Citation
Development of a Bioactive Coating for Platinum Endovascular Coils for the Treatment of Cerebral Aneurysms

Material Information

Title:
Development of a Bioactive Coating for Platinum Endovascular Coils for the Treatment of Cerebral Aneurysms
Creator:
Fernandez, Cristina
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (61 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Materials Science and Engineering
Committee Chair:
Batich, Christopher D.
Committee Members:
Gower, Laurie B.
Goldberg, Eugene P.
Graduation Date:
8/7/2010

Subjects

Subjects / Keywords:
Aneurysms ( jstor )
Arteries ( jstor )
Intracranial aneurysm ( jstor )
Lumens ( jstor )
Magnification ( jstor )
Neck ( jstor )
Platinum ( jstor )
Smooth muscle ( jstor )
Subarachnoid hemorrhage ( jstor )
Thrombosis ( jstor )
Materials Science and Engineering -- Dissertations, Academic -- UF
aneurysm, coils, endovascular, hemorrhage, plga, subarachnoid
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
Materials Science and Engineering thesis, M.S.

Notes

Abstract:
Subarachnoid hemorrhage (SAH), the rupture of a cerebral aneurysm, is the most prevalent form of stroke. Treatment options for cerebral aneurysms aim to prevent future rupture by isolating the aneurysm orifice from the parent artery. Endovascular treatment with platinum coils has become a popular and less invasive alternative to surgical clipping in the past 20 years. Thrombus formed around the coil serves as a scaffold for the formation of fibrous tissue toward the periphery of the aneurysm lumen, ultimately leading to coverage of the aneurysm neck with endothelial tissue. Increasing the efficiency of this healing process is of great interest to avoid unwanted growth of the aneurysm due to blood flow. The purpose of this preliminary study is to identify proteins to be used in a bioactive coating for platinum endovascular coils that will increase the efficiency of fibrous tissue organization and endothelialization in the aneurysm lumen. Platinum endovascular coils were treated with a 50/50 poly (DL-lactide-co-glycolic acid) (PLGA) coating containing proteins such as Connective Tissue Growth Factor (CTGF), Bone Morphogenic Protein-4 (BMP-4), Monocyte Chemoattractant Protein-1 (MCP-1), or Stromal Cell-Derived Factor-1 (SDF-1). Protein release into the aneurysm lumen occurred through initial protein release from hydrophilic channels in the PLGA followed by PLGA degradation. Coatings were characterized by scanning electron microscopy and an in vitro release study, and a murine aneurysm model was employed to characterize the cellular response in vivo. SDF-1, CTGF, and MCP-1 showed favorable results in vivo, with an increase in new smooth muscle cells developing around the aneurysm lumen near the site of the coil over the course of three weeks. A long-term goal of this project to be completed in future studies is to optimize the dosage and delivery of a protein or combination of proteins that would increase the efficiency of the wound-healing response after implantation of endovascular coils. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
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, 2010.
Local:
Adviser: Batich, Christopher D.
Statement of Responsibility:
by Cristina Fernandez.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
10/8/2010
Resource Identifier:
004979971 ( ALEPH )
707467070 ( OCLC )
Classification:
LD1780 2010 ( lcc )

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occlusion percentage. HES is composed of a platinum coil coated with a hydrogel that

swells to three times its original diameter in contact with blood and stops swelling upon

contact with the aneurysm wall. In an in vitro model of a ruptured intracranial aneurysm,

HES eliminated gaps within the aneurysm lumen without inciting changes in aneurysm

size61. HES received a CE mark and FDA 510(k) clearance in 2002. A clinical study

comparing HydroCoil with bare platinum coils found the mean volume percentage

occlusion with HydroCoils to be 70.7%16. An animal study comparing several second-

generation endovascular devices, including HES, found greater occlusion at

angiography, however, the level of organization of fibrous tissue around the aneurysm

necks was similar for all devices tested28.

Controlled Release of Proteins

The FDA has cleared lactic and glycolic acid copolymers (PLGAs) for use in

several pharmaceutical products or medical devices50. PLGA has gained popularity for

use in medical devices due to its excellent biodegradability, mechanical strength, and

biocompatibility25. In body fluids, PLGA undergoes hydrolysis of its ester backbone and

degrades back to lactic and glycolic acid monomers. These monomers are metabolized

and eliminated in vivo as carbon dioxide and water in the Krebs cycle52

PLGA microspheres have been widely studied for applications involving controlled

release of drugs or proteins55. Proteins and peptides may be encapsulated into

microspheres using a water-in-oil-in-water emulsion and solvent evaporation technique,

in which the hydrophilic protein is protected by a hydrophobic polymer layer63. Protein

release from microspheres occurs through two mechanisms: pore diffusion of the

protein through hydrophilic channels, and PLGA degradation. Protein release profiles

are characterized by an initial burst followed by a lag phase, followed by increased









of platinum endovascular coils with proteins38, degradable polymers35, and hydrogels37,

to name a few.

The purpose of this study was to develop a bioactive coating for platinum

endovascular coils that would increase the efficiency of the fibrous tissue organization

of coiled aneurysms. Several objectives were followed in the development of this

bioactive coating:

1. Identify an effective vehicle for controlled release of protein and an

effective coating method for platinum endovascular coils. A water-in-oil

emulsion of 50/50 poly(D,L-lactic-co-glycolic acid) (PLGA) was tested as a

vehicle for protein release. Airbrushing and dip coating were explored as

possible coating application techniques.

2. Characterize the release rate of protein in vitro from a coating for platinum

endovascular coils. A Bradford Protein Assay was used to quantify the

soluble concentration of protein released from coatings.

3. Identify effective proteins that would increase the efficiency of the

recruitment and organization of smooth muscle cells in the aneurysm

lumen and characterize their response in vivo. Bone Morphogenic

Protein-4 (BMP-4), Connective Tissue Growth Factor (CTGF), Stromal

Cell-Derived Factor-1 (SDF-1), and Monocyte Chemoattractant Protein-1

(MCP-1) were released individually through the coatings and tested in a

murine aneurysm model. Their results were examined qualitatively

through immunohistochemical (IHC) staining of 5 pm cross sections of ex

vivo aneurysms.









from the nozzle of the airbrush make it difficult to see how much is accumulating on the

surface of the coil.

Figure 4-3 shows a coil that has been dip-coated with PLGA and SDF-1 at 20X

magnification (A), 50X magnification (B), 100X magnification (C), 200X magnification

(D), 700X magnification (E), and 2000X magnification (F). Platinum coils in these

images were coated using PLGA Coating 2. These images show a porous coating that

tends to be sparser towards the middle of the coils and agglomerate at one end. This is

most likely due to the manner in which the coils are coated with PLGA. Since the coils

are dip-coated, it is likely that during the coating process the PLGA solution would drip

towards the opposite end of the coil and accumulate at the end.

Figure 4-3 (D) and (E) show that the coating is uneven in some parts of the coil.

This is likely due to the fact that the coils are laid flat to dry. The areas where the coil

lays flat against the surface of the Petri dish on which it dries will have less PLGA

coating. Figure 4-3 (E) shows the uneven coverage of the PLGA coating in more detail.

Figure 4-3 (F) shows that the surface morphology is rugged and porous. The lumps in

the coating could be attributed to the magnesium hydroxide that is added to the PLGA

solution as a neutralizing agent. Although these coatings cover the surface of the coil

unevenly, they are suitable for preliminary testing purposes. Future testing may require

more precise coating methods.

Coils were examined ex vivo prior to treatment with paraformaldehyde. Figure 4-4

depicts ex vivo coils at 20X magnification (A), 150X magnification (B), and 200X

magnification (C). The coils in these images had been coated with PLGA Coating 2.

The coil appears thinner than the other coils in Figures 4-1, 4-2, and 4-3 because the









CHAPTER 4
RESULTS AND DISCUSSION

Protein Release Measurements

Bovine serum albumin (BSA) was used as a model protein to determine the

protein release rate from the PLGA and PEC coatings. BSA was chosen for its cost-

effectiveness, as well as because it is the standard for the Bradford assay, therefore the

absorbance of the unknown samples would more closely match that of the standard.

The release rate of BSA from each coating was intended to serve as a model for protein

release.

Ultimately the PEC method coatings were discontinued from testing due to

concerns regarding the solubility of the alginate. Alginate has been attempted as an

embolic agent for cerebral aneurysms; however, the water-soluble nature of the alginate

causes escape of the embolic agent into the parent artery circulation46. Furthermore,

Koji Hosaka in the Department of Neurosurgery at UF attempted unsuccessfully to inject

alginate into a murine aneurysm as an embolic agent and experienced significant

difficulty maintaining the alginate inside the aneurysm lumen due to the water solubility

of the alginate. The PEC coatings were discontinued due to these concerns.

Bradford assays for the coils coated with the PEC method PLGA Coating 1 were

inconclusive, since the absorbance readings for the unknown samples were extremely

close to those for the 0 tg/mL standard, suggesting that the concentration of BSA

released from each coating was on the order of nanograms or picograms. Since the

tested proteins were only available in 10tlg/mL concentrations due to budget concerns,

the BSA was diluted to this concentration in order to maintain uniformity with the rest of

the protein coatings. Although an in vitro release rate could not be determined for














































E
Figure 4-6. H&E Staining of Ex Vivo Arteries with Platinum Coils First Trial. A)
Uncoated coils, B) Coils coated only with PLGA, C) BMP-4 D) CTGF, and E)
SDF-1.


C
,









neck. The stainless steel delivery wire is advanced through a microcatheter into the

lumen of the aneurysm, positioning the platinum coil inside the aneurysm17'19. Figure 2-

1 depicts a cartoon diagram of the positioning of the GDC inside an aneurysm.

A positive direct electric current of 0.5 to 2 mA is applied to the delivery guide wire,

dissolving it through electrolysis and deploying the platinum coil into the aneurysm. An

electrode placed at the patient's groin connects the negative ground pole.

Electrothrombosis is initiated when the positive current is applied, forming a thrombus in

approximately 4 to 12 minutes. Several coils may be deployed to fill the aneurysm

depending on its size and shape. Systemic heparinization is reversed at the end of the

procedure using protamine sulfate. Angiograms are used post-embolization to

determine the level of thrombosis and the placement of the coils 17,19

Thrombus Formation and Healing Process

The progression of thrombosis over time occurs because more blood particles

become entrapped within the coil network in the hours subsequent to the coiling

procedure, and because clot formation within the coils increases after systemic

heparinization is reversed19. GDCs serve as a scaffold by which the thrombus is held

together, allowing it to grow toward the interior of the aneurysm lumen and eventually

seal off the aneurysm neck from the parent artery circulation17

Thrombus formation in aneurysms treated with detachable coils occurs similarly to

that of the wound healing response after tissue injury although the process is delayed

and occasionally incomplete6. A primary difference between normal tissue injury and

thrombus formation in aneurysms is that the aneurysm represents a spatial defect with

a lack of stromal tissue6. Therefore, aneurysm healing occurs from the periphery of the

aneurysm lumen to the center.









The PLGA coatings incorporated proteins in a water in oil emulsion. One type of

protein was encapsulated in each coating sample, and various proteins were tested to

determine their effect on the development of new fibrous tissue and smooth muscle

cells. Proteins involved in the study included Monocyte Chemoattractant Protein-1

(MCP-1) (Sigma, St. Louis, MO), Stromal Cell-Derived Factor-1 (SDF-1) (R&D Systems,

Inc., Minneapolis, MN), Connective Tissue Growth Factor (CTGF) (PeproTech, Inc.,

Rocky Hill, NJ), and Bone Morphogenic Protein-4 (BMP-4) (PeproTech, Inc., Rocky Hill,

NJ). Proteins were reconstituted in Dulbecco's sterile Phosphate Buffered Saline (PBS)

solution without calcium and magnesium (Mediatech, Inc., Herndon, VA). Dr. Hoh's

research group in the UF Department of Neurosurgery provided all proteins.

Methods

Poly (D,L-lactide-co-glycolide) (PLGA) Coatings

PLGA Coating 1 for sutures and platinum coils

A coating solution composed of a water-in-oil emulsion was used to encapsulate

proteins in 50/50 PLGA. The oil phase of the emulsion was composed of 5 (w/v)%

50/50 amorphous PLGA dissolved in methylene chloride. The aqueous phase of the

emulsion was prepared by mixing 10 itg/mL of a given protein solution and 3(w/v)%

magnesium hydroxide in PBS. The magnesium hydroxide was used as neutralizing

agent to protect the protein stability from acidic byproducts of PLGA degradation, and

3(w/v)% magnesium hydroxide has been shown to yield greater protein release66. An

emulsion was created by mixing the aqueous phase with the oil phase in a 1:10 ratio

under an ice bath, forming a milky solution with small white agglomerations of protein.









prayers, and love. I would also like to thank my aunt, Lilian del Pozo, and my

grandmother, Manuela Mourin de Fernandez, for their love and support. Finally, I would

like to thank my parents, Antonio Fernandez and Maria Elena Pozo de Fernandez, for

raising me in an environment where I was nurtured and challenged to always improve

upon myself, and allowing me to experience countless opportunities that have made me

the person I am today. I cannot thank them enough for their continued and relentless

love, support, and encouragement in my education, without which I would not have

been able to accomplish my goals.













coil
/ .


-" fV


guide wire


Figure 2-1. Cartoon Diagram of Platinum Coil Insertion. The coil is inserted into the
aneurysm lumen through the guide wire. Multiple coils are typically required
to completely occlude an aneurysm.









ACKNOWLEDGMENTS

I would like to thank my committee members, Dr. Christopher Batich, Dr. Laurie

Gower, and Dr. Eugene Goldberg. In particular, I would like to thank Dr. Christopher

Batich for welcoming me into his research group, for his support, and for introducing me

to this project. I would like to thank Dr. Brian Hoh for his support, encouragement, and

professional insight, and Dr. Ed Scott for his collaboration. I would also like to thank the

members of the Hoh group in the Department of Neurosurgery at UF, Dr. Koji Hosaka,

Erin Wilmer, and Daniel Downes for their continued efforts and collaboration. I am

sincerely grateful to the members of the Batich group, particularly Titilayo Moloye, Pei

Yu "Pinky" Chung, Sam Popwell, and Brad Willenberg for their technical assistance,

support, and friendship.

I would like to thank the faculty and staff in the College of Engineering, particularly

Dr. Angela Lindner, Dr. Jonathan Earle, Jeff Citty, Margie Williams, and Yolanda

Hankerson for their support throughout my years at the University of Florida. I am

forever grateful for the skills that I have acquired during my years of service in student

organizations in the College of Engineering, and I thank them for providing our

organizations with the funding and administrative support possible to impact the lives of

so many students. I would also like to thank Prinda Wanakule and Chelsea Magin, who

have served as role models for me through the Society of Women Engineers and have

motivated me to test the boundaries of what I could accomplish.

I am extremely thankful for the love and support of the wonderful friends I have

made during my undergraduate and graduate studies at the University of Florida. I feel

truly blessed to be surrounded by such accomplished and motivated people. I would

like to thank my grandmother, Lilia Diaz de Pozo, for her countless words of wisdom,









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

DEVELOPMENT OF A BIOACTIVE COATING FOR PLATINUM ENDOVASCULAR
COILS FOR THE TREATMENT OF CEREBRAL ANEURYSMS

By

Cristina Elena Fernandez

August 2010

Chair: Christopher D. Batich
Major: Materials Science and Engineering

Subarachnoid hemorrhage (SAH), the rupture of a cerebral aneurysm, is the most

prevalent form of stroke. Treatment options for cerebral aneurysms aim to prevent

future rupture by isolating the aneurysm orifice from the parent artery. Endovascular

treatment with platinum coils has become a popular and less invasive alternative to

surgical clipping in the past 20 years. Thrombus formed around the coil serves as a

scaffold for the formation of fibrous tissue toward the periphery of the aneurysm lumen,

ultimately leading to coverage of the aneurysm neck with endothelial tissue. Increasing

the efficiency of this healing process is of great interest to avoid unwanted growth of the

aneurysm due to blood flow.

The purpose of this preliminary study is to identify proteins to be used in a

bioactive coating for platinum endovascular coils that will increase the efficiency of

fibrous tissue organization and endothelialization in the aneurysm lumen. Platinum

endovascular coils were treated with a 50/50 poly (DL-lactide-co-glycolic acid) (PLGA)

coating containing proteins such as Connective Tissue Growth Factor (CTGF), Bone

Morphogenic Protein-4 (BMP-4), Monocyte Chemoattractant Protein-1 (MCP-1), or

Stromal Cell-Derived Factor-1 (SDF-1). Protein release into the aneurysm lumen









CHAPTER 1
INTRODUCTION

Unruptured intracranial aneurysms are suspected to be present in 3.6 6.0% of

the population older than 30 years61. Subarachnoid hemorrhage (SAH), the rupture of

an intracranial aneurysm that causes bleeding into the subarachnoid space of the brain,

is the most common form of stroke27 and accounts for 25% of cerebrovascular deaths61.

The combined morbidity and mortality rate of SAH still reaches 60% despite the

availability of treatment options14. Furthermore, only 40% of SAH patients will recover

enough to regain their independence14. The frequency of SAH in western populations

has been reported between 6 and 8 per 100,000 person years29.

Previously, cerebral aneurysms were treated by performing a craniotomy and

placing a clip at the neck of the aneurysm to prevent blood flow into the aneurysm sac.

Less invasive endovascular treatments emerged in the 1970s with balloon

occlusion20'21. In the past 20 years, treatment with endovascular coils has become

popular, involving the deployment of a platinum coil via a guiding catheter inserted

through the femoral artery17'19. The coil is deployed via a small positive current that

causes electrothrombosis and forms a clot that will eventually organize into fibrous

tissue. The aneurysm is removed from circulation from the parent artery by the

eventual endothelialization of the aneurysm neck.

Recanalization, or growth of the aneurysm remnant, is a significant problem

associated with endovascular coiling45. Increasing the rate of endothelialization of the

aneurysm neck is thought to decrease the recanalization rate of coiled aneurysms.

Several groups have attempted to improve the recanalization rate through modifications





























A


















B
Figure 4-8. IHC Staining of Ex Vivo Arteries Treated with Platinum Coils. A) PLGA
coating with no protein and B) PLGA and SDF-1 coating. Cell nuclei are
depicted in blue and smooth muscle is depicted in red. Fluorescence
microscopy images are at 10X magnification.





















