been examined for use as scaffolds. Martin et al. used porous polyglycolic acid and polyethylene glycol scaffolds, which showed the ability for MSCs to differentiate when loaded on the scaffold [ 17]. Similarly, Holy et al. used porous polylactic acid scaffolds in vitro, demonstrating normal cell activity as well [12]. In vivo a number of studies have been performed using porous ticalcium phosphate, or hydroxyapatite ceramics [ 14,16,18-20]. Using MSCs, these implants show a development of bone tissue that incorporates the degradable biomnaterial. Both polymer and ceramic systems, however, show cells that appear to be randomly oriented. No effort has been made to examine cell orientation in any of these studies.

Though the problem of fatigue and tissue replacement is addressed with the

development of cell based tissue engineering technology, so far there has been no attempt to organize the implanted cells, or direct the ECM and mineralization products they lay






29

down. As reviewed next in the contact guidance section, there have been many studies performed in vitro that indicate the efficacy of this type of cellular and ECM organization. Applying this knowledge to cell based tissue engineering is the next step in this area of research and the focus of this work.

As we have seen, cellular and molecular organization is primarily responsible for the mechanical properties many tissues possess and so accomplishing this organization seems to be a worthwhile goal. A self-healing, or living tissue engineered replacement possessing anisotropic: mechanical properties similar to bone, may be the solution to many bone replacement problems.

Contact Guidance

Cell systems and tissues are influenced by a number of factors during their

development and the course of their existence within an organism. Two of the most prominent factors in vivo appear to be chemistry and topography. Chemical cues have been shown to effect cell activity during the phenomenon of chemotaxis, a cellular movement toward a gradient of a chemo-attractant molecule [391. Cells guided by this type of stimulus will migrate in the direction of a released cell product thus localizing themselves to the site of injury, or need, as in the migration of leukocytes to damaged tissue [69]. Upon fracture, it is likely that bone repair is instigated in an analogous fashion by the release of chemical agents like TGF-0 and other proteinaceous cell products, thus attracting osteoblastic pre-cursors to the site [41,46].

Another form of influence on cells, which may be considered chemical is cell

binding to specific receptors, or arrangements of chemical molecules that are bound to a surface (Figure 2-8). Protein mediated receptor binding to substrates is an event that






30




4W)





Receptors
Fibronectin (Fn) in ECM



Figure 2-8. Receptor mediated adhesion of osteoblasts via fibronectin. Other ECM proteins perform similar functions, though Fn is one of the most prominent. Adapted from Alberts etal. [39].

occurs in numerous cell types within the body including those of endothelial (vascular endothelial cells) [70-72]1, mesenchymal (mesenchymal stem cells and fibroblasts) [20,73,74] and epithelial cells (neural) [75] origin.

Attachment of cells to substrate surfaces is almost always mediated by protein

adsorption [76]. These proteins are components of the serum used for cell culture and include fibronectin and vitronectin among others [76,77]. Adhesion and attachment by cells to these proteins depends on the ability for the proteins to adsorb to the surface, which in turn is dependent on the surface energy of the substrate. These proteins must also interact with the substrate in a way that does not change their conformation, thus remaining recognizable to the cell. Adhesion and attachment have been shown to influence rate of proliferation, or growth [71,72,76-79].

Modulus and stiffness of the substrate also have an effect on cell function and growth. Studies performed using collagen gels as scaffolds for cells of mesenchymal origin show that increased stiffness of the scaffold leads to an increased rate of proliferation. In addition to this increased rate the duration of proliferation persisted longer than that seen





31

in scaffolds with less stiffness [801. In similar studies collagen gels were anchored on one axis leading to a preferential tension development in the direction of this axis by fibroblasts. Cell numbers increased by five times in the anchored gels and ECM generation was greater than that seen in the un-anchored gels, which in contrast demonstrated a five times decrease in cell number [8 1 ].

Another major form of cellular influence: topography, has been shown to direct the function of cells in vivo, as well. Neural cells migrate along the lengdi of radial glial cell fibers until they reach their destination, thus being guided to different regions of the central nervous system [82,83]. Basement membranes, the thin layer that many cell types grow directly on, also have an inherent topography that has been suggested to influence corneal epithelial cells [841 and renal endothelial cells [85).

Introduction of synthetic devices into the body disrupts many of these cell and tissue systems, which then try to recover and re-establish stability in the presence of the implant. Implant design has come to focus on minimization of these disturbances and optimization of the cellular response to these devices once they have been placed in vivo [86]. Ideally cells could be induced to respond to devices in an optimal manner by using their inherent cellular mechanisms and machinery, which allow them to exist and live in their normal environment. Inducing migration of a given cell type, or using microtopography to direct a cell's function are examples of this concept.

Toward this end, many studies have been performed that examine how topography and chemistry influence cell shape [70,87,88], migration [75, 89-911 and function [72, 91,92]. This cellular direction, which is known as contact guidance, has been widely examined and found to occur in many different cell types. Similarly, studies have been performed






32


that use niicropatterned, chemical cues to spatially guide cell growth, in addition to influencing cell function [70,72,77]. There have also been a number of studies that examine the effects of receptors and receptor-like molecules on cell activity.

Contact guidance, the topographical, or chemical control of cellular orientation and

activity, is well supported by many studies in the literature [75,82-91,64,79,93,94]. Some of the earliest experiments studied the influence of topographical cues by examining how cells oriented themselves on glass fibers [85]. Since then the electronics industry has developed viable micro-fabrication methods and a number of studies have shown how topographical cues with specific sizes and patterns can invoke cells to behave in predictable ways [64,79,90,91,93,94].

Using these methods, many cells of mesenchymal origin have been shown to orient

themselves parallel to micron scale topographical features, like ridges [64,86,93,94]. This is also true of osteoblasts, the primary bone forming cells in mammals. Once oriented, osteoblasts and osteoblast-like cells will mineralize and lay down extra-cellular matrix parallel to microtopographical features on a culture substrate [31,32,91]. In fact in vitro mineralization of bone has been directed on a macro-scale merely by scratching the surface of a culture dish with sand paper [3 1].

Since the aforementioned development of micro-fabrication in the electronics industry a number of studies have shown the specific effects that topographical cues can have on cell shape and activity. Cues with specific sizes and patterns, which were created using the micro-machining technology, can invoke cells to behave in predictable ways [64,79,82-91,93-95]. A number of theories have been put forth regarding the mechanisms of contact guidance. The direction of a colloidal exudate from the cell by the grooves






33

[76], avoidance by cells of discontinuities in their paths [29], as well as the thought that focal contacts may only form on the tops of the ridges [32]. All of these theories enjoy some degree of experimental support, though which has the most dominant effect is not clear at present. There does, however, appear to be some correlation of feature size with the influence on cell shape. Groove dimensions that seem most effective across many cell types, are grooves with dimensions on the order of magnitude of the cell [76]. This is not always the case, however. In a recent study by Nealy et al., which used nano-scale features, contact guidance was exhibited by corneal epithelia] cells [96].

In addition to glass fibers, early contact guidance studies often used spider webs [32] to orient fibroblasts. This effect was achieved below a critical diameter of 100 lim. Above 100 jim, the cells were no longer oriented on the fiber, which led to the hypothesis that cells were unable to bend around a certain sharpness of curvature. That is, the cells could not bend around the circumference of a certain fiber once its diameter had gone below a certain point, leaving them no recourse, but to elongate in the direction of the fiber's long axis [29,97]. Fibers have also been shown to orient many other cells types including neurons, schwann cells, macrophages and transformed BHK fibroblasts [75,98]. In these studies the contact guidance effect was shown on other fiber types too, namely carbon filaments and synthesized fibronectin filaments.

Architecture of Fiber Based Scaffolds: Development of Cell-Based Tension

Using the phenomena of contact guidance, which orients not only cells, but also their ECM products, advanced scaffold materials can be designed. Commercial fibers can be woven into textiles of various 3-1) configurations possessing strong mechanical properties. Adjusting the tightness and geometry of the weave, as well as the diameter of






34


the fibers allows a number of control features. Fiber based scaffolds can be designed to allow for initial nutrient exchange, as well as longer term vascular infiltration. In addition the direction of cellular growth and ECM deposition can be influenced so that a decreased remodeling requirement is present after the newly created tissue is formed. That is cells can be aligned to a degree that is close to the alignment and orientation they will exhibit after remodeling and so will be less distant from their equilibrium state.

An extensive review of the literature indicates that one of the key components of connective tissue cellular physiology is the achievement of proper cell spreading, or tension. Cell spreading is a phenomenon influenced by topography, chemistry and stiffness of the substrate the cell is bound to. These are the elements of contact guidance as reviewed earlier. As we saw, surface properties led to proper adhesion through the protein mediated receptor binding (Figure 2-9). Topography and chemical micropattems also influence adhesion and the direction, which cells are able to achieve stretch. Indeed spreading and the achievement of proper cell tension appear to be one of the main requirements for normal cell function.

Without adhesion, contact dependent cells, like those of mesenchymal origin remain rounded without inducing tension and fail to differentiate [54,88,92,99]. On another front inhibition of stretch mediated chloride receptors attenuate response of connective tissue cells to topographical guidance [100], strongly implicating stretch as a requirement in topographical guidance. Mechanical stretching of the substrate cells reside on has also been shown to direct cellular orientation and deposition of ECM proteins in a MSC system [ 10 1 ]. Indeed a translational strain of 10% applied concurrently with 25% rotational strain was responsible for increasing the alignment of these cells 2.5 times






35


when compared to controls, which received no stimulus [ 10 1]. In addition to cellular alignment, collagen fibrils were found aligned as well, when no fibrils were even seen in the control constructs. Interestingly 0.2 grams of tensile stress has also been shown to increase BMP-4 by two times that of control as part of suture development in the mineralization of rat calvaria [102].

So in addition to the development of cellular tension, which is necessary for normal and optimal cell function, cellular stretch plays a role in developmental process. Indeed stretch mediated receptors may be the signaling mechanism responsible for translating cell based tension generation into enhanced cellular function and differentiation. Therefore scaffolds developed for application in connective tissue engineering should include consideration of these phenomena in their design.

In tissue engineering applications, scaffolds loaded with cells are often seen to

contract through the development of cellular based mechanical force [80,81,103-105]. This phenomenon has been strongly associated with the contractile protein "Smooth Muscle Actin" (SMA). SMA is so named because it was first discovered in smooth muscle cells, but it is also found in other cells of mesenchymal origin like fibroblasts and osteoblasts. In fact it has been shown that MSCs and osteoblasts, will contract tissueengineered scaffolds when they are loaded onto them [103,104,106].

Contraction by osteoblasts and other cells of mesenchymal origin occurs in close

relation to the numbers of cells loaded on these scaffolds [ 104,106]. Once loaded onto the scaffold, the force generation is linearly associated with the numbers of cells, which indicates a collective effort by the cells leading a total force generation [105]. This relationship between cell number and contraction also indicates that cellular






36


communication is occurring through gap junctions [ 104]. Gap junctions exist on the physical level and are essentially pores that interconnect adjacent cells, or cells that are in actual contact with each other. Gap junction interactions have been implicated in macroscopic phenomenon of scaffold contraction [ 104], which indicates these cells are acting in a multi-cellular manner. In fact these findings suggest that osteoblasts and connective tissue cells are acting in an aggregate manner with many physically associated cells generating force cooperatively.

The importance of tension generation by connective tissue cells becomes apparent

when its effects on cell function are considered. When seeded on collagen and collagen glycosaminoglycan scaffolds proliferation of cells is markedly enhanced, but only when the gels are secured to the culture dish. Free-floating gels/scaffolds experienced a regression in the number of cells, a fact attributed to their inability to generate tension [81,103]. In a similar manner, when a series of collagen scaffolds with increasing degrees of cross-linking leading to increasing amounts of stiffness are used, the stiffest formulations led to the greatest proliferation rate and duration [80].

In addition to enhanced proliferation, the ability of cells to generate tension led to increased production of collagen [8 1 ], as well as increased production of proteins, calcium and alkaline phosphatase [103]. All these responses indicate increased levels of differentiation by cells, which also has been attributed to the ability to generate tension.

Overall it appears that connective tissue cells lead to contraction of scaffold material as each tries to achieve the an optimal tension, or stretch necessary for proper differentiation and optimal fiction. The sum of these many individual cell tensions leads to a cooperative force causing the whole construct to contract. It also becomes apparent






37


that these cells are not acting individually, but are physically linked to each other in an aggregate manner, allowing cell communication through gap junctions.

This contraction is commonly seen in wound healing and it can lead to scar formation [107]. Similarly it is part of the normal healing cycle seen in tendon healing [ 108]. The same proteins that cause this in other closely related cell types are found in Osteoblasts and MSCs, therefore it seems likely that this phenomenon is a part of normal fracture healing. In fact it follows logically that contraction of cells healing a fr-acture would be useful in bringing separated fragments of bone back together. Given the importance of this cellular activity, it must be considered in the design of a fiber-based scaffold's architecture and spacing.

Proper spacing of fibers is necessary to allow optimal cell tension and stretch, but also it must be optimized for the aggregate activity, or cooperative bridging of cells occurring across gaps in the scaffold. During connective tissue healing, a fibrin clot acts as scaffold allowing cells to migrate from areas of healthy tissue to the area of injury [108]. Without this scaffold, or a synthetic replacement, in vitro studies show cells are unable to bridge gaps as small as 50 pm [ 108].

Autocrine/Paracrine Considerations in Scaffold Design

As mentioned previously, tensile stress increases BMP-4 by a factor of two as part of the developmental cycle [102]. In addition, this stimulus leads to an increased responsiveness of osteoblasts to morphogens and vitamin D [102]. Applying exogenous TGF-3 on the other hand increases the tension produced per cell by a factor of two [ 105]. Increased TGF-3 and BMP-4 precede increased production of collagen, alkaline phosphate and increased MSC differentiation. Production of these growth factors as





38

stimulated by tension occurs in such a way that they have their effects in an autocrine and paracrine manner, thus tying this form of cellular communication to tensile stimulus. Additionally it is seen that exogenously administered TGF-P increases multi-layering of cells within the confines of pores in collagen scaffolds and causes an increase in macroscale contraction of these scaffolds [105].

Inter-cellular communication is a phenomenon that takes on many forms. Autocrine signaling is the ability of a cell to produce cytokines that are then sensed by receptors on the same cell (Figure 2-9). This method of communication was first elucidated in cancer cells, which were seen to produce growth factors independently of environmental stimulus, thus freeing them to grow uncontrollably and form tumors [39]. Since then autocrine signaling has been established as part of the normal physiology of many tissues including bone [69,85].

Paracrine signaling is the intercellular communication that occurs between adjacent cells, confining its action to a local area (Figure 2-1OA)[69]. Some paracrine signaling




0
4 0

0








Figure 2-9. Diagram of autocnine signaling whereby a cell produces a certain cytokine and then releases it. Cell product is then bound by receptors on the cell's own surface. Adapted from McCance and Heuther [69].






39


occurs by growth factors being released in small quantities to the local extra-cellular milieu. In the case of TGF-P and BMP-4, however, the signaling molecules are bound in the matrix and non-soluble. Signaling occurs by actual physical contact between the cells creating the stimulus as cells nearby extend filopod-like extensions that contact the stimulating cells directly (Figure 2-1013)[5].

In order to optimize the cellular microenvironent, paracrine signaling distances must be considered in addition to fiber spacing leading to the most biologically optimal cellular tension. Indeed the two phenomena are inter-related as fiber spacing is essentially a topographical stimulus and surface topography has been shown to stimulate autocrine and paracrine growth factors [ 109]. But how far paracrine signals released into the extra-cellular milieu able to travel and still remain effective is also a highly relevant question. It seems logical that there is a fiber spacing distance that will allow intercellular paracrine communication between adjacent fibers and that it should be optimized in a 3-D scaffold.

Work in the area of paracrine signaling distance is rather sparse, though it is generally accepted that this is a major contributor to osteoblastic development and how it occurs [85,109]. However, some studies have been performed in culture using various cell types. Generally it is seen in culture models using pituitary cells, as well as parathyroid cells that increasing distance between secretory cells leads to a decreased response, or communication between cells [1I10, 111 ]. An apparent critical distance beyond which interaction becomes negligible in pituitary cells is approximately 75 j tm [110]. This distance of course is for diffusible cell products released into the local environment.






40





















Figure 2-10. Diagram of paracrine signaling. A.) shows a cell releasing a diffusible cytokine (square nucleus) that acts over short distances creating a gradient of responses in surrounding cells (note color scale of oval nuclei). B.) shows similar paracrine phenomena, but the cytokine is passed by filopod-like extensions, or actual physical connections. Adapted from Christian [5].

Matrix bound BMPs on the other hand may possess paracrine activity with a more limited distance of interaction, perhaps as low as a few cell diameters, or tens of microns [5].

Distance and spacing between fibers is therefore likely to be important in the

development of proper cell adhesion and tension. This phenomena, as we have seen, is closely related to autocrine and paracrine signaling distance, which must also be considered in the design of fiber based scaffolds.

Overall, it seems apparent that fiber based scaffolds possess strong potential for use in development of hierarchical bone replacement materials. Their design flexibility allows for control of cellular organization, as well as ECM organization, two of the most integral components leading to the mechanical integrity of bone. In addition, weaving and organization of fibers allows for higher level 3-D construct design that allows greater





41

nutrient diffusion and facilitation of vascular ingrowth, while optimizing cell adhesion and induction of tension, as well as intercellular autocrine and paracrine interactions.














CHAPTER 3
SINGLE FIBER STUDIES

Introduction

As mentioned previously, fibers have been used for a number of scaffold-based tissue engineering experiments. Ahmed and Brown used synthetic Fibronectin (Fn) fibers for the direction of Schwann cells, as well as dermal fibroblasts, macrophages and epitenon fibroblasts. These fibers, with diameters of 0.5-7.0 Wi, were organized into a mat leading to a surface composed of fibers oriented in the same direction [75].

Carbon and polymer filaments have also studied for use as scaffold materials in the area of connective tissue engineering. In vitro Kevlar-49, nylon and carbon allow centimeter scale migration of mesenchymal originating cells from tendon explants along their surfaces. In addition to undergoing extensive outgrowth, these cells were oriented with the long axes of these synthetic fibers. Kevlar-49, nylon and carbon fibers in this study were 12 jim, 7.5 p~m and 22 pWm respectively [ 112].

Other tendon outgrowth studies performed using carbon (8 P~m diameter), Dacron (I11 jim diameter), polyethylene (20 p~m diameter) and nylon (102, 52 and 22 g~m diameters) show cells of mesenchymnal origin orient in the direction of their long axes primarily in response to the diameter of the fibers they are grown on. This is true of all fibers, though there is an interesting and important qualification to this trend: Larger diameter fibers on the order of 100 p~m did not orient cells until after they had become confluent on the fiber






42






43

surface [113]. This implies some socially based mechanical interaction is at work as cells become confluent and come into contact with each other.

In vivo, carbon bundles composed of 8 pm thick fibers have been used as a ligament replacement material and exhibit the ability to organize fibroblasts and tendon cells. More than orienting these cells they appear to orient the collagen produced by the oriented cells [301. However, these tows were under biomechanical stress as the subjects used them in the course of everyday living, so it is unclear if the collagen orientation was a result of topographical cellular orientation. It is conceivable that collagen was oriented by the dynamic stresses of walking and muscular contraction that were placed on it in its role as a ligament replacement.

Though these studies provide much qualitative data about the orientation of cells on

the surfaces of fibers, they are lacking in hard quantitative evidence. Overall it is apparent that diameter has an effect and that time, which allows the achievement of confluence, are important factors in contact guidance of mesenchymal cells on fibers.

Some quantitative work has been done on the fiber based contact guidance of

connective tissue cells. Dunn and Heath performed alignment measurements of chick heart fibroblasts on the surfaces of soda glass fibers at 48 hours, showing an order of magnitude increase in NFF on fibers of 40 Lrn diameter compared to fibers of 100 pm [29]. Above 100 gm there was no significant contact guidance. Similarly, Fischer and Tickle quantitatively showed normal BHK rat fibroblasts elongated on the surface of glass fibers, but at only 24 hours [97]. Both studies use quantitative measures of cellular elongation, but are limited to very short time periods. Indeed





44

measures are taken to prevent social interaction between cells even for these short times in culture.

In this study mesenchymal stem cells are studied quantitatively and for time periods that extend beyond those needed to achieve confluence. In fact the time periods used were lengthy enough to observe the characteristic multilayering observed in this cell type. The object of this study was to examine the effects of diameter and time on MSC contact guidance and to do this in a quantitative manner. It is hypothesized that decreasing diameter will lead to an increased level of contact guidance, as will increasing culture times. Knowledge and understanding of this cellular response to fibers will prove invaluable in fiber-based scaffold design.

This section's main hypothesis is: MSCs will have a significantly (alpha = 0.05)

different elongation and cellular activity when grown on -140 pm, 100 pm and 79 pm diameter fibers. This difference in cell activity will exist between each fiber diameter group and inducing contact guidance of these cells will be the primary goal.

Materials

Black Nylon (EthilonTM, Ethicon) sutures of 4-0 (- 140 pm diameter), 9-0 (-79 pm diameter) and 10-0 (- 39 pam diameter) sizes were used for initial SEM studies. Clear polyglactin (MaxonTM, Davis and Geck) fibers of 5-0 (-140 jn diameter), 6-0 (- 99 Ptm diameter) and 7-0 (- 79 pm diameter) were used for light microscopy studies.

Culture materials included a-minimal essential medium (Sigma, M0894) with 15% fetal bovine serum (Sigma, F4135), 50 mg/ml ascorbic acid (Sigma, A4034), 10 mM bglycerophosphate (Sigma, G9891), antibiotics (0.1 mg/ml penicillin G, 0.05 mg/ml gentamicin and 0.3 mg/ml fungizone) and 10-8 M dexamethasone (Sigma, D2915).






45

Bovine Fibronectin (Fn) -25 ug/mI (Sigma, F 114 1) was used to soak constructs prior to cell seeding, thus making fiber surfaces more amenable to cell adhesion. After soaking, En solution was pipetted off and cells were seeded directly onto constructs.

MSCs were collected from both femora of grown, Sprague Dawley rats (-1 50-300 g) that were provided courtesy of Dr. Colin Sumner's lab at the UF brain institute.

Methods

Single Fiber Scaffolds

Single fiber scaffolds were prepared by stringing Maxon and Nylon sutures with the diameters mentioned previously across polystyrene support rings (Figure 3-1). Constructs were designed to suspend the fiber completely above the floor of the culture dish so all cellular response was caused by the topography of the fiber alone.

















Figure 3-1. Examples of Maxon TM single fiber constructs. 5-0 is on the left, 6-0 center and 7-0 on the right.

Cell Culture

After dissecting each femur out, the epiphyses were cut off and the marrow plugs were flushed out of both diaphyses using aliquots of the fully supplemented medium(FSM) to






46

be described next. Plugs were flushed into 30 ml of FSM, which will be: a-minimal essential medium (Sigma, M0894) with 15% fetal bovine serum (Sigma, F4135), 50 mg/ml ascorbic acid (Sigma, A4034), 10 mM b-glycerophosphate (Sigma, G989 1), antibiotics (0.1 mg/ml penicillin G, 0.05 mg/ml gentamicin and 0.3 mg/ml fungizone).

After the primary culture passage of --6 days, cells were trypsinized and passaged using a 0.01% trypsin and 10 M EDTA mixture (Sigma, T3924) in phosphate buffered saline. Released cells were reseeded into three 75 mm2 culture flasks. MSC Seeding

MSC Seeding onto the fibers was done at extremely high concentration by taking a whole flask of trypsinized MSCs and adding -10ml of medium to it. Addition of this small amount of medium served to quench the trypsin reaction, while leaving the cells at a very high density. After EtOH and UV sterilization (24 hours in absolute EtOH followed by drying under 256 nm UV light), fibers were placed in 24 well plates and secured to the walls via melting of the polystyrene support ring to the polystyrene of the dish (heated implement was a small soldering iron). Fibronectin (25 ug/ml) was used to soak constructs (except for 9-0 constructs) for one hour then each construct was covered with a large drop if high density cells in suspension and allowed to incubate for about 2-4 hours while cell adhesion occurred. At this point the wells were flooded with culture medium to the normal level.

SEM Imaging (Nylon sutures)

Cells were fixed in a phosphate buffered saline with 10% formalin at pH 7.4. Each of the constructs were dehydrated by serial ethanol incubation at concentrations of 30%, 50%, 70%, 90% and 100% over periods of about two days (longer dehydration time






47


minimizes shrinkage of cellular material). Following ethanolic dehydration, constructs were critical point dried, coated with -20 A of Pd/Au and examined via SEM. Light Microscopy (Maxon Sutures)

Constructs were removed at 3, 6, 9 and 12 days and passed through serial EtOH

dehydration of 30%, 50%, 70%, 90% and 100%. Upon reaching 100% EtOH, constructs were left in 100% EtOll until all constructs were removed from culture then all were stained simultaneously using Hematoxylin and Eosin stains. Nuclear Form Factor (NFF) and the angle were measured with respect to the fiber's long axis as described in Appendix 1.

Results

SEM Studies

4-0 nylon sutures

Constructs initially showed a robust amount of cell adhesion and coverage under the light microscope during culture. By the last day, every fiber was completely covered with cells and some even appeared to have begun multi-layering.

Cells liberally covered the surface of the fibers, but an interesting phenomena was that these cells were elongated though not parallel to the long axis of the fibers (Figure 32). Instead they were spirally oriented around the surface of the fiber, much like a barber's pole. A similar result was seen in work by Ricci et al. when larger diameter fibers were used [113].

In addition to the spiraling effect of cell orientation, what appeared to be

multilayering was present. Light microscopy during culture demonstrated what appeared to be multilayers, or areas of cell buildup at the periphery of the fibers. Because the fibers were dyed black and used primarily to develop protocols, light microscopy staining was






48










4 i









i ji






Figure 3-2. 4-0 nylon fibers with RMSCs after being cultured for 5 days. A and B) Note elongated and flattened cells that spiral around the long axis of the fiber. C.) is a close up. Arrows indicate some of the most prominent cells. Flattened cells cover nearly the entire not possible and cells were only visible in a x-section-like view where they were not superimposed on the fiber.

Examination of 4-0 fibers showed a definite striation-like appearance (Figure 3-3), which appeared to proceed in two directions (red arrow and green arrow indicate approximate directions). In some areas it appeared that each of these oriented striae were composed of cells and that the cells in one direction were overlapping the cells that were growing in the other spiral direction.






49









'JA
V




Figure 3-3. Examples of MSC growth on the surface of 4-0 nylon fibers. These cells exhibit spiraling and what may be alternating spiral orientations between layers.

Though this result requires further support, there is some indication in the literature

that may explain it. Commonly osteoblast-like cells multilayer during culture, depositing collagen at each level in a manner that is situated in orthogonal directions between each layer. That is cells in one layer are separated by collagen laid down by the next cell in the layer above, this collagen in turn is at a 90 degree angle from the collagen laid down by the cell below (both chicken and rat cell culture models show this) [114-1161. 9-0 nylon sutures

9-0 Nylon sutures shown in Figure 34 exhibit less cell adhesion than the other nylon fibers examined, as they were not pre-soaked in Fn. This illustrates the importance of surface properties and receptor mediated adhesion in MSC settling and growth. Despite the fewer numbers of cells, every cell observed was indeed elongated in the direction of the fiber's long axis.

Images of much more flattened cells that are also extending in the same direction as the fiber are reproduced in Figure 3-5. These cells may be similar to "sheath cells" found in tendon experiments on fibers [30,114];. These studies that found three populations of






50



20
60 JLM








A


E








Figure 34. Examples of RMSC growth on the surface of 9-0 nylon fibers. A.) shows a cell with two long processes essentially parallel to the fiber. B and Q show a cell and a close up of the same cell respectively. D) shows a cell that is much flatter. E.) a close up of the same cell. It is elongated in the direction of the fiber as were all the cells seen on the fiber.

cells grew on their fibers, one a flat sheath like cell, similar to these, that covered the surface of the fiber, another spherical cell type like the one seen in Figure 3-5A and a spindle shaped cell similar to that seen in Figure 3-6. These cells may be very similar as they are derived from the same cells in vivo as the MSCs used in this study. Morphologically the cells in the current study appear very similar. 10-0 nylon sutures

In contrast to the 4-0 fibers, 10-0 fibers show cells that follow the long axis of the

fiber very closely (Figure 3-6). Figures 3-6A, 3-613 and 3-6C show more flattened cells, while 3-613, 3-6E and 3-6F show cells with a more spindle shaped morphology.






51


vow,












............


A FB]


Figure 3-5. Examples of RMSC growth on the surface of 9-0 nylon fibers. This set of images shows flattened cells that are at least qualitatively extended in the direction of the 9-0 fiber they are growing on. On the right is a close up of the cell material showing many filopodia.

Maxon sutures

Images of 5-0, 6-0 and 7-0 sutures were obtained on samples cultured for 12 days under conditions described above. 5-0 fibers were fully covered with MSCs that show spiraling pattern (Figure 3-7). Cells on 5-0 fibers were flattened in a manner that was much more pronounced than that seen on the 6-0 (Figure 3-8) and 7-0 fibers (Figure 3-9). Additionally, cells on the 6-0 and 7-0 fibers were much more elongated than those seen on the 5-0 fibers. 7-0 fibers appeared to exhibit the greatest amount of multi-layering and elongation. These results are supported by the quantitative data taken to measure cellular orientation and elongation, though SEM of these fibers was performed only on the twelfth day.






52






































E A/ F


Figure 3-6. SEM images of 10-0 nylon sutures at various magnifications. A, B and Q show highly flattened and multi-layered cells that are elongated in the direction of the fibers. D, E and F) show a more spindle shaped cell that also is elongated in the same orientation as the fiber.






53






















Figure 3-7. 5-0 Maxon sutures exhibiting RMSC adhesion and growth. A.) Shows MSCs covering the entire surface of the fiber and marked spiraling, or angling with respect to the fiber's long axis. Note cells are rather flattened and not highly elongated. 500x. Arrow indicates general trend of the spiraling behavior exhibited by cells. B) Again shows flattened and angled MSCs, but at I 000x. Q Flattened and angled MSCs at 1500x. Light Microscopy Studies of Maxon

In general the larger diameter 5-0 and 6-0 fibers showed the barber pole spiraling similar to that seen in the SEM images of 4-0 nylon (Figures 3-2 & 3-3), as well as SEM images of 5-0 Maxon (Figure 3-7). 7-0 Maxon however did not show noticeable evidence of this spiraling behavior. Elongation of RMSCs on the surfaces of the fibers was evident and contact guidance of cells was clearly occurring. Differences in nuclear orientation between fiber diameters were visible and confirmed by statistical analysis. Multilayering occurred on all three fiber diameters by day 12. These findings are consistent with SEM data, which is presented below.






54
























Figure 3-8. 6-0 Maxon sutures exhibiting RMSC adhesion and growth. A) shows MSCs covering the entire surface of the fiber. Many flattened cells are present and much more elongated than those seen on the 5-0 fibers. Spindle shaped cells are present as well (blue arrowheads) 500x. B) Shows flattened and elongated MSCs at l000x. C) Shows flattened and elongated MSCs at I 500x. An example of multilayering is clearly evident (red arrowhead) as cells overlap.














Figure 3-9. 7-0 Maxon sutures exhibiting RMSC adhesion and growth. A.) Fibers are completely covered by flattened and elongated MSCs 500x. 13.) shows flattened and elongated MSCs at 1 000x. Examples of multilayering are evident as cells overlap each other (red arrowheads).






55













Figure 3-9 Continued. 7-0 Maxon sutures exhibiting RMSC adhesion and growth. Again flattened and elongated MSCs are evident. Multilayering and overlap are evident (red arrowheads) 1500x.

Nuclear form factor

Figure 3-10 shows a nucleus with all NFF measurements as they were performed in Adobe Photoshop. Originally three day data was slated to be collected, however, there was not sufficient cell growth for any kind of meaningful analysis.


















Figure 3-10. Example of Nuclear form factor measurements. The image shows a representative 5-0 fiber after 6 days in culture.

Figure 3-l1 is a graph of the effects of diameter on NFF independent of time, while Figure 3-12 shows effects of time independent of diameter. Clearly both factors, which were the factors used in the 2 way analysis of variance, show that NFF leads to increased






56


orientation. All differences resulting from diameter were statistically significant with a power P = <0.05. When comparing time factors of 6 days in culture vs. 12 days, there was a statistically significant difference with a power of P<0.05, while the other differences were significant with P = <0.1I (i.e. 6 days vs. 9 days, and 9 days vs. 12 days).


UaTeter Effects on N~iewn (Oien~aon (F

(InepndrXW of lime)
0.35


0.30- lg0T


0.25





0.15


0.10


0.05


0.00
5-0 6-0 7-0

&u'osize(LIsp. sz

Figure 3-11. Graph showing the independent influence of fiber diameter on NFF. Bars are standard errors.

