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Synthesis of Dissolvable Magnetic Microspheres for Tissue Scaffold Applications

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Title:
Synthesis of Dissolvable Magnetic Microspheres for Tissue Scaffold Applications
Creator:
Garcia, Andrew R
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (76 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemical Engineering
Committee Chair:
RINALDI,CARLOS
Committee Co-Chair:
SCHMIDT,CHRISTINE E
Graduation Date:
5/2/2015

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Subjects / Keywords:
Alginates ( jstor )
Emulsions ( jstor )
Ferrofluids ( jstor )
Magnetic fields ( jstor )
Magnetism ( jstor )
Magnets ( jstor )
Scaffolds ( jstor )
Size distribution ( jstor )
Surfactants ( jstor )
Viscosity ( jstor )
Chemical Engineering -- Dissertations, Academic -- UF
b
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemical Engineering thesis, M.S.

Notes

Abstract:
Magnetic microspheres were made by an emulsification process where smaller magnetic nanoparticles are cross-linked with calcium into solid alginate gel matrices. Microspheres were designed to serve as templates that can form tissue-engineered scaffolds with guided hollow channels and voids. The microspheres presented herein were made from a matrix that could be dissolved by the addition of a chemical. They were proven to possess magnetic properties by SQUID measurements and were seen to align with moderate magnetic fields using optical microscopy. The mass fraction of iron oxide inside microspheres was also analyzed by TGA. As an attempt to control microsphere size, experimental results are compared with a theoretical model. SEM was used to characterize the size distribution of microspheres in different samples, where the diameters of about 100 microspheres were measured per experiment. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2015.
Local:
Adviser: RINALDI,CARLOS.
Local:
Co-adviser: SCHMIDT,CHRISTINE E.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-05-31
Statement of Responsibility:
by Andrew R Garcia.

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5/31/2017
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LD1780 2015 ( lcc )

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SYNTHESIS OF DISSOLVABLE MAGNETIC MICROSPHERES FOR TISSUE SCAFFOLD APPLICATIONS By ANDREW RYAN GARCIA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2015

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2015 Andrew Ryan Garcia

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To my parents, Anyela Vera and Carlos Garcia Quiroz

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4 ACKNOWLEDGMENTS I want to thank Dr. Carlos Rinaldi for his dedication and insight in facilitating the progression of th e project present ed in this thesis to its current form. Likewise, I want to thank his research group since their attitudes and initiatives have been essential in advan cing the project. I thank Ana Bohorquez for her sincere advice and willingness to help me in several occasions, as well as for supplying me with magnetic nanoparticles. I also want to thank Andreina Chiu for her instruction on the DLS, and her advice on so me imaging techniques. I thank Catherine Snyder for working in the project with me during the first year of it and for remaining available for characterizing samples up until the end of the thesis work. I thank both Lorena Maldonado and Mythreyi Unni for their advice, assistance and instruction with SQUID measurements. I thank Melissa Cruz for her contributions to our research group and for being so helpful I thank Rohan Dhavalikar for his friendship and advice. I also thank Dr. Isaac Torres for his frien dship and willingness to help. I want to thank Dr. Christine Schmidt for her consideration in allowing our lab to work in a collaborative project with hers and for her generosity on letting me use her research facilities. I want to thank Christopher Lacko for his outstanding dedication and discipline in the research, as well as for his exceptional collaboration. I want to thank Tung Chen for all her motivation and support. I also want to thank Dr. Spyros Svoronos as my former graduate coordinator and for g iving me the opportunity to grade his Process Design class. Likewise, I want to thank Shirley Kelly for her optimistic attitude and her assistance with the MS program Lastly, I t hank my parents brother, and extended family Without their great support an d understanding I would not have been able to make it this far.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 BACKGROUND ................................ ................................ ................................ ...... 16 2.1. Tissue engineered Scaffolds and their Biomedical Applications ...................... 16 2.2. Some Fundamental Ph ysical Properties of Ferrofluids ................................ .... 19 2.2.1. Magnetization ................................ ................................ ......................... 19 2.2.2. Column Formation ................................ ................................ .................. 20 2.2.3. Viscosity ................................ ................................ ................................ 21 2.3. Alginate as a Microsphere Matrix and Cross linking Chemistry ....................... 22 2.4. The Formation of Emulsions in Microsphere Precursors ................................ 23 2.4.1. Definition of an emulsion ................................ ................................ ........ 23 2.4.2. Theory ................................ ................................ ................................ .... 25 3 MATERIALS AND METHODOLOGY ................................ ................................ ...... 34 3.1. Materials Used ................................ ................................ ................................ 34 3.2. Equipment ................................ ................................ ................................ ........ 34 3.3. Synthesis of Magnetite Nanoparticles by Co Precipitation Method Followed by Peptization ................................ ................................ ................................ ...... 34 3.4. Magnetic Alginate Microspheres by Emulsification ................................ .......... 35 3.5. Characterization ................................ ................................ ............................... 36 3.5.1. Analytical Instrumentation ................................ ................................ ...... 36 3.5.1.1 Optica l microscopy ................................ ................................ ......... 36 3.5.1.2. Scanning electron microscopy (SEM) ................................ ........... 36 3.5.1.3. Superconducting quantum interference device (SQUID) magnetometry ................................ ................................ ......................... 37 3.5.1.4. Thermogravimetric analysis (TGA) measurements ....................... 37 3.5.2. Software ................................ ................................ ................................ 38 3.5.2.1. Igor Pro ................................ ................................ ......................... 38 3.5.2.2. ImageJ ................................ ................................ .......................... 38 3.5.2.3. Python ................................ ................................ ........................... 40

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6 4 RESULTS AND DISCUSSION ................................ ................................ ............... 42 4.1. Microsphere Size Control Study ................................ ................................ ...... 42 4.1.1. Effect of Shear Rate on the Size of Alginate Microspheres Using Mineral Oil as the Continuous Phase ................................ ............................ 43 4.1.2. Effect of Shear Rate on the Size of Alginate Microspheres Using 1 Octadecene as the Continuous Phase ................................ .......................... 45 4.1.3. Effect of Surfactant on the Size of Alginate Microspheres ...................... 47 4.2. Effect of Iron Oxide Content on the Size and Magnetization of Microspheres ................................ ................................ ................................ ....... 48 5 CONCLUSION ................................ ................................ ................................ ........ 60 APPENDIX A CALCULATION OF SURFACE TENSIONS AND MIXER PARAMETERS ............. 62 B SEM IMAGES OF MICROSPHERE SAMPLES ................................ ...................... 65 LIST OF REFERENCES ................................ ................................ ............................... 71 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 76

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7 LIST OF TABLES Table page 4 1 Fixed emulsion and process conditions for microspheres made employing different shear rates for emulsion formation. The continuous phase was mineral o il. ................................ ................................ ................................ .......... 51 4 2 Fixed emulsion and process conditions for microspheres made employing different shear rates for emulsion formation. The continuous phase was 1 octadecene. ................................ ................................ ................................ ........ 51 4 3 Fixed emulsion and process conditions for microspheres made using different surfactant amounts in the emulsion. ................................ ..................... 51 4 4 Fixed emulsion and process cond itions for microspheres made with different ferrofluid concentrations used to make the dispersed phase. ............................. 52 4 5 Magnetic properties measurements of microspheres made using different ferrofluid c oncentrations to make the dispersed phase and of ferrofluid ............ 52