A B












C D












E F
Figure 4-3. SEM Images of Coated Platinum Coil. A) 20X magnification, B) 50X
magnification, C) 100X magnification, D) 200X magnification, E) 700X
magnification and F) 2000X magnification. The coating accumulates at one
end of the coil due to vertically dip-coating the coil in the PLGA solution. The
coating does not evenly cover the surface of the coil since it is laid flat to dry.









Modifications to Platinum Endovascular Coils

After bare platinum coils were introduced, several groups have attempted to

modify the surface or the coil technique in order to increase the efficiency of thrombus

formation. Coils have been coated with proteins, polymers, and hydrogels, among

others. The problem of recanalization has been addressed in two major ways: by

attempting to increase the rate of neointimal formation to seal the aneurysm neck, or

increasing the percentage of occlusion of the entire aneurysm volume.

Protein-Coated Coils

In 1999, Tamantani et al. compared the angiographic and histopathologic results

of collagen-coated platinum coils in canine aneurysms, and found that coils coated with

collagen promoted earlier formation of thrombus in the aneurysm lumen in conjunction

with a decline in recanalization rate of occluded aneurysms57. Dawson et al. compared

the results of collagen-coated coils and traditional platinum coils in swine, and found

that treatment of aneurysms with collagen-coated coils yielded a completely occluded

aneurysm with collagen-rich fibrous tissue with no evidence of recanalization2.

Murayama et al. coated platinum coils with type I collagen, fibronectin, vitronectin,

laminin, or fibrinogen using an ion implantation technique intended to retain the

mechanical properties of the coil and improve the adhesion of surface cells during

exposure to shear stresses and enzymes in the aneurysm lumen. Greater scar

formation was reported with type I collagen with new endothelium found at the

aneurysm orifice34

Hino et al. continuously administered Factor VIII, also known as the wound-healing

factor, intravenously for 5 days in swine treated with coil embolization and found

increased endothelialization at the aneurysmal orifice. Factor VIII aids in the formation









most significant predictors of a recurrence are treatment during the acute phase after

rupture, aneurysm size (greater than 10 mm diameter), neck width (greater than 4 mm),

unsatisfactory initial angiographic result and length of follow-up period. In this study,

46.9% of all recurrences were detected by 6 months, while 96.9% were detected by 36

months, indicating that the suggested 6 month follow-up period for angiograms is not

enough to accurately track patients45

The International Subarachnoid Aneurysm Trial (ISAT) was a multicenter,

randomized clinical trial that compared the effects of neurosurgical clipping with

endovascular treatment with platinum coils in patients with ruptured intracranial

aneurysms. This trial recruited and followed 2143 patients who were randomly

assigned to coiling or clipping treatment between the years of 1994 2002. After one

year, the risk of dependence or death was reduced by 22.6% for patients who

underwent endovascular treatment31. A long-term follow-up of the ISAT trial indicated

that rebleeding from coiled aneurysms tends to occur within 5 years of the initial

treatment, and found a significantly smaller death risk for patients treated with coils than

those treated with surgery32.

After the ISAT, endovascular obliteration became much more popular than

surgery. Aneurysm patients now tend to receive surgical procedures only if they are

unsuitable for endovascular treatment. An aneurysm may be unsuitable for

endovascular treatment due to its diameter, its neck size, abnormal intracranial

vasculature, thrombus in the aneurysm lumen or other thrombo-embolic issues,

rebleeding following initial endovascular treatment, and unsuccessful endovascular

treatment.









DEVELOPMENT OF A BIOACTIVE COATING FOR PLATINUM ENDOVASCULAR
COILS FOR THE TREATMENT OF CEREBRAL ANEURYSMS




















By

CRISTINA ELENA FERNANDEZ


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010









CHAPTER 6
FUTURE WORK

The data obtained from these preliminary studies show positive steps forward

toward the development of a bioactive coating for platinum endovascular coils. The

following studies should be performed in order to corroborate and expand upon the

results from this study.

1. Characterization of the release rate of PLGA Coating 2. PLGA Coating 2

uses a significantly higher initial protein concentration in the coating

emulsion than PLGA Coating 1, therefore, the amount of protein released

should be able to be detected with a Bradford assay. A timed-release study

tested at 0.5, 1, 3, 7, 14, and 21 days is necessary to model the release

rate of PLGA Coating 2.

2. Viability of proteins released. The Bradford Protein Assay measures the

concentration of soluble protein released from the coatings. It is of interest

to determine if the proteins released from the coatings are viable by

performing an Enzyme-Linked Immunosorbent Assay (ELISA) on each

protein released from the coatings.

3. Repetition of in vivo testing with CTGF, BMP-4, SDF-1, and MCP-1. These

studies should be repeated in order to corroborate the results from trials 1

and 2. Furthermore, further characterization of the cell types present in the

ex vivo cross-sections of treated aneurysms would be of interest.

4. Improvement of the uniformity of the PLGA Coating. SEM images of the

coils dip-coated with PLGA indicate that a more uniform coating is needed.









60. Vogt RR, Unda R, Yeh LC, et al: Bone morphogenic protein 4 enhances
vascular endothelial growth factor secretion by human retinal pigment epithelial
cells. J Cell Biochem 98:1196-1202, 2006

61. Wardlaw JM, White PM: The detection and management of unruptured
intracranial aneurysms. Brain 123:205-221, 2000

62. Watanabe K, Sugiu K, Tokunaga K, et al: Packing efficacy of hydrocoil embolic
system: in vitro study using ruptured aneurysm model. Neurosurgery Rev
30:127-130, 2007

63. Wong HM, Wang JJ, Wang CH: In vitro sustained release of human
immunoglobulin G from biodegradable microspheres. Ind Eng Chem Res
40:993-948, 2001

64. Yamaguchi J, Kusano KF, Masuo O, et al: Stromal cell-derived factor-1 effects
on ex vivo expanded endothelial progenitor cell recruitment for ischemic
neovascularization. Circulation 107:1322-1328, 2003

65. Zernecke A, Schober A, Bot I, et al: SDF-la/CXCR4 axis is instrumental in
neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ
Res 96:784-791, 2005

66. Zhu G, Schwendeman SP: Stabilization of proteins encapsulated in cylindrical
poly(lactide-co-glycolide) implants: mechanism of stabilization by basic additives.
Pharm Res 17:351-357, 2000

67. Zhu W, Tian Y, Zhou LF, et al: Development of a novel endothelial cell-seeded
endovascular stent for intracranial aneurysm therapy. J Biomed Mat Res Part A
85A:715-721, 2008









Polyelectrolyte complex coating technique

Preliminary coatings were also made using a polyelectrolyte complex (PEC)

composed of acetic acid, chitosan, and protein solution23. Sodium alginate powder was

used to prepare a 1.0 (w/v)% alginate solution in water, and chitosan powder was used

to create a 1.0 (w/v)% chitosan solution in acetic acid. Vicryl sutures were used to test

the PEC coatings. Vicryl sutures were cut into 4 mm sections and rinsed in 70%

ethanol in a Petri dish and allowed to dry as previously described. Sutures were dip-

coated in alginate solution for 15 seconds, dipped into a 10 [tg/mL BSA solution for 15

seconds, and then dipped in chitosan solution for 15 seconds. Sutures were allowed to

dry for several hours and were stored at 4 OC.

Protein Release Measurements

BSA was used as a sample protein to obtain an estimate of the release rate of

both PLGA Coatings 1 and 2 and the PEC coatings. Coated coil or suture samples

were placed in a 1.5 mL micro centrifuge tube containing 1 mL PBS. Samples were

placed in a hybridized incubator at 37 OC, and were tested at 1, 3, 7, 14, and 21 days

after placement in PBS. Separate samples were used for each time point, and n=3

samples of each type were tested each time.

The concentration of BSA released was measured using a Bradford protein assay,

in which BSA was used as a standard. Samples were prepared for the Bradford protein

assay by transferring 0.5 mL of PBS solution from their micro centrifuge tubes to a

cuvette and adding 0.5 mL of Coomassie protein assay reagent. Absorbance of the

samples was measured at 595 nm using a UV-2410PC spectrophotometer (Shimadzu).

Each sample was assayed four times, and the soluble BSA concentration released was









PLGA Coating 1 for sutures and platinum coils....................................... 30
PLGA Coating 2 for platinum coils ................... ............... 31
Coating technique ............... ....... ...... ......... ............... 31
Airbrush coating technique....................... .......................... 31
Polyelectrolyte complex coating technique ............... ....... ................ 32
Protein Release Measurements .......... ............. ......... ................. 32
Scanning Electron M icroscopy ............................. ............ .. ............... 33
In Vivo A na lyses ............................................................. .... ........ 33
M urine aneurysm m odel .................................................... .................. 33
Analysis of ex vivo samples ........... ............ ..................... ............... 34

4 RESULTS AND DISCUSSION .. ... ......................... ..... ............... 37

Protein Release Measurements........................ ..... ............................ 37
Scanning Electron Microscopy............... ............................... 38
Murine Aneurysm Model ............ ........ .......... ................ ............... 40
Vicryl Sutures ........................ ......... ........... 40
Platinum Coils ............................. ............... 41
Platinum coils with PLGA Coating 1 .......... ..... ................ 41
Platinum coils with PLGA Coating 2............... ......................... ......... 42

5 CO NCLUSIO NS .............. ...... .. ... ....... ....... ................. .......... 51

6 FUTURE W ORK ............. ...... .. .. .... ............. ......... ....... ........... 53

LIST O F REFERENCES ........... ................... ........................................... 55

B IO G RA P H IC A L S K ETC H ............. ...................................................... ............... 61
































To my parents


























A

















B
Figure 4-5. IHC Staining of Ex Vivo Arteries Treated with Sutures. A) Suture with only
PLGA coating, and B) Suture with PLGA and CTGF coating. Fluorescence
microscopy images at 10X magnification. DAPI was used to stain cell nuclei
blue and anti-CTGF was used to identify the presence of CTGF.









CHAPTER 2
BACKGROUND

Cerebral Aneurysms and Subarachnoid Hemorrhage (SAH)

Cerebral aneurysms occur because atherosclerosis or vessel wall injury caused

by trauma or inflammation increases the tendency of intradural walls to develop

aneurysms. Sac formation is initiated due to the local degeneration of the internal

elastic lamina of the blood vessel by haemodynamic stresses. Blood flow within this

newly formed aneurysmal sac causes enlargement, leading to potential rupture9

Risk Factors for Subarachnoid Hemorrhage

Juelva et al. deemed smoking and female gender as risk factors for aneurysm

growth27. In fact, 45-75% of SAH patients tend to be smokers, compared to 20-30% of

the general adult population. The number of cigarettes smoked daily had a greater

impact than the amount of years the patient had been a smoker and the age at which

they started. Cigarette smokers have an imbalance of elastase and al-anti trypsin,

which may contribute to aneurysm formation or SAH, because the increased elastase

activity in the artery wall coupled with hemodynamic stress may cause aneurysm

formation or increase the rate of aneurysm growth27.

Although aneurysm growth rate was not found to differ by gender, women with

aneurysms were found to be at a higher risk for the formation of new aneurysms than

men27. Estrogen has an inhibitory effect on aneurysm formation, therefore fluctuations

in estrogen, as well as the decreased collagen content of cerebral arteries after

menopause, may favor aneurysm formation or growth. Women smokers are further at

risk, since cigarette smoking has been shown to decrease estrogen levels27.









release when the polymer degrades63. In this study, this popular double emulsion

technique has been adapted for use as a coating for polymer coils as a water-in-oil

emulsion.

Proteins Released from Bioactive Coating

This study will examine the cellular response elicited by the release of four

proteins into a murine aneurysm lumen over the course of three weeks. Each protein

has been linked to angiogenesis or recruitment of smooth muscle cells. Proteins will be

evaluated for their ability to recruit new smooth muscle cells to the aneurysm lumen.

Monocyte Chemoattractant Protein-1

Monocyte Chemoattractant Protein-1 (MCP-1) is a member of the CC chemokine

family proven to activate monocytes, macrophages, and lymphocytes. MCP-1 has been

shown to promote the development of aneurysms, and is expressed in aneurysms

through endothelial cells and some smooth muscle cells5. Progenitor cell migration in

the brain after ischemic incidents is due in part to MCP-1, since MCP-1 protein is

expressed in ischemic cortex in murine stroke models. Furthermore, elevated levels of

MCP-1 were present in the cerebrospinal fluid of ischemic stroke patients49. Vascular

smooth muscle cells and fibroblasts have specific receptors for MCP-1, and MCP-1 has

induced the migration of smooth muscle cells in vitro. Collagen expression by

fibroblasts was enhanced in vitro due to the production of TGF- 3 produced by MCP-133.

Stromal Cell-Derived Factor-1

Stromal Cell-Derived Factor-1 (SDF-1) is a CXC chemokine whose receptor,

CXCR4, is expressed on the surface of endothelial progenitor cells, and plays an

important role in the regulation of proliferation, mobilization, and angiogenesis64. SDF-1

mediates the recruitment of smooth muscle cell progenitors from the bone marrow65.









Other risk factors for increased rate of aneurysm formation and SAH include

excessive alcohol consumption58, cocaine and amphetamine abuse40, oral

contraceptives26, and high cholesterol4.

Symptoms of SAH

The keynote symptom of SAH is a sudden onset of a robust headache. This

headache may appear in conjunction with one of the following: nausea or vomiting, brief

loss of consciousness, or cranial nerve palsies7. Some patients may experience a

sentinel bleed or warning leak, which is a milder headache lasting several days.

Sentinel bleeds typically occur between 2-8 weeks prior to SAH7.

Diagnosis of SAH

A non-contrast cranial computed tomography (CT) scan is first performed in a

patient presenting with symptoms of SAH. A negative CT scan requires a diagnostic

lumbar puncture for analysis of the cerebrospinal fluid7. A positive result from either of

these tests requires the patient to undergo further analysis. Catheter-based

angiography is typically performed to determine the size and shape of the ruptured

intracranial aneurysm. Magnetic Resonance Angiography (MRA) and dynamic spiral

CT angiography (CTA) may be performed if angiography is not possible due to time

constraints7. CTA is a noninvasive, outpatient procedure that may be used to detect

cerebral aneurysms less than 5 mm in diameter59.

Treatments for Cerebral Aneurysms and SAH

Aneurysm Clipping

Aneurysm clipping is performed with the goal of preventing further aneurysm

growth and rupture. A clip is surgically placed at the aneurysm neck in order to block

the flow of blood into the lumen of the aneurysm. Morbidity and mortality associated









25. Jain RA: The manufacturing techniques of various drug loaded biodegradable
poly(lactide-co-glycolide) (PLGA) devices. Biomat 21:2475-2490, 2000

26. Johnston SC, Colford JM Jr, Gress DR: Oral contraceptives and the risk of
subarachnoid hemorrhage: a meta-analysis. Neurology 51:411-418, 1998

27. Juelva S, Poussa K, Porras M: Factors affecting formation and growth of
intracranial aneurysms: A long-term follow-up study. Stroke 32:485-491, 2001

28. Killer M, Hauser T, Wenger A, et al: Comparison of experimental aneurysms
embolized with second-generation embolic devices and platinum coils. Acta
Neurochir 151:497-505, 2009

29. Linn FH, Rinkel GJ, Algra A, et al: Incidence of subarachnoid hemorrhage: Role
of region year and rate of CT scanning: A meta-analysis. Stroke 27:625-629,
1996

30. Mawad ME, Mawad JK, Cartwright J, et al: Long-term histopathologic changes in
canine aneurysms embolized with Guglielmi detachable coils. AJNR 6:7-13,
1995

31. Molyneux A, Kerr R, Birks J, et al: Risk of recurrent subarachnoid hemorrhage,
death, or dependence and standardized mortality ratios after clipping or coiling of
an intracranial aneurysm in the International Subarachnoid Aneurysm Trial
(ISAT): long-term follow-up. Lancet Neurol 8:427-433, 2009

32. Molyneux A, Kerr R, Stratton I, et al: International Subarachnoid Aneurysm Trial
(ISAT) of neurological clipping versus endovascular coiling in 2143 patients with
ruptured intracranial aneurysms: a randomised trial. Lancet 360:1267-1274,
2002

33. Mukaida N, Harada A, Matsushima K et al: Interleukin-8 (IL-8) and monocyte
chemoattractant and activating factor (mcaf/mcp-1), chemokines essentially
involved in inflammatory and immune reactions. Cytokine and Growth Factor
Reviews 9:9-23, 1998

34. Murayama Y, Suzuki Y, Vihuela F, et al: Development of a biologically active
Guglielmi detachable coil for the treatment of cerebral aneurysms. Part I: In vitro
study. AJNR 20:1986-1991, 1999

35. Murayama Y, Tateshima S, Gonzalez NR, et al: Matrix and bioabsorbable
polymeric coils accelerate healing of intracranial aneurysms: Long-term
experimental study. Stroke 34:2031-2037, 2003









occurred through initial protein release from hydrophilic channels in the PLGA followed

by PLGA degradation. Coatings were characterized by scanning electron microscopy

and an in vitro release study, and a murine aneurysm model was employed to

characterize the cellular response in vivo. SDF-1, CTGF, and MCP-1 showed favorable

results in vivo, with an increase in new smooth muscle cells developing around the

aneurysm lumen near the site of the coil over the course of three weeks. A long-term

goal of this project to be completed in future studies is to optimize the dosage and

delivery of a protein or combination of proteins that would increase the efficiency of the

wound-healing response after implantation of endovascular coils.
























A B















C
Figure 4-4. SEM Images of an Ex Vivo Coil. A) at 20X magnification, B) at 150X
magnification and C) at 200X magnification. The coil appears stretched due
to the manner in which it was removed from the artery cross-section. The coil
was coated with PLGA and SDF-1.









