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Modulating Macrophage Response to Biomaterials

Permanent Link: http://ufdc.ufl.edu/UFE0042899/00001

Material Information

Title: Modulating Macrophage Response to Biomaterials
Physical Description: 1 online resource (168 p.)
Language: english
Creator: Zaveri, Toral D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: biomaterial -- integrin -- macrophage -- nanotopography
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of numerous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macrophage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach involves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, we have investigated the role of integrin Mac-1 and RGD-binding integrins in the in-vivo osteolysis response and macrophage inflammatory processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of fibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac-1 integrin was isolated using a Mac-1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted biomaterial with ELVAX polymer loaded with Echistatin which contains the RGD sequence. For the in-vivo osteolysis study and to study the in-vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in ELVAX and dissolved in macrophage media respectively. By studying the phagocytosis, inflammatory and FBR of macrophages from integrin knockout mice, as well as using various integrin blocking techniques we aim to identify the role of various integrins in macrophage inflammatory response. These integrins can serve as therapeutic targets for mitigating this inflammatory response and improve functional life of implanted biomaterials. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 days. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. Hence although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent. Considering the toxicity of ZnO nanorod surface towards macrophages, their role as an antibacterial surface was explored. Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO and glass substrates. Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. ZnO nanorod surface and sputtered ZnO demonstrated a significant bactericidal effect, killing respectively 2.5x and 1.7x times the number of bacteria dead on glass. A similar bactericidal effect of ZnO substrates on S. epidermidis was also evident, with sputtered ZnO and ZnO nanorod substrates killing respectively 22x and 32x times bacteria dead on glass. These data support the further investigation of ZnO nanorod coatings for bacterial adhesion resistance and bactericidal properties.
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.
Statement of Responsibility: by Toral D Zaveri.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Keselowsky, Ben.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042899:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042899/00001

Material Information

Title: Modulating Macrophage Response to Biomaterials
Physical Description: 1 online resource (168 p.)
Language: english
Creator: Zaveri, Toral D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: biomaterial -- integrin -- macrophage -- nanotopography
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of numerous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macrophage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach involves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, we have investigated the role of integrin Mac-1 and RGD-binding integrins in the in-vivo osteolysis response and macrophage inflammatory processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of fibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac-1 integrin was isolated using a Mac-1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted biomaterial with ELVAX polymer loaded with Echistatin which contains the RGD sequence. For the in-vivo osteolysis study and to study the in-vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in ELVAX and dissolved in macrophage media respectively. By studying the phagocytosis, inflammatory and FBR of macrophages from integrin knockout mice, as well as using various integrin blocking techniques we aim to identify the role of various integrins in macrophage inflammatory response. These integrins can serve as therapeutic targets for mitigating this inflammatory response and improve functional life of implanted biomaterials. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 days. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. Hence although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent. Considering the toxicity of ZnO nanorod surface towards macrophages, their role as an antibacterial surface was explored. Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO and glass substrates. Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. ZnO nanorod surface and sputtered ZnO demonstrated a significant bactericidal effect, killing respectively 2.5x and 1.7x times the number of bacteria dead on glass. A similar bactericidal effect of ZnO substrates on S. epidermidis was also evident, with sputtered ZnO and ZnO nanorod substrates killing respectively 22x and 32x times bacteria dead on glass. These data support the further investigation of ZnO nanorod coatings for bacterial adhesion resistance and bactericidal properties.
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.
Statement of Responsibility: by Toral D Zaveri.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Keselowsky, Ben.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042899:00001


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1 MODULATING MACROPHAG E RESPONSE TO BIOMAT ERIALS By TORAL ZAVERI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOP HY UNIVERSITY OF FLORIDA 2011