All of the data in Figure 3-13 has been shown to be statistically different from each other at a power of P = <0. 1. There appear to be effects on cellular elongation that are related to social interactions of cells, or how they squeeze together when the layers become confluent. 6-0 and 5-0 fibers show what seem to be fluctuations in the degree of







57


Effect of Culture Time on Nuclear Orientation (NFF)
(Independent of Diameter)
0.4




0.3




0.2



OAN N
0.1





0.0
4 6 10 12 14
Tim. in culture Idavsl

Figure 3-12. Graph showing the independent influence of time on NFF. Bars are standard errors.





Effects of Culture Time on 0.4- Nuclear Orientation (NFF)

5-0 NFF MM6-0 NFF 7-0 NFF
0.3




0.2




0.1




0.0
Dav 6 Dav 9 Dav 12
Time in Culture (Days)


Figure 3-13. Graph showing the effects of time on NFF for all diameters studied. Note 70 fibers are the only ones showing a gradual and continuous increase in elongation. Both 5-0 and 6-0 fibers fluctuate with time probably due to the effects of social interactions. Bars are standard errors.





58

nuclear elongation. Multilayering was apparent on all the constructs and imaging of nuclei for analysis was primarily done on what appeared to be the top layer cell coverage. Deeper layers suffered from a loss of contrast thus making it virtually impossible to collect data from them. Fluctuation in nuclear eloRgation as seen in Figure 3-13 may indicate how social interaction is important in cell orientation. Basically each layer of cells, as it becomes confluent on top of the layer before it, may elongate and orient as it nears full confluence. This is followed by the accumulation of the next layer, which is less elongated at first. This is analogous to the phenomena seen by Ricci et al. who observed that tendon outgrowth cells did not elongate until confluence was reached on fibers above 100 pm [ 113].

In the similar vein, 7-0 fibers showed increasing elongation throughout the culture

times examined. These fibers were less than 100 un, the diameter that seems established in the literature as the critical diameter for continuous contact guidance. Nuclear angle analysis

Figure 3-14 shows the same nucleus as seen in figure 3-9. This time the image is

demonstrating the method used to measure nuclear angle. Figures 3-15 and 3-16 show the main effects of the 2 way ANOVA (time and diameter). Both factors exhibit a significant effect with p = <0.00 1, while the interaction between the main effects is significant with p = <0.056. Overall both time and diameter influence the angle of the nucleus with respect to the long axis of the fiber. Also there is an interaction between time and diameter leading to an influence on nuclear angle that is greater than each factor acting alone.






59




















Figure 3-14. Example of nuclear angle measurements. Image of the same nucleus depicted in Figure 3- 10. This micrograph demonstrates the method used to measure the nuclear angle with respect to the fiber's long axis.

Nuclear Angle Effects of Fiber Diameter 100 (independent of Time)


0
1060
I
0
Zu 20

0



5-0 6-0 7-0
Diameter (U.S.P. suture size)

Figure 3-15. Graph showing the effects of diameter independent of time. Bars are standard errors.

Figure 3-17 shows all fiber sizes vs. time in culture. With the exception of the 6-0, 6 day data, nuclear angle behaved exactly as hypothesized by increasing (becoming closer to alignment with the fiber) with diameter and time. When comparing figure 3- 1OC to






60


Nuclear Angle Effects of Time 100 (independent of Diameter)



80
0


40


0


Day 6 Day 9 Dayl12
Time (days) Figure 3-16. Graph showing the effect of time in culture on nuclear orientation as measured via nuclear angle. Bars are standard errors.

Corrected Nuclear Angle Effects of Diameter and Time
100 5-0 fibers

6-0fier
7- ibr
LO
"a 60
2
~40

Z 20


Day 6 Day 9 Day 12

Time (Days)


Figure 3-17. Graph showing the effects of time and diameter together. Bars are standard errors. Figure 3-17 it becomes apparent that NFF and nuclear angle are not directly correlated, which is interesting, as these values were calculated from the very same nuclear images.






61

So nuclear orientation appears to be influenced by the diameter of the fiber in a way that is independent of cellular elongation. Again we see that 7-0 fibers exhibit the greatest influence on cellular activity and lead to elongation that is more robust than either the 60, or 5-0 diameter fibers.

Overall it seems that nuclear orientation measured by the angle of the nucleus is a more predictable method of examining cellular contact guidance. Nuclear orientation looks as if it will require further study and increased understanding.

Discussion

It is abundantly clear that decreasing diameter and increasing culture time led to

greater contact guidance of MSCs in this culture system. These responses by MSCs were exactly as hypothesized. 5-0 and 6-0 diameter fibers showed what is likely to be the effect of the social interaction of cells on their nuclear elongation. 7-0 fibers on the other hand are of small enough diameters that their topographical influence overpowers the need for social interactions. All fiber sizes show a nuclear orientation, however, this orientation appears to be independent of nuclear elongation.

This behavior has implications for fiber-based scaffold design. By quantifying the behavior of these MSCs on various diameters and for various times, the fiber candidate that is most optimal for a given system can now be chosen. Though similar studies have been performed with similar cell types [29,97] an extensive literature review shows they have never been done using the MSC culture system. In addition the level of measurement in this study, using quantitative methods has not been matched for time, or numbers of quantitative measures (i.e. both NFF and nuclear angle).

Though the presence of social interaction between cells has been observed on a qualitative level, it has never been observed and measured as closely as this study.






62


Fluctuations in the nuclear elongation, independent of the angle suggest that as confluence is reached each layer of cells in the multilayered system becomes more and more elongated. This is to say that layers of cells are built up by a period of oriented, but un-elongated cellular morphologies followed by elongation in the direction of the fiber's long axis. After the achievement of confluence, the process begins again on the next level of multilayer.

Larger size fiber diameters show spiraling and as seen in figures 5, 6 and 8. This

spiraling may occur at equal and opposite directions within each layer. ECM~collagen has been linked to cellular orientation and in this cell type it is seen that collagen layers are deposited in orthogonal directions to each other [114-116]. Perhaps this mechanism of cellular layering is related to the mechanism that lays down collagen in alternating layers. As previously noted orientation of ECM is strongly correlated to the orientation of the cells depositing it [31,76,117-120]. Indeed the deposition and orientation of many individual ECM proteins including collagen has been shown to parallel the oriented layers of osteoblasts laying them down [ 12 1].



Conclusions

This study quantitatively shows that RMSCs elongate and orient in response to diameter, as well as time in culture. In the case of both time and diameter contact guidance occurred in an increasing manner when measured via nuclear angle. A critical fiber diameter existed at approximately 100 p.m, however, for the cellular elongation measured by NFF. Below this critical diameter, cells were increasingly oriented in a gradual manner over time. Though hen grown on fibers above the critical diameter, cells






63

were oriented by nuclear angle, but elongation seemed to occur in response to social interactions between cells as each layer achieved confluence.

Overall, given the nature of this cell type and the way it has been shown to orient ECM in a manner that parallels cellular orientation, this form of contact guidance will allow the control of structural ECM proteins. As mentioned in chapter 2, control of ECM proteins; namely collagen, is what creates the anisotropic mechanical properties found in natural bone. This study shows, through rigorous scientific methods coupled to strong statistical support, that controlling cellular orientation and thus ECM orientation is now a capability to be included in the arsenal of design characteristics available to connective tissue engineers.













CHAPTER 4
CELLULAR BRIDGING PHENOMENA

Introduction

The overall goal of this work is to develop an understanding of cellular organization and ECM deposition, then use this knowledge to organize cells and ECM in a desired manner. Developing tissue constructs with multiple layers of cellular and ECM orientation appears to be a promising method of increasing the construct's anisotropic mechanical strength. As reviewed in the background, mechanical anisotropy is a common strategy employed by tissues as part of their physiologic function. This is particularly true of bone and connective tissues, though this fact appears to have been overlooked in the tissue engineering literature when scaffold materials are chosen. Typically randomly organized scaffold materials have been employed despite the fact that substrate topography has been shown to strongly influence cellular orientation and ECM deposition. These induced forms of cellular and ECM organization is very similar to the structure seen in vivo, which is the very same structure and organization responsible for mechanical anisotropy.

In 2-1) MSC systems, cells multi-layer and act cooperatively to produce a collagen

rich ECM, which is organized into orthogonal layers. Orthogonal lamellae of collagen are separated by cellular layers, which are responsible for collagen deposition. Originally this cellular multilayering provided the rationale for the multi-fiber construct experiments discussed in the 5th chapter using 30 ttn as a spacing distance between fibers.




64





65

Multilayering phenomena in MSC culture, by estimates and projection from data in the literature, achieve heights of approximately 15-20 pLm, so 30 pm would be proper for cells on adjacent fibers to grow and eventually join in the space between fibers (Figure 4-1).


















Figure 4-1. Multilayering on adjacent fibers leading to interactions due to their proximity. Possibly these interactions are of a paracrine nature. The correct spacing would allow cells to span the distance between fibers, while using the fibers as a stimulus for cellular and ECM organization.

In the course of the experimental work it became apparent that there was at least one more phenomenon involved in the 3-D cellular organization process beyond that of mere multi-layering, which appears to be the primary process in 2-D systems. The single fiber constructs studied in the last chapter primarily demonstrated MSC multilayering, but when more than one physical surface, or topographical feature is present, another cellular process becomes evident. Cells begin to span distances, or "bridge" between surfaces when these surfaces are found within certain ranges and under the right conditions of proximity.





66

This bridging phenomenon is obviously a prominent behavior contributing to the

cellular interaction with 3-D scaffolds. Furthermore this behavior does not appear to be explicitly addressed in the literature as it currently stands. For both these reasons cellular bridging merits study here, not only to elucidate and characterize it as a previously unobserved phenomenon, but also to understand how it influences cellular development and function.

Materials

Bridging was induced and observed on many different materials including: standard polystyrene culture flasks (CorningTM9, 4-0 monofilament nylon sutures (Ethilon TM), 7-0 monofilament polyglyconate sutures (Maxon TM ), stainless steel screen and bioactive glass fibers synthesized as previously described by Dominguez et al. [ 122]. Culture materials were identical to those used in chapter 3.

VonKossa staining was performed using Silver nitrate 5%- 5 gmn in 100 ml DI water =5 % solution. Sodium thiosulfate 5% 5 gm in 100 ml DI water = 5 % solution nuclear fast red- 5gm aluminum sulfate, 100 ml DI water, nuclear fast red 0. 1 gin.

Methods

Culture Methods

Culture and MSC seeding was performed as described in chapter 3. Nylon sutures were cultured for 12 days, Maxon TM for 13 days and Bioactive glass fibers 6 days before data collection.. Polystyrene studies were performed for 11I days total with data collected on days 7, 8, 10 & 11. Stainless steel screens were followed for 23 days with data collected on days 10, 12, 15, 18 &23.





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SEM Imaging

SEM was performed on Nylon, Maxon TM, stainless steel and bioactive glass fibers with preparation for microscopy as described in chapter 3. Light Microscopy Studies

Polystyrene imaging was performed on live cells in situ as they grew in the culture dish. No staining was performed so viable cells could be maintained allowing the development and documentation of individual bridges. Von Kossa Staining

Von Kossa staining was performed on stainless steel screens to follow the

development of ECM and its subsequent mineralization. This method demonstrates salts of calcium and it works by replacing Ca +2 ions with silver from a silver nitrate solution (5% in DI water). The silver salt that is produced by this is then photosensitive (silver nitrate is the same chemical used in the production of a black and white photographic negative). Exposing the sample to light then causes a photic reduction of the silver salts leading to a black precipitation of silver in the locations where calcium is present.

Von Kossa staining procedure[118,120,1231

1.) Absolute EtOH dehydration, 2 times for 2 minutes each.

2.) 95% EtOH dehydration, 2 times for 2 minutes each.

3.) DI water rinse 2 times.

4.) Silver nitrate 5% incubate 30 minutes under high power light (- 100-150 Watt

bulb leading to photic reduction of silver).

5.) DI water rinse

6.) Sodium thiosulfate 5% incubate 3 minutes (washes away residual silver nitrate)

7.) DI water rinse






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8.) Nuclear Fast Red incubate 5 minutes (stains nuclei dark red and cytoplasm

pink).

9.) 95% EtOH dehydration, 2 times for 2 minutes each

10.) Absolute EtOH dehydration, 2 times for 2 minutes each. Proliferation Studies

Bridging of MSCs between two topographical features is logically dependent on the distance between them. In an effort to understand how spacing between fibers effects



9.17 E0.05 mm






















Figure 4-2. Example of bioactive glass fiber constructs used for the proliferation study. Dimensions of these cylindrical containers allowed the calculation of fiber density and provided a constant volume necessary for the study. cell function we performed proliferation studies using bioactive glass fibers with various packing densities. Various masses of bioactive glass fibers (10, 20, 30 & 40 mg) were gently packed into constructs with a given volume (Figure 4-2). Increasing quantities of





69


fibers within this given volume was assumed to decrease the distance between fibers and thus influence the ability for these cells to perform bridging.

Cells were seeded onto all constructs at an estimated density of 1.10 +!- 0.07 x10 cells/mi and allowed to incubate for 3, 7, 10, 14 and 21 days. As each time point came due, cells were removed from culture, trypsinized and counted using a Beckman-Coulter Multi-sizer III cell counter. Sample sizes were 500 mL and were diluted with Isoton 11 diluent.

Cell counts were analyzed using a two way analysis of variance with time (3, 7, 10, 14 and 21 days) and fiber density (10, 20, 30 & 40 mg) as factors. Post hoc testing was performed using Tukey's multiple comparison.

Results

Light Microscopy of Bioactive Glass Fibers After 6 Days in Culture

Preliminary studies on bioactive glass fibers placed directly into the RMSC culture system for 6 days showed a marked degree of multi-layering in the vicinity of the fibers where they rested on the floor of the dish (Figure 4-3). This degree of multi-layering was particularly evident when comparing cellular behavior on flat areas of the dish where fibers were not present (Figure 4-4). In fact multi-layering was so extensive that four micrographs were necessary to include the entirety of the representative image (Figure 4-3 montage).

In addition to multi-layering in the vicinity of the fibers, RMSCs were oriented toward the fibers as if using it as an anchor for the development of tension.






70



















Figure 4-3. Montage of light micrographs portraying the cellular interaction with bioactive glass fibers placed in standard culture well. Note orientation of cells with respect to the fibers, they appear to be pointed in toward the fibers and multilayering is extensive.










_4






Figure 4-4. Light micrograph of area without fibers in the same culture well as that shown in Figure 4-3. Note the lack of multi-layering particularly when compared to Figure 4-3.






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Scanning Electron Microscopy

Bioactive glass fibers after 6 days in culture

Figure 4-5 shows SEM images of RMSCs grown on bioactive glass fibers. Figure 45A shows cells covering the fibers and growing in a robust manner on the fiber surfaces. In some cases cells grew over fibers, securing them to the bottom of the petri-dish. Figures 4-5 shows a close up example of cellular interactions with the fibers and the dish. Figure 4-6 shows examples of fiber to fiber bridging, a phenomenon that was commonly seen throughout the extent of the specimen's surface. This bridging was often multi-cellular as seen in Figure 4-5, but there were many instances of unicellular bridging as well (Figure 4-6).




















Figure 4-5. Examples of cellular growth on fiber placed in the RMSC system taken with SEM. A.) shows a large aggregate of cells and fibers with the same type of orientation seen under light microscopy. B.) shows a large multi-cellular bridge that spans the distance from the bottom of the dish to the fiber.

Figure 4-513 demonstrates bridging from the dish floor to fibers, another commonly seen cellular behavior. Again the bridge demonstrated is multi-cellular, though there






72

were unicellular examples of this type of bridging as well. This multi-cellular aggregate demonstrates a more columnar, or cylindrical bridging motif, in contrast the the flattened, sheet-like bridging was also seen. Both structures were clearly multi-cellular with their respective morphologies determined by what appears to be the topography, or the shapes presented by the fibers in their vicinity.


I
A










Figure 4-6. Examples of unicellular bridging on bioactive glass over distances of -70 gm. In addition to being unicellular these are also fiber to fiber bridges. Polymer l1bers

Figure 4-7 shows micrographs of RMSCs bridging distances between two adjacent Nylon fibers. These constructs were incubated for 12 days prior to SEM examination and demonstrate what appears to be a multi-cellular bridge (left most bridge) and a unicellular bridge (right most bridge).

Figure 4-8 is a micrograph of RMSC bridging on adjacent Maxon fibers. Though this particular bridge is shorter than that seen on the bioactive glass fibers and the Nylon fibers, bridges were not limited to shorter lengths. This construct was incubated for 13 days in RMSC culture.






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Figure 4-7. Examples of unicellular bridging between nylon fibers. This image demonstrates what appears to be a multi-cellular bridge (left most bridge) and a unicellular bridge (right most bridge).

















Figure 4-8. Bridging between Maxon fibers. Light microscopy of Bridge Development on Polystyrene Culture Flask

In order to classify, or identify the sequence of events involved in the development of MSC bridging, RMSCs were seeded in standard polystyrene culture flasks and individual bridges were observed and documented serially over time.






74

Initially after seeding there were many individual cells that had elongated along the flat surface of the dish, sometimes for hundreds of im. An example of this elongated cellular morphology is shown in Figure 4-9A (green arrow). These highly elongated cells were interspersed with cells that had assumed more flattened morphologies. In addition to elongation on flat surfaces, there were also cells that elongated and had associated with the topography of the polystyrene wall similar to the cellular behavior shown in Figure 4-913 (red arrow). Initial stages of bridging, as demonstrated by single cell bridging, indicated that bridging was possible only when there was an actual physical pathway for bridge formation. It appeared that elongation of cells, which can occur on the order of

















Figure 4-9. Initial RMSC elongation. A.) Representative micrograph of highly elongated cells that seemed to be responsible for initial unicellular bridging. hundreds of microns facilitated bridge formation by traveling the distance initially. In other words it did not seem possible for cells to reach across gaps without there being a pathway for them to elongate on while constantly maintaining adhesion and contact (Figures 4-6 and 4-9).






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4X








Figure 4-9 continued. Initial RMSC unicellular bridging. B.) shows the formation of a unicellular bridge between the floor of the culture dish and the wall of the dish (red arrow).

Bridge development progressed between the floor of the culture flask and the wall. By day 7 large multi-cellular bridges were apparent similar to the one shown in Figure 4-10. Bridges extended for hundreds if not thousands of microns and were clearly multicellular in nature. The areas underneath the bridge, that is the areas of the floor between the site of attachment at the wall and where the bridge was secured to the floor of the dish exhibited few cells. It seemed as if the cells that had once inhabited that region of the dish

had released and become part of the bridge as it pulled off the floor and suspended itself in the medium. This reduced number of cells is apparent in Figure 4- 10 as well. With continued time in culture the cells composing the bridge showed evidence of proliferating within the bridge proper. This was apparent in a thickening in the bridge near its site of attachment to the flask wall (Figure 4-11, red arrow).






76















Flask Wall Flask Floor

Figure 4-10. Multicellular bridge at 7 days in the RMSC culture. Bridge is extending from the floor of a standard polystyrene culture flask to the wall. On the 10th day in culture, there were instances of ECM nodule formation within the body of the bridge in the location where the thickening of the bridge had occurred (Figure 4-12). These nodules were very similar morphologically to those seen on the flat surface of other areas of the culture dish. On the 11Ith day in culture the bridge contracted back from the wall of the dish and formed a large multi-cellular nodule that appeared to be contain a large fraction ECM as part of its composition. Again this












Figure 4-11. Multicellular bridge at 8 days in the RMSC culture. Bridge shows proliferation and thickening within its body. This is the same bridge shown in figure 410.






77











Al
Figure 4-12. Multicellular bridge at 10 days in the RMSC culture. Nodules appear to be forming within the body of the bridge at the site of proliferation. nodule was morphologically very similar to those seen in flat areas of the dish, however, this nodulewas much larger, on the order of hundreds of microns versus approximately fifty microns for nodules on the flat surface. Bridge Development on Stainless Steel Screens (Light Microscopy and SEM)

After classifying, or identifying the sequence of events involved in the development of MSC bridging, RMSCs were seeded onto stainless steel screens and the general















Figure 4-13. Multicellular bridge at I11 days in the RMSC culture. Bridge contracted back from the wall of the dish and formed a large aggregate of cells and ECM. This nodule was morphologically identical to others seen in the center of the dish where no bridging occurred, only much larger.





78

development of bridges was observed and documented serially over time. This study was performed in order to introduce a controlled topography into the RMSC system and examine the bridging phenomenon associated with it. The screens used had a weave that led to square holes with dimensions of approximately 170 tin on a side. The fact that it was a woven fabric was also important in the overall study of fibers and their use as scaffold materials. Using screens, therefore allowed insight into fiber based scaffold applications, though the fibers composing these particular woven screens were steel.

To study the effect of bridging on the development of ECM and mineralization, constructs were stained via the Von Kossa Method. Additionally, to ensure that the bridging effects were the result of the fibers incorporated in the weave of the screen, the screens were suspended above the floor of the petri-dish.

After seeding cells were apparent on the lengths of the fibers and could be visualized at the periphery of the fibers, which, like the Nylon fibers used, were opaque. Numbers of cells increased until by the 10th day then began to form unicellular bridges, or bridges composed of a few cells (Figure 4-14). This bridging is also shown in a 3-D formation of even larger aggregates of cells that assumed flattened bridges and in some cases more cylindrical bridges (Day 12 and Day 15 images Figures 4-14, 4-16A, 4-16B, 4-17A and 4-17B respectively).

By the 18th day entire holes of the screen were covered by flat sheets of cells acting cooperatively and there was even the presence of occasional non-mineralized nodules of ECM (green arrowhead Day 18 image figure 11, also see SEM representation in Figure 4-15A and 4-15B. Continued time in culture led to the cylindrical bridges progressively






79

spanned distances across the square holes until they had completely bridged the diagonals of the square holes.




ay 10 Day 12 Day 15








Bar
-15OPM

Day I ay 23






Figure 4-14. Development of bridging on stainless steel screens. Day 10 shows unicellular bridging, or bridging with only a few cells. As time passes cells proliferate and form more complete sheet-like bridges until, by Day 18 most holes in the screen are filled with cells. At this time early nodule formation has begun, though it is unmineralized (green arrowhead). By Day 23 mineralization is highly apparent as evidenced by nodules stained black with the Von Kossa stain. By the last day of the study, mineralization was seen within the bodies of bridges that were spanning distances across the holes of the screen. Though the presence of flattened sheet-like bridges and cylindrical bridges were both apparent during the course of the study, prior to the 23rd day, flattened sheets appeared to be most dominant in numbers. Later in the study, especially day 23, cylindrical bridges became moreprevalent, which indicated that some of the flattened bridges were becoming cylindrical (Figures 4-14, 419A and 4-19B).






80








-A B







Figure 4-15. SEM of stainless steel screens after 10 days in RMSC culture. Unicellular bridging is prominent.














AB

Figure 4-16. SEM of stainless steel screens after 12 days in RMSC culture. Bridging becomes more noticeably multicellular with cells that are remarkably flattened.

This behavior may be related to the contraction of cells as was seen later in the

bridging sequence documented in the polystyrene flasks. An example of a cylindrical bridge is shown in Figures 4-14 and 4-14B. At the base of the cylindrical bridge seen in Figure 4-14B is a mineralized nodule (red arrow). This was verified visually by light microscopy prior to SEM preparation (data not shown).






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A B

Figure 4-17. SEM of stainless steel screens after 15 days in RMSC culture.












A

Figure 4-18. SEM of stainless steel screens after 18 days in RMSC culture. Proliferation Studies of RMSCs on Bioactive Glass

Two way ANOVA showed both main factors were significant with P< 0.001.

Growth curves for 20 mg, 30 mg and 40 mg constructs displayed a very similar growth kinetic, which was not a strict sigmoidal curve (Figure 4-20). The 10 mg construct on the other hand displayed traditional sigmoidal growth, in fact this fiber density was the only density that showed a statistical difference between cell counts taken on day 7 and day 10 (P<0.05).






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Figure 4-19. SEM of stainless steel screens after 23 days in RMSC culture.

Cell counts for the 10 mg construct were very close to those from the 20 mg construct for much of the time period measured. In fact the differences in cell concentration between these two fiber densities did not become statistically significant until the 2 1 st day (P<0.05). That there are differences between the shapes of the two curves is apparent, however, indicating some sort of critical difference, or cut off point between 10 mg and 20 mg. Of all the curves only the 60 mg construct showed a distinct plateau at the end of the 21 day study indicating it was the only one that had finished proliferating (i.e. day 14 data was not significantly different from day 2 1). In addition to this, the 60 mg curve was markedly higher than any of the other experimental conditions indicating not only that the cells finished their replicative stage sooner, but also that the proliferation had been more robust on this fiber density. On the final day of this study the 20 mg construct displayed a cell count very close to that of the 40 mg construct the two were not significantly different at this point, and it appeared to still be proliferating. Overall the 60 mg curve provided the most robust example of the characteristic growth kinetic and had the highest cell counts as well.





83




RMSC Growth on 77s Bloactive Glass Fibers of Various Densities
2500
--10 mg of Fibers
-0- 20 mg of Fiber's
Z 2000 -w 40 mg of Fibers
--v-- 60 mg of Fibers 0U 0mg of Fibers

1500
0

C 1000
0

=500

0

0 510 15 20 25
Time (days)

Figure 4-20. RMSC growth curves for each fiber density. Star indicates site of 1 st plateau where Schmidt et al. show maximal osteocalcin concentration, the characteristic late differentiation marker. # indicates time point where Schmidt et al. found alkaline phosphatase, the early differentiation marker was highest.

Discussion

The potential of fiber based scaffolds in bone replacement is extensive. Flexible mechanical properties, high porous volume, designable woven architectures, all contribute to the attractiveness of fiber based scaffolds in connective tissue engineering. Single fibers influence cell orientation and elongation as shown in chapter 2. This behavior is very similar to that seen on flat 2-D surfaces with microtopographies of given dimensions [29,31,32,89]. This cell type leads to multi-layering on flat surfaces, a cellular behavior that is essential to the formation of ECM nodules and the





84


mineralization of these nodules [80,115,116]. Similar multi-layering behavior is seen on single fibers leading to similar nodule formation and mineralization.

Multi-layering and nodule formation are functions of many cells, or aggregations of cells, which communicate with each other [104,124] and cooperatively perform. How these cells grow on single fibers, as well as flat micropattemed surfaces is similar in that there is only one substrate for them to interact with, either a fiber, or a flat surface. With the introduction of more than one surface, or macrotopography the cellular response becomes more complex. In this context macrotopographies can be thought of as those created by multiple surface with features on the size order of the multi-cellular aggregates, while microtopographies are those that act at the level of the individual cell.

Bridging acts at the macrotopographical level, the level that 3-D scaffolds necessarily exhibit. Single fibers lead to a microtopographical response, but where two fibers contact, a macrotopographical feature exists. In this sense, 3-D fiber-based scaffolds are hierarchical, combining not only microtopography where cells are in contact with only a single fiber surface, but also macrotopography features where two fibers interact and bridging occurs. Therefore, an understanding of single fiber effects on cell function (i.e. multilayering), as well as multi-fiber effects (bridging) is essential for future fiber based scaffold design, or any other hierarchical 3-D scaffold design. This is particularly so in light of the fact that tissues are 3-D and these interactions, or mechanisms are obviously part of the tissue level machinery necessary for the development of bone, or ligaments. Bridging seems to progress in a generalizable developmental sequence of events as is shown in Figure 4-21. Single cell bridging occurs first (Figure 4-21 A) as a cell encounters macrotopographies like the wall of a culture dish, or the intersection of two





85


fibers. An example of this is shown in Figure 4-9 of the results. Multi-cellular bridging follows as more cells become involved either as they are lifted from their surface in a sheet-like manner, or proliferation that occurs within the body of the bridge itself. Both behaviors appear to be present as seen by the sparsity of cells below the bridge in Figure 4-10, as well as the thickening of the same bridge over time. Eventually multi-cellular



A]








FD F]






Figure 4-2 1. Schematic generalization of multicellular bridging. structure exhibits ECM nodules within its body (Figure 4-2 1 C) that eventually mineralize as seen when MSCs were grown on the stainless steel screens. In the case of the polystyrene culture dish the bridge contracted until it had pulled from the side of the dish and formed a large nodule of ECM and cells (Figure 4-2 1 D).

The context of this bridging phenomenon is to be found in many areas of the literature. Cells of mesenchymal origin grown on 2-D surfaces with [31,32,73,74,89,94,125], show that proliferation and differentiation are strongly influence by the ability for these cells to adhere to their substrate. Adhesion is





86

influenced by the factors of surface roughness [90,121,126,127], or topography [31,32], surface chemistry [128] and surface energy [129].

In addition to the relationship between adhesion and the cellular ability to develop tension, there is the effect of mechanical stretch on cellular activity. Stretch applied externally to MSC leads to increased differentiation, as well as an orientation of ECM in the direction of the force [101]. Similarly, inhibition of chloride sensitive stretch receptors causes a decreased response to topography, drawing a link between the topography and the cellular need for stretch, or tension [100]. It is interesting to note how the strength with which a cell adheres and develops tension, as well as an applied stretch stimulus both result in similar cellular responses in the form of proliferation and differentiation. Overall it seems tension generation is key for proper proliferation and differentiation of mesenchymally derived cells on a 2-D surface.