36. Murayama Y, Vihuela F, Duckwiler G, et al. Embolization of incidental cerebral
aneurysms by using the Guglielmi detachable coil system. J Neurosurg 90:207-
214, 1999

37. Murayama Y, Vihuela F, Ishii A, et al: Initial clinical experience with Matrix
detachable coils for the treatment of intracranial aneurysms. J Neurosurg
105:192-199, 2006

38. Murayama Y, Vihuela F, Suzuki Y, et al: Development of the biologically active
Guglielmi detachable coil for the treatment of cerebral aneurysms. Part II: An
experimental study in a swine aneurysm model. AJNR 20:1992-1999, 1999

39. Murayama Y, Vihuela F, Tateshima S, et al: Cellular responses of bioabsorbable
polymeric materials and Guglielmi detachable coil in experimental aneurysms.
Stroke 33:1120-1128, 2002

40. Oyesiku NM, Colohan AR, Barrow DL, et al: Cocaine-induced aneurysmal
rupture: an emergent factor in the natural history of intracranial aneurysms?
Neurosurgery 32:518-526, 1993

41. Pierot L, Leclerc X, Bonafe A, et al: Endovascular treatment of intracranial
aneurysms with Matrix detachable coils: midterm anatomic follow-up from a
prospective multicenter registry. AJNR 29:57-61, 2008

42. Pierot L, Leclerc X, Bonafe A, et al: Endovascular treatment of intracranial
aneurysms using Matrix coils: short- and mid-term results in ruptured and
unruptured aneurysms. Neurosurgery 63:850-858, 2008

43. Piton J, Billerey J, Constant P, et al: Selective vascular thrombosis induced by a
direct electrical current: animal experiments. J Neuroradiol 5:139-152, 1978

44. Raaymakers TW, Rinkel GJ, Limburg M, et al: Mortality and morbidity of surgery
for unruptured intracranial aneurysms: a meta-analysis. Stroke 29:1531-1538,
1998

45. Raymond J, Guilbert F, Weill A, et al: Long-term angiographic recurrences after
selective endovascular treatment of aneurysms with detachable coils. Stroke
34:1398-1403, 2003

46. Raymond J, Metcalfe A, Desfaits A, et al: Alginate for Endovascular Treatment of
Aneurysms and Local Growth Factor Delivery. AJNR 24:1214-1211, 2003

47. Roth C, Struffert T, Grunwald IQ, et al: Long-term results with Matrix coils vs.
GDC: an angiographic and histopathological comparison. Neuroradiol 50:693-
699,2008









Smooth muscle staining was performed by biotinylating the primary antibody.

Slides were blocked in Serum Free Blocking Solution (DAKOCytomation, Carpinteria,

CA), then avidin and biotin (Vector Labs, Burlingame, CA) for 15 minutes each. Slides

were washed between each step. Mouse anti-actin (Sigma, St.Louis, MO) was applied

for 20 minutes at room temperature using a dilution of 1:600. After washing twice for

five minutes, Streptavidin Alexa Fluor 594 (Molecular Probes, Eugene, OR) was applied

at 1:500 for 45 minutes as a detection agent.

Staining with Fibroblast-Specific Protein-1 (FSP-1) (Abcam, Cambridge, MA), at a

titer of 1:150, required 25-minute heat induced antigen retrieval with 10mM Citra buffer,

pH 6.0 for optimal staining. Slides were blocked in 2% horse serum for 30 minutes prior

to the application of primary antibody overnight at 4 C. Slides were incubated for 45

minutes in 1:500 Alexa Fluor 594 anti-rabbit raised in donkey (Molecular Probes) and

were washed twice for 5 minutes and mounted in VectaShield with 4',6-diamidino-2-

phenylindole (DAPI) prior to imaging. Positive control tissues and concentration-

matched Ig controls were included with each immunoassay.









Thrombus begins to organize approximately one week after aneurysm treatment

with platinum coils. A thin membrane may be found over the aneurysm neck, which will

continue to develop over the course of the next month. Fibrous tissue may be found

along the periphery of the aneurysm lumen after about 1-3 months, and will cover the

coils over the course of a year. New endothelium may be seen covering the aneurysm

neck after approximately one year after treatment with platinum coils2.

Mawad et. al studied the growth of neointima in dogs six months after aneurysm

obliteration with platinum coils and found neointima to be tightly adhered to coils and

covered with endothelium. The aneurysm lumen was filled with organized fibrous tissue

in three layers. The outer layer consisted of endothelium adjoining that of the parent

artery. The second layer contained organized smooth muscle cells, while the innermost

layer contained disorganized smooth muscle cells. Minimal foreign body reaction and

inflammation was noticed30.

Advantages of Endovascular Coiling

Coil embolization has several notable advantages over clipping; the most

paramount being that there is no need for craniotomy and brain manipulation.

Furthermore, the medical condition of the patient has less of an impact on the timing

and performance of the procedure, an important benefit, since it is critical to treat

ruptured aneurysms within 15 days of SAH, most favorably after 2 days10. Since

craniotomy is unnecessary, aneurysm location is of less importance, which is beneficial

to patients with aneurysms in the posterior region of the brain, which is difficult to reach

for clipping. Finally, endovascular coiling may be attempted in cases where clipping has

failed10'8.









in the aneurysms treated with Matrix coils, as the BPM was likely replaced by mature

scar tissue, which retracts in the wound-healing process25

Matrix Coils were Food and Drug Administration (FDA) approved in 2002, and

were the first commercially available bioactive coil for the treatment of cerebral

aneurysms41. Initial clinical trials with Matrix coils indicate moderate improvement in

the recanalization rate compared with bare platinum coils. However, increased friction

in the first-generation Matrix coils made them more difficult to insert, and the packing

density was less than of bare platinum coils. In theory, the strength of the organized

connective tissue produced by the Matrix coils is stronger than unorganized thrombus,

increasing its resistance against the mechanical forces that cause recanalization and

coil remodeling within the aneurysm37

A prospective multicenter registry conducted in France to evaluate the safety and

efficacy of Matrix coils found a recanalization rate of 25.7%, which increased if the

volume percentage of the aneurysm occluded was less than 25%41. Biologic activity

was demonstrated with the Matrix coils due to a 30% rate of progressive thrombosis at

mid-term follow-up42. Another study indicated that long-term results of treatment with

bare platinum coils compared to Matrix coils do not exhibit a difference in occlusion and

recanalization rates, while the increased friction of the Matrix coils adds to complications

with insertion and placement47

Hydrogel-Coated Coils

Platinum coils have been shown to reach a volume occlusion percentage of

approximately 25-33%, despite post-procedural angiography indicating complete

occlusion of the aneurysm57. The HydroCoil Embolic System (HES) is designed to

improve the rate of aneurysm recanalization by addressing the issue of volume



















"A. B










C D










E
Figure 4-7. H&E Staining of Ex Vivo Arteries with Platinum Coils Second Trial. A)
PLGA coating with no protein, B) SDF-1, C) MCP-1, D) BMP-4, E) CTGF.









of granulation tissue by acting as an enzyme to drive fibroblasts from surrounding

tissues to proliferate toward stabilized fibrin22. Zhu et al. coated a stainless steel stent

with a heparinized polymer and canine endothelial cells. The layer of endothelial cells

on the surface of the stent remained largely intact after 48 hours of a high shear stress

brushing test intended to simulate blood flow67. Abrahams et al. coated bare platinum

coils with Vascular Endothelial Growth Factor (VEGF) and found an increased

endothelialization response compared with uncoated coils. VEGF is a glycoprotein

produced by macrophages, endothelial cells, and smooth muscle cells that binds

heparin and has been shown to promote angiogenesis2.

Matrix Coils

Murayama et al. loosely packed swine experimental aneurysms with several types

of bioabsorbable polymer matrix (BPM), and observed a linear relationship between

collagen levels in the experimental aneurysms and the rate of polymer degradation.

The strongest inflammatory reaction was produced by 50/50 poly(lactic-co-glycolic acid)

(PLGA), which had the fastest degradation time. More organized collagen deposits

were found in the neck and lumen of aneurysms embolized with BPMs with a faster

degradation time, particularly compared to standard platinum coils39.

Matrix coils are platinum coils with a 50/50 PLGA coating designed to increase the

efficiency of thrombus organization in order to prevent aneurysm recanalization. Matrix

coils are composed of 70% BPM and 30% platinum35. Initial testing in swine found

more organized thrombus in aneurysms treated with Matrix coils after 14 days than with

bare platinum coils, however, results after 3 months were similar for both GDCs and

Matrix coils. Most importantly, a reduction in the size of the aneurysm sac was noticed









CHAPTER 5
CONCLUSIONS

Several coatings capable of releasing protein were examined, with a water-in-oil

emulsion composed of PLGA and protein showing the greatest promise. Chitosan and

alginate in a polyelectrolyte complex proved to be an unsuccessful technique due to the

solubility of the PEC coating posing problems with migration of the coating into the

parent artery. PLGA Coating 2 provided a greater initial concentration of protein in the

coating solution, and shows promise for use as a coating that would be most cost-

effective, requiring smaller initial protein concentrations.

Airbrushing the PLGA coatings into the coils proved to be an inefficient coating

method due to significant loss of the PLGA solution during the coating process

agglomeration of PLGA on the coil, and concerns regarding contamination of protein

solutions. Dip coating proved to be an effective method for coating the coils that would

maintain the sterility of the protein solution and allow the most control in the coating

process. Further optimization of the dip-coating method is required to ensure that the

coating dries evenly on the surface of the coils.

Release rates for the coatings were unable to be determined in vitro using the

Bradford Protein Assay, although it is likely that PLGA Coating 2 does release a

detectable amount of protein. Further testing is required to determine the in vitro

release rate of this coating. However, both PLGA Coating 1 and PLGA Coating 2 were

proven to release protein based on in vivo studies in a murine aneurysm model due to a

noticeable difference in the cellular response of ex vivo arteries treated with different

protein solutions in comparison with the control coatings.









LIST OF ABBREVIATIONS


pg Micrograms

C Degrees Celsius

BPM Bioabsorbable Polymer Matrix

BMP Bone Morphogenic Protein

BSA Bovine Serum Albumin

CTA Computed Tomography Angiography

CTGF Connective Tissue Growth Factor

DAPI 4'-6-diaminidino-2-phenylindole

ECM Extracellular Matrix

EPC Endothelial Progenitor Cell

FSP-1 Fibroblast Specific Protein-1

GDC Guglielmi Detachable Coil

HEMA 2-hydroxyethylmethacrylate

HES HydroCoil Embolic SystemTM

ISAT International Subarachnoid Hemorrhage Trial

kV kilovolts

mA Milliamperes

MCP-1 Monocyte Chemoattractant Protein 1

mL Milliliters

mm Millimeters

MRA Magnetic Resonance Angiography

PEC Polyelectrolyte complex

PFA Paraformaldehyde

PLGA Poly (D,L-lactide-co-glycolic acid)









The cellular response of murine aneurysms treated with BMP-4, CTGF, SDF-1,

and MCP-1 was tested and compared. CTGF, SDF-1, and MCP-1 elicited a favorable

response in vivo proven by H&E staining of ex vivo aneurysms indicating a greater

presence of smooth muscle cells than control coils. Smooth muscle actin staining of

aneurysms treated with SDF-1 showed a robust response of smooth muscle cells

around the periphery of the aneurysm lumen. This preliminary study proves that CTGF,

SDF-1, and MCP-1 should be explored further as possible proteins incorporated in a

bioactive PLGA coating for platinum endovascular coils.











Standard Curve at 595 nm


0.9
0.8 -
S0.7
S0.6
8 0.5 ---------
S 0.4

S0.3 y = 0.0232x + 0.3423
S0.2
0.1


0 5 10 15 20 25

Concentration of BSA (plg/mL)

Figure 3-1. Standard Curve at 595 Nanometers. Known concentrations of BSA were
used to construct a standard curve based on absorbance at 595 nm.









LIST OF REFERENCES


1. Abrahams JM, Diamond SL, Hurst RW, et al: Topic Review: Surface
modifications enhancing biological activity of Guglielmi detachable coils in
treating intracranial aneurysms. Surg Neurol 54:34-41, 2000

2. Abrahams JM, Forman MS, Grady MS, et al: Delivery of human vascular
endothelial growth factor with platinum coils enhances wall thickening and coil
impregnation in a rat aneurysm model. AJNR 22:1410-1417, 2001

3. Abreu JG, Ketpura NI, Reversade B, et al. Connective-tissue growth factor
(CTGF) modulates cell signaling by BMP and TGF-P. Nat Cell Bio 4:599-604,
2002

4. Adamson J, Humphries SE, Ostergaard JR, et al: Are cerebral aneurysms
atherosclerotic? Stroke 25:963-966, 1994

5. Aoki T, Kataoka H, Ishibashi R, et al: Impact of monocyte chemoattractant
protein-1 deficiency on cerebral aneurysm formation. Stroke 40:942-951, 2009

6. Bavinzski G, Talazoglu V, Killer M, et al: Gross and microscopic histopathological
findings in aneurysms of the human brain treated with Guglielmi detachable coils.
J Neurosurg 91:284-293, 1999

7. Bederson JB, Connolly ES Jr, Batjer HH, et al: Guidelines for the management of
aneurysmal subarachnoid hemorrhage: A statement for healthcare professionals
from a special writing group of the Stroke Council, American Heart Association.
Stroke 40:994-1025, 2009

8. Bendszus M, Bartsch A, Solymosi L: Endovascular occlusion of aneurysms using
a new bioactive coil: A matched pair analysis with bare platinum coils. Stroke
38:2855-2857, 2007

9. Byrne JV, Guglielmi G. Endovascular Treatment of Intracranial Aneurysms.
Berlin; New York: Springer, 1998

10. Byrne JV, Sohn MJ, Molyneux AJ: Five-year experience in using coil
embolization for ruptured intracranial aneurysms: outcomes and incidence of late
rebleeding. J Neurosurg 90: 656-663, 1999

11. Choudhari KA, Ramachandran MS, McCarron MO, et al: Aneurysms unsuitable
for endovascular intervention: Surgical outcome and management challenges
over a 5-year period following International Subarachnoid Haemorrhage Trial
(ISAT). Clinical Neurology and Neurosurgery 109:868-875, 2007

12. Dawson RC, Krisht AF, Barrow DL, et al: Treatment of experimental aneurysms
using collagen-coated microcoils. Neurosurgery 36:133-140, 1995









determined by comparing the absorbance of a sample against a standard curve created

with BSA. The standard curve was constructed by drawing a line of best fit between

the absorbance values of BSA standards of known concentrations. Unknown sample

concentrations were determined using the equation for the line of best fit, where y

indicated sample absorbance and x indicated sample concentration. Figure 3-1 shows

a sample standard curve used.

Scanning Electron Microscopy

The surface morphology of uncoated coils, airbrushed coils, dip-coated PLGA

coils, and ex-vivo dip-coated PLGA coils was examined using a JEOL SM-31010 field

emission scanning electron microscope at 15 kV. Samples were attached to a stub

using double adhesive tape and sputter coated with carbon. SEM images were used to

qualitatively compare the morphology of two coating techniques, as well as to analyze

the progression of the PLGA coating morphology prior to implantation in a murine

aneurysm model and ex-vivo.

In Vivo Analyses

Murine aneurysm model

The cellular response of the sutures and coils was analyzed in vivo using a murine

aneurysm model. In vivo analyses were performed in collaboration with Koji Hosaka,

Erin Wilmer, and Daniel Downes in the Department of Neurosurgery at the University of

Florida. Briefly, the right common carotid artery (RCCA) of a mouse was bathed in 25

mg/mL of diluted and filter sterilized porcine pancreatic elastase for 20 minutes, causing

the artery to swell. The distal end of the artery was cauterized to occlude blood flow,

creating an aneurysm. Animals were closed up, allowed to regain consciousness and

become ambulatory, and were monitored for any post-surgical complications or distress.









CHAPTER 3
MATERIALS AND METHODS

Materials

Coatings

Coatings were primarily composed of ester terminated (nominal) 50/50 Poly (D,L-

lactide-co-glycolide) (PLGA) in hexafluoroisopropanol (HFIP) with an inherent viscosity

of 0.61 dL/g (Lactel Absorbable Polymers, Pelham, AL). PLGA was dissolved in

methylene chloride (Fisher Scientific, Pittsburgh, PA). Anhydrous 95.0% magnesium

hydroxide was used as a neutralizing agent (Sigma Aldrich, St. Louis, MO).

Vicryl Sutures and Endovascular Coils

Dr. Brian L. Hoh in the Department of Neurosurgery at UF provided endovascular

coils and Vicryl sutures for all experiments. Sterile Polyglactin 910 coated Vicryl violet

braided sutures (Ethicon, Inc., Somerville, NJ) were used for preliminary testing of the

coatings. TruFill DCS OrbitTM Detachable Coils (Cordis Neurovascular, Miami, FL)

were used to test the coatings in subsequent experiments.

Proteins

A Coomassie (Bradford) Protein Assay Kit (Pierce, Rockford, IL) was used for

protein release rate measurements of the coatings. Coomassie (Bradford) Protein

Assay Reagent was composed of G-250 dye, methanol, phosphoric acid, and

solubilizing agents in water. The kit also contained Bovine Serum Albumin (BSA)

standard ampoules with a concentration of 2 mg/mL in a solution of 0.9% saline and

0.05% sodium azide. BSA was used as a standard for the Bradford assays as well as

in control experiments as a sample protein to model the in vitro release rate of the

PLGA coatings.

































2010 Cristina Elena Fernandez









LIST OF FIGURES


Figure page

2-1 Cartoon Diagram of Platinum Coil Insertion. ..................................... ...... ........ 28

3-1 Standard Curve at 595 Nanometers.......................... .......................... 36

4-1 SEM Image of an Uncoated Platinum Coil. ................................................. 44

4-2 SEM Image of a Coil Coated by Airbrushing ....... ..... ........................ 44

4-3 SEM Images of Coated Platinum Coil............... ................................ ......... 45

4-4 SEM Im ages of an Ex Vivo Coil.. ......................... .......................... ............ 46

4-5 IHC Staining of Ex Vivo Arteries Treated with Sutures........................................ 47

4-6 H&E Staining of Ex Vivo Arteries with Platinum Coils First Trial ..................... 48

4-7 H&E Staining of Ex Vivo Arteries with Platinum Coils Second Trial ................ 49

4-8 IHC Staining of Ex Vivo Arteries Treated with Platinum Coils.............................. 50









Ideal aneurysms for coiling are saccular aneurysms with a small neck, as larger-

necked aneurysms do not form a complete endothelial layer. Small aneurysms range

between 4 10 mm, large aneurysms range between 11 25 mm, and giant aneurysms

are those greater than 25 mm36. Narrow-necked aneurysms are favored for

endovascular coiling since the goal is to pack coils as densely as possible without

encroaching on the parent vessel. Large-necked aneurysms are susceptible to coil

movement due to arterial blood flow. In a well-packed aneurysm, only about 20-40% of

the volume has been filled, therefore large-necked aneurysms are more likely to be

compacted by blood flow toward the sac and away from the aneurysm neck8.