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2 2011 Toral Zaveri

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3 To Mom, Dad and Khanjan

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4 ACKNOWLEDGMENTS I would like to express my heartiest gratitude to Dr Benjamin Keselowsky for his support and guidance without which none of this work would have b een possible. His encouragements and suggestions made the research more interesting and challenging. His patience and faith made me successfully complete this endeavor even after facing countless hurdles and roadblocks. I would like to acknowledge my super visory committee: Dr Michael Clare Salzler, Dr Steve Ghivizzani, Dr Brandi Ormerod and Dr Yiider Tseng for serving on my supervisory committee, for their valuable time and support /teachings, guidance and support. I am thankful to all my past and prese Abhinav Acharya, Jerome Karpiak, Natalia Dolgova, Jamal Lewis, Matt Carsten and Matt Sines. I would especially like to acknowledge Natalia as she has been a mentor throughout my PhD guiding me through the intri cacies of lab experiments. I really appreciate her help with the mouse surgeries which was the most daunting part of my PhD research owing to my immense fear of mice I would also like to give special thanks to Jamal as he has assisted me with several of my exp eriments and particularly mouse surgeries. I acknowledge the help of the Pathology mouse colony staff: Fred and Ronnie. It is from them that I have learnt the basics of mouse colony breeding and was successfully able to maintain my Mac 1 KO mouse col ony. I take this opportunity to thank Dave Miller from the CGRC ACS facility for his help in maintaining the Mac 1 KO colony and working with my schedule in having a ready supply of mice available. I would also like to thank t he ACS vet technicians who hel p me set up and practice mice surgeries.

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5 Histology experiments were a major part of my research and I would like to thank Marda Jorgensen from the CTAC facility for her support and guidance in teaching me the intricacies of paraffin embedding, sectioning and staining. I would like to offer special thanks to Dr Wronski for guiding me and allowing me to work in his lab for the PMMA embedding procedure. I thank everyone in his lab especially Alicia and Alyssa for guiding me at each step of the PMMA embedding process as well as helping out with the staining process. I had very limited time to finish my experiments and they have been more than cooperative in helping me meet my time line. Before I begin to acknowledge all my friends here in Gainesville, I woul d like to give my biggest thanks to Kamal, without whom I would have never had the courage to come to US in the 1 st place or ever consider switching to the PhD program. I would also like to thank my friends from my undergraduate years Bhagyashree, Kanchan, Reshma, Jatin, Karishma and Kartik who supported me through emails and phone calls during the initial stay in Gainesville when I had made no friends here. During my stay here in Gainesville I made some close friends, which have been my biggest su pport aw ay from home. They have supported me both personally as well as professionally in sharing the frustrations of PhD life. The few people who have been there for me throughout the PhD have been Neetu, Sushant and Dushyant. They have been patient enough to und erstand my research, suggest solutions and share the frustrations on a daily basis. They became my family away from home, stood by me through the toughest times of my life and never left my side even for a single day. I will always remember the countless b each trips that we took and the Gator nights we went to so religiously. Neetu with her hard working and dedicated nature was the biggest

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6 source of inspiration to persevere through all difficulties. Her warm heart, contagious laughter and vivacious nature f illed my days with joy. It is from Sushant that I have learnt the biggest lessons of my life, lessons of patience, understanding and selflessness and it is in him I have found my closest friend. He makes talking to him so easy and with him I have had the m ost memorable days of my life exploring Gainesville like never before. Another person that made a big impact on me in these years at UF has been Sungho, his versatile and multicultural nature helped me understand and cope with the cultural differences that I faced in the US. I have learnt a lot from him about biomedical engineering as well as the world owing to his insatiable thirst for knowledge and interest in history, geography, art and anthropology. I would also like to thank all my other roommates Am ee Mehta, Preeti Sood and Mamta Chahar who became my friends and made the stay at Gainesville an enjoyable experience. For making Gainesville fun and exciting, large number of my friends played a role and I would like to thank, Abhishek, Purushottam, Aniru ddh, Vibhava, Richa, Karam, Arul, Parnitha, Anu, Jaesoek and Ashwini. I would like to thank my cousin Monali who lives in Miami and hence is the nearest family member. Whenever I missed family, Miami was never too far and her warm hospitality m ade me feel I am back in India. This acknowledgment would not be complete without thanking my parents, my brother Jesal, my husband Khanjan and his family. My parents have always been very supportive and encouraging for my education and no price or sacrifice has ever been big enough for them when it came to ensuring that I got the best of education as well as what life had to offer. They visited me in Gainesville every single year and spent a month with me, which turned out to be the best time of the year for me. Khanj an has