Though 2-D culture systems provide a glimpse of the cellular machinery involved in proliferation and differentiation, tissues are 3-D structures composed of cells. It seems therefore that the cellular process of tension generation is designed to operate in the 3-D environment, a theory that is bom out by many examples in the literature. Studies performed using collagen gels as scaffolds for cells of mesenchymal origin show that increased stiffness of the scaffold leads to an increased rate of proliferation. In addition to this increased rate the duration of proliferation persisted longer than that seen in scaffolds with less stiffness [80]. In similar studies collagen gels were anchored on one axis leading to a preferential tension development in the direction of this axis by fibroblasts. Cell numbers increased by 5 times in the anchored gels and ECM generation was greater than that seen in the un-anchored gels, which in contrast demonstrated a 5




Full Text
FIBER BASED SCAFFOLDS IN CONNECTIVE
TISSUE ENGINEERING:
USING THE ARCHITECTURE OF WOVEN SCAFFOLDS
TO INFLUENCE AND CONTROL THE FORMATION
OF ORGANIZED TISSUES
By
CHARLES ALAN SEEGERT
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2002

Copyright 2002
by
CHARLES ALAN SEEGERT

To the Pinnacle

ACKNOWLEDGMENTS
I would like to thank everyone who made it possible for me to perform the research in
this dissertation. In particular I would like to acknowledge Dr. Colin Sumners and the
people in his lab who provided me with the tissues that I used to develop my cell cultures.
I would especially like to thank Dr. Brennan, my major adviser, who allowed me the
time and provided me with the resources I needed to develop my ideas.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES ix
ABSTRACT xiii
CHAPTERS
1 INTRODUCTION 1
Autogeneic Bone Grafting 2
Allogeneic Bone Grafts 3
Synthetic Bone Replacement Materials and Xenogeneic Bone Grafts 5
Problem to be Approached 6
Tissue Engineering 8
The Case for Hierarchical Organization of Fiber Constructs 9
2 BACKGROUND 13
Bone Formation in Utero 13
MSC Differentiation 15
Bone Development and Bone Anatomy 21
Fracture Healing and Ectopic Bone Formation 25
Porosity and Diffusion Properties of Implant Materials 25
Remodeling and Cellular Orientation 28
Contact Guidance 29
Architecture of Fiber Based Scaffolds: Development of Cell-Based Tension 33
Autocrine/Paracrine Considerations in Scaffold Design 37
3 SINGLE FIBER STUDIES 42
Introduction 42
Materials 44
Methods 45
Single Fiber Scaffolds 45
v

Cell Culture 45
MSC Seeding 46
SEM Imaging (Nylon sutures) 46
Light Microscopy (Maxon Sutures) 47
Results 47
SEM Studies 47
4-0 nylon sutures 47
9-0 nylon sutures 49
10-0 nylon sutures 50
Maxon sutures 51
Light Microscopy Studies of Maxon 53
Nuclear form factor 55
Nuclear angle analysis 58
Discussion 61
Conclusions 62
4 CELLULAR BRIDGING PHENOMENA 64
Introduction 64
Materials 66
Methods 66
Culture Methods 66
SEM Imaging 67
Light Microscopy Studies 67
Von Kossa Staining 67
Proliferation Studies 68
Results 69
Light Microscopy of Bioactive Glass Fibers After 6 Days in Culture 69
Scanning Electron Microscopy 71
Bioactive glass fibers after 6 days in culture 71
Polymer fibers 72
Light microscopy of Bridge Development on Polystyrene Culture Flask 73
Bridge Development on Stainless Steel Screens (Light Microscopy and SEM)... 77
Proliferation Studies of RMSCs on Bioactive Glass 81
Discussion 83
Conclusions 92
5 MULTI-FIBER STUDIES 94
Introduction 94
Materials 97
Methods 97
Cell Culture 97
Construct Preparation 97
Maxon Construct Sterilization 99
vi

Stainless Steel Construct Preparation 100
Transmission Electron Microscopy 101
Statistical Analysis 101
Results 104
Maxonâ„¢ Bridging 104
Maxonâ„¢ bridging day 3 104
Maxonâ„¢ bridging day 6 104
Maxonâ„¢ bridging day 9 105
Maxonâ„¢ bridging day 10 105
Maxonâ„¢ bridging day 11 105
Multi-Layer Construct Bridging 107
Cell Angle and Spacing Distance 109
Stainless Steel Bridging 109
Stainless steel bridging day 3 109
Stainless steel bridging day 6 110
Stainless steel bridging day 8 110
Stainless steel bridging day 10 (mineralization analysis) 110
Transmission Electron Microscopy Ill
Transmission electron microscopy of 7-0 single fiber 111
Transmission electron microscopy of 7-0 25 pm spaced parallel array 112
Transmission electron microscopy of contracted 7-0 25 pm parallel
array 114
Discussion 117
Conclusions 123
6 CONCLUSIONS AND FUTURE WORK 125
APPENDIX QUANTITATIVE CONTACT GUIDANCE ANALYSIS METHODS ....132
Conceptual and Illustrated Review of Nuclear Form Factor (NFF) 132
Extension of NFF Correction Concept to Nuclear Angle Measurements 134
Mathematical Derivation 135
LIST OF REFERENCES 138
vii
BIOGRAPHICAL SKETCH
150

LIST OF TABLES
Table page
1-1. List of Design Requirements for More Ideal Bone Replacement Material 6
5-1. Summary of Statistical Results from the Mineralization Study Performed on
Stainless Steel Screens 111
Vlll

LIST OF FIGURES
Figure page
1-1 An example of a femoral reconstruction using a segment of allograft bone 5
1-2 Schematic representation of diaphyseal bone replacement 7
1 -3 A diagram of the general orientation of lamellae in a segment of bone 9
1 -4 Schematic representation of collagen fibril orientation within a segment of bone... 10
1-5 General concept of the hierarchical level of woven fiber based scaffolds 11
2-1 Schematic of the cell cycle 14
2-2 MSC developmental sequence leading to bone producing cells 17
2-3 Lines of force seen in proximal portions of the femur 21
2-4 Examples of lamellar orientation within a section of bone 23
2-5 Fibrillar orientation within bone 23
2-6 Rotated plywood model 24
2-7 Schematic representation of ectopic bone formation around implanted,
demineralized bone chips 26
2-8 Receptor mediated adhesion of osteoblasts via fibronectin 30
2-9 Diagram of autocrine signaling 38
2-10 Diagram of paracrine signaling 40
3-1 Examples of Maxonâ„¢ single fiber constructs 45
3-2 4-0 nylon fibers with RMSCs after being cultured for 5 days 48
3-3 Examples of MSC growth on the surface of 4-0 nylon fibers 49
3-4 Examples of RMSC growth on the surface of 9-0 nylon fibers 50
IX

3-5 Examples of RMSC growth on the surface of 9-0 nylon fibers 51
3-6 SEM images of 10-0 nylon sutures at various magnifications 52
3-7 5-0 Maxon sutures exhibiting RMSC adhesion and growth 53
3-8 6-0 Maxon sutures exhibiting RMSC adhesion and growth 54
3-9 7-0 Maxon sutures exhibiting RMSC adhesion and growth 54
3-9 Continued. 7-0 Maxon sutures exhibiting RMSC adhesion and growth 55
3-10 Example of nuclear form factor measurements 55
3-11 Graph showing the independent influence of fiber diameter on NFF 56
3-12 Graph showing the independent influence of time on NFF 57
3-13 Graph showing the effects of time on NFF for all diameters studied 57
3-14 Example of nuclear angle measurements 59
3-15 Graph showing the effects of diameter independent of time 59
3-16 Graph showing the effect of time in culture on nuclear orientation as measured via
nuclear angle 60
3-17 Graph showing the effects of time and diameter together 60
4-1 Multilayering on adjacent fibers leading to interactions due to their proximity....65
4-2 Example of bioactive glass fiber constructs used for the proliferation study 68
4.3 Montage of light micrographs portraying the cellular interaction with bioactive
glass fibers placed in standard culture well 70
4-4 Light micrograph of area without fibers in the same culture well as that shown in
Figure 4-3 70
4-5 Examples of cellular growth on fiber placed in the RMSC system 71
4-6 Examples of unicellular bridging on bioactive glass over distances of ~70 pm. ..72
4-7 Examples of unicellular bridging between nylon fibers 73
4-8 Bridging between Maxon fibers 73
4-9 Initial RMSC elongation 74
x

4-9 continued. Initial RMSC unicellular bridging 75
4-10 Multicellular bridge at 7 days in the RMSC culture 76
4-11 Multicellular bridge at 8 days in the RMSC culture 76
4-12 Multicellular bridge at 10 days in the RMSC culture 77
4-13 Multicellular bridge at 11 days in the RMSC culture 77
4-14 Development of bridging on stainless steel screens 79
4-15 SEM of stainless steel screens after 10 days in RMSC culture 80
4-16 SEM of stainless steel screens after 12 days in RMSC culture 80
4-17 SEM of stainless steel screens after 15 days in RMSC culture 81
4-18 SEM of stainless steel screens after 18 days in RMSC culture 81
4-19 SEM of stainless steel screens after 23 days in RMSC culture 82
4-20 RMSC growth curves for each fiber density 83
4-21 Schematic generalization of multicellular bridging 85
5-1 Eye shaped bridging phenomenon caused by the weave of the screen 96
5-2 Pictures of mult-fiber micromanipulator 98
5-3 Examples of multi-fiber constructs for RMSC culture 99
5-4 Diagram of stainless steel construct preparation 100
5-5 Diagram of stainless steel statistical test 102
5-6 Examples of single fiber RMSC growth 106
5-7 Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 25 pm. 107
5-8 Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 55 pm. 108
5-9 Micrographs of multi-layer parallel arrays 108
5-10 Examples of bridging between fibers of 25 pm and 55 pm spaced parallel arrays. 109
5-12 Transmission electron micrograph montage of 7-0 single fiber construct in cross-
section 112
xi

5-13 Transmission electron micrograph montage of 7-0 single fiber construct in long-
section 113
5-14 Transmission electron micrograph montage of 7-0 multi-fiber 25 pm spaced
parallel array in cross-section 114
5-15 Transmission electron micrograph montage of contracted 7-0 multi-fiber 25 pm
spaced parallel array in cross-section 115
5-16 TEM images of longitudinal section of the contracted portion of 7-0 25 pm
spaced parallel array 116
5-17 Characteristic banding pattern of collagen fibrils 117
5-18 Schematic representation of flat surface multilayering versus bridging 122
6-1 Surface of a 5-0 maxon fiber imaged with light microscope 127
6-2 Conceptual diagram of bone replacement 128
6-3 A possible sequence of growth factors for release in a bone replacement system. 131
A-1 Diagram of NFF measurement 132
A-2 Diagram of relationship between nuclear dimensions and fiber geometry 133
A-3 Diagram of trigonometric approximations used to determine nuclear dimensions. 133
A-4 Diagrammatic comparison of NFF and nuclear angle 134
A-5 Diagrammatic relationship between NFF measurements and nuclear angle
measurements 135
A-6 Diagrammatic representation of calculated rotational correction 136
Xll

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FIBER BASED SCAFFOLDS IN CONNECTIVE
TISSUE ENGINEERING:
USING THE ARCHITECTURE OF WOVEN SCAFFOLDS
TO INFLUENCE AND CONTROL THE FORMATION
OF ORGANIZED TISSUES
By
Charles Alan Seegert
August 2002
Chair: Anthony B. Brennan Ph.D.
Major Department: Biomedical Engineering
The skeleton generates locomotion and provides mechanical support for the human
body. It is essential in every aspect of normal living, which is most apparent when it is
damaged or rendered useless by disease. The repair of the skeletal system by orthopedists
is rather common, occurring millions of times each year. During the 1990s it was
estimated that nearly a million cases per year were bone graft cases, which makes them
one of the most common forms of transplantation in the United States today.
Orthopedic reconstruction restores function, extending and increasing quality of life.
Though progress has been made toward reaching this ideal, bone grafting is still fraught
with shortcomings. Its weaknesses become most apparent when large skeletal defects like
those seen in osteosarcomal resection are treated. Commonly these reconstructions are
xm

performed using allografts combined with steel rods and other devices. Synthetic
materials may also be used, but full skeletal incorporation never occurs and they remain
inanimate. Because they are non-living, these materials accumulate fatigue and
eventually fail.
Cell-based tissue-engineered replacements are a strong candidate to address these
challenges. This treatment system would be composed of a synthetic material, like
resorbable polymer fibers, combined with cells that are loaded onto the fibers. After
implantation the system would foster development of a living and thus self-repairing
replacement tissue.
This study focuses on synthetic scaffolds and how they interact with the cells loaded
onto them. Established principles of contact guidance were applied to influence
orientation and growth of cells on these scaffolds. Guidance of cells and their extra¬
cellular matrix (ECM) products is shown on the level of single fibers. More important,
however, is the direction of cells and ECM when fibers were organized into regular 3-D
structures. Cellular organization and direction far exceeded what has been seen on flat
surfaces, or single fibers. The goal of this work was to use scaffold architectures to
organize cells and ECM in a 3-D manner as is seen in normal tissues. The results of this
study indicate the success of this concept and represent a large step toward the
development of this technology.
xiv

CHAPTER 1
INTRODUCTION
In addition to providing mechanical support for the human body, the skeleton is
integral in generating locomotion. Indeed the musculoskeletal system is an essential
component in nearly every aspect of a person’s life, a fact that is made most apparent
when a portion of this system is damaged or rendered useless by disease. Unfortunately
the repair of the skeletal system by orthopedists is rather common, occurring millions of
times each year alone. During the 1990s it was estimated that nearly a million cases per
year were bone graft cases [1], making bone grafting one of the most common forms of
transplantation in the United States today.
The intent of orthopedic reconstruction is to restore function, thus extending and
increasing the quality of life an individual experiences after injury. Clearly much
progress has been made toward reaching this goal. However, despite this progress and the
frequency with which bone grafting is used, this treatment method is fraught with
difficulties and shortcomings. Implantation of a bone graft or a synthetic bone
replacement material is performed when a void in the skeleton is created by trauma or
resection for pathological treatment. Another major implementation is when injured
bones fail to re-join as is seen in non-unions at a fracture site [2,3], In these situations,
emplacement of a bone graft, or a synthetic bone replacement material is then performed
to bridge the gap, restoring skeletal integrity.
1

2
When reconstructing skeletal defects, three major graft materials are used: autogeneic
bone, allogeneic bone and xenogeneic bone. Synthetic materials like porous
hydroxyapatite and bioactive ceramics have been used in some cases, as well. A degree
of success has been realized with each of these materials, but there are also significant
characteristic limitations to all of them.
Autogeneic Bone Grafting
Autogeneic bone grafting is considered the gold standard and all other materials are
compared to it when their efficacy is evaluated [4], This type of bone graft is taken from
a site within the patient’s own body and thus it is recognized by the body when implanted
elsewhere. Additionally, autograft bone is a living graft, which contains bone producing
cells. Because of this feature, cells are deposited into the site where new bone growth is
desired, thus stimulating a much more rapid recovery. This type of bone deposition and
growth is “osteogenic,” a classification that includes grafts containing phenotypically
committed osteoblasts, or grafts that stimulate proliferation of committed osteoblasts [4],
Other graft materials depend on the infiltration of cells from the surrounding area; thus
recovery takes longer if it occurs at all. This type of healing, which is characterized by
materials providing a scaffold to direct bone growth, is called “osteoconductive” [4],
One further classification of bone graft materials that is particularly germane to this study
is “osteoinductive” materials. Osteoinductive grafts lead to differentiation of
mesenchymal stem cells, or osteoblastic precursors, thus causing them to become fully
committed bone producing cells [4],
Though autogeneic bone is considered the best material, there are still significant
drawbacks to its use. The quantity of this bone graft material available is very limited.

3
Clearly only so much bone can be removed from one part of the body in order to heal
another part; anything more would only create a new defect in skeletal integrity. In
addition to this, harvest sites often experience lengthy and painful recovery periods. In
many cases the recovery of the harvest site takes longer than the recovery of the original
injury the bone graft was used to treat [4].
Allogeneic Bone Grafts
Allogeneic bone grafts are widely used, as well. Unlike autograft bone, allograft bone
is not as limited by supply. This material is taken from other human bodies, usually
deceased, and then passed through a series of treatments ideally rendering it non-
immunogenic and free of pathogens. Allograft bone is used to repair small skeletal
defects with much success. For this use, it is usually “morselized,” or broken up into
small fragments that are then packed into the fracture site. The volume of most defects
treated this way is relatively small, which is related to the success of this application.
Allograft bone is acellular and osteoconductive; therefore it must be infiltrated with bone
producing cells in order to recover. Additionally, there are a number of growth factors
that are utilized by these cells during recovery. Depths of cellular migration into the graft
material, as well as the distance that growth factors can diffuse without degradation, are
again quite limited [5]. Full integration of allograft bone does occur; however. These
distance related factors seem to prevent integration with the body beyond relatively small
distances on the order of a few centimeters [6].
In addition to treating smaller skeletal defects with morselized allograft material, large
skeletal reconstructions are performed with intact, non-morselized segments of allograft
bone. In situations like this the drawbacks of allograft bone become most apparent.

4
Integration by the body ensures the presence of cells and the formation of a living tissue.
Living tissues are capable of self-repair and are regenerate in the presence of stresses
associated with daily activity. Non-animate replacements, even though of a biological
origin, do not repair themselves and normal fatigue processes are continuously at work.
Treatment of osteosarcomas often requires large resections. Historically, when this
disease was encountered, a patient’s limb was amputated leading to a life long handicap.
In the early 1960s, however, limb salvage became the standard of care in these cases.
Providing a mechanical means of support via implantation of allograft bone allows the
patient to maintain the reconstructed limb and a much higher quality of life [7]. During
limb reconstruction, allogeneic bone grafts are frequently augmented with metal fixation
devices, like inter-medullary nails, or plates. Figure 1-1 shows an example of just such a
construction using allograft bone.
Sometimes, in addition to the fixation devices, bone cements are also used, leading to
large static composite structures. At best, these rigid conglomerates of organic and
inorganic materials are fractionally incorporated into the patient’s skeleton due to the
distance limitations mentioned previously. This limited amount of repair occurs at the
ends of the allograft, where it comes in contact with the living bone allowing cells to
infiltrate [3], The rest of the composite structure remains a rigid mass of dead bone and
inorganic components and even the limited amount of incorporation described above only
occurs about 70 % of the time [8],
This remedy is better than the alternative—no limb—but the allograft is never fully
integrated and the accumulation of fatigue eventually culminates in failure of the device.

5
Figure 1-1. An example of a femoral reconstruction using a segment of allograft bone.
The white structure in the center of the image is a metal rod holding the composite in
place.
This failure tends to occur in areas where a hole has been introduced into the allograft
material, sometimes as soon as 1-2 years after implantation [9].
Synthetic Bone Replacement Materials and Xenogeneic Bone Grafts
Synthetic bone replacement materials and xenogeneic bone grafts, like allograft bone,
are not limited by supply. Examples of this class of materials are porous hydroxyapatite,
choraline hydroxyapatite and bioactive glass materials. Generally these materials are
processed into bulk materials with a porous structure leaving relatively poor allowances
for diffusion of nutrients. For example, porous hydroxyapatite only possesses a porous
volume of approximately 30% [10]. Because of this, when it comes to integration these
materials exhibit distance limitations in a way similar to allograft bone. Xenografts, or
grafts derived from other species, are rarely used due to the high chances of rejection and
the possibility of disease transmission between species [4],

6
Problem to be Approached
Bone grafting, particularly of large diaphyseal segments, is far from ideal. Synthetic
materials can only be used to fill small defects, while large defects are repaired using
allograft material. Allograft bone is useful in that it is of the correct dimensions and
density and does possess some of its original mechanical strength; however, fatigue
problems limit its viability over time. For smaller bone defects autograft bone is the
material of choice, but it is limited in quantity and its removal often leads to a long
painful recovery in the area from which it was taken. A number of synthetic materials
have also been used with some limited success, but no truly suitable substitute yet exists,
especially for large segmental bone defects [4],
After examining the currently used bone graft materials and methods, a number of
deficiencies become apparent that must be overcome in order to develop more viable
synthetic bone replacement systems. Opportunities for improvement are summarized in
table 1-1.
Table 1-1. List of Design Requirements for More Ideal Bone Replacement Material
-Unlimited Quantity
-No Pathogenic Transmission
-No Immunogenicity
-Osteoinductive and Osteoconductive to be Fully
Incorporated over Large Volumes.
-Highly Porous to Allow Proper Diffusion and
Infiltration of Cells.
-Mechanically Robust to Provide Support Until
New Tissue Becomes Self Supporting.

7
Overall, a bone replacement system capable of filling large skeletal defects would be
ideal. Using the replacement of a diaphyseal segment of long bone as a goal, or model
system presents a somewhat simplified bone replacement problem. Figure 1-2 shows a
schematic representation of this idea, which if successfully achieved would pave the way
for more complex problems like joint reconstruction, or tendinous attachments.
Additionally, many of the problems associated with replacement of bone in other areas of
the body would be answered.
Figure 1-2. Schematic representation of diaphyseal bone replacement. A. Damaged, or
diseased segment of a long bone. B. Removal of diseased area with margins of healthy
bone, followed by emplacement of a graft/ engineered synthetic replacement. C. The
reconstructed bone with natural and synthetic components. D. After healing and
subsequent resorption of the synthetic replacement material a new segment of living
tissue persists and the patient is healed.

8
Tissue Engineering
Tissue engineering is defined as: the application of engineering disciplines to either
maintain existing tissue structures, or to enable tissue growth [11]. A tissue engineered
construct would solve many of the problems associated with bone grafting. Indeed there
are already many studies in the literature that implant scaffold materials loaded with cells
taken from the patient, or subject [12-20]. This method, where autogeneic cells are taken
from a patient, loaded onto a scaffold and then implanted, is referred to as a “cell based
approach” to tissue engineering.
Using this approach requires a scaffold, or support matrix that cells can adhere to and
proliferate on prior to implantation. Eventually this scaffold would resorb and be excreted
from the body, leaving behind a living segment of bone tissue that is completely
biological. In this model the scaffold is only present to facilitate and direct the growth of
cells and the deposition of cell products, while its eventual resorption allows the induced
tissue to become completely integrated into a subjects anatomical and physiological
systems. Resorbable polymers, particularly in the fiber form meet this requirement, as
well as all the needs listed in table 1-1. Using appropriate processing methods, individual
fibers can be made with remarkably robust tensile strength [12-23]; more importantly,
however, these fibers can be organized in a woven construct that is very rigid [11]. Once
cells have been induced to adhere and grow on a scaffold they will require adequate
nourishment and gas exchange, which presents another major advantage of fiber based
constructs: diffusive properties [23,24],
As mentioned previously, other materials commonly used to replace, or reconstruct
bone have relatively poor allowances for diffusion of nutrients and thus are limited.

9
Randomly packed fibers, on the other hand, exhibit porosities greater than 95%, allowing
for much greater nutrient inflow and waste matter outflow [25],
The Case for Hierarchical Organization of Fiber Constructs
Bone, like many other tissues in the body, exhibits anisotropic mechanical properties.
This directional difference in stiffness, which depends on orientation with respect to the
bone’s long axis, is a characteristic that is directly tied to its layered molecular and
cellular organization. Bone’s structure is hierarchical [26] with two levels of this
hierarchy that are particularly relevant to its mechanical properties: its lamellae, or
layered organization and the collagen fibril arrays within each lamella. Lamellar units are
approximately 3 pm wide and are oriented in a direction parallel to the long axis of the
bone itself (Figure 1-3).
Figure 1-3. A diagram of the general orientation of lamellae in a segment of bone. Red
arrow points out the plane the lamellae are oriented in is in the direction of the long axis
of the bone. Adapted from Liu et al. [63].
Each of these arrays, though rotated around an axis perpendicular to the bone’s long
axis, remains parallel to that axis in their other dimensions (Figure 1-4). Each layer
represents an oriented collagen fibril array and as these parallel fibrils are stacked each
layer is rotated approximately 30 degrees, as is the case in bone [27,28]. The orientations

10
of these collagen fibrils and the presence of many lamellae are responsible for the
mechanical anisotropy of bone as will be discussed more thoroughly in the background.
Logically, when engineering a replacement for large segments of bone, it is desirable
that these systems introduce a similar anisotropy. Fibers below a diameter of ~100 pm
have been shown to influence the orientation of cells grown on them, a phenomenon
known as contact guidance [29]. Cells that have been oriented using principles of contact
guidance have also been shown to deposit their extra-cellular matrix (ECM) products
parallel to their orientation [30-32], Most notably this has been seen in cases where fibers
are used to replace tendons [30].
Figure 1-4. Schematic representation of collagen fibril orientation within a segment of
bone. The cylinders that are fanning out with respect to each other (red arrows) represent
the orientations of collagen fibrils as they are organized within a single lamellae.
Therefore each lamella is composed of collagen fibril layers organized in this pattern.
Adapted from Weiner and Wagner [26].
By creating parallel arrays of fibers that are then organized into layers it stands to
reason that cells oriented by each lamella would deposit ECM in a manner directed by
that layer (Figure 1-5 A). Many layers sandwiched together with angles of rotation
between adjacent layers conceivably could produce an anisotropic engineered material
(Figure 1-5B). This material would be a laminated composite similar to that of normal

11
bone. Weaving scaffolds with desired orientations would then make it possible to design
large scale constructs with the architecture necessary for replacing large segments of
bone (Figure 1-5C).
Figure 1-5. General concept of the hierarchical level of woven fiber based scaffolds. A.)
represents the effects of the first level, which is composed of the fibers themselves. B.)
Shows the second level of the hierarchy, which is weaving of the individual fibers to
form large woven sheets responsible for cellular orientation on a lamellar basis. C.)
Finally combining these many lamellae together and putting them in to a 3-D structure
provides the highest level of the hierarchy and even more mechanical integrity.
A first step toward developing this technology is understanding how mesenchymal
stem cells, the osteoblastic precursors, interact and are oriented by fibers over time.
Additionally, understanding how MSCs are influenced by the spacing and organization of
fibers in their multi-layer configurations is a key requirement.
The specific aims of this work are designed to elucidate the effects on cellular activity
resulting from some of the levels in the hierarchy described above. Studying and
extending what is known about the effects of fiber diameter on cellular and ECM
orientation will be first. Following the single fiber studies will be multi-fiber studies,
which focus on the effects of fiber spacing within a construct composed of fibers

12
arranged in parallel array. Finally, multi-layered constructs with parallel arrays arranged
as shown in Figure 1-5B will be examined.

CHAPTER 2
BACKGROUND
To engineer any synthetic tissue replacement material, it is essential to have an
understanding of the normal physiology of that tissue. Once a familiarity with the
systems involved and the interplay of these systems is achieved, likely sites of
manipulation become evident. Controlling these critical sites and thus the development of
the desired organ system is the goal of a true tissue engineer.
Bone Formation in Utero
Bone formation begins early on in fetal development and progresses rapidly
throughout normal gestation. The cartilaginous beginnings of the skeletal system are seen
as early as the first month of fetal growth when mesenchymal cells begin to lay down
different forms of collagen in an organized manner. These cells separate into two layers,
the outer layer, which is composed of differentiated fibroblasts, and the inner layer,
which remains undifferentiated mesenchymal cells. These layers together are referred to
as the perichondrium, a structure that later becomes the periosteum [33].
Ossification, or the mineralization of the developing skeleton, begins in the second
month of pregnancy and proceeds via two mechanisms. The first mechanism of
mineralization to occur is known as intra-membranous ossification and it takes place in
sites like the calvarium and the clavicles. Intra-membranous ossification is a direct
mineralization in the connective tissues of the fetus, which contrasts with the other
mechanism of bone formation: endochondral ossification.
13

14
Endochondral ossification occurs after a scaffold of cartilage is laid down. This
scaffold of cartilage is then mineralized and eventually replaced with bone that is guided
by its presence. The only difference between intra-membranous ossification and
endochondral ossification is the latter’s requirement of a cartilaginous scaffold, which
must be deposited first [33].
After the initial mineralization of the cartilaginous scaffold, the development of true
bone begins. This formation of true bone begins following the infiltration of vasculature
and subsequent supply of oxygen and nutrients [33-34], MSCs are the developmental
precursors to nearly all the connective tissues in the body as schematically shown in
Figure 2-1. The presence of oxygen as supplied by the vasculature plays a significant role
in controlling the level of differentiation achieved by MSCs [33-35].
Figure 2-1. Schematic of the cell cycle. Top left corresponds to the diagram of cell
division below it; outlining the proliferative stage of MSC development. With the advent
of correct environmental cues MSCs undergo differentiation and enter the Go phase.
Depending on the cues sensed by MSCs they are capable of becoming many different
tissue types as listed on the right.

15
MSC Differentiation
Transformation of MSCs into mature bone producing osteoblasts is a multi-step
process involving a number of environmental and cell based signals. This differentiation
occurs in the Go phase of the cell cycle (Figure 2-1), which is also known as the quiescent
phase since cells are no longer multiplying [36-39]. The diagram also shows that
differentiation at this point leads to different tissue types depending on the cues provided
by the environment.
It is hypothesized that bone development along this pathway depends in part on one
major environmental cue: the supply of oxygen by newly formed vasculature [33-35].
Indeed it appears that MSCs become fibroblasts if there is a relatively low supply of 02,
but osteoblasts if O2 is readily available. Once neovascularization has occurred and this
signal has been received, passage of MSCs to fully developed bone producing cells
proceeds along an orderly and well-defined path, which is reviewed below. This
development is governed by a cascade of cellular based signals, or cytokines, which act
in close concert with the cellular events seen in the developing tissue.
The differentiation of MSCs that leads to bone occurs in the GO phase of the cell cycle
and is schematized below in Figure 2-2. Osteogenic cells develop in a three stage process
with two point in between called restriction points [36-39]. Differentiation and
advancement through each point are restricted until certain conditions are met; then
development may proceed [36,37].
It is generally held that this progression of cellular events is regulated by an intricate
system of feedback mechanisms and chemical cues among the developing cells
themselves and between the cells and their environment [40-42], The proteinaceous
chemical cues, or cytokines, involved in osteogenic cell development are composed of a

16
family of growth factors called bone morphogenetic proteins (BMPs). This family of
proteins is quite large and includes the TGF-P isoforms. Each protein is numbered; for
example the protein involved in the first stages of MSC differentiation is labeled BMP-2.
The role of the BMPs in bone development has been examined from many different
viewpoints. One type of study involves following bone formation, while examining the
locations and temporal sequence of the individual components of the skeletal system that
are produced [43]. Other types of studies follow the production of growth factors and
BMPs spatially and temporally in vivo. The first type of study shows each cell product is
characteristic of a particular stage of cellular development. The second type of study
shows where and when the growth factors and BMPs are released, allowing the two to be
inter-related by shared temporal and spatial relationships [40,41]
In addition, there are other studies that focus on the function of individual growth
factors and BMPs, which provide corroborating evidence for the conclusions drawn from
the inter-related temporal and spatial studies [44]. Essentially knowledge of the BMP
location in the temporal and spatial sequence can be ascertained by the cellular responses
they have been seen to induce. The validity of correlating specific protein production
with a level of bone cell differentiation, as is described above, has been examined in the
past and found to be acceptable in analogous situations [44], Using this method, the
systemic influences that guide the development of the cells and the location of these
BMPs in the cycle can be understood.
The development of osteogenic cells proceeds in a three stage process with two points
in between called restriction points (Figure 2-2). Differentiation and advancement

17
through each point are prevented until certain conditions are met; then development may
proceed.
Stage one is proliferation. Bone morphogenetic protein-2 (BMP-2) and transforming
growth factor-P (TGF-P) are active during this first and earliest phase of cellular
development. BMP-2 is responsible for the initial levels of differentiation [45]. In fact
BMP-2 is specifically located to MSCs, which are considered its target cell [41]. TGF-P
is largely responsible for proliferation, or expansion, and it also stimulates production of
extra-cellular matrix (ECM) components [35,46], BMP-2 and TGF-P each have an
influence on MSC development when they are present individually, but when they are
both present concurrently, they act in a synergistic manner [47].
PROLIFERATION
1
NEGATIVE FEEDBACK
DOWN REGULATING
PROLIFERATION
**
COLLAGEN/
FIBRONECTIN
SYNTHESIS
1
ECM MATURATION
1
NEGATIVE FEEDBACK
DOWN REGULATING
ECM MATURATION
^ W
ECM MINERALIZATION
Figure 2-2. MSC developmental sequence leading to bone producing cells. This sequence
occurs in the Go phase of the cell cycle as seen in Figure 2-1. Adapted from Stein etal.
[36].
In addition to proliferation, the production of ECM is also an important aspect of the
first stage in bone development [48]. TGF-P stimulates production of collagen I, but
collagen II is also produced in significant portions during this time. Collagen II is
integrally associated with endochondral ossification [44] and a cell product that has been

18
associated with BMP-4 [49,50]. BMP-4 appears to be responsible for the number of
chondrocytes that are recruited into the bone producing pathway [51], and it has been
linked to the production of alkaline phosphatase [52,53]. Alkaline phosphatase indicates
that the cells have progressed past the first restriction point, which implicates the value of
BMP-4 in stage one with its activity beginning after TGF-(3 and BMP-2 have started to
produce their effects.
The first restriction point resides between stage 1 and stage 2 of MSC differentiation.
Proliferation and production of ECM occur simultaneously. During stage one, cells
continue to proliferate until they are closely associated with each other, forcing each
other into a less flattened shape. This change in cell density [36] and shape [54] signals
the cells to further differentiate, forming products that lead to ECM maturation, the next
stage in cell development.
The ECM and developing osteogenic cells also interact enhancing differentiation and
cessation of proliferation, as BMP-4 comes into play. This occurs in a feed forward
mechanism where the ECM influences the cells, which in turn influences the ECM [55].
So cell shape (which depends on proliferation) and the ECM largely dictate passage into
the second stage.
The second stage of differentiation is the maturation and modification of ECM, thus
preparing it for mineralization. The Vgr-1 gene is produced at this time by osteogenic
cells and localized into the ECM surrounding hypertrophic chondrocytes [43]. This
specifically happens around cells that are implicated in mineralization later. Vgr-1 has
been associated with vascularization as bone development continues [56], as well as the
further differentiation of osteogenic cells. Interestingly, Vgr-1 must be integrated into the

19
ECM for it to be active, which suggests as conformational change, or cleavage of a
portion of the molecule by the ECM.
The Vgr-1 gene leads to the production of BMP-6; that is BMP-6 is the gene product
of Vgr-1. BMP-6 leads to the production of the LMP-1 protein, a cell product which is
important and necessary for the final differentiation of MSCs. LMP-1 is not regulated by
BMP-2, or BMP-4, only BMP-6 has influence on this protein [57], which makes it the
main growth factor involved in the second stage of MSC differentiation.
Another aspect of the second developmental stage is the preparation of the ECM for
mineralization. It is thought that mineralization of bone requires nucleation sites for the
hydroxyapatite crystals that compose bone. Bone Sialoprotein, (BSP) a non-collagenous
ECM protein, is expressed in high levels in areas of bone that first begin mineralization,
showing its probable importance as a nucleation [58]. This evidence is further supported
by in vitro studies, which show BSP specifically causes nucleation of hydroxyapatite
crystals, where other non-collagenous ECM proteins do not [59].
The second restriction point is after the ECM maturation of the second stage. BMP-6
and the presence of BSP prepare the cellular environment for this transition, which occurs
when that environment is adequately supplied with the necessary quantity of ECM (BSP,
collagen, etc...) and level of differentiation [36,37].
Mineralization is the last stage in bone development and MSC differentiation. Mature
osteoblasts lead to the production of a number of non-collagenous protein, which are
integral for mineralization. This production largely occurs during the second stage of
development with the activity of BMP-6 and BSP. There are proteins, however, that are

20
produced during this last stage of development. Osteocalcin is one of these proteins and
is a calcium binding protein necessary for mineralization [46].
OP-1 (BMP-7) has been purported to be responsible for mineralization and, in
conjunction with the ECM changes, cause terminal stages of osteogenic cell
differentiation [60]. OP-1 also leads to the up-regulation of BMP-6 and the down-
regulation of BMP-2 and -4, which implies that its activity normally occurs in the later
stages of cell development [61].
OP-1 has been shown to induce differentiation of osteoblasts and production of bone
in a number of studies. Its role, however, appears to be in this later stage of bone
development. Using OP-1 on cells that have varying levels of development, causes many
of them to differentiate before they normally would without its stimulation [44]. Many
more cells can be stimulated to begin mineralization, inducing bone formation. OP-1,
however, does not lead to ECM production [44], Essentially, if OP-1 is present too soon
it forces cells to differentiate before they can produce the necessary ECM for vascular
development and other features of fully formed bone.
Presence of OP-1 can lead to fully developed bone when it is administered alone
[44,45], but important aspects of development that are dependent on ECM and the non-
collagenous proteins are attenuated. This implicates OP-1 as the signal protein that is
most important later in development, whose action is to stimulate final differentiation and
aid mineralization. After the ECM has been developed by the earlier stages of growth,
cells that are growing exist in population with varied levels of differentiation depending
on the specific local environment. Terminal differentiation stimulated by OP-1 seems to
influence all cells at their given stage of development, speeding them to final

21
differentiation and mineralization, bringing the whole tissue to a final differentiated
whole.
Bone Development and Bone Anatomy
Growth occurs rapidly at the ends, or epiphyses of developing bones and more slowly
toward the centers of the shafts, or the diaphyses. Rapid growth primarily leads to spongy
bone, while the relatively slow build up of bone that occurs in the diaphysis is more
compact and dense. Spongy bone is composed of trabeculae, a porous honeycomb of
interconnected bony processes. Trabeculae initially develop with a random orientation,
but with the application of the stresses associated with living the orientation of these
processes assume a pattern (Figure 2-3). In a probabilistic manner, trabeculae oriented at
Force
i
Figure 2-3. Lines of force seen in proximal portions of the femur. Weight bearing
pressures as represented by the arrow are responsible for development of trabecular
organization Adapted from Sinclair [33].
odd angles with the lines of stress imposed on the bone are broken down, while those that
are aligned are relatively unchanged [33].
Bone tissue has been optimized through evolution in ways that are very specific to its
function. During development, as mentioned above, it is remodeled through the stresses
associated with weight bearing and muscular tension, so that it exhibits properties of

22
mechanical anisotropy. The source of this anisotropy is traced to the macromolecular and
cellular level of bone.
There are a many different types of bone to be found in nature, but one of the most
important is lamellar bone. This bone type, named for its organizational structure, is the
most common bone type found in humans and is primarily responsible for the load
bearing function of the skeleton. Lamellar bone consists of many mineralized layers, a
characteristic which was noticed as early as 1906 [27]. The elucidation of the lamellar
structure has taken some time, but now it is generally held that a “rotated plywood” motif
best describes the organization of this bone type [26-28, 62,63].
The structure of bone is hierarchical [27]. Two levels of this hierarchy that are
particularly relevant to its mechanical properties its the lamellae and the collagen fibril
arrays within each lammela. Lamellar units are approximately 3 pm wide and are
oriented in a direction parallel to the long axis of the bone itself (Figure 2-4). The next
step down the hierarchy is the collagen fibril. Each lamella is composed of a number of
collagen fibril arrays rotated at angles of about 30 degrees with each other. That is, each
subsequent collagen array is rotated with respect to the one before it as one imagines,
passing from one lamellar boundary to the next. Each array, though rotated around an
axis perpendicular to the bone’s long axis, remains parallel to that axis in their other
dimension (Figure 2-5). When the rotated plywood structures, which compose lamella,
are viewed in cross section they produce microscopic patterns referred to as “nested arcs”
(Figure 2-6B) [28,62].