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8 LIST OF FIGURES Figure page 2 1 Conceptual representation of magnetic microspheres in the abse nce and in the presence of an applied magnetic field. ................................ ......................... 30 2 2 Plot showing the number of magnetic particles in a magnetic chain with respect to the coupling coefficient ................................ ................................ ... 30 2 3 Molecular structures of the m onomer residues of alginic acid and typical structure of alginic acid in chair conformations ................................ .................. 31 2 4 Draw n representation of the gelation process in making an alginate microsphere by calcium cross linking of an emulsion droplet. ............................ 31 2 5 Oil in water (o/w) and water in oil (w/o) emulsions and recomm ended HLBs for emulsifier combinations in different emulsions. ................................ ............. 32 2 6 Photograph of ferrofluid alginate mixture in mineral oil emulsion. ...................... 32 2 7 Computer 37 study. ............... 33 3 1 Schematic of emulsification process undertaken to make alginate magnetic microspheres. ................................ ................................ ................................ ..... 41 3 2 Photographs of the Quantum Design SQUID MPMS3 Magnetometer and of the sample holder type used for the measurements ................................ .......... 41 4 1 Size distributi on histograms of microspheres made employing different shear rates for emulsion formation. The continuous phase was mineral oil. ................ 53 4 2 Effect of shear rate on microsphere size: correlation of experimental data with theoretical prediction. The continuous phase was mineral oil. .................... 54 4 3 Size distribution histograms of microspheres made employing different shear rates for emulsion format ion. The continuous phase was 1 octadecene. ........... 55 4 4 Effect of shear rate on microsphere size: no correlation observed between experimental data and theory. The continuous phase was 1 octadecene. ......... 56 4 5 Size distribution histograms of microspheres made using different surfactant amounts in the emulsion. ................................ ................................ .................... 57 4 6 Size distribu tion histograms of microspheres made using different ferrofluid concentrations to make the dispersed phase. ................................ .................... 58

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9 4 7 Magnetizations with respect to applied magnetic fields for microspheres made u sing different ferrofluid concentrations and for the pure ferrofluid used. ................................ ................................ ................................ ................... 59 A 1 Images of equipment used to make emulsions: a Fisher Scientific PowerGen 125 homogenizer and a Silver son L5 M A laboratory mixer ............................... 6 4 A 2 Screenshot showing the approach taken to measure mixer H through the Analyze/Measure feature of ImageJ. ................................ ................................ .. 64 B 1 SEM image for a magnetic alginate microsphere sample made under optimized conditions. ................................ ................................ .......................... 66 B 2 SEM images of magnetic microspheres made by employing different shear rate s and using mineral oil as the continuous phase. ................................ ......... 67 B 3 SEM images of magnetic microspheres made by employing different shear rates and using 1 octadecene as the continuous phase. ................................ .... 68 B 4 SEM images of magnetic microspheres made by changing the amount of Span 80 and Tween 80 in the pre microsphere emulsion ................................ 69 B 5 SEM imag es of magnetic microspheres made by using increasing concentrations of ferrofuid to make the dispersed phase. ................................ .. 70

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10 LIST OF ABBREVIATIONS ECM Extracellular matrix EDS Energy Dispersive X ray Spectroscopy EDTA FDA FEG GRAS HA HLB MSDS o/w PLGA SEM SQUID TGA w/o Ethylenediaminetetraacetic acid Food and Drug Administration Field Emission Gun Generally Recognized as Safe Hyaluronic acid Hydrophile lipophile balance Material Safety Data Sheet Oil in water Poly(lactic co glyc olic acid) Scanning Electron Microscope Superconducting Quantum Interference Device Thermogravimetric analysis Water in oil

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for the Degree of Master of Science SYNTHESIS OF DISSOLVABLE MAGNETIC MICROSPHERES FOR TISSUE SCAFFOLD APPLICATIONS By Andrew Ryan Garcia May 2015 Chair: Carlos Rinaldi Major: Chemical Engineering M agnetic microspheres were made by an emulsification process where smaller magnetic nanoparticles are entrapped in alginate matrices crosslinked with calcium These microspheres were designed to serve as templates that can form tissue engineered scaffolds with guided hollow channels and voids. The microspheres presented herein were made from a matrix that could be dissolved by the addition of a chemical. They were proven to possess magnetic properties by magnetic measurements and were seen to align with moderate magnetic fields usin g optical microscopy The mass fraction of iron oxide inside microspheres was also analyzed by thermogravimmetric analysis As an atte mpt to control microsphere size, experimental results are compared with a theoretical model. Scanning electron microscopy was used to characterize the size distribution of microspheres in different samples, where the diameters of about 100 microspheres were measured per experiment.

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12 CHAPTER 1 INTRODUCTION In this thesis, we focus on the development of a protocol to make mag netic microspheres in the micron to sub micron range which can form columnar structures in a hydrogel matrix under the application of a magnetic field, and which can be subsequently dissolved with a chemical once these structures have been formed by the ma gnet, imprinting the desired voids and hollow channel structures in the hydrogel. Magnetically templated hydrogels are a novel technology that has been explored in the labs of Dr. Carlos Rinaldi and Dr. Christine Schmidt since the beginning of this thesi s research project. Due to their compatibility with biological tissue and cells, hydrogels can be used as scaffolds in tissue engineering 1 3 As is the nature of the tissue engineering field, these scaffolds commonly serve the function of repairing tissue by implanting cells into the scaffolds, and perhaps certain growth factors, to stimulate cell pro liferation along specific paths 3 5 Of the many potential biomedical applications that may exist for this technology, peripheral nerve repair appears particul arly promising. Peripheral nerve injuries are commonly associated with substantial functional loss and decreased quality of life 6 The most severe case of nerve damage involves complete transection of the nerve, which results in the loss of sensory and mot or function at the injury site 7 8 Peripheral nerve repair technologies aim to restore and prevent loss of motor/sensory peripheral nerve function. The market for the repair of transected peripheral nerves in the extremities has been estimated to be $1.32 to $1.93 billion dollars per year 9 Thus, the development of new nerve repair technologies can be a very promising and altruistic venture.

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13 What makes this technology distinct from other hydrogel scaffolds is the templating process undertaken to generate certain structural properties that are pertinent to tissue repair and regeneration. A template can be defined in the mechanical sense of the word, where the template for a hydrogel would be a material that is inert with the hydrogel and that is used to sha pe it. In the current case, we employ magnetic microspheres as the hydrogel template. As templates, magnetic microspheres can be used to generate empty voids and channels with magnetically programmed paths within the treated hydrogel. In the present case we have chosen alginate as the matrix used to make magnetic microspheres. Alginate was selected from a generous database of FDA GRAS (Generally Recognized as Safe) chemicals due to its agreement with our criteria for the template of a hydrogel with the pot ential to be applied in biomedical practice. Alginate is a biocompatible material, cited in the literature for numerous biomedical applications 10, 11 while also used as an edible ingredient in a molecular gastronomy technique known as spherification 12 Al ginate was also chosen for its ability to incorporate magnetic nanoparticles in microspheres, rendering these alginate microspheres to become magnetic and capable to form columns and / or other configurations dependent on the application of an external mag netic field. Lastly, alginate was chosen due to its ability to be dissolved with a chelating agent such as EDTA 13 In this way, dissolving the alginate microspheres will leave an imprinted architecture on the magnetically templated hydrogel.

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14 The alginate microspheres have been made through a process known as emulsification, followed by gelation of the dispersed phase of the emulsion through ionic crosslinking. In the emulsification process, a mixture of alginate and ferrofluid solutions is dispersed in an oil phase by comminution, where the dispersed phase can be dispersed phase can be then solidified by gelation, entrapping the magnetic nanoparticles in the newly form ed micro spheres and rendering them certain intrinsic magnetic properties of ferrofluids, where the most critical to our investigation would be the ability to respond to an applied magnetic field by magnetically aligning with such. In this thesis we have u sed a theoretical model to estimate the size of magnetic alginate microspheres from some conditions of the particular emulsification process used to make them. This model has been evaluated by testing trends between microsphere diameter distributions with respect to a manipulated variable from the process. We have also analyzed the magnetic properties of microspheres with increasing ferrofluid concentrations used to make the dispersed phase. Chapter 2 presents brief reviews on tissue engineered scaffolds a nd the cross linking chemistry of alginate for making microspheres. Additionally, theoretical background for the magnetic properties of ferrofluids and for the emulsification process used to make the microspheres is also presented. Some relations that are presented in the theoretical background for the emulsification process are applied in subsequent chapters. Chapter 3 presents the materials and methods employed to make the magnetic nanoparticles and microspheres presented in this thesis. The instrumentat ion used to

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15 analyze microsphere samples and some approaches used to treat the data is also presented in this chapter. Chapter 4 presents the results of this investigation. Herein we have used a model derived from the theory of emulsion formation developed by G.I. Taylor where the emulsion droplets were in the order of a centimeter and characterized by photographs 3 7 In this study we show how the theor y can extend to microscopic systems. In this chapter we also present an analysis of the magnetic properti es of microsphere samples with different f errofluid concentrations. Chapter 5 presents some concluding remarks and suggested approaches for future research.