Clinical Trials with Endovascular Coils

Preliminary clinical studies performed by Guglielmi et al. in 1990 found no

permanent neurological trauma after coil embolization17. In a subsequent study,

Guglielmi et al. was able to achieve 70% to 100% endovascular occlusion in 42/43

posterior fossa aneurysms with respective overall morbidity and mortality rates at 4.8%

and 2.4%18. Byrne et al. studied 317 patients with aneurysmal SAH who were treated

with platinum coils. Aneurysms remained occluded in 86.4% of small and 85.2% of

large aneurysms, while 14.7% of aneurysms experienced rebleeding. Despite complete

initial occlusion of the aneurysm lumen, instability of the occlusion is common,

increasing the likelihood of rebleeding after coil embolization. This indicates the

importance of a follow-up angiography to determine the stability of the occlusion in the

months following the original embolization procedures.

Raymond et al. conducted a statistical analysis on 501 aneurysms in 466 patients

treated with GDCs between August 1992 and May 2002, and found that recanalization,

or growth of the aneurysm remnant, was found in 33.6% of treated aneurysms. The









13. Deckers MML, van Bezooijen RL, van der Horst G, et al: Bone morphogenic
proteins stimulate angiogenesis through osteoblast-derived vascular endothelial
growth factor A. Endocrinology 143:1545-1553, 2002

14. Forget, Jr TR, Benitez R, Veznedaroglu E, et al: A review of size and location of
ruptured intracranial aneurysms. Neurosurgery 49:1322-1326, 2001

15. Frazier K, Williams S, Kothapalli D, et al: Stimulation of fibroblast cell growth,
matrix production, and granulation tissue formation by connective tissue growth
factor. J Invest Dermatol 107,404-411, 1996

16. Gaba RC, Ansari SA, Roy SS, et al: Embolization of intracranial aneurysms with
hydrogel-coated coils versus inert platinum coils: effects on packing density, coil
length and quantity, procedure performance, cost, length of hospital stay, and
durability of therapy. Stroke 37:1443-1450, 2006

17. Guglielmi G, Vihuela F, Dion J, et al: electrothrombosis of saccular aneurysms
via endovascular approach. Part 2: preliminary clinical experience. J Neurosurg
75:8-14, 1991

18. Gugliemi G, Vihuela F, Duckwiler G, et al: Endovascular treatment of posterior
circulation aneurysms by electrothrombosis using electrically detachable coils. J
Neurosurg 77:515-524, 1992

19. Guglielmi G, Vihuela, F, Sepetka I, et al: Electrothrombosis of saccular
aneurysms via endovascular approach. Part 1: electrochemical basis, technique,
and experimental results. J Neurosurg 75:1-7, 1991

20. Heilman CB, Kwan ESK, Wu JK: Aneurysm recurrence following endovascular
balloon occlusion. J Neurosurg 77:260-264, 1992

21. Higashida RT, Halbach LD, Hieshima GB, et al: Detachable balloon embolization
therapy of posterior circulation intracranial aneurysms. J Neurosurg 71:512-159,
1989

22. Hino K, Konishi Y, Shimada A, et al: Morphologic changes in neo-intimal
proliferation in an experimental aneurysm after coil embolization: effect of factor
VIII administration. Neuroradiol 46:996-1005, 2004

23. Ho Y, Mi F, Sung H, et al: Heparin-functionalized chitosan-alginate scaffolds for
controlled release of growth factor. Int J Pharm 376:69-75, 2009

24. Hogan BLM: Bone morphogenic proteins in development. Curr Opin Genet Dev
6:432-438, 1996









control. It is interesting to note that despite the increased concentration of protein

released, SDF-1 yielded a less robust response in the second trial than in the first. The

response shown by CTGF is significantly strong. MCP-1 was not tested in the first trial.

Further trials are necessary to corroborate this data.

Figure 4-8 depicts fluorescence microscopy images at 10X magnification of

immunohistochemical staining of ex vivo arteries treated with PLGA coating with no

protein (A) and PLGA and SDF-1 coating (B). Cell nuclei are stained blue with DAPI,

and smooth muscle actin is stained red. Figure 4-8 (B) shows a significantly greater

presence of smooth muscle actin than Figure 4-8 (A), indicating that SDF-1 yields a

greater presence of smooth muscle cells than the control. Further examination of the

effects of SDF-1, CTGF, and MCP-1 will need to be investigated in future trials to

corroborate this preliminary data.









TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................. ....................... ......... ............... 4

L IS T O F F IG U R E S .......................................................................................................... 8

LIST OF ABBREVIATIONS ................................... ........................... .......... 9

A B S T R A C T ................................................................................................................... 1 1

CHAPTER

1 INT R O D U C T IO N ....................................................... .......... .......... 13

2 BA C KG RO U N D ......................................... ........ .......... ...... 15

Cerebral Aneurysms and Subarachnoid Hemorrhage (SAH)................................ 15
Risk Factors for Subarachnoid Hemorrhage .............. ..... ............... 15
Sym ptom s of SAH ........... ........... .......................... ....... ........ 16
Diagnosis of SA H ........................ ................................... 16
Treatments for Cerebral Aneurysms and SAH.............................................. 16
Aneurysm Clipping ............... ........ .. ...... .... ....... .. ........ 16
Endovascular Aneurysm Occlusion with Balloons................ ............. 17
Guglielmi Detachable Coils .................................. .... ...... ...... ............ 17
Thrombus Formation and Healing Process.............. ...... .................. 18
Advantages of Endovascular Coiling ....................... ...... ..................... 19
Clinical Trials with Endovascular Coils ....... ............... ... ...... ...... .... 20
Modifications to Platinum Endovascular Coils ............... ..... .. ............... 22
Protein-C oated C oils ................. ............. ................................. ............ ..... 22
M atrix C o ils ....................................................... 2 3
Hydrogel-C oated C oils ...................... ....... ......... .. ............................ 24
Controlled Release of Proteins ....... ....... ........ ..................... ...... ......... 25
Proteins Released from Bioactive Coating..................................... .................... 26
Monocyte Chemoattractant Protein-1 ............... .... ..... .................. 26
Strom al C ell-Derived Factor-1 .............. ................. .................................... 26
B one M orphogenic P rote in-4 ................................................... ... ................. 27
Connective Tissue G rowth Factor ........... .............................. ...... ............. 27

3 MATERIALS AND METHODS ............................. ................... ... ............... 29

M a te ria ls .................................................................................................... 2 9
C oatings .................................................................... .. .. ........ ...... 29
V icryl Sutures and Endovascular Coils................................... .................... 29
Proteins .............. .. ......... ..... ......................... 29
M methods ................. .............. ............. ............................. 30
Poly (D,L-lactide-co-glycolide) (PLGA) Coatings............. ....... ............ 30


6









48. Rothhammer T, Bataille F, Spruss T, et al: Functional implication of BMP4
expression on angiogenesis in malignant melanoma. Oncogene 26:4158-4170,
2007

49. Schilling M, Strecker JK, Schabitz WR, et al: Effects of monocyte
chemoattractant protein 1 on blood-borne cell recruitment after transient focal
cerebral ischemia in mice. Neuroscience 161:806-812, 2009

50. Schwendeman SP: Recent advances in the stabilization of proteins encapsulated
in injectable PLGA delivery systems. Crit Rev Ther Drug Carrier Syst 19:73-
98, 2002

51. Serbienenko FA: Balloon catheterization and occlusion of major cerebral vessels.
J Neurosurg 41:125-145, 1974

52. Shimo T, Nakanishi T, Nishida T, et al: Connective tissue growth factor induces
the proliferation, migration, and tube formation of vascular endothelial cells in
vitro, and angiogenesis in vivo. J Biochem 126:137-145, 1999

53. Sinha VR, Trehan A: Biodegradable microspheres for protein delivery. J Contr
Rel 90:261-280, 2003

54. Stellos K, Langer H, Daub K, et al: Platelet-derived stromal cell-derived factor-1
regulates adhesion and promotes differentiation in terms of human CD34+ cells
to endothelial progenitor cells. Circulation 117:206-215, 2008.

55. Sun SW, Jeong YI, Jung SW, et al: Characterization of FITC-albumin
encapsulated poly (DL-lactide-co-glycolide) microspheres and its release
characteristics. J Microencapsulation 20:479-488, 2003

56. Tamatani S, Ito Y, Abe H, et al: Evaluation of the stability of aneurysms after
embolization using detachable coils: Correlation between stability of aneurysms
and embolized volume of aneurysms. AJNR 23:762-767, 2002

57. Tamatani S, Ozawa T, Minakawa T, et al: Radiologic and histopathologic
evaluation of canine artery occlusion after collagen-coated platinum microcoil
delivery. AJNR 20:541-545, 1999

58. Teunissen LL, Rinkel GJ, Algra A, et al: Risk factors for subarachnoid
hemorrhage: a systematic review. Stroke 27:544-549, 1996

59. Villablanca JP, Jahan R, Hooshi P, et al: Detection and characterization of very
small cerebral aneurysms by using 2D and 3D helical CT angiography. AJNR
23:1187-1198, 2002









Three weeks post-surgery, the aneurysms were treated with the PLGA-coated

sutures or coils. An incision was made on the RCCA wall by a microsurgical blade, and

the suture or coil was inserted into the resulting pocket. Sutures were used in

preliminary testing and were later replaced by coated coils since the sutures were more

difficult to manipulate and would dissolve over time inside tissue. Preliminary suture

testing included uncoated sutures, sutures coated with PLGA only, and sutures coated

with PLGA and CTGF. Each type of sample was tested on n=3 mice. Subsequent

studies with coils included coils coated with PLGA and CTGF, SDF-1, BMP-4, or MCP-

1. Uncoated coils and coils coated with only PLGA were used as controls.

Sutures or coils were allowed to remain inside the animal for three weeks, after

which the animal would be euthanized. The right and left common carotid arteries were

immediately excised and placed in a 4% paraformaldehyde solution. After the arteries

were excised, the coils or sutures were removed and the arteries were cut into cross-

sections and analyzed.

Analysis of ex vivo samples

In preliminary suture studies, the presence of CTGF was determined by coupling

with anti-CTGF. Hemotoxylin and eosin (H&E) staining was also used to evaluate

cellular behavior of aneurysms treated with coils. Immunohistochemical (IHC) staining

was performed on the 5 pm cross-sections embedded in OCT cryoembedding media

from treated aneurysms fixed in 4% PFA. Sections were treated with acetone at -20 C

for 5 minutes and air-dried prior to staining. The OCT media was removed from the

sample slides by rinsing with 1X Wash Solution (DAKOCytomation, Carpinteria, CA) for

5 minutes.









PLGA Coating 2 for platinum coils

Vicryl sutures and preliminary platinum coils in trial 1 were treated PLGA Coating

1. PLGA Coating 2 was used for trial 2 with platinum coils, and used 3 (w/v)%

magnesium hydroxide in 50/50 PLGA, with 10[tg of protein in 100[LL of PBS forming the

aqueous phase. The oil and aqueous phases were mixed again in a 1:10 ratio in an ice

bath. Smaller quantities of protein solution and PLGA were mixed in the same ratio in

hopes of increasing protein collisions with the surface of the coil and increasing the

amount of proteins contained in the PLGA coating.

Coating technique

Vicryl sutures were originally used to test coatings due to cost-effectiveness, but

were later discarded in favor of platinum endovascular coils due to fraying of the suture

upon implantation. Coils or sutures were cut into approximately 2 mm samples, rinsed

in 70% ethanol inside a Petri dish, and allowed to air dry inside of a fume hood for 10

minutes. Samples were dip-coated into the PLGA solution five times at 15-second

intervals, and allowed to dry by laying flat for approximately 2 hours inside a Petri dish.

Samples were stored at 4 OC until use.

Airbrush coating technique

Preliminary coatings were applied to the suture and coil surfaces using an

airbrush. PLGA Coating 1 was loaded into a Master Airbrush Brand Model G22

Precision Dual-Action Gravity Feed airbrush (TCPGIobal.com). Airbrushing of coatings

was discontinued due to substantial loss of PLGA solution in the coating process from

airbrush flow and contamination concerns for proteins.









with aneurysm clipping is 2.6% and 10.9%, respectively, due to greater procedural

successes and training in recent years44

Endovascular Aneurysm Occlusion with Balloons

In 1974, Serbinenko reported balloon occlusion with detachable balloons to treat

carotid-cavernous sinus fistulas51, which began a movement toward the endovascular

treatment of cerebral aneurysms with detachable balloons. Balloons were composed of

silicone or latex, although latex exhibited a greater rupture rate20. Silicone balloons

were made opaque with metrizamide, and filled with 2-hydroxyethylmethacrylate

(HEMA) once the balloon was inside the aneurysm sac. After the HEMA solidified,

traction was applied to the catheter and the balloon was detached23 Balloon-embolized

aneurysms could rupture if the balloon was overinflated, and aneurysm recurrence was

a problem due to deflation of balloons over time20

Guglielmi Detachable Coils

In 1989, Guglielmi et al. developed a less invasive method for the occlusion of

saccular aneurysms than clipping. Guglielmi detachable coils (GDCs) are pliable

platinum coils soldered onto a delivery wire made of stainless steel that are deposited

inside the aneurysm lumen using electrothrombosis and electrolysis19.

Electrothrombosis occurs when a positive current attracts negatively charged blood

particles, causing the formation of a clot. Platinum is an ideal metal to use because it is

resistant to electrolysis and produces large clots through electrothrombosis43.

GDCs are placed inside an aneurysm through a relatively non-invasive surgical

procedure during which the patient is awake and systemically heparinized while

angiography is used to track the coil in the patient arteries. First, a guiding catheter

inserted through the femoral artery leads the coil and delivery wire to the aneurysm






















Figure 4-1. SEM Image of an Uncoated Platinum Coil. A) 20X magnification and B)
100X magnification.


B
Figure 4-2. SEM Image of a Coil Coated by Airbrushing. A) 20X magnification and B)
the middle of the coil at 100X magnification.









PLGA Coating 1, it was evident that the coatings were indeed releasing protein, since

the in vivo results with each coating were consistently different.

Bradford assays for the PLGA Coating 2 were also inconclusive. This time, the

coatings were made with a 100 [tg/mL BSA solution instead of a 10 [tg/mL solution.

The emulsions were composed of 100 |tg of protein solution and 1 mL of PLGA and

magnesium hydroxide solution. Although the initial Bradford assay was inconclusive,

the release rate experiment will be repeated to determine a release rate, since the

greater concentration of BSA in these coatings should release at detectable amount of

protein.

Scanning Electron Microscopy

Scanning Electron Microscope (SEM) images were used to examine the

morphology of the PLGA coatings on the platinum coils. A homogeneous coating was

desired that would cover the entire surface of the coil. Figure 4-1 depicts an uncoated

coil at 20X magnification (A) and 100X magnification (B). The uncoated coil is smooth

and has a ribbed appearance due to tight winding of the platinum wire.

The morphology of the airbrushed coils differs from that of the dip-coated coils.

Airbrushing was discarded as a coating technique due to difficulties with controlling the

flow of the airbrush, significant waste of PLGA solution during the coating process, and

contamination concerns for the proteins. The morphology of a coil airbrushed with

PLGA Coating 1 is shown in Figure 4-2 at 20X magnification (A), and at 100X

magnification (B). These images show a ragged coating with agglomerations of PLGA

on the sides of the coil. The size of the coils and the thin stream of PLGA emerging









wire was unwound during the removal process from the sectioned artery. Figure 4-4 (B)

and (C) show what appears to be a mixture of coating as well as tissue growth on the

surface of the coil. In these images there appears to be striated sections that could

possibly be tissue that has remained attached to the coil. Comparing Figure 4-4 (D)

with Figure 4-3 (A), it is evident that most of the coating has dissolved from the coil,

leaving behind mostly uncoated coil.


Murine Aneurysm Model

Vicryl Sutures

Vicryl sutures were used for preliminary testing of PLGA Coating 1 due to their

cost-effectiveness. Since an in vitro analysis yielded inconclusive results due to low

amounts of protein contained in the coating, an in vivo analysis was conducted in order

to determine the effects of the protein released. Vicryl sutures with PLGA, PLGA and

CTGF, and uncoated Vicryl sutures were tested in vivo in a murine aneurysm model as

described in Chapter 3. Sutures were implanted in the aneurysm site and were left in

the mice for three weeks. After three weeks, the mice were euthanized as outlined in

Chapter 3, and then the affected arteries were removed, cross-sectioned, and stained

as described previously.

Fluorescence microscopy was used to analyze ex-vivo cross-sectioned arteries

after the sutures were removed. The nuclei of recruited progenitor cells were stained

blue with 4',6-diamidino-2-phenylindole (DAPI), and anti-CTGF was used to determine

the presence of CTGF. Red fluorescent tags indicated the presence of CTGF when it

coupled with the CTGF present. Since Vicryl sutures are designed to dissolve in the

tissue environment, in vivo results from the uncoated suture could not be examined.









Figure 4-6 (C), which shows an ex vivo artery treated with PLGA and BMP-4, does

not differ significantly in appearance from the controls. However, Figures 4-6 (D) and

(E), which show images of ex vivo arteries with treated with CTGF and SDF-1,

respectively, show a significantly larger amount of what appears to be new smooth

muscle cells, indicated in deep red in the images. In this first trial, it appears that SDF-1

elicits the most favorable cellular response, with CTGF also showing a favorable

although less robust response. Although the results of the first trial were promising,

more data was required in order to prove that SDF-1 and CTGF indeed elicited the

growth of new smooth muscle cells along the periphery of the aneurysm lumen.

Platinum coils with PLGA Coating 2

A second trial with platinum coils was performed to corroborate the data from the

first trial. In the second trial, the PLGA Coating 2 emulsions were composed of smaller

volumes of protein and PLGA in a 1:10 ratio. The purpose of using smaller volumes

was to increase the concentration of proteins present on the coils by having the same

amount of protein present in a smaller volume of solution, increasing the number of

collisions between protein molecules and the coil and thus leading to more protein

embedded in the coil coating. The second trial tested PLGA coatings with CTGF, SDF-

1, BMP-4, or MCP-1. PLGA coatings with no protein were used as a control.