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7 been my biggest support in the last 2 years, especially towards the end when the end though in sight seemed very far. He encouraged me to strive harder and never lose focus. Whenever I was frustrated with work he made me laugh and I got to work with infused vigor. To everyone who made the last 6 years of my life comfortable, enjoyable and worth all the pursuit that goes into this degree

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 19 Biological Response to Biomaterials ................................ ................................ ....... 19 Injury Blood material Interactions and Provisional Matrix Formation .............. 20 Protein Adsorption on Biomaterial Surface ................................ ....................... 21 Acute Inflammatio n ................................ ................................ ........................... 22 Chronic Inflammation ................................ ................................ ....................... 23 Granulation Tissue and Foreign Body Reaction ................................ ............... 24 Approaches to Modulate Foreign Body Response ................................ .................. 24 Macrophage Response to Biomaterials ................................ ................................ .. 25 Integrins ................................ ................................ ................................ .................. 27 Role of Integrins in Host Immune Response to Biomaterials ............................ 28 Macrophage Integrins ................................ ................................ ....................... 29 Mac 1 integrin ................................ ................................ ............................ 29 RGD binding integrins ................................ ................................ ................ 31 Integrin Targeted Therapies ................................ ................................ .................... 31 Mac 1 Targeted Therapies ................................ ................................ ............... 32 Antibody blocking ................................ ................................ ....................... 32 Neutrophil inhibitory factor (NIF) ................................ ................................ 32 Targeted Therapies for RGD Binding Integrin ................................ .................. 33 Polymers for Sustained Release of Integrin targeted Therapies ...................... 34 Clinical Significance ................................ ................................ ................................ 35 Arthritis and Total Joint Replacement ................................ ............................... 35 Failure of Total Hip Replacement: Asept ic loosening ................................ ....... 36 Role of Macrophages in Wear Debris Induced Periprosthetic Osteolysis ......... 37 Role of Osteoclasts and Integrins on Ost eoclasts ................................ ............ 39 Surface Modification Approaches to Modulate FBR ................................ ............... 39 Nanostructured Materials ................................ ................................ ........................ 40 Zinc Oxide (ZnO) Nanorod Surface ................................ ................................ .. 41 Implant Infection ................................ ................................ ............................... 42 Thesis Outline ................................ ................................ ................................ ......... 43 2 ROLE OF INTEGRINS IN MACROPHAGE RESPONSE TO PARTICULATE BIOMATERIALS ................................ ................................ ................................ ..... 4 6