23
Figure 2-4. Examples of lamellar orientation within a section of bone Adapted from Liu
et al. [63],
Figure 2-5. Fibrillar orientation within bone. Fibrils are oriented in one plane which
parallels that of the bone’s long axis, while rotating at 30 degree increments with respect
to each other. Adapted from Weiner [27].
Figure 2-6A shows how the rotated layers, when stacked, produce this effect. Each
layer represents an oriented collagen fibril layer and as they are stacked each layer is
rotated approximately 30 degrees, as is the case in bone [27,28].
One can envision how the orientations of these collagen fibrils and the presence of
many lamellae make bone so mechanically anisotropic. The anisotropic nature of bone
has been studied for some time, particularly on the macroscopic level. Directionally
oriented bone specimens, which are very large compared to the lamellar sub-units, have
been subjected to stress-strain measurements. Tensile and compressive examinations of

24
Figure 2-6. Rotated plywood model. A.) Drawing of many parallel fibril arrays each
rotated 30 degrees with respect to the adjacent layers. The blue arrows highlight the
centers of the nested arcs. B.jNotice how the rotation leads to a nested arc motif as seen
in this cross-section. Adapted from Giraud-Guille [28].
these samples have shown that lamellar bone possesses markedly higher modulus values
when loaded parallel to its long axis than in any other direction [27,64], Extending these
measurements down to the lamellar and fibrillar subunit scale has been difficult,
however, some results have been forthcoming supporting the relationship between
anisotropy and the rotated plywood model.
Using microhardness instruments, the presence of anisotropy on a very small scale
was related to the orientation of the mineralized fibrils [65], Similarly, stress-strain data
using very small scale bending specimens (-160 (am diameter) supports the relationship
between the lamellar structure and its function [63].
Overall, the numerous layers present in lamellar bone confer its highest strength in the
longitudinal direction. In addition to this quality, the multi-angular orientation of the
rotated plywood model makes the bone very resistant to fracture with the application of
lateral stresses [26]. These examples demonstrate a relationship similar to what has been

25
described in aortic leaflets and arterial tissue, namely: the lamellar nature of bone, which
is derived on the macromolecular and cellular level, is a quality integral to its physiologic
function.
Fracture Healing and Ectopic Bone Formation
Skeletal repair after fracture follows a sequence of events that is virtually analogous to
that seen during development [35]. MSCs gather at the fracture site and form a repair
blastema, or fracture callus. If the fracture site is stable and not subject to micro-motion,
the MSCs will directly differentiate and become bone producing cells. If, however, there
is instability that allows small amounts of motion the MSCs will form a more
cartilaginous callus that will stabilize the break. With stability the ability for vasculature
to successfully infiltrate increases, a condition that leads to endochondral ossification
[35].
Ectopic bone formation, or the formation of bone in sites well away from the skeleton
proper, proceeds very similarly to that of fetal bone formation as well. Use of
demineralized bone chips, or polymer carriers loaded with BMPs leads to the
development of ectopic bone [15,35,66]. The initial inflammation associated with
implantation of the bone chips, or carrier is responsible for delivering MSCs to the
ectopic site, which can be subcutaneous, or within a muscle. Figure 2-7 schematically
represents this process, which again is dependent on the infiltration of vasculature.
Porosity and Diffusion Properties of Implant Materials
Proper porosity is an integral quality in a scaffold material as it controls the influx of
nutrients to the cells. Initially the nutrients must be able to diffuse into the scaffold until
such time as new vasculature has established itself. In order for vascularization to occur

26
Demineralized
Bone Chips
n
â–¡
Encysted
by MSCs
Bone Formation /
Marrowization
Vascular
Invasion
Cartilage Differentiation
Cartilage Hypertrophy/
First Bone Formation
Figure 2-7. Schematic representation of ectopic bone formation around implanted,
demineralized bone chips. It is thought that demineralized bone chips lead to ectopic
bone formation because they contain BMPs. Adapted from Caplan [35].
the pore structures must be on the order of 200-500 pm in diameter [14]. This
requirement is easily manipulated by varying the weave, tightness and diameter of the
fibers used in the fabric.
There are many studies in the literature that attempt to use osteoconductive scaffolds
to act as a synthetic bone replacement material [4,6,10,67], Osteoconduction is defined as
a material that allows vascular ingress, cellular infiltration, cartilage formation and
mineral deposition [4]. These properties are indeed required in a bone replacement
material, though they are by no means inclusive. Loading scaffolds with MSCs has also
been used with some success in studies examining bone replacement [10,16,67,68].
Osteoblastic precursors, which differentiate and become fully functioning osteoblasts are
encompassed in a phenomena defined as osteoinduction. Any method or material that

27
induces differentiation of precursor cells into adult osteoblasts is included in this
definition.
Large mechanical supports like allograft material have been shown to be
osteoconductive, but only at the ends of the allograft where it contacts the living bone [3].
Allograft bone has been fully incorporated in some instances where it has been used to
fill areas around a collapsed acetabulum [6]. This was limited to only a few centimeters
of material that was surrounded by living bone on all sides. Tricalcium phosphate
scaffolds with pores ranging from 100 pm to 300 pm and a 36% porous volume show
similar results, with vascular and cellular invasion of only 0.75 cm into the synthetic [10].
Replacing large volumes of bone has never been successful in this respect. Scaffold
materials when used alone; seem to limit the nutrient supply, which prevents continued
ingrowth.
Fibers have also been effective as a substrate in fixed bed bioreactors. Fiber type beds,
or substrates, possess porosity much higher than other types of fixed beds (> 90%
porosity). This porosity facilitates nutrient exchange by increasing the volume of medium
allowed in and out of the scaffolds structure [24,25]. Because of the fiber architecture
versus beads, or some other substrate, the cells in bioreactors with fiber beds produced
0.15 IU of interferon per cell greater than an order of magnitude higher than cells on
other substrates. A lack of diffusive ability has been a major drawback of the porous
scaffolds mentioned previously.
A fixed bed is basically a solid support matrix that is used in bioreactor technology to
maintain contact dependent cells in the culture environment. Its 3-D nature increases the
surface area for cell adhesion beyond that of a 2-D culture dish. Some systems use glass

28
beads to pack the fixed bed, but their geometry limits the void fraction available for cell
growth and diffusion [24], Using 3 mm glass beads the void fraction is as low as 35 %,
but using fibers the void fractions increase to > 90%, providing much better flow medium
flow and thus greater cell growth [25].
Remodeling and Cellular Orientation
During development, as well as fracture healing reorganization of cells and
remodeling of their ECM products occurs. This remodeling and organization is in
response to mechanical stresses and is responsible for the development of anisotropic
strength characteristic of bone [33], This portion of the healing sequence can be quite
lengthy and logically, it depends on how much the cells are organized when remodeling
begins. It stands to reason that cells that start the remodeling process in a more organized
state will have to undergo less change than randomly oriented cells and ECM.
A number of resorbable polymer scaffolds have been examined for use as scaffolds.
Martin et al. used porous polyglycolic acid and polyethylene glycol scaffolds, which
showed the ability for MSCs to differentiate when loaded on the scaffold [17]. Similarly,
Holy et al. used porous polylactic acid scaffolds in vitro, demonstrating normal cell
activity as well [12]. In vivo a number of studies have been performed using porous tri¬
calcium phosphate, or hydroxyapatite ceramics [14,16,18-20]. Using MSCs, these
implants show a development of bone tissue that incorporates the degradable biomaterial.
Both polymer and ceramic systems, however, show cells that appear to be randomly
oriented. No effort has been made to examine cell orientation in any of these studies.
Though the problem of fatigue and tissue replacement is addressed with the
development of cell based tissue engineering technology, so far there has been no attempt
to organize the implanted cells, or direct the ECM and mineralization products they lay

29
down. As reviewed next in the contact guidance section, there have been many studies
performed in vitro that indicate the efficacy of this type of cellular and ECM
organization. Applying this knowledge to cell based tissue engineering is the next step in
this area of research and the focus of this work.
As we have seen, cellular and molecular organization is primarily responsible for the
mechanical properties many tissues possess and so accomplishing this organization seems
to be a worthwhile goal. A self-healing, or living tissue engineered replacement
possessing anisotropic mechanical properties similar to bone, may be the solution to
many bone replacement problems.
Contact Guidance
Cell systems and tissues are influenced by a number of factors during their
development and the course of their existence within an organism. Two of the most
prominent factors in vivo appear to be chemistry and topography. Chemical cues have
been shown to effect cell activity during the phenomenon of chemotaxis, a cellular
movement toward a gradient of a chemo-attractant molecule [39], Cells guided by this
type of stimulus will migrate in the direction of a released cell product thus localizing
themselves to the site of injury, or need, as in the migration of leukocytes to damaged
tissue [69]. Upon fracture, it is likely that bone repair is instigated in an analogous
fashion by the release of chemical agents like TGF-P and other proteinaceous cell
products, thus attracting osteoblastic pre-cursors to the site [41,46].
Another form of influence on cells, which may be considered chemical is cell
binding to specific receptors, or arrangements of chemical molecules that are bound to a
surface (Figure 2-8). Protein mediated receptor binding to substrates is an event that

30
Fibronectin (Fn) in ECM
Figure 2-8. Receptor mediated adhesion of osteoblasts via fibronectin. Other ECM
proteins perform similar functions, though Fn is one of the most prominent. Adapted
from Alberts etal. [39].
occurs in numerous cell types within the body including those of endothelial (vascular
endothelial cells) [70-72], mesenchymal (mesenchymal stem cells and fibroblasts)
[20,73,74] and epithelial cells (neural) [75] origin.
Attachment of cells to substrate surfaces is almost always mediated by protein
adsorption [76]. These proteins are components of the serum used for cell culture and
include fibronectin and vitronectin among others [76,77] . Adhesion and attachment by
cells to these proteins depends on the ability for the proteins to adsorb to the surface,
which in turn is dependent on the surface energy of the substrate. These proteins must
also interact with the substrate in a way that does not change their conformation, thus
remaining recognizable to the cell. Adhesion and attachment have been shown to
influence rate of proliferation, or growth [71,72,76-79],
Modulus and stiffness of the substrate also have an effect on cell function and growth.
Studies performed using collagen gels as scaffolds for cells of mesenchymal origin show
that increased stiffness of the scaffold leads to an increased rate of proliferation. In
addition to this increased rate the duration of proliferation persisted longer than that seen

31
in scaffolds with less stiffness [80]. In similar studies collagen gels were anchored on one
axis leading to a preferential tension development in the direction of this axis by
fibroblasts. Cell numbers increased by five times in the anchored gels and ECM
generation was greater than that seen in the un-anchored gels, which in contrast
demonstrated a five times decrease in cell number [81].
Another major form of cellular influence: topography, has been shown to direct the
function of cells in vivo, as well. Neural cells migrate along the length of radial glial cell
fibers until they reach their destination, thus being guided to different regions of the
central nervous system [82,83]. Basement membranes, the thin layer that many cell types
grow directly on, also have an inherent topography that has been suggested to influence
comeal epithelial cells [84] and renal endothelial cells [85].
Introduction of synthetic devices into the body disrupts many of these cell and tissue
systems, which then try to recover and re-establish stability in the presence of the
implant. Implant design has come to focus on minimization of these disturbances and
optimization of the cellular response to these devices once they have been placed in vivo
[86]. Ideally cells could be induced to respond to devices in an optimal manner by using
their inherent cellular mechanisms and machinery, which allow them to exist and live in
their normal environment. Inducing migration of a given cell type, or using
microtopography to direct a cell’s function are examples of this concept.
Toward this end, many studies have been performed that examine how topography and
chemistry influence cell shape [70,87,88], migration [75, 89-91] and function [72, 91,92],
This cellular direction, which is known as contact guidance, has been widely examined
and found to occur in many different cell types. Similarly, studies have been performed

32
that use micropattemed, chemical cues to spatially guide cell growth, in addition to
influencing cell function [70,72,77]. There have also been a number of studies that
examine the effects of receptors and receptor-like molecules on cell activity.
Contact guidance, the topographical, or chemical control of cellular orientation and
activity, is well supported by many studies in the literature [75,82-91,64,79,93,94]. Some
of the earliest experiments studied the influence of topographical cues by examining how
cells oriented themselves on glass fibers [85]. Since then the electronics industry has
developed viable micro-fabrication methods and a number of studies have shown how
topographical cues with specific sizes and patterns can invoke cells to behave in
predictable ways [64,79,90,91,93,94],
Using these methods, many cells of mesenchymal origin have been shown to orient
themselves parallel to micron scale topographical features, like ridges [64,86,93,94], This
is also true of osteoblasts, the primary bone forming cells in mammals. Once oriented,
osteoblasts and osteoblast-like cells will mineralize and lay down extra-cellular matrix
parallel to microtopographical features on a culture substrate [31,32,91]. In fact in vitro
mineralization of bone has been directed on a macro-scale merely by scratching the
surface of a culture dish with sand paper [31].
Since the aforementioned development of micro-fabrication in the electronics industry
a number of studies have shown the specific effects that topographical cues can have on
cell shape and activity. Cues with specific sizes and patterns, which were created using
the micro-machining technology, can invoke cells to behave in predictable ways
[64,79,82-91,93-95]. A number of theories have been put forth regarding the mechanisms
of contact guidance. The direction of a colloidal exudate from the cell by the grooves

33
[76], avoidance by cells of discontinuities in their paths [29], as well as the thought that
focal contacts may only form on the tops of the ridges [32]. All of these theories enjoy
some degree of experimental support, though which has the most dominant effect is not
clear at present. There does, however, appear to be some correlation of feature size with
the influence on cell shape. Groove dimensions that seem most effective across many cell
types, are grooves with dimensions on the order of magnitude of the cell [76], This is not
always the case, however. In a recent study by Nealy et al., which used nano-scale
features, contact guidance was exhibited by corneal epithelial cells [96].
In addition to glass fibers, early contact guidance studies often used spider webs [32]
to orient fibroblasts. This effect was achieved below a critical diameter of ~ 100 pm.
Above 100 pm, the cells were no longer oriented on the fiber, which led to the hypothesis
that cells were unable to bend around a certain sharpness of curvature. That is, the cells
could not bend around the circumference of a certain fiber once its diameter had gone
below a certain point, leaving them no recourse, but to elongate in the direction of the
fiber’s long axis [29,97]. Fibers have also been shown to orient many other cells types
including neurons, schwann cells, macrophages and transformed BHK fibroblasts
[75,98]. In these studies the contact guidance effect was shown on other fiber types too,
namely carbon filaments and synthesized fibronectin filaments.
Architecture of Fiber Based Scaffolds: Development of Cell-Based Tension
Using the phenomena of contact guidance, which orients not only cells, but also their
ECM products, advanced scaffold materials can be designed. Commercial fibers can be
woven into textiles of various 3-D configurations possessing strong mechanical
properties. Adjusting the tightness and geometry of the weave, as well as the diameter of

34
the fibers allows a number of control features. Fiber based scaffolds can be designed to
allow for initial nutrient exchange, as well as longer term vascular infiltration. In addition
the direction of cellular growth and ECM deposition can be influenced so that a
decreased remodeling requirement is present after the newly created tissue is formed.
That is cells can be aligned to a degree that is close to the alignment and orientation they
will exhibit after remodeling and so will be less distant from their equilibrium state.
An extensive review of the literature indicates that one of the key components of
connective tissue cellular physiology is the achievement of proper cell spreading, or
tension. Cell spreading is a phenomenon influenced by topography, chemistry and
stiffness of the substrate the cell is bound to. These are the elements of contact guidance
as reviewed earlier. As we saw, surface properties led to proper adhesion through the
protein mediated receptor binding (Figure 2-9). Topography and chemical micropattems
also influence adhesion and the direction, which cells are able to achieve stretch. Indeed
spreading and the achievement of proper cell tension appear to be one of the main
requirements for normal cell function.
Without adhesion, contact dependent cells, like those of mesenchymal origin remain
rounded without inducing tension and fail to differentiate [54,88,92,99]. On another front
inhibition of stretch mediated chloride receptors attenuate response of connective tissue
cells to topographical guidance [100], strongly implicating stretch as a requirement in
topographical guidance. Mechanical stretching of the substrate cells reside on has also
been shown to direct cellular orientation and deposition of ECM proteins in a MSC
system [101]. Indeed a translational strain of 10% applied concurrently with 25%
rotational strain was responsible for increasing the alignment of these cells 2.5 times

35
when compared to controls, which received no stimulus [101]. In addition to cellular
alignment, collagen fibrils were found aligned as well, when no fibrils were even seen in
the control constructs. Interestingly 0.2 grams of tensile stress has also been shown to
increase BMP-4 by two times that of control as part of suture development in the
mineralization of rat calvaria [102].
So in addition to the development of cellular tension, which is necessary for normal
and optimal cell function, cellular stretch plays a role in developmental process. Indeed
stretch mediated receptors may be the signaling mechanism responsible for translating
cell based tension generation into enhanced cellular function and differentiation.
Therefore scaffolds developed for application in connective tissue engineering should
include consideration of these phenomena in their design.
In tissue engineering applications, scaffolds loaded with cells are often seen to
contract through the development of cellular based mechanical force [80,81,103-105].
This phenomenon has been strongly associated with the contractile protein “Smooth
Muscle Actin’’ (SMA). SMA is so named because it was first discovered in smooth
muscle cells, but it is also found in other cells of mesenchymal origin like fibroblasts and
osteoblasts. In fact it has been shown that MSCs and osteoblasts will contract tissue-
engineered scaffolds when they are loaded onto them [103,104,106].
Contraction by osteoblasts and other cells of mesenchymal origin occurs in close
relation to the numbers of cells loaded on these scaffolds [104,106]. Once loaded onto the
scaffold, the force generation is linearly associated with the numbers of cells, which
indicates a collective effort by the cells leading a total force generation [105]. This
relationship between cell number and contraction also indicates that cellular

36
communication is occurring through gap junctions [104]. Gap junctions exist on the
physical level and are essentially pores that interconnect adjacent cells, or cells that are in
actual contact with each other. Gap junction interactions have been implicated in
macroscopic phenomenon of scaffold contraction [104], which indicates these cells are
acting in a multi-cellular manner. In fact these findings suggest that osteoblasts and
connective tissue cells are acting in an aggregate manner with many physically associated
cells generating force cooperatively.
The importance of tension generation by connective tissue cells becomes apparent
when its effects on cell function are considered. When seeded on collagen and collagen -
glycosaminoglycan scaffolds proliferation of cells is markedly enhanced, but only when
the gels are secured to the culture dish. Free-floating gels/scaffolds experienced a
regression in the number of cells, a fact attributed to their inability to generate tension
[81,103]. In a similar manner, when a series of collagen scaffolds with increasing degrees
of cross-linking leading to increasing amounts of stiffness are used, the stiffest
formulations led to the greatest proliferation rate and duration [80].
In addition to enhanced proliferation, the ability of cells to generate tension led to
increased production of collagen [81], as well as increased production of proteins,
calcium and alkaline phosphatase [103]. All these responses indicate increased levels of
differentiation by cells, which also has been attributed to the ability to generate tension.
Overall it appears that connective tissue cells lead to contraction of scaffold material
as each tries to achieve the an optimal tension, or stretch necessary for proper
differentiation and optimal function. The sum of these many individual cell tensions leads
to a cooperative force causing the whole construct to contract. It also becomes apparent

37
that these cells are not acting individually, but are physically linked to each other in an
aggregate manner, allowing cell communication through gap junctions.
This contraction is commonly seen in wound healing and it can lead to scar formation
[107]. Similarly it is part of the normal healing cycle seen in tendon healing [108]. The
same proteins that cause this in other closely related cell types are found in Osteoblasts
and MSCs, therefore it seems likely that this phenomenon is a part of normal fracture
healing. In fact it follows logically that contraction of cells healing a fracture would be
useful in bringing separated fragments of bone back together. Given the importance of
this cellular activity, it must be considered in the design of a fiber-based scaffold’s
architecture and spacing.
Proper spacing of fibers is necessary to allow optimal cell tension and stretch, but also
it must be optimized for the aggregate activity, or cooperative bridging of cells occurring
across gaps in the scaffold. During connective tissue healing, a fibrin clot acts as scaffold
allowing cells to migrate from areas of healthy tissue to the area of injury [108]. Without
this scaffold, or a synthetic replacement, in vitro studies show cells are unable to bridge
gaps as small as 50 pm [108].
Autocrine/Paracrine Considerations in Scaffold Design
As mentioned previously, tensile stress increases BMP-4 by a factor of two as part of
the developmental cycle [102], In addition, this stimulus leads to an increased
responsiveness of osteoblasts to morphogens and vitamin D [102], Applying exogenous
TGF-ff on the other hand increases the tension produced per cell by a factor of two [105].
Increased TGF-p and BMP-4 precede increased production of collagen, alkaline
phosphate and increased MSC differentiation. Production of these growth factors as

38
stimulated by tension occurs in such a way that they have their effects in an autocrine and
paracrine manner, thus tying this form of cellular communication to tensile stimulus.
Additionally it is seen that exogenously administered TGF-(3 increases multi-layering of
cells within the confines of pores in collagen scaffolds and causes an increase in macro¬
scale contraction of these scaffolds [105].
Inter-cellular communication is a phenomenon that takes on many forms. Autocrine
signaling is the ability of a cell to produce cytokines that are then sensed by receptors on
the same cell (Figure 2-9). This method of communication was first elucidated in cancer
cells, which were seen to produce growth factors independently of environmental
stimulus, thus freeing them to grow uncontrollably and form tumors [39]. Since then
autocrine signaling has been established as part of the normal physiology of many tissues
including bone [69,85].
Paracrine signaling is the intercellular communication that occurs between adjacent
cells, confining its action to a local area (Figure 2-10A)[69], Some paracrine signaling
Figure 2-9. Diagram of autocrine signaling whereby a cell produces a certain cytokine
and then releases it. Cell product is then bound by receptors on the cell’s own surface.
Adapted from McCance and Heuther [69].

39
occurs by growth factors being released in small quantities to the local extra-cellular
milieu. In the case of TGF-P and BMP-4, however, the signaling molecules are bound in
the matrix and non-soluble. Signaling occurs by actual physical contact between the cells
creating the stimulus as cells nearby extend filopod-like extensions that contact the
stimulating cells directly (Figure 2-10B)[5],
In order to optimize the cellular microenvironment, paracrine signaling distances must
be considered in addition to fiber spacing leading to the most biologically optimal
cellular tension. Indeed the two phenomena are inter-related as fiber spacing is essentially
a topographical stimulus and surface topography has been shown to stimulate
autocrine and paracrine growth factors [109], But how far paracrine signals released into
the extra-cellular milieu able to travel and still remain effective is also a highly relevant
question. It seems logical that there is a fiber spacing distance that will allow inter¬
cellular paracrine communication between adjacent fibers and that it should be optimized
in a 3-D scaffold.
Work in the area of paracrine signaling distance is rather sparse, though it is generally
accepted that this is a major contributor to osteoblastic development and how it occurs
[85,109]. However, some studies have been performed in culture using various cell types.
Generally it is seen in culture models using pituitary cells, as well as parathyroid cells
that increasing distance between secretory cells leads to a decreased response, or
communication between cells [110,111], An apparent critical distance beyond which
interaction becomes negligible in pituitary cells is approximately 75 pm [110]. This
distance of course is for diffusible cell products released into the local environment.

40
~v;
.A A A A.
0
Figure 2-10. Diagram of paracrine signaling. A.) shows a cell releasing a diffusible
cytokine (square nucleus) that acts over short distances creating a gradient of responses in
surrounding cells (note color scale of oval nuclei). B.) shows similar paracrine
phenomena, but the cytokine is passed by filopod-like extensions, or actual physical
connections. Adapted from Christian [5].
Matrix bound BMPs on the other hand may possess paracrine activity with a more limited
distance of interaction, perhaps as low as a few cell diameters, or tens of microns [5].
Distance and spacing between fibers is therefore likely to be important in the
development of proper cell adhesion and tension. This phenomena, as we have seen, is
closely related to autocrine and paracrine signaling distance, which must also be
considered in the design of fiber based scaffolds.
Overall, it seems apparent that fiber based scaffolds possess strong potential for use in
development of hierarchical bone replacement materials. Their design flexibility allows
for control of cellular organization, as well as ECM organization, two of the most integral
components leading to the mechanical integrity of bone. In addition, weaving and
organization of fibers allows for higher level 3-D construct design that allows greater

41
nutrient diffusion and facilitation of vascular ingrowth, while optimizing cell adhesion
and induction of tension, as well as intercellular autocrine and paracrine interactions.

CHAPTER 3
SINGLE FIBER STUDIES
Introduction
As mentioned previously, fibers have been used for a number of scaffold-based tissue
engineering experiments. Ahmed and Brown used synthetic Fibronectin (Fn) fibers for
the direction of Schwann cells, as well as dermal fibroblasts, macrophages and epitenon
fibroblasts. These fibers, with diameters of 0.5-7.0 pm, were organized into a mat leading
to a surface composed of fibers oriented in the same direction [75].
Carbon and polymer filaments have also studied for use as scaffold materials in the
area of connective tissue engineering. In vitro Kevlar-49, nylon and carbon allow
centimeter scale migration of mesenchymal originating cells from tendon explants along
their surfaces. In addition to undergoing extensive outgrowth, these cells were oriented
with the long axes of these synthetic fibers. Kevlar-49, nylon and carbon fibers in this
study were 12 pm, 7.5 pm and 22 pm respectively [112].
Other tendon outgrowth studies performed using carbon (8 pm diameter), Dacron (11
pm diameter), polyethylene (20 pm diameter) and nylon (102, 52 and 22 pm diameters)
show cells of mesenchymal origin orient in the direction of their long axes primarily in
response to the diameter of the fibers they are grown on. This is true of all fibers, though
there is an interesting and important qualification to this trend: Larger diameter fibers on
the order of 100 pm did not orient cells until after they had become confluent on the fiber
42

43
surface [113]. This implies some socially based mechanical interaction is at work as cells
become confluent and come into contact with each other.
In vivo, carbon bundles composed of 8 pm thick fibers have been used as a ligament
replacement material and exhibit the ability to organize fibroblasts and tendon cells.
More than orienting these cells they appear to orient the collagen produced by the
oriented cells [30]. However, these tows were under biomechanical stress as the subjects
used them in the course of everyday living, so it is unclear if the collagen orientation was
a result of topographical cellular orientation. It is conceivable that collagen was oriented
by the dynamic stresses of walking and muscular contraction that were placed on it in its
role as a ligament replacement.
Though these studies provide much qualitative data about the orientation of cells on
the surfaces of fibers, they are lacking in hard quantitative evidence. Overall it is apparent
that diameter has an effect and that time, which allows the achievement of confluence,
are important factors in contact guidance of mesenchymal cells on fibers.
Some quantitative work has been done on the fiber based contact guidance of
connective tissue cells. Dunn and Heath performed alignment measurements of chick
heart fibroblasts on the surfaces of soda glass fibers at 48 hours, showing an order of
magnitude increase in NFF on fibers of 40 pm diameter compared to fibers of 100 pm
[29]. Above 100 pm there was no significant contact guidance. Similarly, Fischer and
Tickle quantitatively showed normal BHK rat fibroblasts elongated on the surface of
glass fibers, but at only 24 hours [97], Both studies use quantitative measures of cellular
elongation, but are limited to very short time periods. Indeed

44
measures are taken to prevent social interaction between cells even for these short times
in culture.
In this study mesenchymal stem cells are studied quantitatively and for time periods
that extend beyond those needed to achieve confluence. In fact the time periods used
were lengthy enough to observe the characteristic multilayering observed in this cell type.
The object of this study was to examine the effects of diameter and time on MSC contact
guidance and to do this in a quantitative manner. It is hypothesized that decreasing
diameter will lead to an increased level of contact guidance, as will increasing culture
times. Knowledge and understanding of this cellular response to fibers will prove
invaluable in fiber-based scaffold design.
This section’s main hypothesis is: MSCs will have a significantly (alpha = 0.05)
different elongation and cellular activity when grown on ~ 140 pm, 100 pm and 79 pm
diameter fibers. This difference in cell activity will exist between each fiber diameter
group and inducing contact guidance of these cells will be the primary goal.
Materials
Black Nylon (Ethilonâ„¢, Ethicon) sutures of 4-0 (~ 140 pm diameter), 9-0 (-79 pm
diameter) and 10-0 (~ 39 pm diameter) sizes were used for initial SEM studies. Clear
polyglactin (Maxonâ„¢, Davis and Geek) fibers of 5-0 (-140 pm diameter), 6-0 (~ 99 pm
diameter) and 7-0 (~ 79 pm diameter) were used for light microscopy studies.
Culture materials included a-minimal essential medium (Sigma, M0894) with 15%
fetal bovine serum (Sigma, F4135), 50 mg/ml ascorbic acid (Sigma, A4034), 10 mM b-
glycerophosphate (Sigma, G9891), antibiotics (0.1 mg/ml penicillin G, 0.05 mg/ml
gentamicin and 0.3 mg/ml fungizone) and 10-8 M dexamethasone (Sigma, D2915).