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16 CHAPTER 2 BACKGROUND 2.1. Tissue engineered Scaffolds and their Biomedical Applicatio ns The area of tissue engineering can be defined as the application of biological, chemical, and engineering principles toward the repair, restoration, or regeneration of living tissue by using biomaterials, cells, and factors alone or in combination 14 Bi odegradable scaffold matrices are of significant interest to this area, since these matrices can be used to fill tissue voids, provide structural support and deliver growth factors and/or cells that have the potential to form tissues within the bod y upon t ransplantation 1 5 Oftentimes, tissue scaffolds are made to emulate the natural extracellular matrix (ECM), as scaffolds are used as templates for three dimensional tissue growth 16 Thus, the ultimate goal for the design of an effective scaffold structure i s to replace the natural ECM until host cells can repopulate and resynthesize a new matrix 17 Electrospinning, molecular self assembly, and thermally induced phase separation are three basic techniques that can be used to make nano fibrous scaffolds 16 Tis sue engineered scaffolds are designed to meet the scale of cells and thus, their structural patterns tend to be in the micron to sub micron range. Li et. al., for instance, developed a novel PLGA structure composed of randomly oriented fibers with diameter s ranging from 500 to 800 nm that was used to investigate the cell activities of fibroblasts and mesenchymal stem cells within the scaffolds 17 ( Figure 2 1 ). The microspheres disclosed in this thesis have been applied as sacrificial templates for developi ng scaffolds with porous architectures and hollow channels grown in a particular direction. Figure 2 2 shows a conceptual representation of the rigid,

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17 columnar structures that would be formed, in an ideal case, by magnetic microspheres. Introducing these m icrospheres in a hydrogel precursor ( Figure 2 2 A ) followed by the application of a magnetic field ( Figure 2 2 B ) would render structural arrangements dependent on the orientation of the magnetic field. After the hydrogel is formed, the microspheres can be dissolved and leave behind imprinted architectures in the hydrogel. Due to the ability to manipulate the microsphere template with a magnetic field, there is significant versatility in making scaffolds with varied porous architectures through this method. The magnetically controlled programmability of magnetic microspheres in forming scaffolds with anisotropic channels can be exploited for application in technologies aiming to rapidly populate large scaffolds with cells, facilitating cell proliferation an d/or cell growth along programmed paths. An important, though not exclusive, biomedical application for this technology falls in the area of peripheral nerve repair. Technologies used for peripheral nerve repair aim to restore and/or prevent significant lo ss of motor and sensory peripheral nerve function. Cases of peripheral nerve loss are not uncommon and the market for repair of transected peripheral nerves in extremities has been estimated to be $1.32 to $1.93 billion dollars per year 9 Modern technique s that are currently implemented for repairing peripheral nerves depend critically on the length of the transected or amputated nerve. In cases where the transection leaves a small gap, a hollow tube or connector device can be placed at the gap as a simple conduit to grossly align the nerve endings, so that peripheral nerve regeneration can be facilitated without the stress of a direct suture between nerves 9

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18 For cases where the gap is significantly large, the hollow tube approach may be ineffective in rep airing the transected nerve and harvested nerves may be required 9 Allografts and autografts are the most common harvested nerve types employed for this procedure. Nerve allografts being conventionally taken from cadaveric tissue, have the potential di sadvantages of risk of disease transmission, possible immunogenicity, and slower incorporation or ligamentization 18, 19 Nerve autografts, on received by the injured tissu inflammation and scarring to the area while providing an optimal regeneration conduit 9 However, autograft harvesting permanently damages the harvested site in the patient, creating new life long morbid ity where there was none 9 Moreover, there is a risk of the autograft not working at the implanted site, in which case the patient would have two sites with transected nerve. Alternatives to harvested tissue such as tissue engineered scaffolds are of par ticular interest to overcome these challenges. Using an oriented inner scaffold that can provide a conducive and induct ive environment for axonal regeneration makes it possible to overcome conduit gap limitations 20 The use of internal fibers and guidance channels in a scaffold has been proposed before as a strategy to mimic the guidance structure of the peripheral nerve autograft 2 1 Such proposition is reminiscent of the current templating criteria for the microspheres that are designed in this thesis U sing bioresorbable material s also attenuates several difficulties in tissue repair Bioresorption can be regarded as the total elimination of polymeric structures by dissolution, assimilation, and excretion of extraneous materials 2 2 Figure 2 3 shows a

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19 tis sue engineered nerve applied to a sciatic nerve injury with a gap of 11 mm. After a period of 12 weeks the artificial nerve gets resorbed by the repaired nerve 2 3 In the current progress of the investigation we have synthesized magnetic microspheres and su microspheres to template hyaluronic acid (HA) hydrogels. Potentially, these microspheres may be used to template scaffolds that facilitate tissue repair by using materials with features reminiscent to the ones exhibited in this section. 2.2 Some Fundamental Physical Properties of Ferrofluids The alginate microspheres made in this investigation contain magnetic nanoparticles incorporated in the form of a ferrofluid, that were retained during the i onic crosslinking process that formed them and thus renders them magnetic. Some fundamental physical properties of ferrofluids that are pertinent to the investigation are presented in this section. 2.2.1. Magnetization Fluids consisting of magnetic nano particles alone fall under the classification of colloidal magnetic fluids, which are fluids consisting of a collection of ferri or ferromagnetic single domain particles with no long range order between particles 2 4 Colloidal magnetic fluids, also known as colloidal ferrofluids, exhibit superparamagnetic behavior, where the magnetization in low to moderate fields is much larger than those obtained for paramagnetic systems 2 4 Colloidal ferrofluids must be synthesized, as they cannot be found in nature, and are expected to retain liquid flowability in the most intense applied magnetic fields 2 4 A study where the magnetization (M) of a ferrofluid is examined as a function of applied magnetic field (H) has the potential to determine some fundamental magnetic

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20 p roperties of said system. A treatment by Rosensweig classical theory and statistical mechanics yields the superparamagnetic magnetization law for a monodisperse ferrofluid 2 4 : (2 1 ) (2 2 ) In the iron oxide content study from Chapter 4 (4.2) the magnetization of microsphere samples are obtaine d a s a function of magnetic field Since the magnetic properties of a ferrofluid are related to and by E quation 2 1, the properties can be obtained by fitting experimental magnetization curves with this theoretical model. 2.2.2. Column Formation Column formation in ferrofluids is a phenomenon attributed to magnetic dipole dipole interactions between nearby particles 2 5 An estimate of the number of magnetic particles in a column was obtained by de Gennes and Pincus 2 6 and by Jordan 2 7 where pair cor relations are investigated under the assumption of low density and a strong applied magnetic field: (2 3 ) (2 4 ) Where is the coupling coefficient

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21 Figure 2 4 shows a MATLAB generated plot of the average number of magnetic particles in a magnetic column with respect to the coupling coefficient ( ) for a low density case where From this figure and E quation 2 4 it can be deduced that increasing the magnetic particle volume ( ) will result in increasing the coupling coefficient. As the volume is increased from a small particle volume it can be seen that the number of particles in the chain will increase and find an optimal particle volume where the number of particles per column approaches infinity. Rosensweig found that magnetite particles with 13 nm magnetic diameter have (1981), forming a chain with a length approaching infinity 2 4 as can be appreciated from Figure 2 4 The apparent discontinuity and drop to negative number of particles in the plot is a flaw from the limiting computing capability of not being able to select an infinite number of x axis points around the range where the y axis reaches values which approach infinity. 2.2.3. Viscosity Einstein developed theoretical models for quantifying the viscosity of a suspension of solid spheres in a liquid 2 8 Thes e models can also be used for determining the viscosity of a ferrofluid 24 In the absence of an external field, the following formula can be used to relate the mixture viscosity to the carrier fluid viscosity and solids fraction for suspensions with small ferromagnetic concentrations and uncoated nanoparticles 2 4 : (2 5 )