Figure 4-7 (A) shows an H&E stain of an ex vivo artery that was treated with a

control coil coated only with PLGA. Figures 4-7 (B-E) show ex vivo arteries treated with

coils coated with PLGA and SDF-1, MCP-1, BMP-4, and CTGF, respectively. Similar to

results from the first trial, BMP-4 once again does not show a favorable cellular

response in terms of the appearance of smooth muscle cells. However, SDF-1, MCP-1,

and CTGF all show greater amounts of smooth muscle cells compared with the PLGA









Bone marrow derived circulating EPCs play an important role in the creation of new

vascular tissue in response to SDF-1 and other cytokines. In particular, EPCs exhibit a

dosage-dependent response to SDF-164. SDF-1 is secreted by activated platelets,

supporting the chemotaxis of EPCs into a thrombus54.

Bone Morphogenic Protein-4

Bone Morphogenic Proteins (BMPs) are secreted growth factors of the

transforming growth factor P (TGFP) family, that have been shown to affect cellular

processes such as proliferation, differentiation, chemotaxis, motility, and cell death24

BMPs stimulate osteoblasts to produce Vascular Endothelial Growth Factor A (VEGF-

A), which in turn stimulates angiogenesis by coupling the process to bone formation13

Furthermore, BMP-4 has been linked to the regulation of ocular angiogenesis by

stimulating VEGF release from retinal pigment epithelial cells60. Finally, malignant

melanomas tend to express BMPs, and BMP-4 is suggested to act as an angiogenic

factor due to its positive effect on the migration of endothelial cells in vitro48.

Connective Tissue Growth Factor

Connective Tissue Growth Factor (CTGF), a member of the CCN family of

secreted proteins, is involved in angiogenesis, skeletogenesis, and wound healing. It

contains a cysteine-rich domain known to bind BMP-43. CTGF plays a significant role in

the production of extracellular matrix (ECM) in conditions of excessive collagen

deposition15. CTGF promotes the adhesion, proliferation, and migration of vascular

endothelial cells in vitro, and induces the tube formation of vascular endothelial cells52.









Figure 4-5 depicts fluorescence microscopy images at 10X magnification of Vicryl

sutures coated with PLGA (A) and Vicryl sutures coated with a PLGA coating containing

10 pg of CTGF (B). Although a significant number of cells may be observed in these

images, it is unclear what type of cells they are, and whether they were recruited to the

area by the CTGF present. These preliminary inconclusive results, in addition to the

fraying of the uncoated suture during implantation efforts, were the driving force behind

the decision to use platinum coils instead of sutures in order to obtain an effective

control.

Platinum Coils

Platinum coils with PLGA Coating 1

The first trial with platinum coils involved PLGA coatings with CTGF, SDF-1, or

BMP-4. Uncoated platinum coils and platinum coils coated with only PLGA were used

as controls. In this trial, the PLGA Coating 1 was used, where 1 mL of 10 [tg/mL protein

was mixed with 10 mL of PLGA to create the emulsions as described in Chapter 3. This

resulted in a small concentration of protein contained in each coating, leading to the

release of a concentration of each protein inside the aneurysm lumen that could not be

determined using the Bradford Assay.

Figure 4-6 depicts hematoxylin and eosin (H&E) staining of the cross-sectioned ex

vivo arteries from this trial. Ideal results would show smooth muscle tissue growth

around the periphery of the aneurysm lumen, indicated by a strong red color. Figures 4-

6 (A) and (B) show ex vivo arteries treated with an uncoated platinum coil, and an ex

vivo artery treated with a platinum coil coated with only PLGA. These images serve as

controls by which to compare the protein-encapsulated coatings.









BIOGRAPHICAL SKETCH

Cristina Elena Fernandez was born in Caracas, Venezuela to Antonio Fernandez

and Maria Elena Pozo de Fernandez in 1986. She moved to the United States when

she was two years old, and was raised in Melbourne, Florida, where she graduated

from West Shore Junior Senior High School in 2004. In 2008, she earned a Bachelor

of Science in materials science and engineering with a concentration in biomaterials

from the University of Florida and was awarded the Dean Joseph Weil Award for

Outstanding Leadership in the College of Engineering.

As an undergraduate and graduate student at the University of Florida, Cristina

served on the executive board of several local and national organizations, including the

Society of Women Engineers, the Benton Engineering Council, the Society for

Biomaterials, Epsilon Lambda Chi, the Engineering Student Advisory Council, and the

National Association of Engineering Student Councils. She developed an interest in

biomaterials for cardiovascular and wound healing applications through her

undergraduate and graduate research experiences. After receiving her Master of

Science from the University of Florida, Cristina will attend Duke University in the fall of

2010 to pursue a PhD in Biomedical Engineering.









SAH Subarachnoid Hemorrhage

SDF-1 Stromal Cell Derived Factor 1

SEM Scanning Electron Microscope

TGF-I3 Tumor Growth Factor 1

VEGF Vascular Endothelial Growth Factor









Mechanical dip-coating methods could be explored in order to achieve more

uniform coatings.

5. Optimization of protein dosage. Determining the optimum dosage for each

protein is necessary to obtain the most favorable cellular response.

Eventually, a combination of proteins may be encapsulated in the PLGA

coating. The release rate of the proteins could be further optimized through

the use of additives that would cause proteins to be released at a slower

rate.

6. Characterization of Long-Term Results. Coated coils were tested in a

murine aneurysm model for 3 weeks, which was long enough for the

appearance of new smooth muscle cells to occur. However, more long-

term studies would show more organized fibrous tissue and eventual

endothelialization of the aneurysm neck.




Full Text

PAGE 1

DEVELOPMENT OF A BIOACTIVE COAT ING FOR PLATINUM ENDOV ASCULAR COILS FOR THE TREATMENT OF CEREBRAL ANEURYSMS By CRISTINA ELENA FERNANDEZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1

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2010 Cristina Elena Fernandez 2

PAGE 3

To my pare nts 3

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ACKNOWLEDGMENTS I would like to thank my committee mem bers, Dr. Christopher Batich, Dr. Laurie Gower, and Dr. Eugene Goldberg. In particula r, I would like to thank Dr. Christopher Batich for welcoming me into his research group, for his support, and for introducing me to this project. I would like to thank Dr Brian Hoh for his support, encouragement, and professional insight, and Dr. Ed Scott for his co llaboration. I would also like to thank the members of the Hoh group in the Department of Neurosurger y at UF, Dr. Koji Hosaka, Erin Wilmer, and Daniel Downes for their continued efforts and collaboration. I am sincerely grateful to the mem bers of the Batich group, parti cularly Titilayo Moloye, Pei Yu Pinky Chung, Sam Popwell, and Brad W illenberg for their technical assistance, support, and friendship. I would like to thank the faculty and staff in the College of Engineering, particularly Dr. Angela Lindner, Dr. Jonathan Earle, Je ff Citty, Margie Williams, and Yolanda Hankerson for their support thro ughout my years at the Univer sity of Florida. I am forever grateful for the skills that I have ac quired during my years of service in student organizations in the College of Engineering, and I thank them for providing our organizations with the funding an d administrative support possi ble to impact the lives of so many students. I would also like to thank Prinda Wanakule and Chelsea Magin, who have served as role models for me through the Society of Women Engineers and have motivated me to test the boundarie s of what I could accomplish. I am extremely thankful for the love and support of the wonder ful friends I have made during my undergraduate and graduat e studies at the University of Florida. I feel truly blessed to be surrounded by such acco mplished and motivated people. I would like to thank my grandmother, Lilia Daz de Pozo, for her countless words of wisdom, 4

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prayers, and love. I would als o like to thank my aunt, Lilian del Pozo, and my grandmother, Manuela Mourn de Fernndez, for their love and support. Finally, I would like to thank my parents, Antonio Fernandez and Maria Elena Pozo de Fernandez, for raising me in an environment where I wa s nurtured and challenged to always improve upon myself, and allowing me to experience c ountless opportunities that have made me the person I am today. I cannot thank them enough for their continued and relentless love, support, and encouragement in my edu cation, without which I would not have been able to accomplish my goals. 5

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TABL E OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF FI GURES..........................................................................................................8 LIST OF ABBR EVIATIONS.............................................................................................9 ABSTRACT ...................................................................................................................11 CHAPTER 1 INTRODUC TION....................................................................................................13 2 BACKGRO UND...................................................................................................... 15 Cerebral Aneurysms and Subar achnoid Hemorr hage (SAH)..................................15 Risk Factors for Subar achnoid Hemorrhage....................................................15 Symptoms of SAH............................................................................................16 Diagnosis of SAH.............................................................................................16 Treatments for Cerebral Aneurysms and SAH ........................................................16 Aneurysm Cli pping...........................................................................................16 Endovascular Aneurysm Occl usion with Ba lloons............................................17 Guglielmi Detac hable Coils.....................................................................................17 Thrombus Formation and Healing Pr ocess......................................................18 Advantages of Endova scular Coi ling................................................................19 Clinical Trials with En dovascular Coils.............................................................20 Modifications to Platinum Endovascu lar Coils........................................................22 Protein-Coat ed Coils ........................................................................................22 Matrix Co ils.......................................................................................................23 Hydrogel-Coated Coils.....................................................................................24 Controlled Release of Prot eins...............................................................................25 Proteins Released from Bioactive Coatin g..............................................................26 Monocyte Chemoattract ant Protei n-1...............................................................26 Stromal Cell-Derived Fact or-1..........................................................................26 Bone Morphogenic Protein4............................................................................27 Connective Tissue Gr owth Fa ctor....................................................................27 3 MATERIALS A ND METHOD S................................................................................29 Materials.................................................................................................................29 Coatings...........................................................................................................29 Vicryl Sutures and En dovascular Coils.............................................................29 Protei ns............................................................................................................29 Methods ..................................................................................................................30 Poly (D,L-lactideco -glycolide) (PLG A) Coat ings..............................................30 6

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PLGA Coat ing 1 for suture s and platinum coils..........................................30 PLGA Coating 2 for platinum coils.............................................................31 Coating tec hnique......................................................................................31 Airbrush coati ng techni que.........................................................................31 Polyelectrolyte complex coating tec hnique................................................32 Protein Release Measurem ents.......................................................................32 Scanning Electron Microscopy.........................................................................33 In Vivo Analyses...............................................................................................33 Murine aneurysm model............................................................................33 Analysis of ex vivo sample s.......................................................................34 4 RESULTS AND DISCUSSION...............................................................................37 Protein Release Measurem ents..............................................................................37 Scanning Electron Microscopy................................................................................38 Murine Aneurysm Model.........................................................................................40 Vicryl Su tures...................................................................................................40 Platinum Coils..................................................................................................41 Platinum coils with PLGA Coating 1...........................................................41 Platinum coils with PLGA Coating 2...........................................................42 5 CONCLUS IONS.....................................................................................................51 6 FUTURE WORK .....................................................................................................53 LIST OF RE FERENCES...............................................................................................55 BIOGRAPHICAL SKETCH ............................................................................................61 7

PAGE 8

LIST OF FIGURES Figure page 2-1 Cartoon Diagram of Pl atinum Coil In sertion...........................................................28 3-1 Standard Curve at 595 Nanomet ers.......................................................................36 4-1 SEM Image of an Unc oated Platinum Coil.............................................................44 4-2 SEM Image of a Coil C oated by Airb rushing. .........................................................44 4-3 SEM Images of Coated Platinum Coil....................................................................45 4-4 SEM Images of an Ex Vivo Coil.............................................................................46 4-5 IHC Staining of Ex Vivo Arteries Treated with Suture s...........................................47 4-6 H&E Staining of Ex Vivo Arteries with Platinum Coils First Trial.........................48 4-7 H&E Staining of Ex Vivo Arteries with Platinum Coils Second Trial....................49 4-8 IHC Staining of Ex Vivo Arteries Treated with Platinum Coils. ...............................50 8

PAGE 9

LIST OF ABBREVIATIONS g Micrograms C Degrees Celsius BPM Bioabsorbable Polymer Matrix BMP Bone Morphogenic Protein BSA Bovine Serum Albumin CTA Computed Tomography Angiography CTGF Connective Tissue Growth Factor DAPI 4-6-diaminidino-2-phenylindole ECM Extracellular Matrix EPC Endothelial Progenitor Cell FSP-1 Fibroblast Specific Protein-1 GDC Guglielmi Detachable Coil HEMA 2-hydroxyethylmethacrylate HES HydroCoil Embolic SystemTM ISAT International Subarachnoid Hemorrhage Trial kV kilovolts mA Milliamperes MCP-1 Monocyte Chemoattractant Protein 1 mL Milliliters mm Millimeters MRA Magnetic Resonance Angiography PEC Polyelectrolyte complex PFA Paraformaldehyde PLGA Poly (D,L-lactideco -glycolic acid) 9

PAGE 10

SAH Subarachnoid Hemorrhage SDF-1 Stromal Cell Derived Factor 1 SEM Scanning Electron Microscope TGFTumor Growth Factor VEGF Vascular Endothelia l Growth Factor 10

PAGE 11

Abstract of Thesis Pres ented to the Graduate School of the University of Fl orida in Partial Fulf illment of the Requirements for t he Degree of Master of Science DEVELOPMENT OF A BIOACTIVE COAT ING FOR PLATINUM ENDOVASCULAR COILS FOR THE TREATMENT OF CEREBRAL ANEURYSMS By Cristina Elena Fernandez August 2010 Chair: Christopher D. Batich Major: Materials Science and Engineering Subarachnoid hemorrhage (SAH), the rupture of a cerebral aneurysm, is the most prevalent form of stroke. Treatment opti ons for cerebral aneurysms aim to prevent future rupture by isolating the aneurysm orif ice from the parent ar tery. Endovascular treatment with platinum coils has become a popular and less invasive alternative to surgical clipping in the past 20 years. Thrombus formed around the coil serves as a scaffold for the formation of fibrous tissue toward the periphery of the aneurysm lumen, ultimately leading to coverage of the aneurysm neck with endothelial tissue. Increasing the efficiency of this healing process is of gr eat interest to avoid unwanted growth of the aneurysm due to blood flow. The purpose of this preliminary study is to identify proteins to be used in a bioactive coating for platinum endovascula r coils that will increase the efficiency of fibrous tissue organization and endothelialization in the aneurysm lu men. Platinum endovascular coils were treated with a 50/50 pol y (DL-lactide-co-glycolic acid) (PLGA) coating containing proteins such as Connec tive Tissue Growth Factor (CTGF), Bone Morphogenic Protein-4 (BMP-4 ), Monocyte Chemoattractant Protein-1 (MCP-1), or Stromal Cell-Derived Factor-1 (SDF-1). Protein release into the aneurysm lumen 11

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12 occurred through initial protein release from hydrophilic channels in the PLGA followed by PLGA degradation. Coatings were charac terized by scanning electron microscopy and an in vitro release study, and a murine aneur ysm model was employed to characterize the cellular response in vivo SDF-1, CTGF, and MC P-1 showed favorable results in vivo with an increase in new smooth mu scle cells developing around the aneurysm lumen near the site of the coil over the course of three weeks. A long-term goal of this project to be completed in fu ture studies is to optimize the dosage and delivery of a protein or combi nation of proteins that would increase the efficiency of the wound-healing response after implantation of endovascular coils.

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CHA PTER 1 INTRODUCTION Unruptured intracranial aneur ysms are suspected to be pr esent in 3.6 6.0% of the populat ion older than 30 years61. Subarachnoid hemorrhage (SAH), the rupture of an intracranial aneurysm that causes bleeding into the subarachnoid space of the brain, is the most common form of stroke27 and accounts for 25% of cerebrovascular deaths61. The combined morbidity and mortality rate of SAH still reaches 60% despite the availability of treatment options14. Furthermore, only 40% of SAH patients will recover enough to regain their independence14. The frequency of SAH in western populations has been reported between 6 an d 8 per 100,000 person years29. Previously, cerebral aneurysms were tr eated by performing a craniotomy and placing a clip at the neck of the aneurysm to prevent blood flow into the aneurysm sac. Less invasive endovascular treatment s emerged in the 1970s with balloon occlusion20,21. In the past 20 years, treatme nt with endovascular coils has become popular, involving the deployment of a plat inum coil via a guiding catheter inserted through the femoral artery17,19. The coil is deployed via a small positive current that causes electrothrombosis and forms a clot t hat will eventually organize into fibrous tissue. The aneurysm is removed from circ ulation from the parent artery by the eventual endothelialization of the aneurysm neck. Recanalization, or growth of the aneurysm remnant, is a significant problem associated with endovascular coiling45. Increasing the rate of endothelialization of the aneurysm neck is thought to decrease the re canalization rate of coiled aneurysms. Several groups have attempted to improve th e recanalization rate through modifications 13

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14 of platinum endovascular coils with proteins38, degradable polymers35, and hydrogels37, to name a few. The purpose of this study was to develop a bioactive coating for platinum endovascular coils that would increase the e fficiency of the fibrous tissue organization of coiled aneurysms. Several objectives we re followed in the development of this bioactive coating: 1. Identify an effective vehicle for c ontrolled release of protein and an effective coating method for platinum endovascular coils. A water-in-oil emulsion of 50/50 poly(D,L-lacticco -glycolic acid) (PLGA) was tested as a vehicle for protein release. Airbru shing and dip coating were explored as possible coating application techniques. 2. Characterize the release rate of protein in vitro from a coating for platinum endovascular coils. A Bradford Prot ein Assay was used to quantify the soluble concentration of protein released from coatings. 3. Identify effective prot eins that would increase the efficiency of the recruitment and organization of smoot h muscle cells in the aneurysm lumen and characterize their response in vivo Bone Morphogenic Protein-4 (BMP-4), Connective Tiss ue Growth Factor (CTGF), Stromal Cell-Derived Factor-1 (SDF-1), a nd Monocyte Chemoattractant Protein-1 (MCP-1) were released individually through the coatings and tested in a murine aneurysm model. Their resu lts were exami ned qualitatively through immunohistochemical (IHC) staining of 5 m cross sections of ex vivo aneurysms.