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9 Background ................................ ................................ ................................ ............. 46 Ex perimental Procedure ................................ ................................ ......................... 50 Disc Preparation for Controlled Release of RGD ............................. 50 Determination of Loading and Release Kinetics o f RGD from Discs ................................ ................................ ................................ ............. 51 Mouse Calvarial Osteolysis Model ................................ ................................ ... 51 Macrophage Generation ................................ ................................ ................... 53 Polystyrene(PS) Microparticle Preparation ................................ ....................... 54 Endotoxin Testing ................................ ................................ ............................. 55 Quantification of Macrophage Phagocyto sis of PS MPs ................................ .. 56 Quantification of Macrophage Cytokine Production upon Phagocytosis of PS MPs ................................ ................................ ................................ ......... 56 Polyethylene (PE) Microparticle Preparation ................................ .................... 57 PE MP preparation for phagocytosis experiments ................................ ..... 57 PE MP preparation for cytokine experiments ................................ ............. 58 Inverted Cell Culture Technique for Phagocytosis of UHMWPE MPs .............. 59 Quantification of Macrophage Phagocytosis of PE MPs ................................ .. 60 Quantification of Macrophage Cytokine Production ................................ .......... 60 Statistical Analysis ................................ ................................ ............................ 61 Results ................................ ................................ ................................ .................... 61 Loading and Release Kinetics of RGD from Discs ........................... 61 Role of RGD binding Integrins in MP induced Osteolysis ................................ 61 Role of Mac 1 Integrins in MP induced Osteolysis ................................ ........... 62 Purity of Macrophage Culture and Mac 1 KO Macrophages ............................ 63 Role of Mac 1 and RGD binding Integrins in Macrophage MP Uptake of PS MPs ................................ ................................ ................................ ............... 63 Role of Mac 1 and RGD binding Integrins in Macrophage Inflammatory Cytokine Secretion in Response to PS MPs ................................ ................. 64 UHMWPE MPs Si ze Distribution ................................ ................................ ...... 66 Role of Mac 1 Integrins in Macrophage MP Uptake of PE MPs ....................... 67 Role of Mac 1 Integrins in Macrophage Inflammato ry Cytokine Secretion in Response to PE MPs ................................ ................................ .................... 67 Impact of the Study ................................ ................................ ................................ 68 3 INTEGRIN DIRECTED MODULATION OF MACROPHAGE RESPONSE TO B ULK BIOMATERIALS ................................ ................................ ........................... 90 Background ................................ ................................ ................................ ............. 90 Experimental Procedure ................................ ................................ ......................... 93 Biom aterial Implantation and Analysis ................................ .............................. 93 Foreign Body Giant Cell (FBGC) Formation ................................ ..................... 94 Determination of Loading and Release Kinetics o f Echistatin from Coating around PET Discs ................................ ................................ ............ 94 Statistical Analysis ................................ ................................ ............................ 95 Results ................................ ................................ ................................ .................... 95 Role of Mac 1 in Foreign Body Response to Implanted Biomaterial ................ 95 Role of Mac 1 in Foreign Body Giant Cell Formation ................................ ....... 96

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10 Loading and Release Kinetics of Echistatin from Discs .................... 96 Role of RGD binding Integrins in Foreign Body Response to Implanted Biomaterial ................................ ................................ ................................ .... 96 Impact of the Study ................................ ................................ ................................ 97 4 CONTRIBUTIONS OF SURFACE TOPOGRAPHY AND CYTOTOXICITY TO THE MACROPHAGE RESPONSE TO ZINC OXIDE NANORODS ...................... 103 Background ................................ ................................ ................................ ........... 103 Experimental Procedure ................................ ................................ ....................... 105 Fabrication of ZnO Nanorods ................................ ................................ ......... 105 Macrophage Gene ration ................................ ................................ ................. 105 Substrate Preparation and Macrophage Culture ................................ ............ 105 In vivo Response to ZnO Nanorod Coating ................................ .................... 108 Statistical Analysis ................................ ................................ .......................... 108 Results ................................ ................................ ................................ .................. 109 ZnO Substrate Characterization ................................ ................................ ..... 109 Macrophage Adhesion, Spreading and Viability on ZnO Nanorods ................ 109 Dissolved Levels of Zn and Non contact Based Toxicity of ZnO .................... 111 Foreign Body Response to Zinc Nanorod Coated PET ................................ .. 112 Impact of the Study ................................ ................................ ............................... 113 5 ANTIBACTERIAL EFFECTS OF ZINC OXIDE NANOROD SURFACES ............. 124 Background ................................ ................................ ................................ ........... 124 Experimental Procedure ................................ ................................ ....................... 126 Fabrication of ZnO Nanorods ................................ ................................ ......... 126 Bacterial Culture ................................ ................................ ............................. 126 Substrate Preparation and Bacterial Ad hesion Studies ................................ ........ 126 Fluorescence Staining and Imaging ................................ ............................... 127 Statistics ................................ ................................ ................................ ......... 127 Results ................................ ................................ ................................ .................. 128 Impact of the Study ................................ ................................ ............................... 130 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 138 LIST OF REFERENCES ................................ ................................ ............................. 141 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 168