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22 F or cases where the ferrofluid has higher ferromag netic concentrations and coated nanoparticles, the more complete expression can be used 2 4 2 9 : (2 6 ) Relati ons for the viscosity of a fer rofluid when an external magnetic field is present have been derived, but are beyond the scope of this thesis. 2.3. Alginate as a Microsphere Matrix and Cross linking Chemistry In this study alginate was used as a precursor for the microparticle matrix th at supports magnetic nano particles Alginate is a linear polysaccharide composed of a binary combination of the two residues shown in Figure 2 5 A : (1 4) D L guluronate (G). These polysaccharides exist as combinations of three different types of blocks as seen from Figure 2 5 B : diequatorial (MM), equatorialaxial (MG), axial equatorial (GM) and diaxial (GG) 30 The sel ection of alginate was based on its particular applicability to a biomedical technology conceived by our research group. One of the most considered features for choosing alginate as the microsphere matrix was its ability to incorporate materials by gelati on. Alginate gelation occurs through the interaction of alginate chains with calcium ions. When a calcium solution is added to alginate, calcium ions hold the alginate polymer together and pack the alginate polysaccharide chains into compact structures whi ch resemble solids at the molecular level, as can be seen from Figure 2 6. This phenomenon is known as cooperative

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23 binding, where the aggregation of several alginate chains induced by calcium ions makes these ions bind more firmly 3 1 The formation of algi nate microspheres is ruled by cooperative binding of calcium ions with alginate chains. In this investigation, microspheres are conventionally made by forming an emulsion where the dispersed phase is present as alginate droplets in an oil based continuous phase. After several alginate droplets are dispersed in the solution, calcium is added to the emulsion in order to make alginate gels by cross linking with the alginate chains in a manner that has been illustrated in Figure 2 7 The benefit of forming the se microsphere gels is the reversibility of the alginate gelation process through the use of an ionic sequestrant such as EDTA that disintegrates the compact packing of calcium ions with the alginate chains by selectively binding to calcium ions. In the f ollowing section we provide some background information on the emulsification process undertaken to make the microspheres and introduce some pertinent variables used to study it. 2.4. The Formation of Emulsions in Microsphere Precursors 2.4.1. Definition of an emulsion An emulsion can be defined as a mixture of two commonly immiscible liquids, usually stabilized by emulsifiers, where one liquid is dispersed in the other. Emulsifiers hinder the coalescence of dispersed droplets by accumulating at the phase boundary between immiscible liquids, lowering the surface tension at the interphase. The emulsifiers used in this project were the nonionic surfactants Span 80 and Tween 80. Conventionally, there are two naturally immiscible phases in an emulsion that a re classified as dispersed and continuous phases. The dispersed phase tends to be the

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24 phase with the lower volume fraction in the emulsion, mostly seen as a collection of wo uld be the phase with the dominant volume fraction in the emulsion, observed as the continuous medium which suspends the dispersed phase. A simple notation system for distinguishing the dispersed phase from the continuous phase exists when describing an em ulsion. In the case of making emulsions using oil and water, a variety of emulsions can be made. An oil in water (o/w) emulsion has oil and water as the dispersed and continuous phase, respectively. The opposite is true for water in oil (w/o) emulsions. Mo re complicated emulsion systems exist, where one phase is dispersed in another phase, which is in turn dispersed in the former one. Oil in water in oil (o/w/o) and water in oil in water (w/o/w) are examples of such systems. The choice of adequate surfact ants depends on the identity of these emulsion phases. Figure 2 8 shows that surfactants or surfactant combinations with different HLB values are used depending on whether the emulsion is w/o or o/w. HLB is a surfactant classification system that stands fo r hydrophile lipophile balance. This system is used to express the relative affinity of the surfactant to water and to oil. Knowing the HLB permits some prediction on the behavior of surfactants in an emulsion and can reduce the amount of work and money in selecting them 3 2 An emulsification process creates an emulsion by dispersing one phase in another one through methods such as ultrasound, electrospraying, condensation, and comminution, among others. In the current progress of this investigation we hav e employed comminution as the emulsification process to make alginate microspheres.

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25 The process of comminution produces an emulsion by disrupting a larger volume into smaller subunits, usually by applying a disruptive, mechanical force 3 3 2.4.2. Theory In this investigation, microspheres are made by this emulsification process, where a polar solution is dispersed in an oil phase (w/o emulsion), followed by solidification of the dispersed phase into microspheres through ionic crosslinking. Because emulsific ation is the preceding step in making microspheres, the dispersed The emulsions from this study were made using two different types of rotor stator mixers: a handheld homog enizer and a laboratory mixer. In order to remove the dependency on the different rotor stator dimensions between mixers, the shear rate is defined as 3 4 : (2 7 ) rotor radius, respectively. Maa and Hsu have claimed eddies arising from turbulent flow are required in order t o break the dispersed phase 3 5 The size scale of the eddies has been deduced to decrease with increasing mixing intensity until reaching a critical point where their energy is lost as heat through viscous dissipation and are rendered unable to further frag ment the droplets 3 5 3 6 Eddies, or vortices, are evidently formed in the ferrofluid alginate in mineral oil emulsions made for this study ( Figure 2 9 ). For this thesis, a classic study made by Sir G.I. Taylor is employed to quantify emulsion formation wi th theoretical estimates 3 7 In that study, the fields of flow

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26 investigated were both easy to produce in an actual fluid and simple to define using the following mathematical equations: (2 8 ) The streamline s pertaining to these flow fields were rectangular hyperbolas (Figure 2 10) When the fields of flow are represented in this way, the velocity of a particle in the x direction can be represented by (2 9 ) Where can be reasoned to express the shear rate by rearrangement. The average radii of the emulsion precursors (a) can then be derived from 3 7 as: In the current case we may make the assumption that the average microsphere size is approximately equal to the average microsphere precursor size, supposing the crosslinking does not significant ly change size d ue to solidification into microspheres. The emulsion systems that have been studied in this investigation involve dispersed phases that are significantly more viscous than their respective continuous phase (i.e. ). For such cases, E quation 2 10 ca n be applied by modifying the eccentricity in the capillary number to the following approximation 3 7 : (2 10 ) (2 11 )

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27 (2 12 ) In the current case, the continuous phase is composed of a carrier fluid alone, making its viscosity simpl e to estimate by surveying the properties found on its respective MSDS. However, the dispersed phase is a mixture of solids in water as the carrier fluid. Thus, the viscosity of the dispersed phase must be estimated by using some theoretical relations. Ein stein developed theoretical models that can be used for determining the viscosity of a mixture 2 8 In such study, Einstein found that the intrinsic viscosity of solid spheres in a stationary liquid approaches 5/2. By analogy with E quation 2 5 the viscosity of a mixture can be calculated as a function of its intrinsic viscosity by the following formula: (2 13 ) Where is the carrier fluid and is the solids fraction. As this relationship is only valid for cases where the solids concentration is relatively low, the latter equation can be used to estimate the viscosity of the alginate solutions made throughout this investigation, since the mass fraction of alginate used in solution does not go beyond 5%. Even though it is realized that the dispersed phase is a combination of ferrofluid with alginate, a ferrofluid solution with low volume fractio n will approach the viscosity of water. Moreover, since the viscosity of the alginate used in this investigation is significantly larger than the viscosity of the ferrofluids used, it will be assumed that the

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28 viscosity of alginate will dominate over that o f the ferrofluid in determining the viscosity of the dispersed phase. As mentioned in the beginning of section 3.4, surfactants are known to influence the surface tension ( ) of an emulsion and thus will consequently influence the size of microspheres ( E quation 2 10). The affinity of a surfactant to water and to oil can be expressed as the HLB (hydrophile lipophile balance) value. If two surfactants are used in an emulsion, the HLB of the surfactant blend can be calculated from the HLB values and mass f ractions from the separate surfactants 3 2 : (2 14 ) The HLB value of the emulsion can then be compared with its required HLB value, which is the ideal HLB value generated from an optimum surfactant ratio tha t yields effective blending in an emulsion 3 2 Even though useful in predicting the quality of an emulsion, the HLB value of an emulsion is a qualitative measure. Thus, HLB values are not known to have a definite trend with the surface tension of the emulsi on. The Gibbs adsorption isotherm and the definition of chemical potential for a surfactant species in solution 38 can be used to reason the relation between the surface tension ( ) of an emulsion and the amount of surfactants present in it: (2 15) (2 16)

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29 Where and are the surface excess concentration and the chemical potential for an The surface excess concentration has been defined as the amount of surfactant in excess from a reference amount divided by the area of the interface 39 : (2 17) Differentiating E quation 2 16 and substitution of its differentiated form into E quation 2 15 results in: (2 18) The equation sug gests an increase in the concentration of a surfactant ), would cause a decrease in surface tension. However, quantifying surface tensions from this expression is challenging because the surface excess values and activity coefficients for the different surfactants present in the emulsions made herein are not so easily determined.