45
Bovine Fibronectin (Fn) ~25 ug/ml (Sigma, FI 141) was used to soak constructs prior to
cell seeding, thus making fiber surfaces more amenable to cell adhesion. After soaking,
Fn solution was pipetted off and cells were seeded directly onto constructs.
MSCs were collected from both femora of grown, Sprague Dawley rats (-150-300 g)
that were provided courtesy of Dr. Colin Sumner’s lab at the UF brain institute.
Methods
Single Fiber Scaffolds
Single fiber scaffolds were prepared by stringing Maxon and Nylon sutures with the
diameters mentioned previously across polystyrene support rings (Figure 3-1). Constructs
were designed to suspend the fiber completely above the floor of the culture dish so all
cellular response was caused by the topography of the fiber alone.
Figure 3-1. Examples of Maxonâ„¢ single fiber constructs. 5-0 is on the left, 6-0 center
and 7-0 on the right.
Cell Culture
After dissecting each femur out, the epiphyses were cut off and the marrow plugs were
flushed out of both diaphyses using aliquots of the fully supplemented medium(FSM) to

46
be described next. Plugs were flushed into 30 ml of FSM, which will be: a-minimal
essential medium (Sigma, M0894) with 15% fetal bovine serum (Sigma, F4135), 50
mg/ml ascorbic acid (Sigma, A4034), 10 mM b-glycerophosphate (Sigma, G9891),
antibiotics (0.1 mg/ml penicillin G, 0.05 mg/ml gentamicin and 0.3 mg/ml fungizone).
After the primary culture passage of ~6 days, cells were trypsinized and passaged
using a 0.01% trypsin and 10 M EDTA mixture (Sigma, T3924) in phosphate buffered
'y
saline. Released cells were reseeded into three 75 mm culture flasks.
MSC Seeding
MSC Seeding onto the fibers was done at extremely high concentration by taking a
whole flask of trypsinized MSCs and adding ~10ml of medium to it. Addition of this
small amount of medium served to quench the trypsin reaction, while leaving the cells at
a very high density. After EtOH and UV sterilization (24 hours in absolute EtOH
followed by drying under 256 nm UV light), fibers were placed in 24 well plates and
secured to the walls via melting of the polystyrene support ring to the polystyrene of the
dish (heated implement was a small soldering iron). Fibronectin (25 ug/ml) was used to
soak constructs (except for 9-0 constructs) for one hour then each construct was covered
with a large drop if high density cells in suspension and allowed to incubate for about 2-4
hours while cell adhesion occurred. At this point the wells were flooded with culture
medium to the normal level.
SEM Imaging (Nylon sutures)
Cells were fixed in a phosphate buffered saline with 10% formalin at pH 7.4. Each of
the constructs were dehydrated by serial ethanol incubation at concentrations of 30%,
50%, 70%, 90% and 100% over periods of about two days (longer dehydration time

47
minimizes shrinkage of cellular material). Following ethanolic dehydration, constructs
were critical point dried, coated with ~ 20 Á of Pd/Au and examined via SEM.
Light Microscopy (Maxon Sutures)
Constructs were removed at 3, 6, 9 and 12 days and passed through serial EtOH
dehydration of 30%, 50%, 70%, 90% and 100%. Upon reaching 100% EtOH, constructs
were left in 100% EtOH until all constructs were removed from culture then all were
stained simultaneously using Hematoxylin and Eosin stains. Nuclear Form Factor (NFF)
and the angle were measured with respect to the fiber’s long axis as described in
Appendix 1.
Results
SEM Studies
4-0 nylon sutures
Constructs initially showed a robust amount of cell adhesion and coverage under
the light microscope during culture. By the last day, every fiber was completely covered
with cells and some even appeared to have begun multi-layering.
Cells liberally covered the surface of the fibers, but an interesting phenomena was
that these cells were elongated though not parallel to the long axis of the fibers (Figure 3-
2). Instead they were spirally oriented around the surface of the fiber, much like a
barber’s pole. A similar result was seen in work by Ricci et al. when larger diameter
fibers were used [113].
In addition to the spiraling effect of cell orientation, what appeared to be
multilayering was present. Light microscopy during culture demonstrated what appeared
to be multilayers, or areas of cell buildup at the periphery of the fibers. Because the fibers
were dyed black and used primarily to develop protocols, light microscopy staining was

48
Figure 3-2. 4-0 nylon fibers with RMSCs after being cultured for 5 days. A and B) Note
elongated and flattened cells that spiral around the long axis of the fiber. C.) is a close up.
Arrows indicate some of the most prominent cells. Flattened cells cover nearly the entire
surface.
not possible and cells were only visible in a x-section-like view where they were not
superimposed on the fiber.
Examination of 4-0 fibers showed a definite striation-like appearance (Figure 3-3),
which appeared to proceed in two directions (red arrow and green arrow indicate
approximate directions). In some areas it appeared that each of these oriented striae were
composed of cells and that the cells in one direction were overlapping the cells that were
growing in the other spiral direction.

49
Figure 3-3. Examples of MSC growth on the surface of 4-0 nylon fibers. These cells
exhibit spiraling and what may be alternating spiral orientations between layers.
Though this result requires further support, there is some indication in the literature
that may explain it. Commonly osteoblast-like cells multilayer during culture, depositing
collagen at each level in a manner that is situated in orthogonal directions between each
layer. That is cells in one layer are separated by collagen laid down by the next cell in the
layer above, this collagen in turn is at a 90 degree angle from the collagen laid down by
the cell below (both chicken and rat cell culture models show this) [114-116],
9-0 nylon sutures
9-0 Nylon sutures shown in Figure 3-4 exhibit less cell adhesion than the other nylon
fibers examined, as they were not pre-soaked in Fn. This illustrates the importance of
surface properties and receptor mediated adhesion in MSC settling and growth. Despite
the fewer numbers of cells, every cell observed was indeed elongated in the direction of
the fiber’s long axis.
Images of much more flattened cells that are also extending in the same direction as
the fiber are reproduced in Figure 3-5. These cells may be similar to “sheath cells” found
in tendon experiments on fibers [30,114];. These studies that found three populations of

50
Figure 3-4. Examples of R.MSC growth on the surface of 9-0 nylon fibers. A.) shows a
cell with two long processes essentially parallel to the fiber. B and C) show a cell and a
close up of the same cell respectively. D) shows a cell that is much flatter. E.) a close up
of the same cell. It is elongated in the direction of the fiber as were all the cells seen on
the fiber.
cells grew on their fibers, one a flat sheath like cell, similar to these, that covered the
surface of the fiber, another spherical cell type like the one seen in Figure 3-5 A and a
spindle shaped cell similar to that seen in Figure 3-6. These cells may be very similar as
they are derived from the same cells in vivo as the MSCs used in this study.
Morphologically the cells in the current study appear very similar.
10-0 nylon sutures
In contrast to the 4-0 fibers, 10-0 fibers show cells that follow the long axis of the
fiber very closely (Figure 3-6). Figures 3-6A, 3-6B and 3-6C show more flattened cells,
while 3-6D, 3-6E and 3-6F show cells with a more spindle shaped morphology.

51
Figure 3-5. Examples of RMSC growth on the surface of 9-0 nylon fibers. This set of
images shows flattened cells that are at least qualitatively extended in the direction of the
9-0 fiber they are growing on. On the right is a close up of the cell material showing
many filopodia.
Maxon sutures
Images of 5-0. 6-0 and 7-0 sutures were obtained on samples cultured for 12 days
under conditions described above. 5-0 fibers were fully covered with MSCs that show
spiraling pattern (Figure 3-7). Cells on 5-0 fibers were flattened in a manner that was
much more pronounced than that seen on the 6-0 (Figure 3-8) and 7-0 fibers (Figure 3-9).
Additionally, cells on the 6-0 and 7-0 fibers were much more elongated than those seen
on the 5-0 fibers. 7-0 fibers appeared to exhibit the greatest amount of multi-layering and
elongation. These results are supported by the quantitative data taken to measure cellular
orientation and elongation, though SEM of these fibers was performed only on the
twelfth day.

52
Figure 3-6. SEM images of 10-0 nylon sutures at various magnifications. A, B and C)
show highly flattened and multi-layered cells that are elongated in the direction of the
fibers. D, E and F) show a more spindle shaped cell that also is elongated in the same
orientation as the fiber.

53
Figure 3-7. 5-0 Maxon sutures exhibiting RMSC adhesion and growth. A.) Shows MSCs
covering the entire surface of the fiber and marked spiraling, or angling with respect to
the fiber’s long axis. Note cells are rather flattened and not highly elongated. 500x.
Arrow indicates general trend of the spiraling behavior exhibited by cells. B) Again
shows flattened and angled MSCs, but at lOOOx. C) Flattened and angled MSCs at 1500x.
Light Microscopy Studies of Maxon
In general the larger diameter 5-0 and 6-0 fibers showed the barber pole spiraling
similar to that seen in the SEM images of 4-0 nylon (Figures 3-2 & 3-3), as well as SEM
images of 5-0 Maxon (Figure 3-7). 7-0 Maxon however did not show noticeable evidence
of this spiraling behavior. Elongation of RMSCs on the surfaces of the fibers was evident
and contact guidance of cells was clearly occurring. Differences in nuclear orientation
between fiber diameters were visible and confirmed by statistical analysis. Multilayering
occurred on all three fiber diameters by day 12. These findings are consistent with SEM
data, which is presented below.

54
Figure 3-8. 6-0 Maxon sutures exhibiting RMSC adhesion and growth. A) shows MSCs
covering the entire surface of the fiber. Many flattened cells are present and much more
elongated than those seen on the 5-0 fibers. Spindle shaped cells are present as well (blue
arrowheads) 500x. B) Shows flattened and elongated MSCs at lOOOx. C) Shows flattened
and elongated MSCs at 1500x. An example of multilayering is clearly evident (red
arrowhead) as cells overlap.
Figure 3-9. 7-0 Maxon sutures exhibiting RMSC adhesion and growth. A.) Fibers are
completely covered by flattened and elongated MSCs 500x. B.) shows flattened and
elongated MSCs at lOOOx. Examples of multilayering are evident as cells overlap each
other (red arrowheads).

55
Figure 3-9 Continued. 7-0 Maxon sutures exhibiting RMSC adhesion and growth. Again
flattened and elongated MSCs are evident. Multilayering and overlap are evident (red
arrowheads) 1500x.
Nuclear form factor
Figure 3-10 shows a nucleus with all NFF measurements as they were performed in
Adobe Photoshop. Originally three day data was slated to be collected, however, there
was not sufficient cell growth for any kind of meaningful analysis.
Figure 3-10. Example of Nuclear form factor measurements. The image shows a
representative 5-0 fiber after 6 days in culture.
Figure 3-11 is a graph of the effects of diameter on NFF independent of time, while
Figure 3-12 shows effects of time independent of diameter. Clearly both factors, which
were the factors used in the 2 way analysis of variance, show that NFF leads to increased

56
orientation. All differences resulting from diameter were statistically significant with a
power P = <0.05. When comparing time factors of 6 days in culture vs. 12 days, there
was a statistically significant difference with a power of P<0.05, while the other
differences were significant with P = <0.1 (i.e. 6 days vs. 9 days, and 9 days vs. 12 days).
Diameter Effects on Nuclear Orientation (NFF)
(Independent of Time)
Sutine size (U.S.P. sizes)
Figure 3-11. Graph showing the independent influence of fiber diameter on NFF. Bars
are standard errors.
All of the data in Figure 3-13 has been shown to be statistically different from each
other at a power of P = <0.1. There appear to be effects on cellular elongation that are
related to social interactions of cells, or how they squeeze together when the layers
become confluent. 6-0 and 5-0 fibers show what seem to be fluctuations in the degree of

57
Effect of Culture Time on Nuclear Orientation (NFF)
4 6 8 10 12 14
Time in culture (days)
Figure 3-12. Graph showing the independent influence of time on NFF. Bars are standard
errors.
0.4 -i
0.3 -
0.2 -
0.1 -
0.0
Effects of Culture Time on
Nuclear Orientation (NFF)
5-0 NFF t
I 1 6-0 NFF
Dav 6 Dav 9 Dav 12
Time in Culture (Days)
Figure 3-13. Graph showing the effects of time on NFF for all diameters studied. Note 7-
0 fibers are the only ones showing a gradual and continuous increase in elongation. Both
5-0 and 6-0 fibers fluctuate with time probably due to the effects of social interactions.
Bars are standard errors.

58
nuclear elongation. Multilayering was apparent on all the constructs and imaging of
nuclei for analysis was primarily done on what appeared to be the top layer cell coverage.
Deeper layers suffered from a loss of contrast thus making it virtually impossible to
collect data from them. Fluctuation in nuclear elongation as seen in Figure 3-13 may
indicate how social interaction is important in cell orientation. Basically each layer of
cells, as it becomes confluent on top of the layer before it, may elongate and orient as it
nears full confluency. This is followed by the accumulation of the next layer, which is
less elongated at first. This is analogous to the phenomena seen by Ricci et al. who
observed that tendon outgrowth cells did not elongate until confluence was reached on
fibers above 100 pm [113].
In the similar vein, 7-0 fibers showed increasing elongation throughout the culture
times examined. These fibers were less than 100 pm, the diameter that seems established
in the literature as the critical diameter for continuous contact guidance.
Nuclear angle analysis
Figure 3-14 shows the same nucleus as seen in figure 3-9. This time the image is
demonstrating the method used to measure nuclear angle. Figures 3-15 and 3-16 show the
main effects of the 2 way ANOVA (time and diameter). Both factors exhibit a significant
effect with p = <0.001, while the interaction between the main effects is significant with
p = <0.056. Overall both time and diameter influence the angle of the nucleus with
respect to the long axis of the fiber. Also there is an interaction between time and
diameter leading to an influence on nuclear angle that is greater than each factor acting
alone.

59
Figure 3-14. Example of nuclear angle measurements. Image of the same nucleus
depicted in Figure 3-10. This micrograph demonstrates the method used to measure the
nuclear angle with respect to the fiber’s long axis.
Nuclear Angle
Effects of Fiber Diameter
(Independent of Time)
5-0 6-0 7-0
Diameter (U.S.P. suture size)
Figure 3-15. Graph showing the effects of diameter independent of time. Bars are
standard errors.
Figure 3-17 shows all fiber sizes vs. time in culture. With the exception of the 6-0, 6
day data, nuclear angle behaved exactly as hypothesized by increasing (becoming closer
to alignment with the fiber) with diameter and time. When comparing figure 3-10C to

60
Nuclear Angle
Effects of Time
Figure 3-16. Graph showing the effect of time in culture on nuclear orientation as
measured via nuclear angle. Bars are standard errors.
Corrected Nuclear Angle
Effects of Diameter and Time
100 T
In 80
0)
E
O)
0)
â– a 60
40
U1
c
<
L_
IB
0)
o
3
Z 20
5-0 fibers
6-0 fibers
7-0 fibers
Day 6 Day 9
Time (Days)
Day 12
Figure 3-17. Graph showing the effects of time and diameter together. Bars are standard
errors.
Figure 3-17 it becomes apparent that NFF and nuclear angle are not directly correlated,
which is interesting, as these values were calculated from the very same nuclear images.

61
So nuclear orientation appears to be influenced by the diameter of the fiber in a way that
is independent of cellular elongation. Again we see that 7-0 fibers exhibit the greatest
influence on cellular activity and lead to elongation that is more robust than either the 6-
0, or 5-0 diameter fibers.
Overall it seems that nuclear orientation measured by the angle of the nucleus is a
more predictable method of examining cellular contact guidance. Nuclear orientation
looks as if it will require further study and increased understanding.
Discussion
It is abundantly clear that decreasing diameter and increasing culture time led to
greater contact guidance of MSCs in this culture system. These responses by MSCs were
exactly as hypothesized. 5-0 and 6-0 diameter fibers showed what is likely to be the
effect of the social interaction of cells on their nuclear elongation. 7-0 fibers on the other
hand are of small enough diameters that their topographical influence overpowers the
need for social interactions. All fiber sizes show a nuclear orientation, however, this
orientation appears to be independent of nuclear elongation.
This behavior has implications for fiber-based scaffold design. By quantifying the
behavior of these MSCs on various diameters and for various times, the fiber candidate
that is most optimal for a given system can now be chosen. Though similar studies have
been performed with similar cell types [29,97] an extensive literature review shows they
have never been done using the MSC culture system. In addition the level of
measurement in this study, using quantitative methods has not been matched for time, or
numbers of quantitative measures (i.e. both NFF and nuclear angle).
Though the presence of social interaction between cells has been observed on a
qualitative level, it has never been observed and measured as closely as this study.

62
Fluctuations in the nuclear elongation, independent of the angle suggest that as
confluence is reached each layer of cells in the multilayered system becomes more and
more elongated. This is to say that layers of cells are built up by a period of oriented, but
un-elongated cellular morphologies followed by elongation in the direction of the fiber’s
long axis. After the achievement of confluence, the process begins again on the next level
of multilayer.
Larger size fiber diameters show spiraling and as seen in figures 5, 6 and 8. This
spiraling may occur at equal and opposite directions within each layer. ECM/collagen has
been linked to cellular orientation and in this cell type it is seen that collagen layers are
deposited in orthogonal directions to each other [114-116]. Perhaps this mechanism of
cellular layering is related to the mechanism that lays down collagen in alternating layers.
As previously noted orientation of ECM is strongly correlated to the orientation of the
cells depositing it [31,76,117-120], Indeed the deposition and orientation of many
individual ECM proteins including collagen has been shown to parallel the oriented
layers of osteoblasts laying them down [121].
Conclusions
This study quantitatively shows that RMSCs elongate and orient in response to
diameter, as well as time in culture. In the case of both time and diameter contact
guidance occurred in an increasing manner when measured via nuclear angle. A critical
fiber diameter existed at approximately 100 pm, however, for the cellular elongation
measured by NFF. Below this critical diameter, cells were increasingly oriented in a
gradual manner over time. Though hen grown on fibers above the critical diameter, cells

63
were oriented by nuclear angle, but elongation seemed to occur in response to social
interactions between cells as each layer achieved confluence.
Overall, given the nature of this cell type and the way it has been shown to orient
ECM in a manner that parallels cellular orientation, this form of contact guidance will
allow the control of structural ECM proteins. As mentioned in chapter 2, control of ECM
proteins; namely collagen, is what creates the anisotropic mechanical properties found in
natural bone. This study shows, through rigorous scientific methods coupled to strong
statistical support, that controlling cellular orientation and thus ECM orientation is now a
capability to be included in the arsenal of design characteristics available to connective
tissue engineers.

CHAPTER 4
CELLULAR BRIDGING PHENOMENA
Introduction
The overall goal of this work is to develop an understanding of cellular organization
and ECM deposition, then use this knowledge to organize cells and ECM in a desired
manner. Developing tissue constructs with multiple layers of cellular and ECM
orientation appears to be a promising method of increasing the construct’s anisotropic
mechanical strength. As reviewed in the background, mechanical anisotropy is a common
strategy employed by tissues as part of their physiologic function. This is particularly true
of bone and connective tissues, though this fact appears to have been overlooked in the
tissue engineering literature when scaffold materials are chosen. Typically randomly
organized scaffold materials have been employed despite the fact that substrate
topography has been shown to strongly influence cellular orientation and ECM
deposition. These induced forms of cellular and ECM organization is very similar to the
structure seen in vivo, which is the very same structure and organization responsible for
mechanical anisotropy.
In 2-D MSC systems, cells multi-layer and act cooperatively to produce a collagen
rich ECM, which is organized into orthogonal layers. Orthogonal lamellae of collagen are
separated by cellular layers, which are responsible for collagen deposition. Originally this
cellular multilayering provided the rationale for the multi-fiber construct experiments
discussed in the 5th chapter using 30 pm as a spacing distance between fibers.
64

65
Multilayering phenomena in MSC culture, by estimates and projection from data in
the literature, achieve heights of approximately 15-20 pm, so 30 pm would be proper for
cells on adjacent fibers to grow and eventually join in the space between fibers (Figure
4-1).
Figure 4-1. Multilayering on adjacent fibers leading to interactions due to their
proximity. Possibly these interactions are of a paracrine nature. The correct spacing
would allow cells to span the distance between fibers, while using the fibers as a
stimulus for cellular and ECM organization.
In the course of the experimental work it became apparent that there was at least one
more phenomenon involved in the 3-D cellular organization process beyond that of mere
multi-layering, which appears to be the primary process in 2-D systems. The single fiber
constructs studied in the last chapter primarily demonstrated MSC multilayering, but
when more than one physical surface, or topographical feature is present, another
cellular process becomes evident. Cells begin to span distances, or “bridge” between
surfaces when these surfaces are found within certain ranges and under the right
conditions of proximity.

66
This bridging phenomenon is obviously a prominent behavior contributing to the
cellular interaction with 3-D scaffolds. Furthermore this behavior does not appear to be
explicitly addressed in the literature as it currently stands. For both these reasons cellular
bridging merits study here, not only to elucidate and characterize it as a previously
unobserved phenomenon, but also to understand how it influences cellular development
and function.
Materials
Bridging was induced and observed on many different materials including: standard
polystyrene culture flasks (Comingâ„¢), 4-0 monofilament nylon sutures (Ethilonâ„¢), 7-0
monofilament polyglyconate sutures (Maxonâ„¢), stainless steel screen and bioactive
glass fibers synthesized as previously described by Dominguez et al. [122], Culture
materials were identical to those used in chapter 3.
VonKossa staining was performed using Silver nitrate 5%- 5 gm in 100 ml DI water
= 5 % solution. Sodium thiosulfate 5% - 5 gm in 100 ml DI water = 5 % solution
nuclear fast red- 5gm aluminum sulfate, 100 ml DI water, nuclear fast red 0.1 gm.
Methods
Culture Methods
Culture and MSC seeding was performed as described in chapter 3. Nylon sutures
were cultured for 12 days, Maxonâ„¢ for 13 days and Bioactive glass fibers 6 days
before data collection.. Polystyrene studies were performed for 11 days total with data
collected on days 7, 8, 10 & 11. Stainless steel screens were followed for 23 days with
data collected on days 10, 12, 15, 18 & 23.

67
SEM Imaging
SEM was performed on Nylon, Maxonâ„¢, stainless steel and bioactive glass fibers
with preparation for microscopy as described in chapter 3.
Light Microscopy Studies
Polystyrene imaging was performed on live cells in situ as they grew in the culture
dish. No staining was performed so viable cells could be maintained allowing the
development and documentation of individual bridges.
Von Kossa Staining
Von Kossa staining was performed on stainless steel screens to follow the
development of ECM and its subsequent mineralization. This method demonstrates salts
of calcium and it works by replacing Ca ions with silver from a silver nitrate solution
(5% in DI water). The silver salt that is produced by this is then photosensitive (silver
nitrate is the same chemical used in the production of a black and white photographic
negative). Exposing the sample to light then causes a photic reduction of the silver salts
leading to a black precipitation of silver in the locations where calcium is present.
Von Kossa staining procedurefi 18,120,123]
1.) Absolute EtOH dehydration, 2 times for 2 minutes each.
2.) 95% EtOH dehydration, 2 times for 2 minutes each.
3.) DI water rinse 2 times.
4.) Silver nitrate 5% incubate 30 minutes under high power light (~ 100-150 Watt
bulb leading to photic reduction of silver).
5.) DI water rinse
6.) Sodium thiosulfate 5% incubate 3 minutes (washes away residual silver nitrate)
7.) DI water rinse

68
8.) Nuclear Fast Red incubate 5 minutes (stains nuclei dark red and cytoplasm
pink).
9.) 95% EtOH dehydration, 2 times for 2 minutes each
10.) Absolute EtOH dehydration, 2 times for 2 minutes each.
Proliferation Studies
Bridging of MSCs between two topographical features is logically dependent on the
distance between them. In an effort to understand how spacing between fibers effects
9.17 ± 0.05 mm
Figure 4-2. Example of bioactive glass fiber constructs used for the proliferation study.
Dimensions of these cylindrical containers allowed the calculation of fiber density and
provided a constant volume necessary for the study.
cell function we performed proliferation studies using bioactive glass fibers with various
packing densities. Various masses of bioactive glass fibers (10, 20, 30 & 40 mg) were
gently packed into constructs with a given volume (Figure 4-2). Increasing quantities of

69
fibers within this given volume was assumed to decrease the distance between fibers and
thus influence the ability for these cells to perform bridging.
Cells were seeded onto all constructs at an estimated density of-1.10 +/- 0.07 x 105
cells/ml and allowed to incubate for 3, 7, 10, 14 and 21 days. As each time point came
due, cells were removed from culture, trypsinized and counted using a Beckman-Coulter
Multi-sizer III cell counter. Sample sizes were 500 mL and were diluted with Isoton II
diluent.
Cell counts were analyzed using a two way analysis of variance with time (3, 7, 10,
14 and 21 days) and fiber density (10, 20, 30 & 40 mg) as factors. Post hoc testing was
performed using Tukey’s multiple comparison.
Results
Light Microscopy of Bioactive Glass Fibers After 6 Days in Culture
Preliminary studies on bioactive glass fibers placed directly into the RMSC culture
system for 6 days showed a marked degree of multi-layering in the vicinity of the fibers
where they rested on the floor of the dish (Figure 4-3). This degree of multi-layering was
particularly evident when comparing cellular behavior on flat areas of the dish where
fibers were not present (Figure 4-4). In fact multi-layering was so extensive that four
micrographs were necessary to include the entirety of the representative image (Figure
4-3 montage).
In addition to multi-layering in the vicinity of the fibers, RMSCs were oriented
toward the fibers as if using it as an anchor for the development of tension.

70
Figure 4-3. Montage of light micrographs portraying the cellular interaction with
bioactive glass fibers placed in standard culture well. Note orientation of cells w ith
respect to the fibers, they appear to be pointed in toward the fibers and multilayering is
extensive.
Figure 4-4. Light micrograph of area without fibers in the same culture well as that
shown in Figure 4-3. Note the lack of multi-layering particularly when compared to
Figure 4-3.

71
Scanning Electron Microscopy
Bioactive glass fibers after 6 days in culture
Figure 4-5 shows SEM images of RMSCs grown on bioactive glass fibers. Figure 4-
5A shows cells covering the fibers and growing in a robust manner on the fiber surfaces.
In some cases cells grew over fibers, securing them to the bottom of the petri-dish.
Figures 4-5 shows a close up example of cellular interactions with the fibers and the
dish. Figure 4-6 shows examples of fiber to fiber bridging, a phenomenon that was
commonly seen throughout the extent of the specimen's surface. This bridging was often
multi-cellular as seen in Figure 4-5, but there were many instances of unicellular
bridging as well (Figure 4-6).
Figure 4-5. Examples of cellular growth on fiber placed in the RMSC system taken with
SEM. A.) shows a large aggregate of cells and fibers with the same ty pe of orientation
seen under light microscopy. B.) shows a large multi-cellular bridge that spans the
distance from the bottom of the dish to the fiber.
Figure 4-5B demonstrates bridging from the dish floor to fibers, another commonly
seen cellular behavior. Again the bridge demonstrated is multi-cellular, though there

72
were unicellular examples of this type of bridging as well. This multi-cellular aggregate
demonstrates a more columnar, or cylindrical bridging motif, in contrast the the
flattened, sheet-like bridging was also seen. Both structures were clearly multi-cellular
with their respective morphologies determined by what appears to be the topography, or
the shapes presented by the fibers in their vicinity.
Figure 4-6. Examples of unicellular bridging on bioactive glass over distances of ~70
pm. In addition to being unicellular these are also fiber to fiber bridges.
Polymer fibers
Figure 4-7 shows micrographs of RMSCs bridging distances between two adjacent
Nylon fibers. These constructs were incubated for 12 days prior to SEM examination
and demonstrate what appears to be a multi-cellular bridge (left most bridge) and a
unicellular bridge (right most bridge).
Figure 4-8 is a micrograph of RMSC bridging on adjacent Maxon fibers. Though this
particular bridge is shorter than that seen on the bioactive glass fibers and the Nylon
fibers, bridges were not limited to shorter lengths. This construct was incubated for 13
days in RMSC culture.

73
Figure 4-7. Examples of unicellular bridging between nylon fibers. This image
demonstrates what appears to be a multi-cellular bridge (left most bridge) and a
unicellular bridge (right most bridge).
Figure 4-8. Bridging between Maxon fibers.
Light microscopy of Bridge Development on Polystyrene Culture Flask
In order to classify, or identify the sequence of events involved in the development of
MSC bridging, RMSCs were seeded in standard polystyrene culture flasks and
individual bridges were observed and documented serially over time.

74
Initially after seeding there were many individual cells that had elongated along the
flat surface of the dish, sometimes for hundreds of pm. An example of this elongated
cellular morphology is shown in Figure 4-9A (green arrow). These highly elongated
cells were interspersed with cells that had assumed more flattened morphologies. In
addition to elongation on flat surfaces, there were also cells that elongated and had
associated with the topography of the polystyrene wall similar to the cellular behavior
shown in Figure 4-9B (red arrow). Initial stages of bridging, as demonstrated by single
cell bridging, indicated that bridging was possible only when there was an actual
physical pathway for bridge formation. It appeared that elongation of cells, which can
occur on the order of
Figure 4-9. Initial RMSC elongation. A.) Representative micrograph of highly elongated
cells that seemed to be responsible for initial unicellular bridging.
hundreds of microns facilitated bridge formation by traveling the distance initially. In
other words it did not seem possible for cells to reach across gaps without there being a
pathway for them to elongate on while constantly maintaining adhesion and contact
(Figures 4-6 and 4-9).

75
Figure 4-9 continued. Initial RMSC unicellular bridging. B.) shows the formation of a
unicellular bridge between the floor of the culture dish and the wall of the dish (red
arrow).
Bridge development progressed between the floor of the culture flask and the wall.
By day 7 large multi-cellular bridges were apparent similar to the one shown in Figure
4-10. Bridges extended for hundreds if not thousands of microns and were clearly
multicellular in nature. The areas underneath the bridge, that is the areas of the floor
between the site of attachment at the wall and where the bridge was secured to the floor
of the dish exhibited few cells. It seemed as if the cells that had once inhabited that
region of the dish
had released and become part of the bridge as it pulled off the floor and suspended itself
in the medium. This reduced number of cells is apparent in Figure 4-10 as well. With
continued time in culture the cells composing the bridge showed evidence of
proliferating within the bridge proper. This was apparent in a thickening in the bridge
near its site of attachment to the flask wall (Figure 4-11, red arrow).

76
/
Flask Wall
Flask Floor
250um
-r; ..
iA\3- r>
Figure 4-10. Multicellular bridge at 7 days in the RMSC culture. Bridge is extending
from the floor of a standard polystyrene culture flask to the wall.
On the 10th day in culture, there were instances of ECM nodule formation within the
body of the bridge in the location where the thickening of the bridge had occurred
(Figure 4-12). These nodules were very similar morphologically to those seen on the flat
surface of other areas of the culture dish. On the 11th day in culture the bridge
contracted back from the wall of the dish and formed a large multi-cellular nodule that
appeared to be contain a large fraction ECM as part of its composition. Again this
Figure 4-11. Multicellular bridge at 8 days in the RMSC culture. Bridge shows
proliferation and thickening within its body. This is the same bridge shown in figure 4-
10.

77
Figure 4-12. Multicellular bridge at 10 days in the RMSC culture. Nodules appear to be
forming within the body of the bridge at the site of proliferation.
nodule was morphologically very similar to those seen in flat areas of the dish, however,
this nodulewas much larger, on the order of hundreds of microns versus approximately
fifty microns for nodules on the flat surface.
Bridge Development on Stainless Steel Screens (Light Microscopy and SEM)
After classifying, or identifying the sequence of events involved in the development
of MSC bridging. RMSCs were seeded onto stainless steel screens and the general
Figure 4-13. Multicellular bridge at 11 days in the RMSC culture. Bridge contracted
back from the wall of the dish and formed a large aggregate of cells and ECM. This
nodule was morphologically identical to others seen in the center of the dish w here no
bridging occurred, only much larger.

78
development of bridges was observed and documented serially over time. This study
was performed in order to introduce a controlled topography into the RMSC system and
examine the bridging phenomenon associated with it. The screens used had a weave that
led to square holes with dimensions of approximately 170 pm on a side. The fact that it
was a woven fabric was also important in the overall study of fibers and their use as
scaffold materials. Using screens, therefore allowed insight into fiber based scaffold
applications, though the fibers composing these particular woven screens were steel.
To study the effect of bridging on the development of ECM and mineralization,
constructs were stained via the Von Kossa Method. Additionally, to ensure that the
bridging effects were the result of the fibers incorporated in the weave of the screen, the
screens were suspended above the floor of the petri-dish.
After seeding cells were apparent on the lengths of the fibers and could be visualized
at the periphery of the fibers, which, like the Nylon fibers used, were opaque. Numbers
of cells increased until by the 10th day then began to form unicellular bridges,
or bridges composed of a few cells (Figure 4-14). This bridging is also shown in a 3-D
formation of even larger aggregates of cells that assumed flattened bridges and in some
cases more cylindrical bridges (Day 12 and Day 15 images Figures 4-14, 4-16A, 4-16B,
4-17A and 4-17B respectively).
By the 18th day entire holes of the screen were covered by flat sheets of cells acting
cooperatively and there was even the presence of occasional non-mineralized nodules of
ECM (green arrowhead Day 18 image figure 11, also see SEM representation in Figure
4-15A and 4-15B. Continued time in culture led to the cylindrical bridges progressively

79
spanned distances across the square holes until they had completely bridged the
diagonals of the square holes.
Figure 4-14. Development of bridging on stainless steel screens. Day 10 shows
unicellular bridging, or bridging with only a few cells. As time passes cells proliferate
and form more complete sheet-like bridges until, by Day 18 most holes in the screen are
filled with cells. At this time early nodule formation has begun, though it is
unmineralized (green arrowhead). By Day 23 mineralization is highly apparent as
evidenced by nodules stained black with the Von Kossa stain.
By the last day of the study, mineralization was seen within the bodies of bridges that
were spanning distances across the holes of the screen. Though the presence of flattened
sheet-like bridges and cylindrical bridges were both apparent during the course of the
study, prior to the 23rd day, flattened sheets appeared to be most dominant in numbers.
Later in the study, especially day 23, cylindrical bridges became moreprevalent, which
indicated that some of the flattened bridges were becoming cylindrical (Figures 4-14. 4-
19A and 4-19B).