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CHA PTER 2 BACKGROUND Cerebral Aneurysms and Subarachnoid Hemorrhage (SAH) Cerebral aneurysms occur bec ause atherosclerosis or vessel wall injury caused by trauma or inflammation increases the tendency of intradural walls to develop aneurysms. Sac formation is initiated due to the local degeneration of the internal elastic lamina of the blood ve ssel by haemodynamic stresses. Blood flow within this newly formed aneurys mal sac causes enlar gement, leading to potential rupture9. Risk Factors for Subarachnoid Hemorrhage Juelva et al. deemed smoking and fema le gender as risk factors for aneurysm growth27. In fact, 45-75% of SAH patients t end to be smokers, compared to 20-30% of the general adult population. The number of cigarettes smoked daily had a greater impact than the amount of y ears the patient had been a smok er and the age at which they started. Cigarette smoker s have an imbalance of elastase and 1-anti trypsin, which may contribute to aneurysm formation or SAH, because the increased elastase activity in the artery wall coupled with hemodynamic stress may cause aneurysm formation or increase the rate of aneurysm growth27. Although aneurysm growth rate was not found to differ by gender, women with aneurysms were found to be at a higher risk fo r the formation of new aneurysms than men27. Estrogen has an inhibitory effect on aneur ysm formation, therefore fluctuations in estrogen, as well as the decreased collagen content of cerebral arteries after menopause, may favor aneurysm formation or gr owth. Women smokers are further at risk, since cigarette smoking has been shown to decrease estrogen levels27. 15

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Other risk factors for increased rate of aneurysm formation and SAH include excess ive alcohol consumption58, cocaine and amphetamine abuse40, oral contraceptives26, and high cholesterol4. Symptoms of SAH The keynote symptom of SAH is a sudden onset of a robust headache. This headache may appear in conjunction with one of the following: nausea or vomiting, brief loss of consciousness or cranial nerve palsies7. Some patients may experience a sentinel bleed or warning leak, which is a milder headache lasting several days. Sentinel bleeds typically occur between 2-8 weeks prior to SAH7. Diagnosis of SAH A non-contrast cranial com puted tomography (CT) scan is first performed in a patient presenting wit h symptoms of SAH. A negative CT scan requires a diagnostic lumbar puncture for analysis of the cerebrospinal fluid7. A positive result from either of these tests requires the patient to under go further analysis. Catheter-based angiography is typically performed to deter mine the size and shape of the ruptured intracranial aneurysm. Magnet ic Resonance Angiography (MRA) and dynamic spiral CT angiography (CTA) may be performed if a ngiography is not possible due to time constraints7. CTA is a noninvasive, outpatient procedure that may be used to detect cerebral aneurysms less than 5 mm in diameter59. Treatments for Cerebral Aneurysms and SAH Aneurysm Clipping Aneurysm clipping is performed with t he goal of preventing further aneurysm growth and rupture. A clip is surgically placed at the aneurysm neck in order to block the flow of blood into the lumen of the aneurysm. Morbidity and mo rtality associated 16

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with aneury sm clipping is 2.6% and 10.9%, respectively, due to greater procedural successes and training in recent years44. Endovascular Aneurysm Occlusion with Balloons In 1974, Serbinenko reported balloon occlus ion with detachable balloons to treat carotid-cavernous sinus fistulas51, which began a movement toward the endovascular treatment of cerebral aneurysm s with detachable balloons. Balloons were composed of silicone or latex, although latex exhibited a greater rupture rate20. Silicone balloons were made opaque with metrizamide, and fill ed with 2-hydroxyethylmethacrylate (HEMA) once the balloon was inside the aneurysm sac. After the HEMA solidified, traction was applied to the cat heter and the balloon was detached23 Balloon-embolized aneurysms could rupture if t he balloon was overinflated and aneurysm recurrence was a problem due to deflation of balloons over time20. Guglielmi Detachable Coils In 1989, Guglielmi et al. developed a less invasive method for the occlusion of saccular aneurysms than clipping. Guglie lmi detachable coils (GDCs) are pliable platinum coils soldered onto a delivery wire made of stainl ess steel that are deposited inside the aneurysm lumen using el ectrothrombosis and electrolysis19. Electrothrombosis occurs when a positive current attracts negatively charged blood particles, causing the formation of a clot. Platinum is an ideal metal to use because it is resistant to electrolysis and produces large clots through electrothrombosis43. GDCs are placed inside an aneu rysm through a relatively non-invasive surgical procedure during which the patient is awake and systemically heparinized while angiography is used to track the coil in the patient arteries. Fi rst, a guiding catheter inserted through the femoral artery leads the coil and delivery wire to the aneurysm 17

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neck. The stainless steel delivery wire is advanced through a microcatheter into the lumen of the aneurysm, positioning t he platinum coil inside the aneurysm17,19. Figure 21 depicts a cartoon diagram of the positi oning of the GDC inside an aneurysm. A positive direct electric curr ent of 0.5 to 2 mA is appli ed to the delivery guide wire, dissolving it through electrolysis and deploying the platinum coil into the aneurysm. An electrode placed at the patients groin connects the ne gative ground pole. Electrothrombosis is initiat ed when the positive current is applied, forming a thrombus in approximately 4 to 12 minutes. Several co ils may be deployed to fill the aneurysm depending on its size and shape. Systemic hepar inization is revers ed at the end of the procedure using protamine sulfate. A ngiograms are used post-embolization to determine the level of thrombosis and the placement of the coils 17,19. Thrombus Formation and Healing Process The progression of thrombosis over time occurs because more blood particles become entrapped within the coil network in the hours subsequent to the coiling procedure, and because clot formation wit hin the coils increases after systemic heparinization is reversed19. GDCs serve as a scaffold by which the thrombus is held together, allowing it to grow toward the interior of the aneurysm lumen and eventually seal off the aneurysm neck from the parent artery circulation17. Thrombus formation in aneurysms treated with detachable coils occurs similarly to that of the wound healing response after tissue injury although the process is delayed and occasionally incomplete6. A primary difference between normal tissue injury and thrombus formation in aneurysms is that t he aneurysm represents a spatial defect with a lack of stromal tissue6. Therefore, aneurysm healing occu rs from the periphery of the aneurysm lumen to the center. 18

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Thrombus begins to organize approxim ately one week after aneurysm treatment with platinum coils. A thin membrane may be found over the aneurysm neck, which will continue to develop over the course of the next month. Fibrous tissue may be found along the periphery of the aneurysm lumen after about 13 months, and will cover the coils over the course of a year. New endothelium may be seen covering the aneurysm neck after approximately one year afte r treatment with platinum coils2. Mawad et. al studied the growth of neoint ima in dogs six months after aneurysm obliteration with platinum coils and found neointima to be tightly adhered to coils and covered with endothelium. The aneurysm lum en was filled with organized fibrous tissue in three layers. The outer layer consisted of endothelium adjoining that of the parent artery. The second layer contained organize d smooth muscle cells, while the innermost layer contained disorganized smooth muscle cells. Minimal foreign body reaction and inflammation was noticed30. Advantages of Endovascular Coiling Coil embolization has several notable advantages over clipping; the most paramount being that there is no need for craniotomy and brain manipulation. Furthermore, the medical condition of the patient has less of an impact on the timing and performance of the procedure, an important benefit, since it is critical to treat ruptured aneurysms within 15 days of SAH most favorably after 2 days10. Since craniotomy is unnecessary, aneurysm location is of less importance, which is beneficial to patients with aneurysms in the posterior region of the brain, which is difficult to reach for clipping. Finally, endovascular coiling ma y be attempted in cases where clipping has failed10,18. 19

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Ideal aneur ysms for coiling are saccular ane urysms with a small neck, as largernecked aneurysms do not form a complete endothelial layer. Small aneurysms range between 4 10 mm, large aneurysms range between 11 25 mm, and giant aneurysms are those greater than 25 mm36. Narrow-necked aneurysm s are favored for endovascular coiling since the goal is to pack coils as densely as possible without encroaching on the parent vessel. Large-necked aneurysms are susceptible to coil movement due to arterial bl ood flow. In a well-packed aneurysm, only about 20-40% of the volume has been filled, therefore lar ge-necked aneurysms are more likely to be compacted by blood flow toward the sac and away from the aneurysm neck18. Clinical Trials with Endovascular Coils Preliminary clinical studies performed by Guglielmi et al. in 1990 found no permanent neurologic al trauma after coil embolization17. In a subsequent study, Guglielmi et al. was able to achieve 70% to 100% endovascular occlusion in 42/43 posterior fossa aneurysms with respective overall morbidity and mortality rates at 4.8% and 2.4%18. Byrne et al. studied 317 patients with aneurysmal SAH who were treated with platinum coils. Aneurysms remained occluded in 86.4% of small and 85.2% of large aneurysms, while 14.7% of aneurysms experienc ed rebleeding. Despite complete initial occlusion of the aneur ysm lumen, instability of the occlusion is common, increasing the likelihood of rebleeding after coil embolization. This indicates the importance of a follow-up angiog raphy to determine the stabilit y of the occlusion in the months following the origin al embolization procedure10. Raymond et al. conducted a statistical analysis on 501 aneurysms in 466 patients treated with GDCs between August 1992 and May 2002, and found that recanalization, or growth of the aneurysm remnant, was fou nd in 33.6% of treat ed aneurysms. The 20

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most significant predictors of a recurrence are treatment during the acute phase after rupture, aneurysm size (great er than 10 mm diameter), ne ck width (greater than 4 mm), unsatisfactory initial angiograp hic result and length of followup period. In this study, 46.9% of all recurrences were detected by 6 months, w hile 96.9% were detected by 36 months, indicating that the suggested 6 mont h follow-up period for angiograms is not enough to accurately track patients45. The International Subarachnoid Aneur ysm Trial (ISAT) was a multicenter, randomized clinical trial that compared the effects of neurosurgical clipping with endovascular treatment with platinum coils in patients with ruptured intracranial aneurysms. This trial recruited and followed 2143 patients who were randomly assigned to coiling or clipping treatment bet ween the years of 1994 2002. After one year, the risk of dependence or death was reduced by 22.6% for patients who underwent endovascular treatment31. A long-term follow-up of the ISAT trial indicated that rebleeding from coiled aneur ysms tends to occur within 5 years of the initial treatment, and found a significantly smaller death risk for patients treated with coils than those treated with surgery32. After the ISAT, endovascular obliterat ion became much more popular than surgery. Aneurysm patients now tend to receive surgical procedures only if they are unsuitable for endovascular treatment. An aneurysm may be unsuitable for endovascular treatment due to its diameter, its neck size, abnormal intracranial vasculature, thrombus in the aneurysm lu men or other thrombo-embolic issues, rebleeding following initia l endovascular treatment, and unsuccessful endovascular treatment11. 21

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Modifications to Platinum Endovascular Coils After bare platinum coils were introduc ed, several groups have attempted to modify the surface or the coil technique in order to increase the efficiency of thrombus formation. Coils have been coated with proteins, polymers, and hydrogels, among others. The problem of recanalization has been addressed in two major ways: by attempting to increase the rate of neointimal formation to seal the aneurysm neck, or increasing the percentage of occlus ion of the entire aneurysm volume. Protein-Coated Coils In 1999, Tamantani et al. compared t he angiographic and hist opathologic results of collagen-coated platinum coils in canine aneurysms, and found that coils coated with collagen promoted earlier formati on of thrombus in the aneurysm lumen in conjunction with a decline in recanalization rate of occluded aneurysms57. Dawson et al. compared the results of collagen-coated coils and traditional platinum coils in swine, and found that treatment of aneurysms with collagen-coated co ils yielded a completely occluded aneurysm with collagen-rich fibrous tiss ue with no evidence of recanalization12. Murayama et al. coated platinum coils with type I collagen, fibr onectin, vitronectin, laminin, or fibrinogen using an ion implantation techniq ue intended to retain the mechanical properties of the coil and improve the adhesion of surface cells during exposure to shear stresses and enzymes in the aneurysm lumen. Greater scar formation was reported with type I coll agen with new endothelium found at the aneurysm orifice34. Hino et al. continuously administered Factor VIII, also known as the wound-healing factor, intravenously for 5 days in swine treated with coil embolization and found increased endothelialization at the aneurysmal orifice. Factor VI II aids in the formation 22

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of granulation tissue by acting as an enzyme to drive fibroblasts from surrounding tissues to proliferate toward stabilized fibrin22. Zhu et al. coated a stainless steel stent with a heparinized polymer and canine endothelial cells. The layer of endothelial cells on the surface of the stent remained largely intact after 48 hours of a high shear stress brushing test intended to simulate blood flow67. Abrahams et al. coated bare platinum coils with Vascular Endothelial Growth Factor (VEGF) and found an increased endothelialization response compared with unc oated coils. VEGF is a glycoprotein produced by macrophages, endothelial cells, and smooth muscle cells that binds heparin and has been show n to promote angiogenesis2. Matrix Coils Murayama et al. loosely packed swine ex perimental aneurysms with several types of bioabsor bable polymer matrix (BPM), and observed a linear relationship between collagen levels in the experimental aneurysms and the rate of polymer degradation. The strongest inflammatory reaction was produced by 50/50 poly(lacticco -glycolic acid) (PLGA), which had the fastest degradation time. More organized collagen deposits were found in the neck and lumen of aneur ysms embolized with BPMs with a faster degradation time, particularly compared to standard pl atinum coils39. Matrix coils are platinum coils with a 50/ 50 PLGA coating designed to increase the efficiency of thrombus organiza tion in order to prevent aneurysm recanalizati on. Matrix coils are composed of 70% BPM and 30% platinum35. Initial testing in swine found more organized thrombus in aneurysms treated with Matrix coils after 14 days than with bare platinum coils, however, results after 3 months were similar for both GDCs and Matrix coils. Most importantly, a reduction in the size of the aneurysm sac was noticed 23

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in the aneurysms treated with Ma trix coils, as the BPM was likely replaced by mature scar tissue, which retracts in the wound-healing proces s25. Matrix Coils were Food and Drug Admi nistration (FDA) approved in 2002, and were the first commercially available bioactive coil for the treatment of cerebral aneurysms41. Initial clinical trials with Matrix coils indicate moderate improvement in the recanalization rate compar ed with bare platinum coils. However, increased friction in the first-generation Matrix coils made them more difficult to insert, and the packing density was less than of bare platinum coils. In theory, the streng th of the organized connective tissue produced by the Matrix coils is stronger than unorganized thrombus, increasing its resistance against the mechanical forces that cause recanalization and coil remodeling within the aneurysm37. A prospective multicenter registry conduct ed in France to evaluate the safety and efficacy of Matrix coils found a recanalizat ion rate of 25.7%, which increased if the volume percentage of the aneurysm occluded was less than 25%41. Biologic activity was demonstrated with the Matrix coils due to a 30% rate of progressive thrombosis at mid-term follow-up42. Another study indicated that long-term results of treatment with bare platinum coils compared to Matrix coils do not exhibit a difference in occlusion and recanalization rates, while the increased friction of the Matrix coils adds to complications with insertion and placement47. Hydrogel-Coated Coils Platinum c oils have been shown to r each a volume occlusion percentage of approximately 25-33%, despite post-procedural angiography indicating complete occlusion of the aneurysm57. The HydroCoil Embolic System (HES) is designed to improve the rate of aneurysm recanalizat ion by addressing the issue of volume 24

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occlusion percentage. HES is composed of a platinum coil coated with a hydrogel that swells to three times its original diameter in contact with blood and stops swelling upon contact with the aneurysm wall. In an in vitro model of a ruptured intracranial aneurysm, HES eliminated gaps within t he aneurysm lumen without inci ting changes in aneurysm size61. HES received a CE mark and FDA 510(k) clearance in 2002. A clinical study comparing HydroCoil with bare platinum coils found t he mean volume percentage occlusion with HydroCoils to be 70.7%16. An animal study comparing several secondgeneration endovascular devices, includi ng HES, found greater occlusion at angiography, however, the level of organiza tion of fibrous tissue around the aneurysm necks was similar for all devices tested28. Controlled Release of Proteins The FDA has cleared lactic and glycolic acid copolymers (PLGAs) for use in several pharmaceutical products or medical devices50. PLGA has gained popularity for use in medical devices due to its excellent biodegradability, mechanical strength, and biocompatibility25. In body fluids, PLGA undergoes hy drolysis of its ester backbone and degrades back to lactic and glycolic acid mo nomers. These mono mers are metabolized and eliminated in vivo as carbon dioxide and water in the Krebs cycle52. PLGA microspheres have been widely studied for applications involving controlled release of drugs or proteins55. Proteins and peptides may be encapsulated into microspheres using a water-in-oil-in-water emulsion and solvent evaporation technique, in which the hydrophilic protein is pr otected by a hydrophobic polymer layer63. Protein release from microspheres occurs thr ough two mechanisms: pore diffusion of the protein through hydrophilic c hannels, and PLGA degradation. Protein release profiles are characterized by an initial burst follo wed by a lag phase, followed by increased 25

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26 release when the polymer degrades63. In this study, this popular double emulsion technique has been adapted for use as a coating for polymer coils as a water-in-oil emulsion. Proteins Released from Bioactive Coating This study will examine the cellular res ponse elicited by the release of four proteins into a murine aneurysm lumen over the course of th ree weeks. Each protein has been linked to angiogenesis or recruitment of smooth muscle cells. Proteins will be evaluated for their ability to recruit new smooth muscle cells to the aneurysm lumen. Monocyte Chemoattractant Protein-1 Monocyte Chemoattractant Protein-1 (MCP1) is a member of the CC chemokine family proven to activate monocytes, ma crophages, and lymphocytes. MCP-1 has been shown to promote the development of aneurysms, and i s expressed in aneurysms through endothelial cells and so me smooth muscle cells5. Progenitor cell migration in the brain after ischemic incidents is due in part to MCP-1, since MCP-1 protein is expressed in ischemic cortex in murine stro ke models. Furthermore, elevated levels of MCP-1 were present in the cerebrospinal fluid of ischemic stroke patients49. Vascular smooth muscle cells and fibroblasts have spec ific receptors for MCP-1, and MCP-1 has induced the migration of smooth muscle cells in vitro Collagen expression by fibroblasts was enhanced in vitro due to the production of TGFproduced by MCP-133. Stromal Cell-Derived Factor-1 Stromal Cell-Derived Factor-1 (SD F-1) is a CXC chemokine whose receptor, CXCR4, is expressed on the surface of endothelial progenitor cells, and plays an important role in the regulation of pr oliferation, mobilization, and angiogenesis64. SDF-1 mediates the recruitment of smooth mu scle cell progenitors from the bone marrow65.