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30 Figure 2 1 Conceptual representation of magnetic microspheres in the absence A) and in the presence B) of an applied magnetic field Observables: ~2,500 microspheres per slide. Figure 2 2 Plot showing the number of magnetic particles in a magnetic chain with respect to the coupling coefficient A ) B )

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31 Figure 2 3 Molecular structures of the monomer residues of alginic acid A) and typical structure of alginic acid in chair conformations B). Figure 2 4. Drawn representation of the gelation process in making an alginate microsphere by cal cium cross linking of an emulsion droplet. A ) B )

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32 Figure 2 5. Oil in water (o/w) and water in oil (w/o) emulsions and recommended HLBs for emulsifier combinations in different emulsions. Figure 2 6 Photograph of ferrofluid alginate mixture in minera l oil emulsion.

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33 Figure 2 7 Computer 3 7 study.

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34 CHAPTER 3 MATERIALS AND METHODOLOGY 3.1. Materials Used with a viscosity of 35 0 500 mPas at 1 % (w/w) solution and M:G ratio of 1.5 1.6 as reported by the supplier was purchased from Thermo Fisher Scientific. Calcium Chloride dehydrate (f.w. 147.01 Da as reported by the supplier ), Span 80, Tween 80, mineral oil and 1 Octadecene w ere also purchased from Fisher Scientific. Magnetite nanoparticles peptized with tetramethyl ammonium hydroxide were made following a synthesis adapted from Massart 40 Herrera et. al. 41 and Creixell et. al. 42 These nanoparticles were provided by Ana Boho rquez 3.2. Equipment Two different mixers were used to make alginate microspheres. The first mixer was a Fisher Scientific PowerGen 125 homogenizer ( Figure A 1 A ) with a frequency range of 8,000 30,000 rpms. The other mixer used was a Silverson L5M A laboratory mixer ( Figure A 1 B s laboratory In order to consolidate the results of the different mixers, the operating frequencies generated by both mixers were transformed to shear rates through a treatment described in detail in the Appendix (A.1). 3.3. Synthesis of M agnetite N anoparticles by C o P recipitation M ethod F ollowed by P eptization This synthesis leads to clusters of magnetite nanoparticles of about 30 40 nm with both Brownian and Neel relaxation. Iron oxide co precipitated nanoparticles are inherently unstable in water and thus an additional peptization process is needed in order to stabilize them in water. The synthesis was revised by Mar Creixell and Ana

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35 Bohorquez from Dr. in 2011 and was adapted from syntheses developed by Massart 4 0 Herrera et. al. 4 1 and Creixell et. al. 4 2 3.4. Magnetic A lginate M icrospheres by E mulsification This is a protocol that makes use of an emulsification process to make magnetic alginate microspheres with controlled diameters in the micron to sub micron range, that can form columnar structures under a moderate magnetic field, and dissolve after templating a hydrogel scaffold matrix. The protocol was adapted from Jay et. al. 4 3 and Jay and Saltzman 4 4 In short, alginate microspheres were made by mixing a viscous alginate solution with a ferrofluid prepared by the synthesis from section 3.2 having a 1:1 alginate solution:ferrofuid volume ratio and emulsifying the resulting mixture in a nonpolar continuous phase by adding surfactant(s) and using a mixer. Conventionally, 1 mL of a 40 mg/mL alginate were mixed with 1 mL 16 mg ferrofluid/mL solution and subsequently added to an 8 mL continuous phase with 5% Span 80. This was co nventionally followed by the addition of 0.5 mL of a Tween 80 solution to the emulsion that resulted in an HLB blend of 7.22 A surfactant index system is used herein, where is Span 80, is Tween 80, is the volume of solution used to mix a surfactant and is the surfactant volume fraction in such solution. Thus, in the conventional protocol: and After the alginate ferrofluid mixture was properly dispersed in the continuous phase, the r esulting dispersed phase droplets were crosslinked into microspheres by adding 2 mL of a 700 mM CaCl2 crosslinking solution to the emulsion while mixing. After allowing some time (~3 min) for proper crosslinking, the mixing was stopped and

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36 ethanol was adde d with a volume that was 5 times the volume of the initial alginate ferrofluid mixture. The experiments presented in Chapter 4 explain the deviations made from the conventional procedure to make the microspheres. The new solution was then centrifuged at 4 ,000 rpm for 1 min, after which the magnetic microspheres were decanted by removing the top oil layer and the surfactant emulsion layer while keeping a strong magnet at the bottom of the centrifuge tube to keep the magnetic bottom layer. A washing procedur e then followed, where a moderate amount of ethanol was added to the magnetic bottom layer, followed by centrifugation at 4,000 rpm for 1 min and repeated multiple times until excess amounts of oil and surfactant were removed. The washed microspheres were then air dried and lyophilized. 3.5. Characterization 3.5.1. Analytical Instrumentation 3.5.1.1 Optical microscopy Optical microscopy images of microsphere samples in this thesis were taken with the Axio Imager.Z2 from Zeiss. Images were taken by Christ opher Lacko from Dr. 3.5.1.2. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) images of the microsphere samples made for this thesis were obtained through the use of two different microscopes: A Phenom Pro Desktop Scanning Electron Microscope (SEM) and a FEI Phillips XL40 Field Emission Gun Scanning Electron Microscope (FEG SEM). The Phenom Pro Desktop SEM had light optical magnification from 20 120 x using the ProX / Pro settings, and fixed at 20 x using the Pure setting. This SEM has an

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37 electron optical magnification range of 80 100,000x, with a digital zoom of 12 x using the ProX / Pro settings, and a magnification range and digital zoom of 70 20,000x, and 12 x using the Pure setting, respect ively. Its resolutions are 17 nm using the ProX /Pro settings and 30 nm using the Pure setting. The FEI Phillips XL40 FEG SEM uses a field emission filament and has a 50nm resolution. It is also equipped with a solid state EDS detector to provide elementa l maps, line scans or point analysis. The software also provides the ability to perform phase mapping. SEM images were taken by Catherine Snyder. 3.5.1.3. Superconducting quantum interference device (SQUID) magnetometry Superconducting quantum interferenc e device (SQUID) magnetic measurements were performed in order to quantify the magnetization of microsphere samples under an applied magnetic field. A Quantum Design SQUID MPMS3 Magnetometer (Figure 3 2 A ) from Dr. Carlos e the SQUID analysis. The magnetometer is extremely sensitive (~10 8 emu), with a capability of measuring sample magnetization up to 300 emu with controllable temperature range from 1.9 400 K. The microsphere samples were analyzed by mounting the lyophil ized microspheres inside a sample holder that handles solids (Figure 3 2 B ), inserting the sample holder inside the magnetometer, and programming the magnetometer to run a Magnetization (M) versus Applied Field (H) curve. Samples were run by Lorena Maldona do and Mythreyi Unni from Dr. Carlos 3.5.1.4. Thermogravimetric a nalysis (TGA) m easurements TGA measurements were performed in order to determine the iron oxide mass fraction of the microspheres prepared for the Magnetization study in sectio n 4.3. The