80
Figure 4-15. SEM of stainless steel screens after 10 days in RMSC culture. Unicellular
bridging is prominent.
Figure 4-16. SEM of stainless steel screens after 12 days in RMSC culture. Bridging
becomes more noticeably multicellular with cells that are remarkably flattened.
This behavior may be related to the contraction of cells as was seen later in the
bridging sequence documented in the polystyrene flasks. An example of a cylindrical
bridge is shown in Figures 4-14 and 4-14B. At the base of the cylindrical bridge seen in
Figure 4-14B is a mineralized nodule (red arrow). This was verified visually by light
microscopy prior to SEM preparation (data not shown).

81
Figure 4-17. SEM of stainless steel screens after 15 days in RMSC culture.
Figure 4-18. SEM of stainless steel screens after 18 days in RMSC culture.
Proliferation Studies of RMSCs on Bioactive Glass
Two way ANOVA showed both main factors were significant with P< 0.001.
Growth curves for 20 mg, 30 mg and 40 mg constructs displayed a very similar growth
kinetic, which was not a strict sigmoidal curve (Figure 4-20). The 10 mg construct on
the other hand displayed traditional sigmoidal growth, in fact this fiber density was the
only density that showed a statistical difference between cell counts taken on day 7 and
day 10 (P<0.05).

82
Figure 4-19. SEM of stainless steel screens after 23 days in RMSC culture.
Cell counts for the 10 mg construct were very close to those from the 20 mg construct
for much of the time period measured. In fact the differences in cell concentration
between these two fiber densities did not become statistically significant until the 21st
day (P<0.05). That there are differences between the shapes of the two curves is
apparent, however, indicating some sort of critical difference, or cut off point between
10 mg and 20 mg. Of all the curves only the 60 mg construct showed a distinct plateau
at the end of the 21 day study indicating it was the only one that had finished
proliferating (i.e. day 14 data was not significantly different from day 21). In addition to
this, the 60 mg curve was markedly higher than any of the other experimental conditions
indicating not only that the cells finished their replicative stage sooner, but also that the
proliferation had been more robust on this fiber density. On the final day of this study
the 20 mg construct displayed a cell count very close to that of the 40 mg construct, the
two were not significantly different at this point, and it appeared to still be proliferating.
Overall the 60 mg curve provided the most robust example of the characteristic growth
kinetic and had the highest cell counts as well.

83
RMSC Growth on 77s Bioactive
Glass Fibers of Various Densities
Figure 4-20. RMSC growth curves for each fiber density. Star indicates site of 1st
plateau where Schmidt et al. show maximal osteocalcin concentration, the characteristic
late differentiation marker. # indicates time point where Schmidt et al. found alkaline
phosphatase, the early differentiation marker was highest.
Discussion
The potential of fiber based scaffolds in bone replacement is extensive. Flexible
mechanical properties, high porous volume, designable woven architectures, all
contribute to the attractiveness of fiber based scaffolds in connective tissue engineering.
Single fibers influence cell orientation and elongation as shown in chapter 2. This
behavior is very similar to that seen on flat 2-D surfaces with microtopographies of
given dimensions [29,31,32,89]. This cell type leads to multi-layering on flat surfaces, a
cellular behavior that is essential to the formation of ECM nodules and the

84
mineralization of these nodules [80,115,116]. Similar multi-layering behavior is seen on
single fibers leading to similar nodule formation and mineralization.
Multi-layering and nodule formation are functions of many cells, or aggregations of
cells, which communicate with each other [104,124] and cooperatively perform. How
these cells grow on single fibers, as well as flat micropattemed surfaces is similar in that
there is only one substrate for them to interact with, either a fiber, or a flat surface. With
the introduction of more than one surface, or macrotopography the cellular response
becomes more complex. In this context macrotopographies can be thought of as those
created by multiple surface with features on the size order of the multi-cellular
aggregates, while microtopographies are those that act at the level of the individual cell.
Bridging acts at the macrotopographical level, the level that 3-D scaffolds necessarily
exhibit. Single fibers lead to a microtopographical response, but where two fibers
contact, a macrotopographical feature exists. In this sense, 3-D fiber-based scaffolds are
hierarchical, combining not only microtopography where cells are in contact with only a
single fiber surface, but also macrotopography features where two fibers interact and
bridging occurs. Therefore, an understanding of single fiber effects on cell function (i.e.
multilayering), as well as multi-fiber effects (bridging) is essential for future fiber based
scaffold design, or any other hierarchical 3-D scaffold design. This is particularly so in
light of the fact that tissues are 3-D and these interactions, or mechanisms are obviously
part of the tissue level machinery necessary for the development of bone, or ligaments.
Bridging seems to progress in a generalizable developmental sequence of events as is
shown in Figure 4-21. Single cell bridging occurs first (Figure 4-21 A) as a cell
encounters macrotopographies like the wall of a culture dish, or the intersection of two

85
fibers. An example of this is shown in Figure 4-9 of the results. Multi-cellular bridging
follows as more cells become involved either as they are lifted from their surface in a
sheet-like manner, or proliferation that occurs within the body of the bridge itself. Both
behaviors appear to be present as seen by the sparsity of cells below the bridge in Figure
4-10, as well as the thickening of the same bridge over time. Eventually multi-cellular
Figure 4-21. Schematic generalization of multicellular bridging,
structure exhibits ECM nodules within its body (Figure 4-21C) that eventually
mineralize as seen when MSCs were grown on the stainless steel screens. In the case of
the polystyrene culture dish the bridge contracted until it had pulled from the side of the
dish and formed a large nodule of ECM and cells (Figure 4-2 ID).
The context of this bridging phenomenon is to be found in many areas of the
literature. Cells of mesenchymal origin grown on 2-D surfaces with
[31,32,73,74,89,94,125], show that proliferation and differentiation are strongly
influence by the ability for these cells to adhere to their substrate. Adhesion is

86
influenced by the factors of surface roughness [90,121,126,127], or topography [31,32],
surface chemistry [128] and surface energy [129].
In addition to the relationship between adhesion and the cellular ability to develop
tension, there is the effect of mechanical stretch on cellular activity. Stretch applied
externally to MSC leads to increased differentiation, as well as an orientation of ECM in
the direction of the force [101]. Similarly, inhibition of chloride sensitive stretch
receptors causes a decreased response to topography, drawing a link between the
topography and the cellular need for stretch, or tension [100]. It is interesting to note
how the strength with which a cell adheres and develops tension, as well as an applied
stretch stimulus both result in similar cellular responses in the form of proliferation and
differentiation. Overall it seems tension generation is key for proper proliferation and
differentiation of mesenchymally derived cells on a 2-D surface.
Though 2-D culture systems provide a glimpse of the cellular machinery involved in
proliferation and differentiation, tissues are 3-D structures composed of cells. It seems
therefore that the cellular process of tension generation is designed to operate in the 3-D
environment, a theory that is bom out by many examples in the literature. Studies
performed using collagen gels as scaffolds for cells of mesenchymal origin show that
increased stiffness of the scaffold leads to an increased rate of proliferation. In addition
to this increased rate the duration of proliferation persisted longer than that seen in
scaffolds with less stiffness [80]. In similar studies collagen gels were anchored on one
axis leading to a preferential tension development in the direction of this axis by
fibroblasts. Cell numbers increased by 5 times in the anchored gels and ECM generation
was greater than that seen in the un-anchored gels, which in contrast demonstrated a 5

87
times decrease in cell number [81]. Tension generation leads to increased proliferation
and ECM deposition, but it also organizes the ECM that is deposited. Osteoblasts grown
on anchored collagen gels also elongated in this direction and had deposited ECM so
that it was oriented on the same axis [103].
Overall it is apparent that tension development is necessary for proper proliferation
and ECM production in 3-D cultured scaffold systems. In this study the probable
mechanism of this tension development is revealed. As we have shown, bridging on a
unicellular and a multi-cellular level appears to be the mechanism that allows tension to
be generated in a 3-D matrix. In addition to providing the tension necessary for proper
differentiation and proliferation, this process allows the development of tissues
throughout the 3-D scaffold. That is, bridging allows for filling the spaces of the matrix
and thus creating a complete cellular composite with cells that interact with adjacent
cells in all spatial directions.
The elements of tension generation, as have been reviewed above, are the amenability
of a surface for cellular adhesion, as well as the modulus of that surface, or substrate. In
other words, how well a cell can bind to a substrate, as well as how much that substrate
resists deformation by cells leads to a commensurate proliferative and differentiative
response. Both of these factors contribute the ability to generate tension, but it seems
unlikely that they are the only factors involved. Adhesion and stiffness are indeed
important, but it seems logical that a mechanical advantage as provided by the
architecture of a matrix would have an effect also. This is particularly true when we
consider bridging and its role of spanning macro-topographical distances from the 3-D
point of view.

88
Bridging as described in this study is a phenomenon that encompasses all these
aspects of tension generation. Because bridges are composed of cells we hypothesized
that there would be an effect of spacing, an architectural characteristic, on proliferation.
Though we have seen multi-cellular structures of relatively enormous sizes (i.e. bridges
in polystyrene flasks Figures 4-9 and 4-10) it is likely that multi-cellular bridging occurs
on smaller length scales as provided by a few cells acting in concert. This assertion
seems reasonable when one considers the first stages of unicellular bridging, which
seem to require an actual physical route for the initial unicellular bridge, thus limiting
distances to the order of less than a few hundred microns.
To test the theoretical effect of spacing on cell function, we performed a growth study
of RMSCs on increasing fiber densities. We hypothesized that increasing fiber density
would lead to an increased proliferative response, which was indeed the case. 3-D
matrices with average pore sizes of -165 pm have been shown to increase proliferation
per unit surface area when compared to that of 2-D matrices [1]. This result was not
thoroughly explained, though the authors theorized that it was an effect of increased
multi-layering. In light of the current results their increase, particularly given that it was
normalized for surface area, may have been due to bridging.
60 mg bioactive glass fiber constructs showed a maximal proliferative response
higher than all other fiber densities, as well as a plateau at day 21 indicating that
proliferation was finished and the cells had moved onto later stages of differentiation.
Indeed cells taken from scaffolds on the 14th day failed to proliferate to any significant
degree when seeded onto tissue culture polystyrene.

89
The dynamic of the proliferative curve was at first perplexing, but its consistent
appearance across many, but not all, fiber densities suggested it was a true cellular
response. The unexpected decrease in proliferation rate was seen between the 7th and
1 Oth days and with close inspection of the literature, proved to correspond with the
growth curves published in many similar studies [1,130,131,132], Interestingly none of
these studies addressed this trend in their growth curves in any significant detail.
Schmidt et al. performed a study that was nearly identical to ours in cell type, time
course and methodology on various metal orthopedic materials. Their results demonstrate
a group of curves that parallel each other, as ours do, with the same decreased rate at the
same time period. They attributed differences in growth rates to the varied roughness of
their materials.
In the same study biochemical testing was performed for the differentiation markers
alkaline phosphatase and osteocalcin. Alkaline phosphatase is a marker associated with
early osteoblatic differentiation, while osteocalcin is associated with the later stages of
differentiation. An intriguing result was that osteocalcin peaked and achieved its highest
levels coincident with the decreased growth between days 7 and 10, while alkaline
phosphatase peaked near the end of the study. At first blush, these results are contrary to
genetic and biochemical studies, which are widely supported in the literature. However,
Sharpe et al. showed in 1984 that human marrow derived cells were composed of two
sub-populations of cells, one with a very rapid proliferative rate, the other with a
relatively slow rate [133]. In a similar study Kramvis and Garnett showed that there
were two biochemically identical, but morphologically distinct sub-populations in the
marrow-derived culture of monkey cells [134], These morphological sub populations

90
were either fusiform, or polygonal and exhibited different proliferation rates. The one
that was most rapid, interestingly, was the fusiform, or elongated morphology. Armed
with these facts our growth curves and the results of others seen in the literature make
much more sense.
A population composed of two sub-populations with differing proliferative rates
accounts for the decreased rate between day 7 and 10 and the increased osteocalcin
concentration seen in Schmidt et al.. At this time the more rapidly proliferating
population has achieved differentiation thus producing osteocalcin around the 7th day.
This inverse interaction between proliferation and differentiation is a response that is
genetically coupled and would explain the presence of osteocalcin as the fast
proliferative cells differentiated relatively early on in the growth of the total population
[36,37],
By the 10th day the second sub-population of marrow derived cells begins its
proliferative stage. This second population only achieves its early stages of
differentiation near the end of the study when the growth curves plateau, supported by
the increased alkaline phosphatase levels near day 19 in Schmidt et al.. Presumably,
continuing the study of osteocalcin for longer time periods would show another increase
when the second subpopulation achieved its full differentiation [131].
Considering these statements and how they apply to the proliferative curves
evidenced here leads to more interesting conclusions. Cells grown in empty constructs
show a fast proliferative increase and subsequent dropping off that occurs between the
3rd and 6th day, which is to say sooner than the other curves. This is the first effect of

91
the 3-D scaffolds, they allow proliferation of the first cell population to occur longer and
more robustly in a way that parallels fiber mass.
The differences in curve shape between the 10 mg and 20 mg constructs were noted
previously in the results. The fast proliferative increase did not occur on the 10 mg
constructs indicating that the spacing, or density was not sufficient to foster proliferation
of this cell population. In fact it appears that the fast proliferative response was inhibited
when this curve is compared to the control. The late proliferative response on the other
hand occurred in a manner that paralleled the 20 mg constructs nearly identically for the
time period between the 10th and 15th day. Overall it seems that in the spacing
difference between the 10 mg and 20 mg fiber constructs indicated a critical point where
the fast proliferative cells were influenced by the spacing and architecture of the 3-D
matrix.
In general we see that the presence of the bioactive glass scaffold leads to an increase
in proliferation that plateaus near the end of the study. This plateau indicates that the
RMSCs have achieved a level of differentiation, which was supported by unsuccessful
attempts to trypsinize and re-plate cells after day 14. Cells never re-attached and began
proliferating in any significant manner indicating that differentiated had proceeded
beyond the proliferative stage. On this bioactive glass scaffold, where it is likely that an
increased mechanical advantage is achieved, the mechanism of differentiation may be
related to bridging and the development of tension, which in turn influences growth
factor and BMP production.
As mentioned in chapter 2, tensile stress increases BMP-4 by a factor of two as part
of the developmental cycle [102]. In addition, this stimulus leads to an increased

92
responsiveness of osteoblasts to morphogens and vitamin D [102], Applying exogenous
TGF-P on the other hand increases the tension produced per cell by a factor of two
[105]. Increased TGF-p and BMP-4 precede increased production of collagen, alkaline
phosphate and increased MSC differentiation. Production of these growth factors as
stimulated by tension occurs in such a way that they have their effects in an autocrine
and paracrine manner, thus tying this form of cellular communication to tensile stimulus.
Additionally it is seen that exogenously administered TGF-P increases multi-layering of
cells within the confines of pores in collagen scaffolds and causes an increase in macro¬
scale contraction of these scaffolds [105].
The increased proliferation and evidence of differentiation that we see on the
bioactive glass scaffolds used in this study may then be a result of endogenous
enhancement of these growth factors. Providing proper spacing and architecture,
therefore, may lead to increased proliferation and differentiation of RMSCs via the
upregulation of growth factors and BMPs. If this tension induced upregulation of natural
cytokines and growth factors indeed occurs, then optimizing fiber spacing and
architecture would to some degree lead to an optimization of growth factor production.
Indeed it seems highly likely that the two phenomena are inter-related as fiber spacing is
a topographical stimulus and topography has been shown to stimulate
autocrine/paracrine growth factors [109].
Conclusions
Overall we see that bridging occurs on a number of different materials and thus is a
property of this cell type. This phenomenon occurred on many different geometries as
well and appeared to require a direct physical pathway when large features, or distances

93
were spanned. Bridging appears to be involved in proliferative processes, as well as, the
deposition and mineralization of ECM.
Using a bioactive glass scaffold, proliferation was enhanced, which showed the
compatibility of this material, as well as the likelihood that this effect was a result of
bridging. Spacing, or fiber density seemed to be the factor influencing proliferation
optimizing cell based tension development by increasing bridge formation appears
critical to proper cell function in 3-D scaffold materials. Most importantly, spacing and
thus tension development is a factor that can be controlled and used by tissue engineers
during the design of scaffold materials.

CHAPTER 5
MULTI-FIBER STUDIES
Introduction
Chapter 3 of this work focused on examining the effects of single fibers on the
orientation and elongation of RMSCs. In chapter 4 the phenomenon of bridging was
studied, showing how RMSCs cooperatively deal with the presence of more complex
geometries. This chapter addresses systems that combine both the orientation and multi¬
layering effects seen on the microtopographies of single fibers, as well as the bridging
and interactions seen on macrotopographies.
Among other things chapter 3 showed that 7-0 fibers had the greatest and most
predictable influence on RMSC morphology. RMSCs when grown on 7-0 Maxon for 12
days experienced a consistent increase in elongation and orientation. Multilayering was
present under these conditions and these multi-cellular structures were oriented in the
directions of the fibers they were residing on.
Chapter 4 demonstrated the development of multi-cellular structures on a 3-D level, a
phenomenon referred to as bridging. Bridging developed in a number of situations and
on many materials in response to the presence of many topographical surfaces. This
cellular process was related to ECM nodule formation and mineralization, as well as
proliferation. Spacing was studied on the macro-scale (i.e. a whole population of cells in
a culture system), and created by various fiber densities, which led to an overall
influence on cell growth. These bioactive glass fibers, however, were randomly oriented,
94

95
while the overall goal of this dissertation was to show the effects of organized scaffolds
on the tissue development, particularly ECM.
Toward this end it is important to understand how the spacing of organized fibers
effects the ability for cells to multi-layer and bridge. Knowledge of how multi-layering
and bridging occurs in the face of controlled macrotopographies, namely those found at
the junctions between fibers, is also key. With this knowledge firmly in hand it will be
possible to design hierarchical scaffolds that organize and direct the ECM and
mineralization products of MSCs. This control of design and directed tissue growth,
once achieved, promises to be a powerful method for developing anisotropic tissues like
bone.
The approach outlined in chapter 1 discussed multi-layer constructs, which would
then direct cellular organization in a particular way for each lamella. Prior to this,
studies will be performed on individual lamellae with different spacings. These lamellae
will be composed of parallel 7-0 fiber arrays with spacings of either 55 pm, or 25 pm
between each fiber. 7-0 fibers were chosen because they were shown in chapter 3 to
orient the cells the most and will perform most adequately in the pursuit of optimizing
cellular direction. Organizing many fibers in a parallel array will allow the influence of
their cooperative presence to be shown, whether they enhance ECM deposition and
organization compared to a single fiber.
Furthermore, using different spacing will help make clear the relative importance of
bridging in the whole process. 25 pm spacing will allow bridging to occur sooner than
55 pm and may allow the ECM organization and hierarchical effects of multi-fiber
constructs to have a greater effect than the wider spaces. In addition to spacing, the angle

96
between adjacent fibers is a factor that needs to be studied. This aspect of influence on
cellular bridging became apparent during the bridging studies performed in chapter 4.
Bridging similar to that shown in figure 1 was very common and when considered in
light of the SEM data shown in chapter 4 it was apparent that cells were bridging in
response to the woven architecture of the screen.
Figure 5-1. Eye shaped bridging phenomenon caused by the weave of the screen. Eye
shaped bridges alternate orientation based on what appears to be the tendency to bridge
the shortest distance. The shortest distance alternates with the weave of the screen
leading to regular and alternating bridging pattern (arrows). (Magnification = 200x)
Cells bridged at the comers of the holes created by the interwoven fibers and
appeared to be bridging preferably at sites where the distance between fibers was least.
Because the fibers were woven in an alternating motif, the bridging occurred in a regular
manner leading to eye shapes that alternated in orientation (Figure 5-1). With this
evidence the study of angles on cellular bridging and its effect on cellular function
makes logical sense. For this aspect of weaving, stainless steel screens will again be
used in a manner similar to that seen in chapter 4, only the angles between fibers will be
altered from their current 90 degrees to more acute angles.

97
Finally, multi-layer constructs with each layer, or lamella being a parallel array of 7-0
fibers that is situated at different angles, will be seeded, cultured and studied. Overall the
goals of this chapter are to examine the effects of controlled spacing distance on the
bridging phenomena and the organization of ECM products. In addition how the cells
interact at particular angles, or sites of inter-lamellar junctions, and how this effects
ECM organization will be studied.
Materials
Cell culture materials were the same as those used in chapters 3 and 4. Von Kossa
stain was performed using the same materials as chapter 4, while multi-fiber constructs
were made from 7-0 Maxonâ„¢ sutures (Davis & Geek). Prior to being incorporated into
constructs fibers were exposed to a 50 watt Argon plasma for 2 minutes at 50 milliTorr.
Stainless steel screens used in this chapter were the same as in chapter 4.
Methods
Cell Culture
Cell Culture was performed in the same manner as described in chapters 3 and 4,
though one notable difference was the seeding density used in this chapter. Constructs
were seeded at ~1.5-2.0 x 105 cells/ml vs. chapters 3 and 4 where the constructs were
seeded with ~1.0 x 105 cells/ml. This was done to increase the numbers of cells initially
settling on the construct, which would allow a more rapid development of cell layers and
overall a more timely series of experiments.
Construct Preparation
In order to insure that proper spacing was achieved between fibers, a special device
was constructed. Pictures of this micromanipulator are shown in Figure 5-2 and it is

98
merely a simple lever that translates the relatively large movements of the handle (a
syringe barrel) through the pivot point to the actual manipulating portion of the device (a
Figure 5-2. Pictures of mult-fiber micromanipulator. A) Microscope with
micromanipulator on stage, note hanging weights attached to fibers by yellow thread
(red arrow). B) Front view of micromanipulator showing handle and yellow threads used
to maintain tension on fibers. C) Manipulator with pivot point shown. The pivot point
was composed of silicone rubber, which the actual manipulator (30 gauge needle) was
pushed through. D) Front view of polystyrene support ring and actual manipulator.
1 inch 30 gauge needle). Because the handle of the device was much longer than that of
the manipulator, the movements of the manipulator were much reduced with respect to
the handle’s movement.
The micromanipulator was mounted onto an inverted microscope with a calibrated
micrometer eyepiece (Figure 5-2A). Through an opening in the stage of the
micromanipulator it was possible to view fibers strung across the device. Polystyrene

99
support rings, to which the fibers were glued, could be inserted underneath the fibers,
while fibers were kept under constant tension by weights of ~ 29 grams on each side.
Using the micromanipulator, fibers were positioned to the correct spacing (either 25
pm, or 50 pm) and cemented to the polystyrene support ring using a-
methylcyanoacrylate. Once secured to the support ring, excess fiber material was clipped
from the multi-fiber construct. A parallel array multi-fiber construct prepared with this
method is shown in Figures 5-3A and 5-3B. Figure 5-3C shows a multi-layer multi-fiber
construct with four parallel arrays of 7-0 fibers overlapping each other at a central point.
Multi-fiber constructs were made with 45-degree angles between each successive layer.
Figure 5-3. Examples of multi-fiber constructs for RMSC culture. A) Shows parallel
array multi-fiber construct with 25 pm spacing and ruler for size reference. B) 25 pm
parallel array with fibers visible. C) Multi-layer, multi-fiber array with four lamellae.
Maxon Construct Sterilization
After assembly constructs were left in a laminar flow hood overnight to allow
complete polymerization of cyanoacrylate. Constructs were then mounted in 24 well

100
plates using heated implement and immersed in 10% bleach for 30 minutes followed by
two rinses in absolute ethanol. Rinsing with ethanol was followed by a 24 hour soak in
ethanol under a 254 nm UV lamp.
Stainless Steel Construct Preparation
Stainless steel specimens were cut into rectangles with the dimensions of 7 mm x 12
mm then clamped in a pair of hemostats in the middle of their longest dimension and
one side was pulled with a pair of pliers (Figure 5-4A). Applying tension to one half of
the construct led to the weave of the screens being elongated in the direction of the force
creating parallelograms with the desired acute and obtuse angles (Figure 5-4B). Angled
Figure 5-4. Diagram of stainless steel construct preparation. A) Stainless steel construct
schematic. Screens were clamped in the center and pulled with pliers (i.e. red arrow). B)
Elongated squares,or parallelograms produced by force application and imaged with
light microscopy. C) 90 degree control section of screen (Magnification = 40x).
screens were evaluated to determine the resulting acute angles leading to a mean value
of 63.8 degrees with a standard deviation of 9.8 degrees (n = 90 measurements).
Identical measurements performed on the unstretched portions of the screens (Figure 5-

101
4C) led to a mean value of 89.3 degrees with a standard deviation of 2.1 (n = 90
measurements). Screens were then autoclaved and placed in culture for 10 days.
Transmission Electron Microscopy
Specimens were cultured for 21 days then fixed in 3% glutaraldehyde in phosphate
buffered saline at pH 7.4 for 3 hours. 4% OSO4 was used for post-fixation and samples
were placed in this solution for 10 minutes followed directly rinse with de-ionized water
and immersion in 30% EtOH the first stage in a serial ethanolic dehydration passing
through 50%, 70%, 90% and finally 100% EtOH. Sometimes OSO4 precipitated on the
surfaces of samples leading to granular artifacts.
After fixation and dehydration, samples were embedded in epon 828 hardened with
Jeffamine D-230 in a stoichimetric ratio of 1.1:1.0, Jeffamine D-230: epon 828. Epon
with specimens was allowed to cure at room temperature overnight followed by oven
curing at 60 degree Celsius for 24 hours.
Embedded samples were sectioned using a Leica ultracut microtome at ~250 nm,
stained with uranyl acetate and lead citrate, then viewed with a JEOL 200 CX
transmission electron microscope at 200 kV.
Von Kossa Staining
Performed as detailed in chapter 4.
Statistical Analysis
The effect of weaving angle on mineralization was analyzed using the binomial
parameter z-test. This is a test designed to measure the probabilities associated with
experiments that are quantified in a yes, or no manner. Mineralization, or nodule
formation within bridges formed at the locations of the angles made by interwoven
fibers was the measured dependent variable. To perform these measurements, each

102
parallelogram in the case of the stretched portions of the screens was divided into
quadrants (Figure 5-5). Similarly the square holes of the unstretched portions of the
Figure 5-5. Diagram of stainless steel statistical test. Schematic representation of
parallelograms, in the case of the angled sections of the screen, and squares in the 90
degree portions. Data was collected from the same orientation within the total specimen.
Green quadrants (finely hatched) are equal to “Yes,” or positive experiments in the
binomial parameter test, red areas (coarser hatching) equal “No.”
screens were divided into quadrants. If mineralized nodules were seen in the quadrants
associated with acute angles, it was considered a “Yes,” or positive result, while nodules
in the obtuse regions were considered “No.” Measurements in the unstretched regions
were performed in the same orientation as the long axis of the parallelograms as
indicated by the arrow in Figure 5-5. In the 90 degree region the same criteria were used
to determine “Yes,” or “No” data, that is the presence of mineralization was measured in
quadrants that corresponded to the quadrants in the stretched portions of the screens
(This is indicated by the hatching seen in the quadrants in Figure 5-5).
In the 90 degree control screens, though bridging is somewhat regular in the
alternating eye shape motif, it should occur just as often in one direction as the other

103
direction. Under this experimental condition the probability (7to) of bridging and
therefore mineralization occurring is just as likely in the “Yes” quadrants as the “No.”
Since the holes are divided into four quadrants, two that equal yes and two that equal no,
the probability (7to) that bridging and mineralization will occur in each is the same and
is 7Co = 0.5 the control probability.
If bridging and hence mineralization is not affected by the angles of the fibers then
the likelihood that it would occur on acute angles is equal to the probability seen on
obtuse angles. This would be the same as that seen in the controls. The hypothesis,
however, is that bridging and hence mineralization will occur preferentially in the
quadrants associated with the acute angles, or more often than that seen on the control
portions of the screen (7tacute > 7To)- The complete statistical test is reproduced below.
Null Hypothesis: 7i = 7t0 Where:^ â–  0.5
Alternate Hypothesis: 7iangle>7i0
Test Statistic: z - -.lnglc-710 Where: CT*angie= ("o-fi-no)
CT
Wangle ^ n
Reject Null Hypothesis if:
z > z
a
Where: za is the probability
a of type 1 error.

104
Results
Maxonâ„¢ Bridging
Maxonâ„¢ bridging day 3
Single fiber constructs exhibited many cells adhering to their surfaces indicating they
were well seeded.
25 micron parallel arrays all showed at lease one case of bridging between fibers
sometimes 3 or 4 examples of bridging were present.
55 micron parallel arrays had a level of cellular attachment that was similar to the
single fiber constructs. It appeared that each fiber was acting independently of the
others. There was one example of bridging near the periphery of the constructs where
the fibers were bound to the support ring.
Maxonâ„¢ bridging day 6
Single fiber constructs were completely covered with cells and some had developed
thick areas of multi-layering.
25 micron parallel arrays showed some examples of inter-fiber bridging across the
whole length of the construct from one end of the fibers to the other. There were
spontaneous inter-fiber bridges forming mid-fiber on every one of the constructs. These
inter-fiber bridges were abundantly evident and not the result of any physical pathway
between fibers.
55 micron parallel arrays had become fully covered with cells and exhibited some
bridging, but only at the ends of the fibers near where the constructs were bound to the
support ring. This bridging appeared to be due to the physical pathway provided by the
support ring and was not very extensive.

105
Maxonâ„¢ bridging day 9
Single fiber constructs had developed very thick cells layers that were approximately
20-25 pm thick as measured by micrometer eyepiece, which led to a thickness of fiber
plus cell layer totaling -120-125 pm. One construct demonstrated a contraction of cells
along its length that led to a thick cellular layer -35 pm thick around the entire
circumference of the fiber (Figure 5-6B). This contraction proceeded from both ends of
the construct toward the center.
25 micron parallel arrays an estimated 75-90% of total volume available between
fibers was bridged on all constructs.
55 micron parallel arrays had developed thick multi-layering on individual fibers with
bridging at the ends of the constructs. Only about 20% of total available volume was
bridged between fibers and only at the ends of the fibers.
Maxonâ„¢ bridging day 10
Single fiber constructs demonstrated no significant change, except the lone, previously
noted contraction along the center of one of the fibers had progressed.
25 micron parallel arrays 3 of 4 constructs were completely bridged. One area of one
construct had undergone a contractile process similar to that seen on the single fiber
construct (data not shown).
55 micron parallel arrays exhibited little change except the development of contractile
processes on some fibers.
Maxonâ„¢ bridging day 11
Single fiber constructs showed no remarkable change except contraction on the
aforementioned fiber had progressed to such a degree that all the cells were balled at the

106
center of the fiber (image in Figure 5-6B was taken at this time point). One remarkable
feature was the bridging that had developed where the single fiber constructs were
attached to the support ring. This response to the topography of the ring and fiber
combination led to a bridge that transitioned from the topography of the ring to that of
the fiber in a continuous and gradual manner (Figure 5-6A).
Figure 5-6. Examples of Single Fiber RMSC Growth. A) A single fiber construct with
heavy multi-layers of cells that are oriented in the direction of the fiber. Note the way
cells bridge from the support ring to the fiber orienting in the direction of the ring and
gradually, in a continuous manner changing their orientation in response to the fiber
until they are aligned with it. B) Contracted area of cells on a single fiber. Cells were
originally like those seen in image A. This contraction was not seen on the other single
fiber in the same well.
25 micron parallel arrays By this time, the spacing of the parallel arrays had been
altered near the center, or farthest away from where the fibers were secured to the
support ring. Apparently the bridging between fibers had led to a cell based contraction
that caused the fibers to be pulled together as seen in Figure 5-7B. Figure 5-7A shows
bridging near the support ring where fibers still maintain the original spacing of 25 pm.
55 micron parallel arrays 1 construct had developed a thick multi-cellular bridge
where a contractile balling of cells had gotten near to the adjacent fiber (Figure 5-8C).
There was no physical pathway for the cells to follow and this instance occurred mid¬
fiber, but only on one construct. This single example occurred by a different process
related to the

107
Figure 5-7. Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 25
pm. A) 25 pm parallel array with dense cell coverage. Cell are oriented in the direction
of the fibers and spaces between fibers are completely filled. B) Construct near the
center of the fibers showing contraction and the drawing of fibers closer together. C)
Attachment site shows continuity of multi-cellular bridging similar to that seen on the
single fiber constructs (bar =250 pm).
balling up of cells that had contracted in the direction of the fiber’s long axis. The sites
of bridging that did occur between fibers where they were bound to the support ring did
not lead to any notable contraction, or alteration in the fiber’s spacing (Figure 5-8A).
Multi-Layer Construct Bridging
Bridging between fibers progressed in a manner analogous to that seen on parallel
arrays. Bridging between layers was very robust and showed a marked propensity for
forming at the sites where fibers formed acute angles (Figure 5-9). Figure 5-9B shows
examples of bridging between layers, where it was possible for bridging to occur in

108
Figure 5-8. Representative micrographs of 7-0 multi-fiber parallel arrays spaced at 55
pm. A) Example of bridging originating from the support ring attachment on a 55 pm
parallel array. B. Top fiber shows a contractile process with the beginning of a bridge to
the next lower fiber. Bridging to the right is from attachment site just outside the frame
of the picture. C) Multi-cellular bridge that formed after formation of a contractile
process on the second fiber from the top. This image is taken 2 days after the contraction
occurred and cells have begun to reorganize in the direction of the fibers.
Figure 5-9. Micrographs of multi-layer parallel arrays. A) Multi-layer array with 45-
degree angles of rotation between layers. Bridging between fibers is apparent, as is
bridging between layers. B) Bridging preferentially forming on acute angles.