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Bone marrow derived circulating EPCs play an im portant role in the creation of new vascular tissue in response to SDF-1 and other cytokines. In particu lar, EPCs exhibit a dosage-dependent response to SDF-164. SDF-1 is secreted by activated platelets, supporting the chemotaxis of EPCs into a thrombus54. Bone Morphogenic Protein-4 Bone Morphogenic Proteins (BMPs) are secreted growth factors of the transformin g growth factor (TGF ) family, that have been shown to affect cellular processes such as proliferation, differ entiation, chemotaxis, motility, and cell death24. BMPs stimulate osteoblasts to produce Vasc ular Endothelial Growth Factor A (VEGFA), which in turn stimulates angiogenesis by coupling the process to bone formation13. Furthermore, BMP-4 has been linked to the regulation of ocular angiogenesis by stimulating VEGF release from retinal pigment epithelial cells60. Finally, malignant melanomas tend to express BMPs, and BMP-4 is suggested to act as an angiogenic factor due to its positive effect on the migration of endothelial cells in vitro48. Connective Tissue Growth Factor Connectiv e Tissue Growth Factor (CTG F), a member of the CCN family of secreted proteins, is involved in angiogenes is, skeletogenesis, and wound healing. It contains a cysteine-rich domain known to bind BMP-43. CTGF plays a significant role in the production of extracellular matrix (E CM) in conditions of excessive collagen deposition15. CTGF promotes the adhesion, pro liferation, and migration of vascular endothelial cells in vitro and induces the tube formation of vascular endothelial cells52. 27

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28 Figure 2-1. Cartoon Diagram of Platinum Coil Insertion. The coil is inserted into the aneurysm lumen through the guide wire. Mu ltiple coils are typically required to completely occlude an aneurysm.

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CHA PTER 3 MATERIALS AND METHODS Materials Coatings Coatings were primarily composed of es ter terminated (nominal) 50/50 Poly (D,Llactideco glycolide) (PLGA) in hexafluoroisopr opanol (HFIP) with an inherent viscosity of 0.61 dL/g (Lactel Absorbable Polymers, Pelham, AL). PLGA was dissolved in methylene chloride (Fisher Scientific, Pi ttsburgh, PA). Anhydrous 95.0% magnesium hydroxide was used as a neutralizing agent (Sigma Aldrich, St. Louis, MO). Vicryl Sutures and Endovascular Coils Dr. Brian L. Hoh in the Department of N eurosurgery at U F provided endovascular coils and Vicryl sutures for all experiments. Sterile Polyglactin 910 coated Vicryl violet braided sutures (Ethicon, Inc., Somerville, NJ) were used for preliminary testing of the coatings. TruFill DCS OrbitTM Detachable Coils (Cordis Neurovascular, Miami, FL) were used to test the coati ngs in subsequent experiments. Proteins A Coomas sie (Bradford) Pr otein Assay Kit (Pierce, Rockford, IL) was used for protein release rate measurements of the coatings. Coomassie (Bradford) Protein Assay Reagent was composed of G-250 dye, methanol, phosphoric acid, and solubilizing agents in water. The kit also contained Bovine Serum Albumin (BSA) standard ampoules with a concentration of 2 mg/mL in a solution of 0.9% saline and 0.05% sodium azide. BSA was used as a standard for the Bradford assays as well as in control experiments as a sample protein to model the in vitro release rate of the PLGA coatings. 29

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The PLGA coatings incorporated proteins in a water in oil emulsion. One type of protein was encapsulated in each coating sample, and various proteins were tested to determine their effect on the development of new fibrous tissue and smooth muscle cells. Proteins involved in the study in cluded Monocyte Chemoattractant Protein-1 (MCP-1) (Sigma, St. Louis, MO), Stromal Ce ll-Derived Factor-1 (SDF-1) (R&D Systems, Inc., Minneapolis, MN), Connective Tissue Grow th Factor (CTGF) (PeproTech, Inc., Rocky Hill, NJ), and Bone Morphogenic Protein-4 (BMP-4) (PeproTech, Inc., Rocky Hill, NJ). Proteins were reconstituted in Dul beccos sterile Phosphate Buffered Saline (PBS) solution without calcium and magnesium (Medi atech, Inc., Herndon, VA). Dr. Hohs research group in the UF Department of Neurosurgery provided all proteins. Methods Poly (D,L-lactideco-gl ycolide) (PLGA) Coatings PLGA Coating 1 for sutures and platinum coils A coating solution com posed of a water-in-oil emulsion was used to encapsulate proteins in 50/50 PLGA. The oil phase of the emulsion was composed of 5 (w/v)% 50/50 amorphous PLGA dissolved in methylene chloride. The aqueous phase of the emulsion was prepared by mixing 10 g/mL of a given prot ein solution and 3(w/v)% magnesium hydroxide in PBS. The magnesium hydroxide was used as neutralizing agent to protect the protein stability from acidic byproducts of PLGA degradation, and 3(w/v)% magnesium hydroxide has been show n to yield greater protein release66. An emulsion was created by mixing the aqueous phase with the oil phase in a 1:10 ratio under an ice bath, forming a milky solution with small white agglomerati ons of protein. 30

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PLGA Coating 2 for platinum coils Vicryl sutures and preliminary platinum co ils in trial 1 were treated PLGA Coating 1. PLGA Coating 2 was used for trial 2 with platinum coils, and used 3 (w/v)% magnesium hydroxide in 50/50 PLGA, with 10 g of protein in 100 L of PBS forming the aqueous phase. The oil and aqueous phases were mixed again in a 1:10 ratio in an ice bath. Smaller quantities of protein solution and PLGA were mixed in the same ratio in hopes of increasing protein co llisions with the surface of the coil and increasing the amount of proteins contai ned in the PLGA coating. Coating technique Vicryl sutures were originally used to te st coatings due to cost-effectiveness, but were later discarded in favor of platinum en dovascular coils due to fraying of the suture upon implantation. Coils or sutures were cu t into approximately 2 mm sampl es, rinsed in 70% ethanol inside a Petri dish, and allow ed to air dry inside of a fume hood for 10 minutes. Samples were dip-coated into the PLGA solution five times at 15-second intervals, and allowed to dry by laying flat for approximately 2 hours inside a Petri dish. Samples were stored at 4 C until use. Airbrush coating technique Preliminary coatings were applied to the suture and coil surfaces using an airbrush. PLGA Coating 1 was loaded in to a Master Airbrush Brand Model G22 Precision Dual-Action Gravity Feed airbrush (TCPGlobal.com). Airbrushing of coatings was discontinued due to substantial loss of PL GA solution in the coating proc ess from airbrush flow and contamination concerns for proteins. 31

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Polyelectrolyte complex coating technique Preliminary coatings were also made using a polyelectrolyte complex (PEC) composed of acetic acid, chitosan, and protein solution23. Sodium alginate powder was used to prepare a 1.0 (w/v)% alginate soluti on in water, and chitosan powder was used to create a 1.0 (w/v)% chitosan solution in acetic acid. Vicr yl sutures were used to test the PEC coatings. Vicryl sutures were cut into 4 mm sections and rinsed in 70% ethanol in a Petri dish and allo wed to dry as previously de scribed. Sutures were dipcoated in alginate solution for 15 seconds, dipped into a 10 g/mL BSA solution for 15 seconds, and then dipped in chitosan solution for 15 seconds. Sutures were allowed to dry for several hours and were stored at 4 C. Protein Release Measurements BSA was u sed as a sample protein to obtai n an estimate of the release rate of both PLGA Coatings 1 and 2 and the PEC coatings. Coated coil or suture samples were placed in a 1.5 mL micro centrif uge tube containing 1 mL PBS. Samples were placed in a hybridized incubator at 37 C, and were tested at 1, 3, 7, 14, and 21 days after placement in PBS. Separate samples were used for each time point, and n=3 samples of each type were tested each time. The concentration of BSA released was m easured using a Bradfor d protein assay, in which BSA was used as a standard. Sample s were prepared for the Bradford protein assay by transferring 0.5 mL of PBS solution from their micro centrifuge tubes to a cuvette and adding 0.5 mL of Coomassie pr otein assay reagent. Absorbance of the samples was measured at 595 nm using a UV -2410PC spectrophotometer (Shimadzu). Each sample was assayed four times, and the soluble BSA concentration released was 32

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determined by comparing the absorbance of a sample against a standard curve created with BSA. The standard curve was construct ed by drawin g a line of best fit between the absorbance values of BSA standards of known concentrations. Unknown sample concentrations were determined using the equat ion for the line of best fit, where y indicated sample absorbance and x indicated sample concentration. Figure 3-1 shows a sample standard curve used. Scanning Electron Microscopy The surface morphology of uncoated coils airbrushed coils, dip-c oated PLGA coils, and ex-vivo dip-coated PLGA coils was examined using a JEOL SM-31010 field emission scanning electron microscope at 15 kV. Samples were attached to a stub using double adhesive tape and sputter coated wit h carbon. SEM images were used to qualitatively compare t he morphology of two coating techniques, as well as to analyze the progression of the PLGA coating morphology prior to implantation in a murine aneurysm model and ex-vivo In Vivo Analyses Murine aneurysm model The cellular response of the sutures and coils was analyzed in viv o using a murine aneurysm model. In vivo analyses were performed in co llaboration with Koji Hosaka, Erin Wilmer, and Daniel Downes in the Department of Neurosurgery at the University of Florida. Briefly, the right common carotid artery (RCCA) of a mouse was bathed in 25 mg/mL of diluted and filter sterilized porcine pancreatic elasta se for 20 minutes, causing the artery to swell. The di stal end of the artery was c auterized to occlude blood flow, creating an aneurysm. Animals were closed up, allowed to regain consciousness and become ambulatory, and were moni tored for any post-surgical complications or distress. 33

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Three weeks post-surgery, the aneurysm s were treated with the PLGA-coated sutures or coils. An incision was made on the RCCA wall by a microsurgical blade, and the suture or coil was inserted into the resulting pocket. Sutures were used in preliminary testing and were later replaced by coated coils since the sutures were more difficult to manipulate and would dissolve over time inside tissue. Preliminary suture testing included uncoated sutures, sutures coated with PLGA only, and sutures coated with PLGA and CTGF. Each type of sample was tested on n=3 mice. Subsequent studies with coils included coils coated with PLGA and CTGF, SDF-1, BMP-4, or MCP1. Uncoated coils and coils coated wit h only PLGA were used as controls. Sutures or coils were allowed to remain inside the animal for three weeks, after which the animal would be euthanized. The right and left common carotid arteries were immediately excised and placed in a 4% parafo rmaldehyde solution. After the arteries were excised, the coils or sutures were re moved and the arteries were cut into crosssections and analyzed. Analysis of ex vivo samples In preliminary suture studies, the presence of CTGF was determined by coupling with anti-CTGF. Hemotoxylin and eosin (H&E) stainin g was also used to evaluate cellular behavior of aneurysms treated with coils. Immunohistochemical (IHC) staining was performed on the 5 m cross-sections embedded in OCT cryoembedding media from treated aneurysms fixed in 4% PFA. Sections were treated with ac etone at -20 C for 5 minutes and air-dried prior to staining. The OCT media was removed from the sample slides by rinsing with 1X Wash Solution (DAKOCytomation, Carpinteria, CA) for 5 minutes. 34

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Smooth muscle staining was performed by biotinylating the primary antibody. Slides wer e blocked in Serum Free Blocking Solution (DAKOCytomation, Carpinteria, CA), then avidin and biotin (Vector Labs, Bu rlingame, CA) for 15 minutes each. Slides were washed between each step. Mouse anti-actin (Sigma, St.Louis, MO) was applied for 20 minutes at room temper ature using a dilution of 1:600. After washing twice for five minutes, Streptavidin Alexa Fluor 594 (Molecular Probes, Eugene, OR) was applied at 1:500 for 45 minutes as a detection agent. Staining with Fibroblast-Specific Protein1 (FSP-1) (Abcam, Cambridge, MA), at a titer of 1:150, required 25-minute heat induc ed antigen retrieval with 10mM Citra buffer, pH 6.0 for optimal staining. Slides were blocked in 2% horse serum for 30 minutes prior to the application of primary antibody overnight at 4 C. Slides were incubated for 45 minutes in 1:500 Alexa Fluor 594 anti-rabbit raised in donkey (Molecular Probes) and were washed twice for 5 minutes and mount ed in VectaShield with 4,6-diamidino-2phenylindole (DAPI) prior to imaging. Posi tive control tissues and concentrationmatched Ig controls were included with each immunoassay. 35

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36 Figure 3-1. Standard Curve at 595 Nanometer s. Known concentrations of BSA were used to construct a standard curve based on absorbance at 595 nm.

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CHA PTER 4 RESULTS AND DISCUSSION Protein Release Measurements Bovine ser um albumin (BSA) was used as a model protein to determine the protein release rate from the PLGA and PEC coatings. BSA was chosen for its costeffectiveness, as well as because it is the st andard for the Bradford assay, therefore the absorbance of the unknown samples would more closely match that of the standard. The release rate of BSA from each coating wa s intended to serve as a model for protein release. Ultimately the PEC method coatings were discontinued from testing due to concerns regarding the solubi lity of the alginate. Alginate has been attempted as an embolic agent for cerebral aneurysms; however the water-soluble nature of the alginate causes escape of the embolic agent into the parent artery circulation46. Furthermore, Koji Hosaka in the Department of Neurosurger y at UF attempted unsuccessfully to inject alginate into a murine aneurysm as an embolic agent and experienced significant difficulty maintaining the alginate inside t he aneurysm lumen due to the water solubility of the alginate. The PEC coatings we re discontinued due to these concerns. Bradford assays for the coils coated with the PEC method PLGA Coating 1 were inconclusive, since the absorbance readings for the unknown samples were extremely close to those for the 0 g/mL standard, suggesting that the concentration of BSA released from each coating was on the order of nanograms or picograms. Since the tested proteins were only available in 10 g/mL concentrations due to budget concerns, the BSA was diluted to this concentration in order to maintain uniformity with the rest of the protein coatings. Although an in vitro release rate could not be determined for 37

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PLGA Coat ing 1, it was evident that the coatings were indeed releasing protein, since the in vivo results with each coating were consistently different. Bradford assays for the PLGA Coating 2 were also inconclusive. This time, the coatings were made with a 100 g/mL BSA solution instead of a 10 g/mL solution. The emulsions were composed of 100 g of protein solution and 1 mL of PLGA and magnesium hydroxide solution. Although the initial Bradfor d assay was inconclusive, the release rate experiment will be repeated to determine a release rate, since the greater concentration of BSA in these coatings should release at detectable amount of protein. Scanning Electron Microscopy Scanning Electron Microscope (SEM) images were used to examine the morphology of the PLGA coat ings on the platinum coils. A homogeneous c oating was desired that would cover the entire surface of the coil. Figure 41 depicts an uncoated coil at 20X magnification (A) and 100X magnification (B). The uncoated coil is smooth and has a ribbed appearance due to tight winding of the platinum wire. The morphology of the airbrushed coils differs from that of the dip-coated coils. Airbrushing was discarded as a coating techni que due to difficulties with controlling the flow of the airbrush, signific ant waste of PLGA solution during the coating process, and contamination concerns for the proteins. The morphology of a coil airbrushed with PLGA Coating 1 is shown in Figure 4-2 at 20X magnification (A), and at 100X magnification (B). These images show a ragged coating with agglomerations of PLGA on the sides of the coil. T he size of the coils and the th in stream of PLGA emerging 38

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from the nozzle of the airbrush make it difficult to see h ow much is accumulating on the surface of the coil. Figure 4-3 shows a coil that has been di p-coated with PLGA and SDF-1 at 20X magnification (A), 50X magnification (B), 100 X magnification (C), 200X magnification (D), 700X magnification (E), and 2000X magnification (F). Platinum coils in these images were coated using PLGA Coating 2. These images show a porous coating that tends to be sparser towards the middle of t he coils and agglomerate at one end. This is most likely due to the manner in which the coils are coated with PLGA. Since the coils are dip-coated, it is likely t hat during the coating process the PLGA solution would drip towards the opposite end of the coil and accumulate at the end. Figure 4-3 (D) and (E) show that the coati ng is uneven in some parts of the coil. This is likely due to the fact that the coils are laid flat to dry. The areas where the coil lays flat against the surface of the Petri dish on which it dries will have less PLGA coating. Figure 4-3 (E) shows the uneven cover age of the PLGA coating in more detail. Figure 4-3 (F) shows that the surface mor phology is rugged and porous. The lumps in the coating could be attribut ed to the magnesium hydroxide that is added to the PLGA solution as a neutralizing agent. Although these coatings co ver the surface of the coil unevenly, they are suitable for preliminary test ing purposes. Future testing may require more precise coating methods. Coils were examined ex vivo prior to treatment with paraformaldehyde. Figure 4-4 depicts ex vivo coils at 20X magnification (A), 150X magnification (B), and 200X magnification (C). The coils in these images had been coated with PLGA Coating 2. The coil appears thinner than t he other coils in Figures 41, 4-2, and 4-3 because the 39

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wire was unwound during the removal process fr om the sectioned arte ry. Figure 4-4 (B) and (C) show what appears to be a mixture of coating as well as tissue growth on the surface of the coil. In these images t here appears to be striated sections that could possibly be tissue that has remained attac hed to the coil. Compari ng Figure 4-4 (D) with Figure 4-3 (A), it is evident that most of the coating has di ssolved from the coil, leaving behind mostly uncoated coil. Murine Aneurysm Model Vicryl Sutures Vicryl sutures were used for preliminary testi ng of PLGA Coating 1 due to their cost-effectiveness. Since an in vitro analysis yielded inconclusive results due to low amounts of protein contai ned in the coating, an in vivo analysis was conducted in order to determine the effects of the protein released. Vicryl sutures with PLGA, PLGA and CTGF, and uncoated Vicryl sutures were tested in vivo in a murine aneurysm model as described in Chapter 3. Sutures were impl anted in the aneurysm site and were left in the mice for three weeks. After three weeks, the mice were euthanized as outlined in Chapter 3, and then the affect ed arteries were removed, cross-sectioned, and stained as described previously. Fluorescence microscopy was used to analyze ex-vivo cross-sectioned arteries after the sutures were removed. The nucle i of recruited progenitor cells were stained blue with 4,6-diamidino-2-phenylindole (DAPI ), and anti-CTGF was used to determine the presence of CTGF. Red fluorescent tags indicated the presence of CTGF when it coupled with the CTGF present. Since Vicryl sutures are designed to dissolve in the tissue environment, in vivo results from the uncoated suture could not be examined. 40