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11 LIST OF FIGURES Figure page 1 1 Schematic of the process of aseptic loosening. ................................ .................. 45 2 1 Release kinetics of RGD from polymer disc ................................ ....... 77 2 2 RGD binding integrins modulates osteolysis in response to particulate biomaterials ................................ ................................ ................................ ........ 78 2 3 Mac 1 integ rin modulates osteolysis in response to particulate biomaterials ..... 79 2 4 Purity of the macrophage culture ................................ ................................ ........ 80 2 5 Presence of endo toxin plays a role in cytokine production upon phagocytosis of LPS coated PS MPs however it does not play a role in MP uptake. ............... 80 2 6 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages at cell : MP ratio of 1:1 0 ................................ ............................... 81 2 7 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages at cell : MP ratio of 1:20 ................................ ............................... 82 2 8 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages at cell : MP ratio of 1:40 ................................ ............................... 83 2 9 Phagocytosis of protein opsonized PS MPs by macrophage is modulated by blocking RGD binding int egrins with soluble RGD peptide ................................ 84 2 10 Integrin Mac 1 modulates inflammatory cytokine secretion from macrophages upon exposure to protein and LPS coated PS MPs ................................ ............ 84 2 11 Macrophage cytokine secretion upon exposure to protein coated PS MPs is modulated by blocking RGD binding integrins ................................ .................... 85 2 12 Particle size distribution of protein coated UHMWPE MPs. ................................ 86 2 13 Integrin Mac 1 modulates phagocytosis of protein opsonized PE MPs by macrophages at cell : M P ratio of 1:20 ................................ ............................... 87 2 14 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages at cell : MP ratio of 1:40 ................................ ............................... 88 2 15 Detail of a single well for inverted culture phagocytosis assay ........................... 89 2 16 Integrin Mac 1 modulates inflammatory cytokine secretion from macrophages upon exposure to protein and LPS c oated UHMWPE MPs ................................ 89

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12 3 1 Integrin Mac 1 modulates foreign body response to subcutaneously implanted biomaterials. ................................ ................................ ..................... 100 3 2 Integrin Mac 1 does not play a role in fusion of macrophages to form foreign body giant cells ................................ ................................ ................................ 101 3 3 Release kinetics of Echistatin from polymer coating on PET discs .. 101 3 4 RGD binding integrins modulates foreign body response to subcutaneously implanted biomaterials. ................................ ................................ ..................... 102 4 1 Surface topography of sputtered ZnO and ZnO nanorods. ............................... 117 4 2 Time lapse images of adherent macrophage seeded on ZnO nanorods .......... 118 4 3 Macrophage adhesion and viability on Z nO substrates ................................ .... 119 4 4 Dissolved levels of zinc in culture media when macrophages are cultured on zinc oxide substrates ................................ ................................ ........................ 120 4 5 S etup to determine cytotoxicity of ZnO when cells are not present in contact with the substrates and viability of macrophages in this setup with ZnO substrates ................................ ................................ ................................ ......... 121 4 6 Foreign body response to zinc oxide coated PET discs implanted subcutaneously in mice ................................ ................................ .................... 122 4 7 Correlation between dissolved zinc levels in media and macrophage viability. 123 5 1 Pseudomonas aeruginosa adhesion and viability is re duced on ZnO nanorod substrates ................................ ................................ ................................ ........ 134 5 2 Pseudomonas aeruginosa demonstrate decreased adhesion and viabi lity of bac teria on ZnO nanorod ................................ ................................ ................. 135 5 3 Staphylococcus epidermidis viability is reduced on ZnO substrates. ................ 136 5 4 Staphylococcus epidermi dis demonstrate decreased viability, but comparable adhesion on ZnO nanorod. ................................ ................................ ............... 137