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3 8 apparatus used for thermogravimetric analysis (TGA) was a SDT Q600 V20.9 Build 20 TGA instrument. In order to perform the analyses, microsphere samples in lyophilized form were mounted in a TGA pan. The samples were analyzed using a temperature scan rate of approximately 20 C/min, with a temperature range of 0 1000C. The 3.5.2. Software 3.5.2.1. Igor Pro Igor Pro ver. 6.1.2.1 was used in order to process the data obtained from SQUID measurements. The Langevin function relates the magnetization of a ferrofluid to an applied magnetic field H and some fundamental magnetic properties of the system: ( 3 1 ) ( 3 2 ) A regression algorithm for the experimental v ersus plot developed by Dr. Rinaldi in Igor Pro wa s used to determine such properties automatically. 3.5.2.2. ImageJ ImageJ 1.48v, developed by Wayne Rasband from the National In stitutes of Health (NIH), was used as an analytical tool to process and analyze microsphere images obtained through SEM. One image processing initiative taken was adding a scale bar to an image, which was done by declaring the known size of a reference o bject in the image (i.e. an old

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39 scale bar) using the Analyze/Set Scale feature, followed by the Analyze/Tools/Scale Bar feature. This initiative was most useful in setting a global scale bar in a SEM image ges. For this thesis, montages have been created where multiple images are arranged in a gradient of an alterated parameter, such as shear rate. SEM image montages were feat observed region (e.g. 400 x 400 m). After doing so, all treated images were cropped to a same target size of interest. A global scale bar that would relate the scale of all SEM images present in the montage was then added to the last image in the series (i.e. the last image opened in ImageJ) by the process outlined above. All images were then stacked to a single archive by the Image/Stacks/Images to Stack feature, scaling all im ages to either the image with the largest or the smallest virtual size. After this, the montage was created by the Image/Stacks/Make Montage feature. Size measurements of the microspheres seen in different SEM images were also done using ImageJ. In order to obtain a reasonable size distribution, about 100 microspheres were sampled per experiment. Due to the high density of microspheres in several images, microspheres were mainly measured by a manual approach. In this approach, an image was first scaled to actual size by the processes described above, encircling the microsphere, it could then be measured by the Analyze/Measure feature, following by labelling the specific part icle in order to keep track of it being measured

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40 through the Analyze/Label feature. This would yield an area measurement of the particle. After repeating the procedure about 100 times, the resulting microsphere area measurements were transformed to microsp here diameters using Excel. 3.5.2.3. Python Python was run using Canopy version 1.4.1, a cross platform environment for scientific computing. Scripts were made in order to provide for a facile approach to store, process and analyze data. A Python script t hat contains the size distribution and magnetization data for all of the microsphere experiments done for this thesis, for instance, has a size of only 170 KB. The easy storage and processing of data allows for the production of high quality images that ca n be generated in a matter of a few seconds. All plots and histograms from Chapter 4, as well a s a couple of figures from Chapter 2, were made running Python in the Canopy editor.

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41 Figure 3 1. Schematic of emulsification process undertaken to make algi nate magnetic microspheres. Figure 3 2. Photographs of the Quantum Design SQUID MPMS3 Magnetometer A ) and of the sample holder type used for the measurements B ). A ) B )

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42 CHAPTER 4 RESULTS AND DISCUSSION The sizes of magnetic alginate microspheres and their magnetic p roperties were two features considered important to their application in tissue engineering. The microsphere sizes were controlled in the micron and sub micron ranges. Likewise, their magnetic properties were investigated, though not optimized. Studies were conducted where a variable hypothesized to have an effect on either the size or magnetization of the microspheres is manipulated while all other conditions are kept constant. The effects of the manipulated variables are described in this chapt er. The microspheres presented herein were made by an emulsification process described in Chapter 3. The following size control studies were inspired by a paper written by Taylor where emulsion formation was quantified by mathematically defined flow field s 3 7 4.1. Microsphere Size Control Study The microspheres presented in this thesis have been made by crosslinking the dispersed phase, which was initially present as emulsion droplets. Herein it has been assumed that the sizes of microspheres do not differ significantly from the size of the pre microsphere emulsion droplets. Thus, the expressions from Chapter 2 for calculating emulsion droplet size w ere used in order to test for the trends between microsphere size with respect to some of the ir variables. ( 4 1 ) ( 4 2 )

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43 Since the viscosity of the dispersed phase ( was greater than the viscosity of the continuous phase used ( in all the experiments presented in this thesis, E quation 2 12 was used to approximate the eccentricity term ( in the capillary number ( E quation 4 2 ) throughout the chapter. In this section trends in the microsphere size distributions with respect to a changed condition will be evaluated. The first subsection (4.1.1) shows a series of microsphere samples made using different shear rates and using mineral oil as the continuous phase. The second subsection (4.1.2) shows a series of samples made in a way an alogous to those in the first subsection using 1 octadecene as the continuous phase. The third subsection (4.1.3) shows a series of microsphere samples made using different surfactant concentrations. 4.1.1. Effect of Shear Rate on the Size of Alginate Mic rospheres Using Mineral Oil as the Continuous Phase Throughout this chapter the eccentricity and capillary number for an 37 approximation Equation 2 12 and Equation 4 2 Likewise, the shear rate and HLB were calculated by Equations 2 7 and 2 14, respectively. The dispersed phase viscosity was estimated from the known viscosity of alginate at a particular mass fraction (section 3.1) and Equation 2 13. The continuous phase viscosity was obtained from the MSDS of the oil used. Lastly, the surface tension was estimated through a treatment described in Appendix A. The effect of shear rate on the microsphere size using mineral oil as the continuous phase was investigated in this s ection. The conditions that were kept constant throughout microsphere samples are listed in Table 4 1

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44 Figure 4 1 shows a series of histograms representing the size distributions of alginate magnetic microspheres made under the application of different sh ear rates The histograms were made by sampling a set of microspheres and measuring their diameters from SEM images, the details of this can be found in Appendix B The histograms are stacked in order of decreasing shear rate from top to bottom and show a tre nd between size and shear rate, where an increasing shear rate results in the production of smaller microspheres. The uncertainty in the measured diameter (D) is expressed as 1 standard deviation from the mean of each sample. An increase in standard devia tion with a decrease in shear rate can be noted, making drastic increases below the 4,700 rad/s shear rate experiment (increasing 1.5 below the 1,200 rad/s shear rate experiment. From Figure 4 1 it can be observed that the microsphere samples that were made employing relatively low shear rates ( 2,300 rad/ s) have significantly polydisperse distributions, which may have been the result of lower power mixing not being able to disperse the microsphere precursor effectively in the continuous phase. The plot shown in Figure 4 2 compares the experimental values for the microsphere size distributions in terms of shear rate with the theoretical fit made by using E quation 4 1 The mean microsphere sizes from the experimental data are plotted as green triangles, with error bars that measure 1 standard deviation fro m the mean. From the plot we can see the hyperbolic trend (blue line) between shear rate and size from E quation 4 1 comes into close agreement with the microsphere samples obtained employing moderate to high shear rates (current samples done in the range of 2,300

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45 13,000 rad/s) shear rates, though the agreement is not as good at the lower shear rates due to the larger polydispersity of those samples. diameter with high precision (i.e. narrow standard deviations). From looking at the measured microsphere diameter values from Figure 4 1 it was realized that an attempt to make microsp conditions would result in generating microspheres with a large standard deviation and thus, low precision. As it was discussed previously, an increase in the polydispersity at low shear rates wa s considered to be the result of low power mixing. Thus, it was hypothesized employing moderate to high shear rates to generate microspheres with relatively narrow distributions while changing a different variable from E quation 4 1 with the objective to increase microsphere size. 4.1.2. Effect of Shear Rate on the Size of Alginate Microspheres Using 1 Octadecene as the Continuous Phase An attempt to make larger microspheres with relatively high precision was made by chang ing the continuous phase used to make the emulsions. Such approach was taken since E quation 4 1 shows that the microsphere size is inversely proportional to the viscosity of the continuous phase. In this sectio n the results are presented for a series of microsphere samples made employing different shear rates and using 1 octadecene as the continuous phase. Because 1 O ctadecene used in this study had a significantly lower viscosity (4.32 cP) to the dispersed phas e, assumption 37 E quation 2 12