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Figure 4-5 depicts fluorescence microscopy images at 10X magnif ication of Vicryl sutures coated with PLGA (A) and Vicryl su tures coated with a PLGA coating containing 10 g of CTGF (B). Although a significant nu mber of cells may be observed in these images, it is unclear what type of cells they are, and whether they we re recruited to the area by the CTGF present. These preliminary inconclusive results, in addition to the fraying of the uncoated suture during implanta tion efforts, were the driving force behind the decision to use platinum coils instead of sutures in order to obtain an effective control. Platinum Coils Platinum coils with PLGA Coating 1 The first tri al with platinum coils involv ed PLGA coatings with CTGF, SDF-1, or BMP-4. Uncoated platinum coils and platinum coils coated with only PLGA were used as controls. In this trial, the PLGA Coating 1 was used, where 1 mL of 10 g/mL protein was mixed with 10 mL of PLGA to create the emulsions as descr ibed in Chapter 3. This resulted in a small concentration of protei n contained in each coating, leading to the release of a concentration of each protein inside the aneurysm lumen that could not be determined using the Bradford Assay. Figure 4-6 depicts hematoxylin and eosin (H &E) staining of the cross-sectioned ex vivo arteries from this trial. Ideal results would show smooth muscle tissue growth around the periphery of the aneurysm lumen, indica ted by a strong red color. Figures 46 (A) and (B) show ex vivo arteries treated with an uncoat ed platinum coil, and an ex vivo artery treated with a platinum coil coated with only PLGA. These images serve as controls by which to compare t he protein-encapsulated coatings. 41

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Figure 4-6 (C), which shows an ex vivo artery treated with PLGA and BMP-4, does not differ si gnificantly in appearance from th e controls. However, Figures 4-6 (D) and (E), which show images of ex vivo arteries with treated with CTGF and SDF-1, respectively, show a significantly larger amount of what appears to be new smooth muscle cells, indicated in deep red in the images. In this first trial, it appears that SDF-1 elicits the most favorable cellular res ponse, with CTGF also showing a favorable although less robust response. Although the resu lts of the first trial were promising, more data was required in order to prov e that SDF-1 and CTGF indeed elicited the growth of new smooth muscle cells al ong the periphery of the aneurysm lumen. Platinum coils with PLGA Coating 2 A second trial with platinum coils was performed to corroborat e the data from the first trial. In the second tr ial, the PLGA Coating 2 emulsions were composed of smaller volumes of protein and PLGA in a 1:10 rati o. The purpos e of using smaller volumes was to increase the concentration of proteins present on the coils by having the same amount of protein present in a smaller volume of soluti on, increasing the number of collisions between protein molecules and t he coil and thus leading to more protein embedded in the coil coating. The second tr ial tested PLGA coatings with CTGF, SDF1, BMP-4, or MCP-1. PLGA coatings wit h no protein were used as a control. Figure 4-7 (A) shows an H&E stain of an ex vivo artery that was treated with a control coil coated only with PLGA. Figures 4-7 (B-E) show ex vivo arteries treated with coils coated with PLGA and SDF-1, MCP-1, BM P-4, and CTGF, respectively. Similar to results from the first tria l, BMP-4 once again does not show a favorable cellular response in terms of the appearance of smooth muscle cells. However, SDF-1, MCP-1, and CTGF all show greater amounts of sm ooth muscle cells compared with the PLGA 42

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43 control. It is interesting to note that despite the increased concentration of protein released, SDF-1 yielded a less robust response in the second trial than in the first. The response shown by CTGF is significantly strong. MCP-1 was not tested in the first trial. Further trials are necessary to corroborate this data. Figure 4-8 depicts fluorescence microsc opy images at 10X magnification of immunohistochemical staining of ex vivo arteries treated with PLGA coating with no protein (A) and PLGA and SDF-1 coating (B). Cell nuclei are stained blue with DAPI, and smooth muscle actin is stained red. Figure 4-8 (B) shows a si gnificantly greater presence of smooth muscle actin than Figure 48 (A), indicating that SDF-1 yields a greater presence of smooth musc le cells than the control. Further examination of the effects of SDF-1, CTGF, and MCP-1 will need to be investigated in future trials to corroborate this preliminary data.

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A B Figure 4-1. SEM Image of an Uncoated Plat inum Coil. A) 20X magnification and B) 100X magnification. A B Figure 4-2. SEM Image of a Coil Coated by Airbrushing. A) 20X magnification and B) the middle of the coil at 100X magnification. 44

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A B C D E F Figure 4-3. SEM Images of Coated Plati num Coil. A) 20X magnification, B) 50X magnification, C) 100X magnification, D) 200X magnification, E) 700X magnification and F) 2000X magnification. The coating accumulates at one end of the coil due to vertically dip-coating the coil in the PLGA solution. The coating does not evenly cover the surface of the coil since it is laid flat to dry. 45

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A B C Figure 4-4. SEM Images of an Ex Vivo Coil. A) at 20X m agnification, B) at 150X magnification and C) at 200X magnification. The coil appears stretched due to the manner in which it was removed fr om the artery cross-section. The coil was coated with PLGA and SDF-1. 46

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A B Figure 4-5. IHC Staining of Ex Vivo Arteries Treated with Suture s. A) Suture with only PLGA coating, and B) Suture with PL GA and CTGF coating. Fluorescence microscopy images at 10X magnification. DAPI was used to stain cell nuclei blue and anti-CTGF was used to identify the presence of CTGF. 47

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A B C D E Figure 4-6. H&E Staining of Ex Vivo Arteries with Platinum Co ils First Trial. A) Uncoated coils, B) Coils coated only wit h PLGA, C) BMP-4 D) CTGF, and E) SDF-1. 48

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A B C D E Figure 4-7. H&E Staining of Ex Vivo Arteries with Platinum Coils Second Trial. A) PLGA coating with no protein, B) SDF-1, C) MCP-1, D) BMP-4, E) CTGF. 49

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50 A B Figure 4-8. IHC Staining of Ex Vivo Arteries Treated with Plat inum Coils. A) PLGA coating with no protein and B) PLGA and SDF-1 coating. Cell nuclei are depicted in blue and smooth muscle is depicted in red. Fluorescence microscopy images are at 10X magnification.

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CHA PTER 5 CONCLUSIONS Several coatings capable of releasing pr otein were examined, with a waterinoil emulsion c omposed of PLGA and protein show ing the greatest promise. Chitosan and alginate in a polyelectrolyte complex prov ed to be an unsuccessful technique due to the solubility of the PEC coating posing problem s with migration of the coating into the parent artery. PLGA Coating 2 provided a great er initial concentration of protein in the coating solution, and shows promise for use as a coating that would be most costeffective, requiring smaller initial protein concentrations. Airbrushing the PLGA coatings into the coils proved to be an inefficient coating method due to significant loss of the PL GA solution during the coating process agglomeration of PLGA on the coil, and concer ns regarding contamination of protein solutions. Dip coating proved to be an effect ive method for coating the coils that would maintain the sterility of the protein solution and allow the most control in the coating process. Further optimization of the dip-coating method is required to ensure that the coating dries evenly on the surface of the coils. Release rates for the coati ngs were unable to be determined in vitro using the Bradford Protein Assay, alt hough it is likely that PLGA Coating 2 does release a detectable amount of protein. Further testing is required to determine the in vitro release rate of this coating. However, both PLGA Coating 1 and PLGA Coating 2 were proven to release protein based on in vivo studies in a murine aneurysm model due to a noticeable difference in the cellular response of ex vivo arteries treated with different protein solutions in comparison with the control coatings. 51

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52 The cellular response of murine aneurysm s treated with BMP-4, CTGF, SDF-1, and MCP-1 was tested and compared. CTGF, SDF-1, and MCP-1 elicited a favorable response in vivo proven by H&E staining of ex vivo aneurysms indicating a greater presence of smooth muscle cells than control coils. Smooth muscle actin staining of aneurysms treated with SDF-1 showed a robust response of smooth muscle cells around the periphery of the aneurysm lumen. This preliminary study proves that CTGF, SDF-1, and MCP-1 should be explored further as possible proteins incorporated in a bioactive PLGA coating for platinum endovascular coils.

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CHA PTER 6 FUTURE WORK The data obtained from these preliminary studies sho w positive steps forward toward the development of a bioactive coating for plat inum endovascular coils. The following studies should be performed in order to corroborate and expand upon the results from this study. 1. Characterization of the release rate of PLGA Coating 2. PLGA Coating 2 uses a significantly hig her initial protein concen tration in the coating emulsion than PLGA Coating 1, therefore, the amount of protein released should be able to be detected with a Bradf ord assay. A timed-release study tested at 0.5, 1, 3, 7, 14, and 21 da ys is necessary to model the release rate of PLGA Coating 2. 2. Viability of proteins released. The Bradford Pr otein Assay measures the concentration of soluble protein released fr om the coatings. It is of interest to determine if the proteins releas ed from the coatings are viable by performing an Enzyme-Linked Immunosor bent Assay (ELISA) on each protein released fr om the coatings. 3. Repetition of in vivo testing with CTGF, BMP-4, SDF-1, and MCP-1. These studies should be repeated in order to corroborate the results from trials 1 and 2. Furthermore, further characteriza tion of the cell types present in the ex vivo cross-sections of treated aneurysms would be of interest. 4. Improvement of the uniformity of t he PLGA Coating. SEM images of the coils dip-coated with PLGA indicate that a more uniform coating is needed. 53

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Mechanical dip-coating met hods could be explored in or der to achieve more uniform coatings. 5. Optimization of protein dosage. Determining the optimum dosage for each protein is necessary to obtain the mo st favorable cellular response. Eventually, a combination of proteins may be encapsulated in the PLGA coating. The release rate of the proteins could be further optimized through the use of additives that would cause proteins to be released at a slower rate. 6. Characterization of Long-Term Results. Coated coils were tested in a murine aneurysm model for 3 weeks, which was long enough for the appearance of new smooth muscle cells to occur. However, more longterm studies would show more organized fibrous tissue and eventual endothelialization of the aneurysm neck. 54

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LIST OF REFERENCES 1. Abrahams JM, Diamond SL, Hurst RW et al: Topic Review: Surface modifications enhancing biological activity of Guglielmi detachable coils in treating intracranial aneurysms. Surg Neurol 54 :34-41, 2000 2. Abrahams JM, Forman MS, Grady MS, et al: Delivery of human vascular endothelial growth factor with platinum coils enhances wall thickening and coil impregnation in a rat aneurysm model. AJNR 22 :1410-1417, 2001 3. Abreu JG, Ketpura NI, Reversade B, et al. Connective-tissue growth factor (CTGF) modulates cell signaling by BMP and TGF. Nat Cell Bio 4 :599-604, 2002 4. Adamson J, Humphries SE, Ostergaar d JR, et al: Are cerebral aneurysms atherosclerotic? Stroke 25 :963-966, 1994 5. Aoki T, Kataoka H, Ishibashi R, et al: Impact of mono cyte chemoattractant protein-1 deficiency on cerebral aneurysm formation. Stroke 40 :942-951, 2009 6. Bavinzski G, Talazoglu V, Killer M, et al: Gross and microscopic histopathological findings in aneurysms of the human brain tr eated with Guglielmi detachable coils. J Neurosurg 91 :284-293, 1999 7. Bederson JB, Connolly ES Jr, Batjer HH, et al: Guidelines for the management of aneurysmal subarachnoid hemorrhage: A stat ement for healthcare professionals from a special writing group of the Stroke Council, American Heart Association. Stroke 40 :994-1025, 2009 8. Bendszus M, Bartsch A, Solymosi L: Endovascular occlusion of aneurysms using a new bioactive coil: A matched pair analysis with bare platinum coils. Stroke 38:2855-2857, 2007 9. Byrne JV, Guglielmi G. Endovascular Treatment of Intrac ranial Aneurysms. Berlin; New York: Springer, 1998 10. Byrne JV, Sohn MJ, Molyneux AJ: Five-year experience in using coil embolization for ruptured in tracranial aneurysms: outco mes and incidence of late rebleeding. J Neurosurg 90: 656-663, 1999 11. Choudhari KA, Ramachandran MS, McCa rron MO, et al: Aneurysms unsuitable for endovascular intervention: Surgic al outcome and management challenges over a 5-year period following International Subarachnoid Haemorrhage Trial (ISAT). Clinical Neurology a nd Neurosurgery 109 :868-875, 2007 12. Dawson RC, Krisht AF, Barrow DL, et al: Treatment of experimental aneurysms using collagen-coated microcoils. Neurosurgery 36 :133-140, 1995 55

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13. Deckers MML, van Bezooijen RL, van der Horst G, et al: Bone morphogenic proteins stimulate angiogen esis through osteoblast-derived vascular endothelial growth factor A. Endocrinology 143 :1545-1553, 2002 14. Forget, Jr TR, Benitez R, Veznedaroglu E, et al: A review of size and location of ruptured intracranial aneurysms. Neurosurgery 49 :1322-1326, 2001 15. Frazier K, William s S, Kothapalli D, et al: Stimul ation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107 ,404-411, 1996 16. Gaba RC, Ansari SA, Roy SS, et al: Em bolization of intrac ranial aneurysms with hydrogel-coated coils versus inert platinum coils: effects on packing density, coil length and quantity, procedure performance, cost, lengt h of hospital stay, and durability of therapy. Stroke 37 :1443-1450, 2006 17. Guglielmi G, Viuela F, Dion J, et al : electrothrombosis of saccular aneurysms via endovascular approach. Part 2: pr eliminary clinical experience. J Neurosurg 75:8-14, 1991 18. Gugliemi G, Viuela F, Duckwiler G, et al: Endovascular treatment of posterior circulation aneurysms by electrothrombosis using electrically detachable coils. J Neurosurg 77 :515-524, 1992 19. Guglielmi G, Viuela, F, Sepetka I, et al: Electrothrombosis of saccular aneurysms via endovascular approach. Part 1: electrochemic al basis, technique, and experimental results. J Neurosurg 75:1-7, 1991 20. Heilman CB, Kwan ESK, Wu JK: Aneur ysm recurrence following endovascular balloon occlusion. J Neurosurg 77:260-264, 1992 21. Higashida RT, Halbach LD, Hieshima GB, et al: Detachable balloon embolization therapy of posterior circulat ion intracranial aneurysms. J Neurosurg 71 :512-159, 1989 22. Hino K, Konishi Y, Shimada A, et al: Morphologic changes in neo-intimal proliferation in an experim ental aneurysm after coil emboliz ation: effect of factor VIII administration. Neuroradiol 46 :996-1005, 2004 23. Ho Y, Mi F, Sung H, et al: Heparin -functionalized chitosan-alginate scaffolds for controlled release of growth factor. Int J Pharm 376 :69-75, 2009 24. Hogan BLM: Bone morphogenic proteins in development. Curr Opin Genet Dev 6 :432-438, 1996 56

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25. Jain RA: The manufacturing techni ques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomat 21 :2475-2490, 2000 26. Johnston SC, Colford JM Jr, Gress DR: Oral contraceptives and the risk of subarachnoid hemorrhage: a meta-analysis. Neurology 51 :411-418, 1998 27. Juelva S, Poussa K, Porras M: Fa ctors affecting formation and growth of intracranial aneurysms: A long-term follow-up study. Stroke 32:485-491, 2001 28. Killer M, Hauser T, Wenger A, et al: Comparison of experimental aneurysms embolized with second-generation embo lic devices and platinum coils. Acta Neurochir 151:497-505, 2009 29. Linn FH, Rinkel GJ, Algra A, et al: Incidence of subarachnoid hemorrhage: Role of region year and rate of CT scanning: A meta-analysis. Stroke 27 :625-629, 1996 30. Mawad ME, Mawad JK, Cartwright J, et al: Long-term histopathologic changes in canine aneurysms embolized with Guglielmi detachable coils. AJNR 6 :7-13, 1995 31. Molyneux A, Kerr R, Bir ks J, et al: Risk of recurr ent subarachnoid hemorrhage, death, or dependence and standardized mortality ratios after clipping or coiling of an intracranial aneurysm in the Inter national Subarachnoi d Aneurysm Trial (ISAT): long-term follow-up. Lancet Neurol 8 :427-433, 2009 32. Molyneux A, Kerr R, Stra tton I, et al: International Subarachnoid Aneurysm Trial (ISAT) of neurological clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneur ysms: a randomised trial. Lancet 360 :1267-1274, 2002 33. Mukaida N, Harada A, Matsushima K et al: Interleukin-8 (IL-8) and monocyte chemoattractant and activating factor (mcaf/mcp-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine and Growth Factor Reviews 9 :9-23, 1998 34. Murayama Y, Suzuki Y, Viuela F, et al: Development of a biologically active Guglielmi detachable coil for the treatment of cerebr al aneurysms. Pa rt I: In vitro study. AJNR 20 :1986-1991, 1999 35. Murayama Y, Tateshima S, Gonzal ez NR, et al: Matrix and bioabsorbable polymeric coils accelerate healing of intracranial aneurysms: Long-term experimental study. Stroke 34:2031-2037, 2003 57

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BIOGRAPHICAL SKETCH Cristina Elena Fernandez was born in Ca racas, Venezuela to Antonio Fernandez and Maria Elena Pozo de Fernandez in 1986. She moved to the United States when she was two years old, and was raised in Melbourne, Florida, where she graduated from West Shore Juni or Senior High School in 2004. In 2008, she earned a Bachelor of Science in materials science and engineer ing with a concentration in biomaterials from the University of Florida and was awarded the Dean Joseph Weil Award for Outstanding Leadership in the College of Engineering. As an undergraduate and graduate student at the University of Florida, Cristina served on the executive board of several loca l and national organizations, including the Society of Women Engineers, the Benton Engineering Council, the Society for Biomaterials, Epsilon Lambda Chi, the Engi neering Student Advisory Council, and the National Association of Engi neering Student Councils. She developed an interest in biomaterials for cardiovascular and wound healing applications through her undergraduate and graduate research experiences. After receiving her Master of Science from the University of Florida, Cris tina will attend Duke University in the fall of 2010 to pursue a PhD in Bi omedical Engineering. 61