109
either direction on the fiber. Bridging occurred more prominently on the acute side of
the intersection of the fibers in these situations versus obtuse ( Figure 5-9B blue
arrowheads).
Cell Angle and Spacing Distance
Cell Angle and Spacing Distance seemed to be related. In Figure 5-10 the cells were
oriented in the directions of the fibers, but also seemed angled to a degree necessary to
span the distance between fibers. Because the fibers were farther apart on the 55 pm
constructs, the cells seemed to be at a more obtuse angle than that seen for bridging
between 25 pm constructs.
Figure 5-10. Examples of bridging between fibers of 25 pm and 55 pm spaced parallel
arrays. A) An examples of spontaneous multi fiber bridging on a 25 pm parallel array.
Note the way cells are oriented between fibers, their angle is very slight with respect to
the fibers (Blue arrowheads). B) Bridging on 55 pm parallel arrays near site of
attachment to the support ring. This bridging developed using the support ring then
separated. Note much sharper angles of the cells with respect to the fibers (Blue
arrowheads).
Stainless Steel Bridging
Stainless steel bridging day 3
Many cells were evident on the stainless steel fibers composing the weave of the
mesh and unicellular bridges had begun to form.

110
Stainless steel bridging day 6
Cell growth was robust and obvious with many examples of unicellular bridging.
Bridging occurred at the junctions of the fibers and in the case of the angled portions of
the screens, the bridging was subjectively much greater on the acute angles versus the
obtuse. Bridging also began to occur sooner than that seen on the 90 degree portions of
screen.
Stainless steel bridging day 8
Cell growth and bridging continued in a very rapid manner and there were instances
of holes being completely filled w ith bridged cells. Occasional mineralized nodules were
seen in the bodies of the bridges.
Figure 5-11. Example of von Kossa stained, angled stainless steel screens with
characteristic bridging on the acute angles. Mineralization is seen within the bodies of
bridges formed in the angled junctions between fibers (red arrowheads).
Stainless steel bridging day 10 (mineralization analysis)
Screens were removed from culture then fixed in 3% glutaraldehyde. After Von
Kossa staining, screens were analyzed for mineralization. A representative image of
bridging and mineralization on the angled portions of the screens is shown in figure 11.
The probability of mineralization was shown to be significantly greater on the acute
angles than mineralization seen on the obtuse angles (P < 0.0001). The probability of

Ill
mineralization seen on the 90 degree portions of the screen, however, was not
significantly different for either orientation.
Overall, the probability of mineralization was shown to be greater on the quadrants of
screen that included acute angles versus obtuse, while on the 90 angle portions it was
Table 5-1. Summary of Statistical Results from the Mineralization Study Performed on
Stainless Steel Screens.
Yes
No
Probabilitv
(7T)
CT7t
“z”
value
2a
(for
a=0.99)
Significant
Angled
65
21
0.76
0.054
4.74
2.57
Yes
Sauares
35
31
0.53
0.062
0.48
2.57
No
equally likely to occur in any quadrant (data summarized in table 1). Mineralization
preferentially occurred on the acute angles of stainless steel screens after being cultured
in a RMSC system for 10 days.
Transmission Electron Microscopy
Transmission electron microscopy of 7-0 single fiber
Figure 5-12 shows a cross sectional view of a single 7-0 fiber cultured for 21 days in the
RMSC system. This montage of micrographs reveals multi-layering composed of a
depth of about 3-5 cells. Cellular interconnections are evident.
Lengthwise sections of the same single fiber construct are shown in Figure 5-13.
Again cellular interconnections are evident and what appears to be collagen is being
deposited between cell layers in the extra-cellular space. Cells are much elongated
indicating contact guidance effect on the 7-0 fiber diameter. The cell multilayer
appeared to be very uniform throughout the length that was examined.

112
Figure 5-12. Transmission electron micrograph montage of 7-0 single fiber construct in
cross-section. Cell multi-layer on the surface of a single 7-0 fiber construct
(magnification = 3750x). Thickness of multi-layer is approximately 3-5 cells and the
inset shows an inter-cellular communication, or what appears to be a pseudopod-like
connection between cells (inset magnification = 15,000 x).
Transmission electron microscopy of 7-0 25 micron spaced parallel array
The sample shown in Figure 5-7C was cross-sectioned near the ends of the fibers
where they were secured to the support ring and had not been contracted, or pulled
together as occurred nearer the center of the construct. Figure 5-14 shows an electron
micrograph montage of a representative bridge that occurred between fibers. The curve
of the fibers is evident on either side of the bridge, which formed on the top of the fiber

113
\
\ Fiber Side
\
\
\
- 4 um
Figure 5-13. Transmission electron micrograph montage of 7-0 single fiber construct in
long-section. 7-0 single fiber construct cultured for 21 days fibrillar deposits
morphologically similar to that of collagen are present between cell layers (blue arrows,
magnification = 1825 x). In the inset cellular interconnections are also seen between cell
layers (red arrowheads, magnification = 5,000x).
array, or on the upper part of the array as it sat in the culture well. This formation of
bridges on the tops of the fibers may indicate a gravitational effect of cell settling during
seeding. There were very few cells in the space below the bridge as it is shown
indicating that a strong majority of cells were present were involved in the inter-fiber
bridging in the area pictured. The thickness of the bridge appeared greater than that seen
on single fibers and the cells did not seem as tightly associated in their multilayering.
There appeared to be more space between cells than that seen on the surfaces of the
single fibers. In addition, cells appeared to be more flattened and thinner in nature than
that seen on the single fiber.

114
Figure 5-14. Transmission electron micrograph montage of 7-0 multi-fiber 25 pm
spaced parallel array in cross-section. Bridging between fibers shows cells that are
multi-layered and seem to be less densely associated than that seen on a single 7-0 fiber
(magnification lOOOx, bar =4 pm). Inset shows flattened cells (blue arrow) and cellular
communication pseudopods are present as well (red arrowheads, magnification = 7500x,
bar = 1 pm).
Transmission electron microscopy of contracted 7-0 25 micron parallel array
The sample shown in Figure 5-8C was cross-sectioned near the center of the
construct in an area where the cells had contracted, or pulled together the fibers. The
fibers of the construct were pulled virtually all the way together and the intervening
space between them was completely filled with cells and ECM (Figure 5-15). There
appeared to be two main influences on cell orientation and multi-layering, the influence
of bridging which was seen on the cells composing the upper most portions of the
cellular aggregate and the influence of fibers. Fiber induced cellular orientation followed

115
a similar pattern to that seen on single fiber samples with a cellular multilayer that was
3-5 cells thick. Where the cells forming the bridge and the cells associated with the fiber
met, there were multilayers as deep as 10-12 cells. In the center of the bridge cell layers
extended the entire distance to the location where the fibers were brought together
(Figure 5-15). Cells were highly flattened and again appeared to have more space
between them than the cells seen on single fiber constructs. A direct comparison can be
made by looking at cells on the surface of the fibers versus those suspended between the
fibers composing the body of the bridge. Fibrillar ECM deposits are seen between the
Figure 5-15. Transmission electron micrograph montage of contracted 7-0 multi-fiber 25
pm spaced parallel array in cross-section. This parallel array that was contracted, or had
the fibers pulled together by the cells. Note the thickness of multi-layering particularly
in the center of the image. There are also increased amounts of extra-cellular space vs.
that see on the cells directly associated with the fibers (magnification 1000X). Inset
shows ECM development between cells (magnification = 4000x)

116
cell layers and is abundant in the large areas of space between cells in the bridge (Figure
5-15 inset).
Cellular interconnection was again prominent, perhaps even more than any other
experimental condition examined. Longitudinal sections of the same sample taken from
the area immediately adjacent to that shown in Figure 5-15 revealed the most organized
ECM and cellular orientation seen of all samples studied. Collagen fibrils are abundant
in the extra-cellular space between the cells (Figure 5-16 blue arrows). Insets show
collagen fibrils at high magnification. Cell orientation is also highly apparent indicating
Figure 5-16 TEM images of longitudinal section of the contracted portion of 7-0 25 pm
spaced parallel array (magnification lOOOx). Highly ordered cell layers are shown (red
arrowheads). Oriented collagen fibrils are being deposited between layers (blue arrows).
Insets are higher magnification views of collagen fibrils (magnification = 18,750x, bars
~ 500 nm).

117
that cells involved in the bridging process were flattened and extended in the direction of
the fiber constructs in a very ordered and oriented manner (red arrowheads). Fibrils were
confirmed to be collagen by the presence of the characteristic banding pattern that
existed on the scale of 60-80 nm (Figure 5-17).
Figure 5-17. Characteristic banding pattern of collagen fibrils. Collagen fibrils found in
the extracellular space between cell layers (magnification of small image = lOOOx, bar ~
8 pm). Inset shows close up of collagen fibrils, which demonstrate the characteristic
banding pattern widely associated with collagen. Arrows highlight two examples of the
many bands visible (magnification = 100,000, bar -300 nm).
Discussion
As mentioned earlier in this work, woven fibers when used as tissue scaffolds offer a
number of advantages. Using appropriate processing methods, individual fibers can be
made with remarkably robust tensile strength [21-23], more importantly, however, these
fibers can be organized in a woven construct that is very rigid [11], It was hypothesized
that controlled arrays of fibers would be capable of organizing cells and their ECM

118
products directionally over large surface areas, or volumes. The results of this chapter
provide strong evidence that this hypothesis is indeed correct.
In Chapter 3 cells were shown to experience a strong contact guidance in response to
the diameter of fibers they were grown on. This effect was seen yet again on the fibers
composing the parallel arrays in this study. Elongation of cells on the fibers was
apparent on all constructs regardless of the spacing between their fibers. This
phenomenon, however, was especially apparent on the constructs with 55 pm spacing as
bridging on these constructs was sparse and each fiber acted virtually independently
from the others. In many ways the constructs with 55 pm spacing acted as a collection of
single fibers.
Though there is little material in the literature addressing the topic of bridging the
ability of cells of mesenchymal origin to migrate across spacing has received some
study. Murray et al. studied the migration of anterior cruciate ligament cells onto
synthetic scaffold materials and showed that migration was inhibited by the presence of
gaps between the ligamentous tissue used as a cell source and the scaffold [108]. These
gaps were effective at preventing migration when they as little as 50 pm wide. This
matches well with the current results, which showed bridging on 55 pm constructs, but
only as long as a physical path was present like the one found at the ends of the
constructs where the fibers were secured. There were no cases of spontaneous bridging
across the 55 um gaps between fibers like that seen on the parallel arrays with 25 pm
spacing.
The cells elongated and oriented on individual fibers of parallel arrays with 25 pm
spacing, as well. However, spontaneous bridging between fibers became a significant

119
and added factor as early as the third and sixth days. Effectively cells were being guided
not only by individual fibers they were growing on as seen initially, but also by the way
they were able to bridge and fill the distance between fibers (Figure 5-7A). 25 pm
spacing allowed the development of what was essentially a layer, or lamella of cells and
ECM, which extended for multi-millimeter length scales (5-6 mm, the length of the
multi-fiber constructs as seen in Figure 5-3), covered fiber surfaces and filled inter-fiber
spaces.
As expected, single fibers themselves directed cell growth and organization of the
cells and ECM (Figures 5-12 and 5-13). More importantly we see from the electron
micrographs that cells composing the bridges were also oriented and directed in the
spaces between fibers (Figures 5-14 and 5-15). This cellular orientation appeared to be
even greater than that seen on single fibers as the cells were more noticeably flattened in
their cross sectional view. This shows bridging, which is a hierarchical cellular activity
in that it occurs in addition to the normal multi-layering seen on fibers, is directed by the
macrotopographies provided by the combination of two or more single fibers.
This bold statement is further supported by the data obtained by RMSC growth on
angled stainless steel screens. We see from the robust statistical results that formation of
mineralized nodules within bridges occurs preferentially on the inter-fiber angles that
are acute in nature. 90 degree angle inter-fiber junctions on the other hand led to a
random formation of mineralized nodules. Bridging preferentially occurred on the acute
angles, or distances that were shorter than their 90 degree counterparts in such a way
that it led to mineralization that was far greater. In addition to showing another aspect of

120
controlled bridging as a controllable phenomenon, these results show the ability to direct
and control mineralization within the spaces of a scaffold or matrix.
Controlled collagen deposition is also achieved in this system using the
macrotopography of multi-fiber parallel arrays. The MSC type used in this study
deposits collagen in the extra-cellular spaces existing between cell layers [114-116,134].
It has been shown repeatedly that this deposition occurs in alternating orthogonal
orientations between adjacent cell layers, or layers that are built up on top of each other
when grown on flat surfaces [114-116]. This is not the case in this bridging system.
Orientation of fibrils at 21 days of culture in a RMSC system showed collagen that was
in parallel orientations with the fibers between every extra-cellular layer (figures 15 and
16).
Overall with the evidence of these studies it is quite apparent that the direction of
cellular orientation, collagen deposition and mineralization are capable of being
controlled by designed fiber scaffolds. The spacing of fibers influences the ability of
cells to bridge between them. Bridging between fibers of appropriate spacing is seen to
be a highly directed activity leading to the deposition of collagen in a manner that
appears more oriented than that seen on flat surface, or surfaces with microtopographies.
Furthermore the angle between fibers, a critical aspect of fiber weaving, has been shown
to influence the bridging and hence the spatial location of mineralization. All these
aspects of control are possible through an understanding of the multi-layering seen in
this system and more importantly the bridging phenomenon this cell type undergoes.
Not only do these studies show the feasibility of fiber-based scaffolds in bone

121
replacement tissue engineering, they provide deep insight into the bridging process
itself, which appears to be primarily responsible for these effects.
RMSCs we saw were interconnected in every instance of specimen studied via TEM,
which indicates these cells form interconnected networks. This is a fact supported by
many studies in the literature including the studies of the cellular interconnections
known as gap junctions, which are actual physical connections between cells [104,105].
In addition, the autocrine and paracrine interactions that govern many aspects of bone
cell communication indicate they are interconnected networks. It is not apparent from
this data if the cellular interconnections are sites for cellular communication, though
they are almost certainly sites of mechanical attachment that allow the networks to form.
Studies performed on flat surfaces match up well with the multilayering behavior we
see in this work. Layers formed on the surfaces of single fibers with thickness of about
3-5 cells with little space between layers. These aspects of multilayering and
extracellular space have also been seen by in a virtually identical manner many times in
the literature [114-116,135]. Organization of collagen by the single fiber surface,
however, is not consistent with these studies in that it orients collagen on each layer
parallel to the fiber it is grown on. Flat surfaces showed the alternating pattern
mentioned previously.
Bridging data diverges even more significantly from these trends. Though the
formation of physical inter-cellular connections remains leading to an interconnected
cellular network, the space between cell layers is much greater. This result makes sense
if one considers the differences in topographies available between the flat surface and
that which exhibits a macrotopography. On a flat surface, all cells must bind to that

122
surface, either directly, or indirectly (i.e. indirectly by upper layers binding to the lower
layers which in turn bind that surface. Figure 5-18 bottom). Given the fact that these
cells require a tension generation for their proper development and differentiation, the
only way it can be achieved is by creating force in the directions they are flattened. It
seems quite likely that on a 2-D surface this well documented contractile force
generation leads to a compressive component, decreasing space between cells.
In the bridged MSCs on the other hand we see that cellular elongation and flattening
is greater (Figures 5-14 and 5-15, as well as the SEM data seen in Figures 4-15 through
4-19). This indicates that perhaps cells involved in bridging are capable of achieving
greater tension and thus more flattening with the advantage of a second topographical
binding site. Also, the presence of more than one surface for attachment provides a way
of developing tension without compressing extracellular spaces (Figure 5-18 upper).
Force
Figure 5-18. Schematic representation of flat surface multilayering versus bridging.
Upper portion shows the increased elongation of cells achieved within a multi-cellular
bridge with increased amounts of available extracellular space (brown hatching). Lower
portion illustrates the effect of lateral tension generation which leads to compression of
cells involved in the multi-layer. Note cells are not as elongated and there is not as much
available extracellular space.

123
This space is the site of collagen deposition as we seen from the TEM data, therefore
this increased space may be available for the deposition of collagen. 3-D scaffolds used
in tissue engineering studies have shown increased amounts of collagen and ECM
production when compared to flat surfaces [103]. This increased amount of space may
be the reason, or at least part of the reason for this increased production ability.
Scaffolds where tension development is not possible (i.e. scaffolds that contract but
are not anchored so there is no resistence to contraction and therefore no tension
development) also show markedly less ECM production when compared to anchored
scaffolds [81]. This fact fits with the current theory of the role extracellular space plays
in that the contraction seen in unanchored scaffolds would eliminate excess extra¬
cellular space, as well.
Conclusions
In this chapter a number of concrete facts defining the bridging process of RMSCs
are elucidated. Studies using parallel Maxonâ„¢ arrays showed that spontaneous bridging
occurred below a given spacing distance. This bridging was substantially reduced,
however, when these distances were as high as 55 pm. In this way it was shown that
bridging and cell growth could be directed across large volumes of a parallel fiber array
when they were spaced at about 25 pm. Cell growth on the fibers themselves was
directed as shown in chapter 3, but more importantly TEM data here shows that it was
directed between fibers, as well. This inter-fiber direction of cells was characterized by
cell layers that were thicker and composed of more cells than that seen on a single fiber.
In addition to these qualities, the cells appeared to be more flattened and directed than
on single fibers.

124
Collagen orientation was also directed as seen most prominently in the contracted
areas of the 25 pm parallel arrays. This orientation implies that collagen organization is
influenced by the presence of the fibers, which allow bridging and cell growth. This
growth and organization of cells leads to a more flattened morphology and an increased
extracellular space that may explain why increased amounts of ECM are achieved when
culture is performed on 3-D scaffolds.
Studies performed in chapter 4 gave an indication of the effects that weave has on the
formation of bridging and that it could be manipulated as desired. In this study using the
angled stainless steel screens this fact was confirmed and shown to be a significant
factor in the deposition of mineralization, namely at the location of acute angles. This
trend, or propensity for bridging on acute angles was seen on the multi-layer, multi-fiber
parallel arrays, as well.
Overall the spacing distance and angle of weave, two manipulate factors in woven
scaffold design, have been shown to influence and direct the orientation of cell growth,
ECM organization and the deposition of mineral.

CHAPTER 6
CONCLUSIONS AND FUTURE WORK
The goal of the research documented in this dissertation was to show the potential for
using fiber-based scaffolds in engineering anisotropic tissues, particularly connective
tissues like bone. Overall, using different weaving strategies and fiber types, scaffolds
can be created that direct ECM and mineralization which are the major mechanical
components of connective tissues. Using different weaves this direction of ECM can be
used to design mechanical properties of engineered tissues.
In chapter 3 the direction of cells by single fibers was studied in a quantitative
manner that extended and moved beyond any study performed thus far in the literature.
We saw that organization of MSCs was a process that depended on time and the
diameter of the fiber. Over extended periods of time and with a small enough diameter
MSCs elongated in a continuous and predictable manner, which is important since
organization of ECM has been correlated with this type of behavior in the past. Indeed in
chapter 5 of this work we confirm this organization of ECM on single fiber specimens.
In addition to the effects on MSCs, the method of calculated rotational correction
used for the measurement of nuclear angle and cellular orientation was novel and
derived specifically for this study. Correction in this manner allowed two aspects of
cellular orientation to be simultaneously studied increasing the perspective on the
elongation and orientation processes. When this method was applied we saw that
elongation and orientation do not follow the same dynamic trends.
125

126
In chapter 4 bridging was presented and characterized as a phenomenon involved in
3-D cell-scaffold interactions. Bridging, on a multi-cellular level, was shown to occur on
many different materials including various polymers (polyglyconate, Nylon and
polystyrene), ceramics, or glasses (77s bioactive glass fibers) and metal (stainless steel),
thus showing that the process of bridging was a characteristic of the cell type not a
specific surface, or material. Multi-cellular bridging led to deposition of mineral and
ECM within the bodies of the bridges themselves, thus demonstrating that
mineralization could be controlled as bridging could be predictably induced. In chapter 5
we saw that this bridging preferentially occurred on acute angles and led to
mineralization that was statistically greater than that seen on 90 degree angles.
Proliferation studies performed on bioactive glass fibers showed marked effects on
proliferation dynamics, as well as the overall increases in cell numbers. These increases
were strongly tied to the density, or spacing of fibers that cells were grown on. Similar
effects of spacing on bridging were seen in chapter 5 where the bridging of RMSCs on
multi-fiber parallel arrays occurred robustly on those spaced with 25 pm. Arrays spaced
at 55 pm on the other hand showed only minimal bridging at the ends of the constructs
where they were bound to the support ring. 25 pm spacing distance allowed spontaneous
bridging between fibers and led eventually to an organized ECM deposition between the
fibers. Organization of ECM on single fibers and within the multicellular bridging seen
on arrays of fibers appeared to be parallel to the direction of these topographies on all
layers. This is in contrast to what has been seen before in the literature on flat surfaces.
In addition to bridging between parallel fibers, the bridging at junctions of fibers
showed a propensity for acute angles. Angled multi-layered constructs showed virtually

127
all acute angles were bridged leading to smooth continuous arcs of cells, while obtuse
angles showed a cellular response that was nowhere comparable.
Overall this work addresses cellular interaction to some of the major aspects of
woven scaffold design, namely the diameters of fibers the scaffold is woven from, the
spacing between fibers and angles that the fibers cross each other. It is plain that each of
these factors influences MSC growth and organization in important and predictable
ways, which can be manipulated in woven scaffold design. One further aspect of control
that may be added to the fiber design would be the incorporation of micropattems on the
surfaces of the fibers themselves. Fibers below a certain diameter were shown to direct
cell growth, but there are effects of micropatteming that may add to the ability for cells
to adhere to fiber surfaces. Figure 6-1 shows a 5-0 Maxon fiber
Figure 6-1- Surface of a 5-0 maxon fiber imaged with light microscope. Diameter -140
pm. Features are 5 pm wide, 60pm long and 20 pm apart in the direction perpendicular
to the lines. Each row of lines is separated by 40 pm.
micropatterned with 5 pm wide lines that are 60 pm long. Micropatteming motifs like
this one developed in our labs may have potential for use in woven scaffolds.
Unfortunately this technique was developed too late to be used in the experiments of this
dissertation.

128
It may also be interesting to see how bridging develops using time-lapse microscopy,
or cinemicroscopy. Development of bridging may have been influenced by the settling
of cells during seeding and filming this may allow further insight into the process these
cells follow.
In the larger arena of cell-based tissue engineering this technology of scaffold design
falls into the earlier stages of the cell-based bone replacement model. In chapter 1 the
conceptual process of cell based bone replacement was described and is depicted in
Figure 6-2. Prior to surgery and the removal of damaged, or diseased tissue as seen in
Figure 6-2b, the woven scaffold would be seeded with autologous MSCs taken from a
marrow sample provided by the patient and cultured for a given amount of time. This
pre-surgery preparation would allow the cells to gain a strong foothold on the scaffold,
depositing ECM as guided by the scaffold’s architectures and giving the future tissue a
head start toward the mechanical properties desired.
Figure 6-2- Conceptual diagram of bone replacement. Step B in this model would be
performed using a woven scaffold after it had been seeded and cultured with autologous
MSCs.

129
As often stated, the differences between the in vitro and in vivo environments are
significant and many. In this work cells were usually allowed to continue to a state of
differentiation that allowed the study of mineralization. It seems most likely, however,
that implantation would have to occur before differentiation had become complete. The
in vitro environment, though its complexity is significant, is nothing like that seen on the
level of the organism, or patient and their living physiology. An engineered bone
replacement would need to be capable of entering into the physiologic milieu and once
entering into it being included in the interactions of the relevant systems. Though this
study gives an indication of the usefulness that woven scaffolds may have, projection
into the in vivo environment should be done with caution and certainly after the
performance of further experimentation.
Placing woven constructs with their partially differentiated cells would allow those
cells to respond to their environment in a way that more specialized and differentiated
cells could not. Essentially choosing the right time for implantation involves discovering
when the cells are most flexible and thus receptive to cues from the environment.
Determining when this level of differentiation is reached would be an important step
toward bringing this technology to the clinic.
One particularly important factor to consider in the decision of when to implant is
the infiltration of vasculature, which performs gas exchange and supplies nutrients to the
cells in the scaffold. A certain period of time is necessary for this infiltration to occur,
while the cells on the scaffold would be forced to survive on diffusive properties alone.
MSCs have the capability of withstanding environments with reduced oxygen
concentrations, but it is likely that if too many cells were allowed to accumulate on the

130
scaffold prior to implantation then many would be starved for nutrients and die, thus
setting back the healing process.
In addition to cell numbers, the porosity in the construct itself will be a factor for
vascular infiltration. Common figures in the literature place pore diameters in the range
of 100-400 pm for proper infiltration [18,20], This aspect must be incorporated into the
final weave of the scaffold, which is a relatively modest requirement. A scaffold with
areas designated for cell growth alternating with areas possessing wider woven holes to
allow for vascular infiltration may be capable of meeting this need. Using surface
modification techniques it may even be possible to encourage vascular infiltration in
those areas while discouraging MSC growth and vice versa.
Scaffold design has been the focus of this work and it is an essential part of the cell
based tissue engineering approach, however, it is not the only critical component. Bone
replacement and tissue engineering technologies require a scaffold to facilitate tissue
development, but this will only be part of a whole tissue replacement system. As
mentioned in chapter 2 current bone replacement materials including allograft bone and
synthetic materials are not fully incorporated into living bone. It seems likely that this is
due to the transient nature of the fracture healing cycle, which is invoked during
recovery. During this cycle cells are drawn to the area from the bone and probably can
only migrate finite distances during that time. Also this healing cascade involves the
sequential release of growth factors and the BMPs. This aspect of healing is composed
of autocrine and paracrine signaling, which has distance limitations confining their
activities to the tens of microns, as well as the time limitations inherent in healing.

131
This work addresses the need for cells in the body of the large segmental bone
replacement by seeding them onto the scaffold and then culturing them ex-vivo for a
time prior to implantation. The required cascade of growth factors, however, is still
absent. One solution is to provide an artificial growth factor cascade as part of the
system. In chapter 2 the sequence of cellular differentiation was extensively reviewed, as
were the growth factors associated with each phase of development. Organizing that
information leads to a possible cascade profde shown in figure 3. Units of growth factor
concentration, as well as the release times will need to be filled in with experimentation,
though the literature gives hints of where to start. The order of the growth factors shown,
as well as the curves of the profiles themselves, are derived from an extensive survey of
the literature and are very likely candidates for further work as they are.
Proposed Growth Factor Release
Profile
Time (arbitrary units)
Figure 6-3 A possible sequence of growth factors for release in a bone replacement
system. BMP-2 and TGF-p begin release immediately after implantation, other factors
are delayed for specific amounts of time correlated with the levels of cellular
development where they are to have their desired effect.

APPENDIX
QUANTITATIVE CONTACT GUIDANCE ANALYSIS METHODS
Conceptual and Illustrated Review of Nuclear Form Factor (NFF)
This method was performed in a manner that was very similar to that described by
Dunn and Heath 1976 [29], Figure A-l shows diagrammatically how NFF
measurements were obtained.
Figure A-l. Diagram of NFF measurement.
Dunn and Heath collected data on nuclei that were no more than 1/2 a radius from the
center of the fiber. In this study, however, only nuclei within 1/3 of a radius from the
center were used. This decreased the distortion from foreshortening that we experienced
in our study, which is to say that our study was less variable (more accurate).
Viewing figure 1, La was the measured, or “actual” value of the nucleus as it was
seen in 2-D projection through the microscope. Lc is the “calculated” or corrected value
of the nucleus, which was the true width of the nucleus. Equation A-l is how the Lc was
calculated.
132

133
L c = r- sin
- sin
v v r ;
V r J J
Equation A-l.
Essentially this equation gives the small angle subtended by the width of the
nucleus. Its main function is to find the difference of the two angles that equate to the
two points of the edges of the nucleus (Figure A-2).
B-R = 0
/
©
Figure A-2. Diagram of relationship between nuclear dimensions and fiber geometry. 0
is the difference between Blue angle “B” and Red angle “R.”
This small angle “0,” the angle subtended by the nucleus, is then used to calculate the
chord along the surface of the fiber, which is a close approximation of the nuclear width
(Figure A-3).
L
r(B-R)« Chord “Lc”
Figure A-3. Diagram of trigonometric approximations used to determine nuclear
dimensions.

134
Extension of NFF Correction Concept to Nuclear Angle Measurements
Figure A-4 shows two forms of quantitative measurement used in this study: NFF and
the angular measurement of the nucleus with respect to the fiber.
NFF Angle
Figure A-4. Diagrammatic comparison of NFF and nuclear angle.
There are similar problems associated with the fiber’s radial geometry when
measuring the nuclear angle. Specifically, the farther out on the fiber a nucleus is, the
less pronounced its angle with the respect to the fiber will appear by 2-D projection.
To combat this problem, I derived an extension to the NFF correction concept that
applies to angular measurements.
Nuclear angles were measured as shown in Figure A-4 using the Adobe Photoshop
software package. However, measurement of the base of the triangle in Figure A-4 was
not performed, as it would have been redundant and time consuming. Instead this
derivation is dependent on a robust assumption: The trigonometric identity of similar
triangles (Figure A-5). The actual angle measured corresponds to the blue triangle
(hypotenuse to be exact), however, using the larger, black triangle allows the use of Lc
from the NFF calculation as a base.

135
I —C I
Figure A-5. Diagrammatic relationship between NFF measurements and nuclear angle
measurements.
Harking back to Figure A-l, with a trivial amount of inspection one can see that Lc
will always be greater than or equal to La (equal in the event that the nucleus is perfectly
centered on the fiber). As will become evident shortly, this results in a reduced apparent
nuclear angle.
Mathematical Derivation
This derivation is based on elementary algebra and elementary trigonometry and will
refer to Figure A-6. Imagine a triangle whose hypotenuse bisects the longest elliptical
axis of a nucleus adhered to a fiber. This nucleus is off center on the fiber, which is to
say it is rotated somewhere along the circumference, or surface of the fiber. Figure A-6 A
shows this triangle when viewed through a microscope; note the length of the actual
base of the triangle.

136
Calculated Rotational
Correction.
6A 6B
Figure A-6. Diagrammatic representation of calculated rotational correction.
The second assumption in the derivation is that “H” is equal in the triangles depicted
in both Figure A-6A and Figure A-6B. The triangle shown in both figures is in reality
the same triangle; the only difference is that the second shows the triangle with the
corrected base “Lc” (as if the fiber had been rotated around the long axis). If rotation
occurs only around the long axis of the fiber, the height of the triangle should remain the
same in reality and appear the same in the 2-D projection shown in both figures. If one
finds these assumptions acceptable, the following derivation will readily follow:
= tan0a
leads to: H = tan0a La
leads to: H = tan0e Lc
Through the Equality of “H”
tan0 a L a = tan0 C L c = H

137
Leads to:
tan0 c = tan0 a •
'La'
VLcJ
Equation A-2. Calculated rotational correction.
Taking the arctangent of tan 0C, which is equal to the correction function shown in
Equation 2, will then provide the corrected angle 0C.