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46 was also used to estimate the eccentricity term in E quation 4 2 The conditions that were thus constant throughout these samples are presented in Table 4 2 Figure 4 3 shows the size dis tributions for a series of microsphere samples made employing different shear rates. The histograms in this figure are stacked in order of decreasing shear rate from top to bottom, and the uncertainties in the measured diameters are also expressed as 1 sta ndard deviation from the mean. For the present case, a relation between shear rate and microsphere size is not evident. Consequently, there is no trend for the plot in Figure 4 4 a nd the experimental results do not appear in agreement with the theoretica l model for microsphere size ( E quation 4 1 ). In this plot the error bars also measure 1 standard deviation from the mean. The standard deviations in this plot are about the same magnitude as those from the most polydisperse samples in Figure 4 2 Thus, th ere is no apparent improvement on the size distribution of microspheres using this method. During the emulsification processes undertaken to produce the se microspheres it was observed that the dispersed phase was not capable of mixing well with the 1 oct adecene continuous phase. In these cases, the dispersed phase settl ed and quickly localiz ed at the bottom of the container, even as the mixing rod of the m ixer/homogenizer was constantly being translated throughout. It is thought that there may be a thres hold in the viscosity difference between the two phases that would make a uniform emulsion. One way to study this in the future would be by comparing the size distributions of microspheres prepared using different continuous phases composed of a series of mineral oil/1 octadecene mixtures with

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47 different oil fractions. Another idea to study this would be to vary the viscosity of the dispersed phase by decreasing the alginate concentration. 4.1.3. Effect of Surfactant on the Size of Alginate Microspheres I n Chapter 2, a relation between surfactant concentrations ) and the surface tension of an emulsion was shown: ( 4 3 ) Equation 2 18 shows that surfactant concentration is inversely propor tional to surface tension, whereas E quation 4 3 shows that the relation between surface tension and microsphere size is directly proportional. By direct comparison of these equations it was hypothesized that higher surfactant concentrations would result i n the production of smaller microspheres. This section presents the results of microsphere samples made using different surfactant concentrations. The conditions that we re kept fixed are listed in Table 4 3 and are representative of those used to make a m icrosphere sample in section 4.1.1 that gives a relatively uniform size distribution ( Figure 4 1 teal colored histogram). The HLB of the surfactant blend has been shown in Chapter 2 as: ( 4 4 ) Since the surfactants that are used, Span 80 and Tween 80, have about the same density, their mass fractions are approximately equal to their respective volume fractions, such that

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48 Figure 4 5 shows the size distributions of micr osphere samples made using different surfactant concentrations In order to manipulate the surfactant concentration exclusively, the HLB was kept constant at 7.22. This was done for the first three microsphere samples by keeping the volume fraction proport ion of Tween 80 to Span 80 constant. Even though microsphere size is seen to increase slightly with decreasing surfactant concentrations, more samples may be required to notice a definite trend. The last microsphere sample was made by changing the HLB val ue to 6. The more uniform size distribution for this sample ( Figure 4 5 blue histogram) is consistent with the required HLB value for a water in oil emulsion of mineral oil (HLB 6). 4.2. Effect of Iron Oxide Content on the Size and Magnetization of Micros pheres The following experiments make use of the most optimal conditions found to make alginate magnetic microspheres in the current progress of the research. In such experiments, the magnetic properties and size distributions of magnetic alginate microsp heres are investigated with respect to their different magnetic nanoparticle loadings. SQUID measurements were complemented with TGA data. The effect of iron oxide content on pertinent properties of magnetic microspheres was investigated. The conditions t hat we re kept fixed are listed in Table 4 4 and are representative of those used to make a microsphere sample in section 4.1.1 that gives a narrow size distribution ( Figure 4 1 gold colored histogram). Up to this point in the discussion it has been known that 16 mg/mL of the stock ferrofluid were used to mix with the alginate solution in a 1:1 volume ratio to make the dispersed phase of the emulsion. In order to study the effects of iron oxide content in this section, however, the concentration of this fer rofluid was varied for making each microsphere sample to the tabulated values in the first column of Table 4 5

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49 From these different ferrofluid concentrations used to make the dispersed phase we calculated their corresponding iron oxide volume fractions. S uch values have been referred to in this thesis as theoretical magnetic volume fract ions The se values are convenient for comparison with experimental results because they represent the volume fractions of iron oxide in the alginate microspheres assu ming all iron oxide used is incorporated in them The last row in this table concerns to an analysis done for the ferrofluid used to make the microspheres. Figure 4 6 shows histograms for the size distribution of the microsphere experiments listed above in order of increasing theoretical magnetic volume fraction Even though there appears to be no evident trend in size progression, the SEM images on the Appendix corresponding to these size distributions ( Figure B 5) show a perceivable increase in agglomeration with increasing ferrofluid concentration used to make the dispersed phase This increase in agglomeration makes it progressively harder to measure circular particles and thus introduces a sampling bias to select and measure these particular structures in more agglomerated microsphere experiments, a bias which becomes well noticed on the bottom sample (i.e. the most agglomerated of all the experiments) as it shows a significantly narrow distribution. Figure 4 7 shows the magnetization of these microsphere experiments obtained from SQUID and normalized by their corresponding total sample masses. In this figure the saturation magnetization increases with from 12% to 30%. After the sample with there appears to be a threshold between this sample and the sample with

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50 where the magnetizat ion signal ceases to increase appreciably, and starts to decrease at higher concentrations. A close comparison of the magnetic properties of the microsphere experiments in Table 4 5 is consistent with this argument. In this table it can be seen that the microsphere sample with comes in close proximity with its actual magnetic volume fraction value obtained from a regression algorithm of the SQUID data, which gives about 28%. In turn, samples with higher theoretical volume fractions exhibit a noticeable departure between their theoretical and actual magnetic volume fractions where their actual volume fractions are significantly lower. These results appear to be indicative of not being able to load more magnetic nanoparticles inside the alginate microspher es made at the current conditions being employed. In order to increase the iron oxide content inside the microspheres it may be necessary to change some variables in the process, such as reducing the amount of alginate used for the dispersed phase. The TG A measurements made for the microsphere experiments are presented in Table 4 5 and show no apparent trend between the iron oxide mass fractions in microspheres and increased ferrofluid loading. Since several mounted samples had initial weights of less than 10 mg, they may not have given accurate mass fraction measurements

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51 Table 4 1. Fixed emulsion and process conditions for microspheres made employing different shear rates for emulsion formation. The continuous phase was mineral oil. Condition Value ecc entricity ) 0.0791 capillary number ( 0.0673 concentration in stock ferrofluid 16 mg/mL continuous phase viscosity ( 56 cP dispersed phase viscosity ( 885 cP surface tension 3.2346 dyn/cm HLB 7.22 Table 4 2. Fixed emulsion and process con ditions for microspheres made employing different shear rates for emulsion formation. The continuous phase was 1 octadecene. Condition Value eccentricity ) 0.0061 capillary number ( 0.0051 concentration in stock ferrofluid 16 mg/mL continuous phas e viscosity ( 4.32 cP dispersed phase viscosity ( 885 cP surface tension 20.4123 dyn/cm HLB 7.22 Table 4 3. Fixed emulsion and process conditions for microspheres made using different surfactant amounts in the emulsion. Condition Value eccentricity ) 0.0791 capillary number ( 0.0673 concentration in stock ferrofluid 16 mg/mL continuous phase viscosity ( 56 cP dispersed phase viscosity ( 885 cP shear rate 4700 rad/s

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52 Table 4 4. Fixed emulsion and process co nditions for microspheres made with different ferrofluid concentrations used to make the dispersed phase. Condition Value eccentricity ) 0.0791 capillary number ( 0.0673 continuous phase viscosity ( 56 cP dispersed phase viscosity ( 885 cP surface tension 3.2346 dyn/cm shear rate 9400 rad/s HLB 7.22 Table 4 5. Magnetic properties measurements of microspheres made using different ferrofluid concentrations to make the dispersed phase (first row). The last entry summarizes the magnetic properties analysis for the ferrofluid used to make the microspheres. concentration in stock ferrofluid, mg/mL theoretical magnetic volume fraction magnetic mass fraction in microspheres magnetic volume fraction magnetic diamet er ( ), nm standard deviation ( ) GIVEN CALCULATED TGA SQUID SQUID SQUID 16 12% 31.9% 7. 7 0. 1 % 8.6 0. 1 0.30 0.02 50 30% 21.3% 27.7 0.3 % 8. 5 0. 1 0.31 0.02 100 45% 31.5% 24.9 0.2 % 8.4 0. 1 0.30 0.0 2 200 62% 22.4% 20.7 0.2 % 8.5 0. 1 0.30 0.02 300 71% 15.3% 10.6 0.1 % 8. 6 0. 1 0.30 0.02 (pure ferrofluid) 100% N/A 43.7 0.5 % 8. 3 0. 1 0.31 0.02

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53 Figure 4 1. Size distribution histograms of microspheres made employing different sh ear rates for emulsion formation. The continuous phase was mineral oil.

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54 Figure 4 2. Effect of shear rate on microsphere size: correlation of experimental data with theoretical prediction. The continuous phase was mineral oil.

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55 Figure 4 3. Size distribu tion histograms of microspheres made employing different shear rates for emulsion formation. The continuous phase was 1 octadecene.

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56 Figure 4 4. Effect of shear rate on microsphere size: no correlation observed between experimental data and theory. The continuous phase was 1 octadecene.

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57 Figure 4 5. Size distribution histograms of microspheres made using different surfactant amounts in the emulsion.

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58 Figure 4 6. Size distribution histograms of microspheres made using different ferrofluid concentrations to make the dispersed phase.

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59 Figure 4 7. Magnetizations with respect to applied magnetic fields for microspheres made using different ferrofluid concentrations and for the pure ferrofluid used. Each magnetization has been normalized wit h respect to the total mass of its corresponding sample used for SQUID measurements.

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60 CHAPTER 5 CONCLUSION In t his investigation an approach has been developed to prepare magnetic alginate microspheres with controllable sizes in the micron to sub micron range by an emulsion based comminu tion method. The studies that investigated the effect of shear rate on the size of showed that the theor etical fit came in close agreement with experimental values when mineral oil was used as the continuous phase. In c ontrast, when 1 octadecene was used as the continuous phase the relation between theory and experimental values was not met. It was concluded that this was the result of a viscosity mismatch between the dispersed and continuous phases. It was suggested tha t either increasing the continuous phase viscosity or decreasing the dispersed phase viscosity may resolve this issue in the future. A study that investigated the effect of surfactant quantities on the size of microspheres was made under the assumption th at the microsphere size would increase with decreasing surfactant concentrations. In the study this relation seemed to be met, but it was concluded that more samples would be needed to prove a definite trend. T he microsphere samples that are used to test t he effect of iron oxide content with respect to size and magnetic properties show that microsphere size s remains relatively constant as ferrofluid loading is increased, though agglomeration is seen to increase at high ferrofluid concentrations.

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61 The magne tization values from these samples determined from SQUID suggest there may be excess iron oxide inside the microspheres of samples that were made with higher ferrofluid concentrations in the dispersed phase. In order to increase iron oxide content inside t he microspheres it is suggested to change certain parameters that would affect the amount of ferrofluid that can be loaded inside the microspheres such as the amount of alginate used for the dispersed phase. Finally, TGA measurements showed no trend bet ween measured iron oxide mass fraction in the microspheres and ferrofluid concentration used to make the dispersed phase. However, this may be the case because the initial sample weights mounted for TGA were low.

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62 APPENDIX A CALCULATION OF SURFACE TENSION S AND MIXER PARAMETERS As mentioned in Chapters 2.4 and Chapter 3, two different types of rotor stator mixers were used to make the alginate microspheres. The Fisher Scientific PowerGen 125 homogenizer will be hereinto referred as mixer H (Figure A 1 A) w hile the Silverson laboratory mixer will be hereinto referred as mixer M (Figure A 1 B) Both pieces of equipment produce shear rates that can be calculated by: ( A 1 ) The stator ( ) and rotor radii for mixer H were measured through two methods: 1) Using a Vernier caliper : The diameter of the inner cylinder of the stator of mixer M was measured to be 4 mm, and the outer diameter of its rotor was approximately 3.50 mm. 2) Using ImageJ: The high resolution image shown below was cropped and scaled with ImageJ, setting the scale by declaring the aforementioned stator diameter to be 4 mm by a method comparable to the scale setting methods described in section 3.5.2. After scaling the image, the rotor diameter was measured with the Analyze/Measure feature to be 3.563 mm (Figure A 2) The radii from mixer M were measured in a different manner. After the Ri and R radii for mixer H were measu red, E quations A 1 and 2 10 were used to solve for the such that:

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63 (A 2 ) Where is the average microparticl e radius. experiment with the most narrow size dispersion were used to solve for the abovementioned surface tension, since the average size of such sample would deviate the least. In the case of the current experiments, the samples made with the higher shear rates had more narrow size distributions. Since the stator radius of mixer M was large enough to be measured by ruler, its rotor radius was calculated by Excel solver where the objective function to be minimized (i.e. set to approach 0) was: (A 3 ) Where and were the su rface tensions calculated for the experiments with the most statistically narrow size distributions using mixers H and M, respectively. Since J approaches 0, the surface tension of the emulsion has been estimated to be equal to the calculated

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64 Figure A 1. Images of equipment used to mak e emulsions : a Fisher Scientific PowerGen 125 homogenizer mixer H A ) and a Silverson L5M A laboratory mixer mixer M B) Figure A 2. Screenshot showing the approach taken to measure mixer H through the Analyze/Measure feature of ImageJ. A ) B )

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65 APPENDIX B SEM IMAGES OF MICROSPHERE SAMPLES In the Results section (Chapter 4) we had determined the size distributions of different magnetic alginate microsphere samples by making manual size measurements of about 100 microspheres per sample from taken SEM images through ImageJ, as explained in the Methods section (Chapter 3). Herein a compilation of SEM images are shown for simple qualitative inspection that shows how the size and morphology may be affected by the different variables being manipulated in each study that was discussed in Chapter 4.

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66 Figure B 1 SEM image for a magnetic alginate microsphere sample made under optimized conditions that yield microspheres with relatively narrow size distributions and reasonable microsphere diameters close to a micron. Made using 16 mg/mL of the stock ferrofluid and following the conditions in Table 4 4.

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67 Figure B 2. SEM images of magnetic microspheres made by employing different shear rates and using mineral oil as t he continuous phase (fixed conditions listed in Table 4 1) All images share the same scale bar in the last image. The scale

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68 Figure B 3. SEM images of magnetic microspheres made by employing different shear rates and using 1 octadecene as the continuous phase (fixed conditions listed in Table 4 2) All images share the same scale bar in the last image. The

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69 Figure B 4. SEM images of magnetic microspheres made by changing the amount of Span 80 ( ) and Tween 80 ( ) in the pre microsphere emulsion. As explained in t he methods (3.4 ) and are a water or oil solution used to mix a particular surfactant and its corresponding volume fraction in this solution, respectively (fixed conditions listed in Table 4 3) All images share the same scale bar in the

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70 Figure B 5. SEM images of magnetic microspheres made by using increasing concentrations of ferrofuid to make the dispersed phase Samples were represented by their theoretical volume fractions which dictat e the volume fraction s of iro n oxide in the microspheres for case s where all iron oxide provided is loaded inside the microspheres Such values are practical for comparison with SQUID measurements (fixed conditions listed in Table 4 4 ) All images share th e same scale bar in the last image. The scale bar length is

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76 BIOGRAPHICAL SKETCH Andrew Garcia received his Bachelo r of Science degree in c hemistry from the University of Miami in 2011. In the fall of 2012, Andrew enrolled at University of Florida to obtain an engineering degree. In D ecember of 2013, he joined Dr. Carlos Rinaldi's group to pursue a thesis based Master of Science degree in c hemical e ngineering.