LIST OF REFERENCES
1. Laurencin C, El-Amin S, Ibim S, Willoughby D, Attawia M, Allcock H, Ambrosio
A. A Highly Porous 3-Dimensional Matrix for Skeletal Tissue Regeneration. Journal
of Biomedical Materials Research 1996;30:133-138.
2. Heckman J, Boyan B, Aufdemorte T, Abbott J. The Use of Bone Morphogenetic
Protein in the Treatment of Non-union in a Canine Model. The Journal of Bone and
Joint Surgery 1991;73-A(5):750-764.
3. Hanson P, Warner C, Kofroth R, Osmond C, Bogdanske J, Kalscheur F, Frassica,
F, Markl M. Effect of Intramedullary Polymethylmethacrylate and Autogenous
Cancellous Bone on Healing of Frozen Segmental Allografts. Journal of Orthopedic
Research 1998;16:285-292.
4. Kenley R, Yim J, Ron E, Turek T, Marden L, Hollinger J. Biotechnology and
Bone Graft Substitutes. Pharmaceutical research 1993;10(10):1393-1401.
5. Christian J. BMP, Wnt and Hedgehog Signals: How Far Can They Go? Current
Opinion in Cell Biology 2000;12:244-249.
6. Buma P, Schreurs B, Gardeniers J, Versleyen D, Sloof T. Impacted Graft
Incorporation After Cemented Acetabular Revision. Acta Orthopedica Scandinavica
1996;67(6):536-540.
7. Gunterberg B. Tumor Resection and Skeletal Reconstruction in the Extremities.
In: Branemark P, Ryderik B and Skalak R. eds. Osseointegration in Skeletal
Reconstruction and Joint Replacement. Chicago, IL: Quintessence books; 1997. p
111-119.
8. Ortiz C, Gebhardt M, Jennings L. Springfield, D., Mankin, H., The Results of
Transplantation of Intercalary Allografts After Resection of Tumors. A Long Term
Follow-up Study. Journal of Bone and Joint Surgery 1997;79:97-106.
9. San Julian M, Canadell J. Fractures of Allografts Used in Limb Preserving
Operations. International Orthopaedics 1998;22(l):32-36.
10. Cameron H. Evaluation of a Biodegradable Ceramic. Journal of Biomedical
Materials Research 1977;11:179-186.
11. Wintermantel E, Mayer J, Eckert K, Luscher P, Mthery M. Tissue Engineering
Scaffolds Using Superstructures. Biomaterials 1996;17(2):83-91.
138

139
12. Holy C, Shoichet M, Davies J. Engineering Three-Dimensional Bone Tissue In
Vitro Using Biodegradable Scaffolds: Investigating Initial Cell-Seeding Density and
Culture Period. Journal of Biomedical Materials Research 2000;51:376-382.
13. Ohgushi H, Goldberg V, Caplan A. Repair of Bone Defects with Marrow Cells and
Porous Ceramic. (Experiments in rats). Acta Orthopaedica Scandinavia
1989;60(3):334-339.
14. Ohgushi H, Goldberg V, Caplan A. Heterotopic Osteogenesis in Porous Ceramics
Induced by Marrow Cells. Journal of Orthopaedic Research 1989;7:568-578.
15. Nakahara H, Bruder S, Goldberg V, Caplan A. In Vivo Osteochondrogenic
Potential of Cultured Cells Derived From the Periosteum. Clinical Orthopaedics and
Related Research 1990;259:223-232.
16. Nakahara H, Goldberg V, Caplan A. Culture-Expanded Periosteal-Derived Cells
Exhibit Osteochondrogenic Potential in Porous Calcium Phosphate Ceramics In Vivo.
Clinical Orthopaedics and Related Research 1992;276:291-298.
17. Martin S, Langer, R, Vunjak-Novakovic G, Freed L. Bone Marrow Stromal Cell
Differentiation on Porous Polymer Scaffolds. Proceedings of the 45th Annual
Meeting of the Orthopedic Research Society, 1999.
18. Goshima J, Goldberg V, Caplan A. The Osteogenic Potential of Culture Expanded
Rat Marrow Mesenchymal Cells Assayed In Vivo in Calcium Phosphate Ceramic
Blocks. Clinical Orthopaedics and Related Research 1991;262:298-311.
19. Goshima J, Goldberg V, Caplan A. The Origin of Bone Formed in Composite
Grafts of Porous Calcium Phosphate Ceramic Loaded with Marrow Cells. Clinical
Orthopaedics and Related Research 1991;269:274-283.
20. Dennis J, Caplan A. Porous Ceramic Vehicles for Rat-Marrow Derived (Rattus
Norvegicus) Osteogenic Cell Delivery: Effects of Pre-Treatment with Fibronectin or
Laminin. Journal of Oral Implantology 1993;106(2):106-115.
21. Von Falkai B. Dry-Spinning Technology. In: Masson J. editor. Acrylic Fiber
Technology and Applications. New York: Marcel Dekker; 1995. p 127-135.
22. Postema A, Luiten A, Pennings A. High-Strength Poly(L-Lactide) Fibers by a Dry-
Spinning/ Hot Drawing Process. I. Influence fo the Ambient Temperature on the Dry-
Spinning Process. Journal of Applied Polymer Science 1990;39:1265-1274.

140
23. Postema A, Luiten A, Oostra H, Pennings A. High-strength Poly(L-lactide) Fibers
by a Dry-spinning/ Hot-drawing Process. II. Influence of the Extrusion Speed and
Winding Speed on the Dry-spinning Process. Journal of Applied Polymer Science
1990;39:1275-1288.
24. Kurosawa H, Markl H, Neibuhr-Redder C, Matsumura M. Dialysis Bioreactor with
Radial-Flow Fixed Bed for Animal Cell Culture. Journal of Fermentation and
Bionegineering 1991;72(l):41-45.
25. Perry S, Wang D. Fiber Bed Reactor Design for Animal Cell Culture.
Biotechnology and Bioengineering 1989;34:1-9.
26. Weiner S, Wagner H. The Material bone: Structure-Mechanical Function Relations.
Annual Review of Materials Science 1998;28:271-298.
27. Weiner S. Rotated Plywood Structure of Primary Lamellar Bone in the Rat:
Orientations of the Collagen Fibril Arrays. Bone 1997;20(6):509-514.
28. Giraud-Guille M. Liquid Crystalline Order of Biopolymers in Cuticles and Bones.
Microscopy Research and Technique 1994;27:420-428.
29. Dunn G, Heath J. A New Hypothesis of Contact Guidance in Tissue Cells.
Experimental Cell Research 1976;101:1-14.
30. Mendes D, Iusim M, Angel D, Rotem A, Roffman M, Grishkan A, Mordohohovich
D, Boss J. Histologic Pattern of Biomechanic Properties of the Carbon Fiber-
Augmented Ligament and Tendon. Clinical Orthopedics and Related Research
1984;196:51-60.
31. Matsuzaka K, Walboomers X, Ruijter J, Jansen, J. The Effect of Poly-L-Lactic Acid
with Parallel Surface Micro Grooves on Osteoblast-like Cells In Vitro. Biomaterials
1999;20:1293-1301.
32. Matsuzaka K, Walboomers F, de Ruijter A, Jansen J. Effect of Microgrooved Poly-
L-Lactic (PLA) Surfaces on Proliferation, Cytoskeletal Organization and Mineralized
Matrix Formation of Rat Bone Marrow Cells. Clinical and Oral Implants Research
2000;ll(4):325-333.
33. Sinclair D. Human Growth After Birth. New York: Oxford Medical Publications;
1991.259 p.
34. Lennon D, Edmison J, Caplan A. Cultivation of Rat Marrow Derived Mesenchymal
Stem Cells in Reduced Osygen Tension: Effects on In Vitro and In Vivo
Osteochondrogenesis. Journal of Cellular Physiology 2001;187:345-355.

141
35. Caplan A. Cartilage Begets Bone vs. Endochondral Myelopoiesis. Clinical
Orthopaedics and Related Research 1990;261:257-267.
36. Stein G, Lian J. Molecular Mechanisms Mediating Proliferation/Differentiation
Interrelationships During Progressive Development of the Osteoblast Phenotype.
Endocrine Reviews 1993;14(4):424-441.
37. Stein G, Lian H, Stein J, Van Wijnen A, Montecino M. Transcriptional Control of
Osteoblast Growth and Differentiation. Physiological Reviews 1996;76(2):593-629.
38. Owen T, Aronow M, Shalhoub V, Barone L, Wilming L, Tassinari M, Kennedy M,
Pockwise S, Lian J, Stein G. Progressive Development of Rat Osteoblast Phenotype
In Vitro: Reciprocal Relationships in Expression of Genes Associated with Osteoblast
Proliferation and Differentiation During Formation of the Bone Extracellular Matrix.
Journal of Cellular Physiology 1990;143:420-430.
39. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson J. Molecular Biology of
The Cell. New York: Garland Publishing; 1994.1294 p.
40. Meyer R, Meyer M, Phieffer L, Banks D. Bone Morphogenetic Proteins and
Osteocalcin mRNA Expression During Fracture Healing in the Young and Older Rat.
In: Proceedings of the 45th Annual Meeting of the Orthopedic Research Society,
1999. p 457.
41. Lyons K, Pelton R, Hogan B. Patterns of Expression of Murine Vgr-1 and BMP-2a
RNA Suggest that Transforming Growth Factor-B-like Genes Coordinately Regulate
Aspects of Embryonic Development. Genes and Development 1989;3:1657-1668.
42. Lyons K, Hogan B. TGF-(3 like Genes in Mammalian Development. In Anthony P.
Mahowald, ed. Genetics of Pattern Formation and Growth Control. New York:
Wiley-Liss, 1990; 137-156.
43. Bruder S, Fink D, Caplan A. Mesenchymal Stem Cells in Bone Development, Bone
Repair, and Skeletal Regeneration Therapy. Journal of Cellular Biochemistry
1994;56:283-294.
44. Li I, Cheifetz S, McCulloch C, Sampath K, Sodek J. Effectsof Osteogenic Protein-1
(OP-1, BMP-7) on one Matrix Protein Expression by Fetal Rat Calvarial Cells are
Defferentiation Stage Specific. Journal of Cellular Physiology 1996;169:115-125.
45. Geesink R, Bulstra S, Hoefnagels N. The Use of Osteogenic Protein-1 in Clinical
Cases with Severe Bone Loss: 5 Case Reports. Skeletal Reconstruction and
Bioimplantation, 1997:193-211.

142
46. Cunningham N, Paralkar V, Reddi A. Osteogenin and Recombinant Bone
Morphogenetic Protein 2B are Chemotactic for Human Monocytes and Stimulate
Transforming Growth Factor B-l mRNA Expression. Proceedings of the National
Academy of Science 1992;89:11740-11744.
47. Jin. Y., Y., L., Si., Synergistic Effects of Bone Morphogenetic Protein-2 and Tumor
Growth Factor B on Bone Formation., in Skeletal Reconstruction and
Bioimplantation. 1997. p. 137-148.
48. Stein G, Lian J, Owen T. Relationship of Cell Growth to the Regulation of Tissue-
Specific Gene Expression During Osteoblast Differentiation. FASEB 1990;4:3111-
3123.
49. Li G, Berven S, Simpson H, Triffitt T. Expression of BMP-4 m RNA During
Distraction Osteogenesis in Rabbits. Acta Orthopedica Scandinavica 1998;69(4):420-
425.
50. Nakase T, Nomura S, Yoshikawa H, Hashimoto J, Hirota S, Kitamura Y, Oikawa S,
Ono K, Takaoka K. Transient and Localized Expression of Bone Morphogenetic
Protein 4 Messenger RNA During Fracture Healing. Journal of Bone and Mineral
Research 1994;9(5):651-659.
51. Duprez D, Bell E, Richardson M, Archer C, Wolpert L, Brickell P,
Francis-West P. Overexpression of BMP-2 and BMP-4 Alters the Size and Shape
of Developing Skeletal Elements in the Chick Limb. Mechanisms of
Development 1996;57:145-157.
52. Chen P, Carrington J, Hammonds R, Reddi A. Stimulation of Chondrogenesis in
Limb Bud Mesoderm Cells by Recombinant Human Bone Morphogenetic Protein 2B
(BMP-2B) and Modulation by Transforming Growth Factor-Bl and B2. Experimental
Cell Research 1991;195:509-515.
53. Hughes F, Collyer J, Stanfield M, Goodman S. The Effects of Bone
Morphogenetic Protein-2, -4, and -6 on Differentiation of Rat Osteoblast Cells In
Vitro,. Endocrinology 1995;136(6):2671-2677.
54. Glowacki J, Trepman E, Folkman J. Cell Shape and Phenotypic Expression in
Chondrocytes. Proceedings of the Society for Experimental Biology and Medicine
1983;172:93-98.
55. Shi S, Kirk M, Kahn A. The Role of Type 1 Collagen in the Regulation of the
Osteoblast Phenotype. Journal of Bone and Mineral Research 1996;11(8):1139-1145.
56. Gitelman S, Kirk M, Ye J, Filvaroff E, Kahn A, Derynck R. Vgr-1 / BMP-6 Induces
Osteoblastic Differentiation of Pluripotential Mesenchymal Cells. Cell Growth and
Differentiation 1995;6:827-836.

143
57. Boden S, Liu Y, Hair G, Helms J, Hu D, Radine M, Nanes M, Titus L. LMP-1, A
LIM-Domain Protein, Mediates BMP-6 Effects on Bone Formation. Endocrinology
1998;139(12):5125-5134.
58. Ogata Y, Yamauchi M, Kim R, Li J, Freedman L, Sodek J. Glucocorticoid
Regulation of Bone Sialoprotein (BSP) Gene Expression Identification of a
Glucocorticoid Response Element in the Bone Sialoprotein Gene Promoter. European
Journal of Biochemistry 1995;230:183-192.
59. Hunter G, Goldberg H. Nucleation of Hydroxyapatite by Bone Sialoprotein.
Proceedings of the National Academy of Sciences 1993;90:8562-8565.
60. Sampath T, Maliakal J, Hauschka P, Jones W, Sasak H, Tucker R, White K,
Coughlin J, Tucker M, Pang R, Corbett C, Ozkayanak E, Oppermann H, Rueger D.
Recombinant Human Osteogenic Protein-1 (hOP-1) Induces New Bone Formation In
Vivo with a Specific Activity Comparable with Natural Bovine Osteogenic Protein
and Stimulates Osteoblast Proliferation and Differentiation In Vitro. The Journal of
Biological Chemistry 1992;267(28):20352-20362.
61. Honda Y, Knutsen R, Strong D, Sampath T, Baylink D, Mohan S. Osteogenic
Protein-1 Stimulates mRNA level of BMP-6 and Decreases mRNA Levels of BMP-2
and -4 in Human Osteosarcoma Cells. Calcified Tissue International 1997;60:297-
301.
62. Bouligand Y. Twisted Fibrous Arrangements in Biological Materials and
Cholesteric Mesophases. Tissue and Cell 1972;4(2): 189-217.
63. Liu D, Weiner S, Wagner H. Anisotropic Mechanical Properties of Lamellar Bone
Using Miniature Cantilever Bending Specimens. Journal of Biomechanics
1999;32:647-654.
64. Cowin S. Mechanics of Materials. In: Cowin S, editor. Bone Mechanics. CRC
press: Boca Raton; 1989. p 147-162.
65. Ziv V, Wagner H, Weiner S. Microstructure-Microhardness Relations in Parallel-
Fibered and Lamellar Bone. Bone 1996;18(5):417-428.
66. Yamazaki Y, Oida S, Ishihara K, Nkabayashi N. Ectopic Induction of Cartilage and
Bone by Bovine Bone Morphogenetic Protein Using a Biodegradable Polymeric
Reservoir. Journal of Biomedical Materials Research 1996;30:1-4.
67. Caplan A. Mesenchymal Stem Cells. Journal of Orthopaedic Research 1991;9:641-
650.

144
68. Haynesworth S, Goshima J, Goldberg V, Caplan A. Characterization of Cells with
Osteogenic Potential from Human Marrow. Bone 1992;13:81-88.
69. McCance K, Huether S. Pathophysiology: The Biologic Basis for Disease in Adults
and Children. St. Louis: Mosby; 1994. 1577 p.
70. Matsuda T, Sugawara T. Development of Surface Photochemical Modification
Method for Micropatterning of Cultured Cells. Journal of Biomedical Materials
Research 1995;29:749-756.
71. Kaibara M, Iwata H, Wada H, Kawamoto Y, Iwaki M, Suzuki Y. Promotion and
Control of Selective Adhesion and Proliferation of Endothelial Cells on Polymer
Surface by Carbon Deposition. Journal of Biomedical Materials Research
1995;31:429-435.
72. Chen C, Mrksich M, Huang S, Whitesides G, Ingber D. Micropatterned Surfaces for
Control of Cell Shape, Position and Function. Biotechnology Progress 1998;14:356-
363.
73. Puleo, D., Bizios, R., Formation of Focal Contacts by Osteoblasts Cultured on
Orthopoaedic Biomaterials. Journal of Biomedical Materials Research 1992;26:291-
301.
74. Puleo D, Bizios R. RGDS Tetrapeptide Binds to Osteoblasts and Inhibits
Fibronectin-Mediated Adhesion. Bone 1996;12:271-276.
75. Ahmed Z, Brown R. Adhesion, Alignment, and Migration of Cultured Schwann
Cells on Ultrathin Fibronectin Fibers. Cell Motility and the Cytoskeleton
1999;42:331-343.
76. Rahul S, Stephanopoulos G, Wang D. Review: Effects of Substratum Morphology
on Cell Physiology. Biotechnology and Bioengineering 1993;43:764-771.
77. Mcfarland C, Thomas C, DeFilippis C, Steele J, Healy K. Protein Adsorption and
Cell Attachment to Patterned Surfaces. Journal of Biomedical Materials Research
2000;49:200-210.
78. Goto T, Wong K, Brunette D. Observation of Fibronectin Distribution on the Cell
Undersurface Using Immunogold Scanning Electron Microscopy. Journal of
Histochemistry and Cytochemistry 1999;47(11): 1487-1494.
79. den Braber E, Ruijter J, Smits H, Ginsel L, von Recum A, Jansen J. Effect of
Parallel Surface Microgrooves and Surface Energy on Cell Growth. Journal of
Biomedical Materials Research 1995;29:511-518.

145
80. Lee C, Grodzinsky A, Spector M. The Effects of Cross-Linking of Collagen-GAG
scaffolds on Compressive Stiffness, Chondrocyte-Mediated Contraction, Proliferation
and Biosynthesis. Biomaterials 2001;22:3145-3154.
81. Nakagawa S, Pawelek P, Grinnell F. Long-Term Culture of Fibroblasts in
Contracted Collagen Gels: Effects on Cell Growth and Biosynthetic Activity. Journal
of Investigative Dermatology 1989;93:792-798.
82. Kandel E, Sckwartz J, Jessell T. Principles of Neural Science. Norwalk, CT:
Appleton & Lange; 1991.1135 p.
83. Bayer S, Altman J, Russo R, Dai X, Simmons J. Cell Migration in the Rat
Embryonic Neocortex. Journal of Comparative Neurology 1991;307(3):499-516.
84. Flemming R, Murphy C, Abrams G, Goodman S, Nealey P. Effects of Synthetic
Micro- and Nano-structures Surfaces on Cell Behavior. Biomaterials 1999;20:573-
588.
85. Goodman S, Sims P, Albrecht R. Three-Dimensional Extracellular Matrix Textured
Biomaterials. Biomaterials 1996;17(21):2087-2095.
86. Ratner B, Hoffman A, Schoen F, Lemons J. Biomaterials Science: An Introduction
to Materials in Medicine. New York: Academic Press; 1996. 484 p.
87. Caille N, Tardy Y, Meister J. Assessment of Strain Field in Endothelial Cells
Subjected to Unaxial Deformation of Their Substrate. Annals of Biomedical
Engineering 1998;26:409-416.
88. Ingber D. Fibronectin Controls Capillary Endothelial Cell Growth by Modulationg
Cell Shape. Proceeding of the National Academy of Sciences 1990;87:3579-3583.
89. Chesmel K, Clark C, Brighton C, Black J. Cellular Responses to Chemical and
Morphological Aspects of Biomaterials Surfaces. II. The Biosynthetic and Migratory
Response of Bone Cell Populations. Journal of Biomedical Materials Research
1995;29:1101-1110.
90. Lampin M. Warocquier-Clerout R, Legris C, Degrange M, Sigot-Luizard M.
Correlation Between Substratum Roughness and Wettability, Cell Adhesion, and Cell
Migration. Journal of Biomedical Materials Research 1997;36:99-108.
91. Moses M, Sudhalter J, Langer R. Identification of an Inhibitor of
Neovascularization from Cartilage. Science 1990;248:1408-1410.
92. Ingber D, Prusty D, Sun Z, Betensky H, Wang N. Cell Shape, Cytoskeletal
Mechanics and Cell Cycle Control in Angiogenesis. Journal of Biomechanics
1995;28(12):1471-1484.

146
93. Absolom D, Hawthorn L, Chang G. Endothelialization of Polymer Surfaces.
Journal of Biomedical Materials Research 1988;22:271-285.
94. Rezania K, Healy K. The Effect of Peptide Surface Density on Mineralization of
Matrix Deposited by Osteogenic Cells. In: Proceedings of the Sixth World
Biomaterials Congress, 2000. p 57.
95. Legtenberg R, Jansen H, de Boer M, Elwenspoek M. Anisotropic Reactive Ion
Etching of Silicon Using SF6/02/CHF3 Gas Mixtures. Journal of the Electrochemical
Society 1995; 142(6):2020-2028.
96. Nealy P, Teixeira A, Abrams G, Murphy C. Effect of Nanostructured Surfaces on
the Behavior of Human Corneal Epithelial Cells. In: 221st National Meeting of the
American Chemical Society, 2001. p 137.
97. Fisher P, Tickle C. Differences in Alignment of Normal and Transformed Cells on
Glass Fibres. Experimental Cell Research 1981;131:407-410.
98. Neelima B, Chauhan H, Figlewicz H, Khan T. Carbon Filaments Direct the Growth
of Postlesional Plastic Axons After Spinal Cord Injury. International Journal of
Developmental Neuroscience 1999;17(3):255-264.
99. Benya P, Shaffer J. Dedifferentiated Chondrocytes Reexpress The Differentiated
Collagen Phenotype When Cultured in Agarose Gels. Cell 1982;30:215-224.
100. Tobasnick C, Curtis A. Chloride Channels and the Reactions of Cells to
Topography. European Cells and Materials 2001;2:49-61.
101. Altman G, Horan R, Martin I, Farhadi J, Stark P, Volloch V, Vunjak-
Novakovic G, Richmond J, Kaplan D. Cell Differentiation by Mechanical Stress.
FASEB Journal 2002;16:12-13.
102. Ikegame M, Ishibashi O, Yoshizawa T, Shimonura J, Komori T, Ozawa H,
Kawashima H. Tensile Stress Induces Bone Morphogenetic Protein 4 in
Preosteoblastic and Fibroblastic Cells, which Later Differentiate into Osteoblasts
Leading to Osteogenesis in the Mouse Calvariae in Organ Culture. Journal of Bone
and Mineral Research 2001;16(l):24-32.
103. Akhouayri O, Lafage-Proust M, Rattner A, Laroche N, Caillot-Augusseau A,
Alexandre C, Vico L. Effects of Static of Dynamic Mechanical Stresses on Osteoblast
Phenotype Expression in Three-Dimensional Contractile Collagen Gels. Journal of
Cellular Biochemistry 1999;76:217-230.

147
104. Bowman N, Donahue H, Ehrlich H. Gap Junctional Inercellular Communication
Contributes to the Contraction of Rat Osteoblast Populated Collagen Lattices. Journal
of Bone and Mineral Research 1998;13(11):1700-1706.
105. Zaleskas J, Kinner B, Freyman T, Yannas I, Gibson L, Spector M.
Growth Factor Regulation of Smooth Muscle Actin Expression and Contraction of
Human Articular Chondrocytes and Meniscal Cells in a Collagen-GAG Matrix.
Experimental Cell Research 2001;270:21-31.
106. Awad H, Butler D, Harris M, Ibrahim R, Wu Y, Young R, Kadiyala S, Biovin G.
In Vitro Characterization of Mesenchymal Stem Cell-Seeded Collagen Scaffolds for
Tendon Repair: Effects of Initial Seeding Density on Contraction Kinetics. Journal of
Biomedical Materials Research 2000;51:233-240.
107. Menard C, Mitchell S, Spector M. Contractile Behavior of Smooth Muscle Actin-
Containing Osteoblasts in Collagen-GAG Matrices In Vitro: Implant Related Cell
Contraction. Biomaterials 2000;21:1867-1877.
108. Murray M, Martin S, Spector M. Migration of Cells from Human Anterior Cruciate
Ligament Explants into Collagen-Glycosaminoglycan Scaffolds. Journal of
Orthopaedic Research 2000;18:557-564.
109. Sisk M, Lohman C, Cochran D, Sylvia V, Simpson J, Dean D, Boyan B, Schwartz
Z. Inhibition of Cyclooxygenase by Indomethacin Modulates Osteoblast Response to
Titanium Surface Roughness In a Time Dependent Manner. Clinical Oral Implant
Research 2001;12:52-61.
110. Jia L, Canny B, Leong D. Paracrine Communication Regulates
Adrenocortocotropin Secretion. Endocrinology 1992;130:534-539.
111. Sun F, Maercklein P, Fitzpatrick L. Paracrine Interactions Among Parathyroid
Cells: Effect of Cell Density on Cell Secretion. Journal of Bone and Mineral Research
1994;9(7):971-976.
112. Zimmerman M, Gordon K. Tendon Cell Outgrowth Rates and Morphology
Associated with Kevlar-49. Journal of Biomedical Materials Research
1988;22(A3):339-350.
113. Ricci J, Gona A, Alexander H, Parsons J. Morphological Characteristics of Tendon
Cells Cultured on Synthetic Fibers. Journal of Biomedical Materials Research
1984;18:1073-1087.
114. Nefussi J, Boy-Lefevre M, Boulekbache H, Forest N. Mineralization In Vitro of
Matrix Formed by Osteoblasts Isolated by Collagenase Digestion. Differentiation
1985;29:160-168.

148
115. Gerstenfeld, L., Chipman, S., Kelly, C, Hodgens, K., Lee, D.., Collagen
Expression, Ultrastructural Assembly, and Mineralization in Cultures of Chicken
Embryo Osteoblasts. Journal of Cell Biology 1988;106:979-989.
116. Aronow M, Gerstenfeld L, Owen T, Tassinari M, Stein G, Lian J.
Factors that Promote Progressive Development of the Osteoblast Phenotype in
Culture Fetal Rat Calvaria Cells. Journal of Cellular Physiology 1990;143:213-221.
117. Walboomers X, Croes H, Ginsel L, Jansen J. Contact Guidance of Rat Fibroblasts
on Various Implant Materials. Journal of Biomedical Materials Research
1999;47:204-212.
118. Gomi K, Davies J. Guided Bone Tissue Elaboration by Osteogenic Cells in vitro.
Journal of Biomedical Materials Research 1993;27:429-431.
119. Chesmel K, Black J. Cellular Responses to Chemical and Morphologic Aspects of
Biomaterial Surfaces. I. A Novel in vitro Model System. Journal of Biomedical
Materials Research 1995;29:1089-1099.
120. den Braber E, de Ruijter J, Ginsel L, von Recum A, Jansen J. Orientation of ECM
Protein Deposition, Fibroblast Cytoskeleton, and Attachment Complex Components
on Silicone Microgrooved Surfaces. Journal of Biomedical Materials Research
1998;40:291-300.
121. Anselme K, Bigerelle M, Noel B, Dufresne E, Judas D, lost A, Hardouin P.
Qualitative and Quantitative Study of Human Osteoblast Adhesion on Materials with
Various Surface Roughnesses. Journal of Biomedical Materials Research
2001;49:155-166.
122. Domingues R, Clark B, Brennan A. A sol-gel Derived Bioactive Fibrous
Mesh. Journal of Biomedical Materials Research 2001;55(4):468-474.
123. Thompson S, Hunt R. Selected Histochemical and Histopathological Methods.
Springfield, Ill: Charles C. Thomas Publishing; 1966. 1639 p.
124. Romanello M, Moro L, Pirulli D, Crovella S, D’andrea P. Effects of cAMP on
Intercellular Coupling and Osteoblast Differentiation. Biochemical and Biophysical
Research Communications 2001;282:1138-1144.
125.Webb K, HladyV, Tresco, P. Relationships Among Cell Attachment, Spreading,
Cytoskeletal Organization, and Migration Rate for Anchorage-Dependent Cells on
Model Surfaces. Journal of Biomedical Materials Research 2000;49:362-368.

149
126. Deligianni D. Katasala N, Koutsoukos P, Missirlis Y. Effect of Surface Roughness
of Hydroxyapatite on Human Bone Marrow Cell Adhesion, Proliferation,
Differentiation and Detachment Strength. Biomaterials 2001;22:87-96.
127. Martin J, Schwartz Z, Hummert T, Schraub D, Simpson J, Lankford J, Dean D,
Cochran D, Boyan B. Effect of Titanium Surface Roughness on Proliferation,
Differentiation, and Protein Synthesis of Human Osteoblast-like Cells (MG63).
Journal of Biomedical Materials Research 1995;29:389-401.
128. Heinegard D, Klinge B, Wendel M. Matrix Molecules in Connective Tissues with a
Potential for Use in Guiding Osseointegration. In: Branemark P, Ryderik B and
Skalak R, eds. Osseointegration in Skeletal Reconstruction and Joint Replacement.
Chicago, IL: Quintessence books; 1997; p 73-78.
129. Gugala Z, Gogolewski S. Attachment, Growth and Activity of Rat Osteoblasts on
Poly (L/DL- lactide) Membranes Treated with Various Low-Temperature RF
Plasmas. In: Proceedings of the 6lh World Biomaterials Congress; 2000. p 100.
130. Elgendy H, Norman M, Keaton A, Laurencin C. Osteoblast-like Cell (MC3T3-E1)
Proliferation on Bioerodible Polymer Composite Material. Biomaterials
1993;14(4):263-269.
131. Schmidt C, Ignatius, A, Claes L. Proliferation and Differentiation Parameters of
Human Osteoblasts on Titanium and Steel Surfaces. Journal of Biomedical Materials
Research 2001;54:209-215.
132. Hanley D, McCabe R, Doherty M, Nolan P. McAlinden, M., Nelson, J., Wilson,
D., Enhancement of Human Osteoblast Proliferation and Phenotypic Expression
when Cultured in Human Serum. Acta Orthopedica Scandinavica 2001;72(4):395-
403.
133. Sharpe, P., MacDonald, B., Gallagher, J., Treffry, T., Russell, R., Studies of the
Growth of Human Bone-Derived Cells in Culture Using Aqueous Two Phase
Partition. Bioscience Reports 1984;4(5):415-419.
134. Kramvis A, Garnett H. Proliferative Activity of Vervet Monkey Bone Marrow
Derived Adherent Cells. Experimental Hematology 1987;15:1022-1027.
135. Luegmayr E, Varga F, Roschger P, Klaushofer K. Effects of Triidothyronine on
Morphology, Growth Behavior, and the Actin Cytoskeleton in Mouse Osteoblastic
Cells (MC3T3-E1). Bone 1996;18(6):591-599.

BIOGRAPHICAL SKETCH
Charles Seegert was born in Santa Barbara, CA, in 1971. He spent his childhood in
rural northern California, graduating from Las Plumas High School in 1989. After a brief
stint in the United States Army, he attended the University of California at Davis where
in 1996 he completed a B.S. in Exercise Science with emphasis in exercise physiology. In
1998 he graduated from the Roswell Park Cancer Institute, a graduate division of the
State University of New York at Buffalo, where he earned an M.S. in cellular and
molecular biophysics. Upon graduation from the University of Florida, Charles will be
starting his career at Ethicon incorporated in Somerville, NJ, a subsidiary of Johnson and
Johnson.
150

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Anthony B. Brerfnan, Chairman
Professor of Biomedical Engineering
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Christopher Batich,
Professor of Biomedical Engineering
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
Eugene Goldberg,
Professor of Biomedical Engineering
1 certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
onald Baney, (/
Ronald
Associate Engineer in Materials Science
and Engineering
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully ad^qyfete, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Richard
Professo/ o
ker,
Biomedical Engineering

This dissertation was submitted to the Graduate Faculty of the College of
Engineering and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
August 2002
Pramod Khargonekar
Dean, College of Engineering
Winfred Phillips
Dean, Graduate School

LD
1780
20-C-2
,5 H5I
UNIVERSITY OF FLORIDA




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