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13 LIST OF ABBREVIATION S AAD Aminoactinomycin D ANOVA Analysis of variance AFM Atomic force microscopy BMM Bone marrow derived Macrophages BSA B ovine serum albumin CD Cluster of differentiation CR3 Complement receptor 3 C Degrees Celsius DMEM ECM Extracellular matrix proteins ELISA Enzyme linked immunosorbent assay EU Endotoxin units FAK Focal adhesion kinase F BGC Foreign Body Giant Cells FBR Foreign Body Reaction FBS Fetal bovine serum Fg Fibrinogen FN Fibronectin ICAM Intercellular Adhesion Molecule ICP Inductively Coupled Plasma IFN Interferon IL Interleukin LCCM L 929 cell conditioned medium LPS Lipopolysa ccharide

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14 MAPK Mitogen activated protein kinase MCSF Macrophage Colony Stimulating Factor MIP Macrophage inflammatory protein NR Nanorods NF Nuclear factor kappa beta PBS Phosphate Buffered Saline PDGF Platelet derived growth factor PET Polyethylene Terephthalate PGE Prostaglandin E RANKL Receptor activator for NF RANTES Regulated upon Activation, Normal T cell Expressed, and Secreted ROS Reactive Oxygen Species SEM Scanning electron microscopy Ser Serum TGF Transforming growth factor TLR Toll like receptors TNF Tumor necrosis factor alpha TRAP Tartrate Resistant Acid Phosphatase UHMWPE Ultra high molecular weight polyethylene VN Vitronectin ZnO Zinc oxide

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15 Abstract of Dissertation Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODULATING MACROPHAGE RESPONSE TO BIOMATERIALS By Toral Zaveri December 2011 Chair: Benjamin Keselow sky Major: Biomedical Engineering Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of nu merous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macro phage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach invol ves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, w e have investigated the role of integrin Mac 1 and RGD binding integrins in the in vivo osteolysis response and macrophage inflam matory

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16 processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of f ibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac 1 integrin was isolated using a Mac 1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted bio material with polymer loaded with Echistatin which contains the RGD sequence. For the in vivo osteolysis study and to study the in vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in and dissolved in macrophage media respectively. By studying the phagocytosis, inflammatory and FBR of macrophages from integrin knockout mice, as well as using various integrin blocking techniques we aim to identify the role of various integrins in macrophage inflammatory response. These integrins can serve as therapeutic targets for mitigating this inflammatory response and improve functional life of implanted biomaterials. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenti ng well controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively fla t sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to Z nO flat substrate and glass. While these data

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17 suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. In order to determine long term ph ysiological responses, ZnO nanorod coated and sputtered ZnO coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 d ays Upon implantation, both ZnO coated discs resulted in a discontinuous cellular fibrous capsule indic ative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body ca psule formation. Hence although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topogra phy dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent. Considering th e toxicity of ZnO nanorod surface towards macrophages, their role as an antibacterial surface was explored. Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant as sociated infection. To assess the efficacy of ZnO nanorod surfaces as an anti bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO and glass substrates. Common implant associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. ZnO nanorod surface and sputtered ZnO demonstrated a significant bactericidal effect, killing respectively 2.5x and 1.7x times the number of bacteria dead on glass. A similar bactericidal effect of ZnO substra tes on S. epidermidis was also evident, with sputtered ZnO and ZnO nanorod

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18 substrates killing respectively 22x and 32x times bacteria dead on glass. These data support the further investigation of ZnO nanorod coatings for bacterial adhesion resistance and bactericidal properties

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19 CHAPTER 1 INTRODUCTION Biological Response to Biomaterials The N ational I nstitutes of H ealth defines combination of substances natural or synthetic in origin that have been designed to interact wit h biological systems for any period of time to treat, augment or replaces any tissue, organ, or function of the body [1] Biomaterials are used in medical devices, tissue engineering constructs as well as biotechnological applications. The biomaterials product industry is rapidly expanding; it had a market size of $25.5 billion in 2008 and has been projected to grow 15% annually until 2013 [2] Some common application of biomaterials are artificial joints, bone plates for fracture fixation, dental implants, artificial heart valves, intraocular lenses as well as skin grafts [3] A variety of materials are being used as biomaterials including metals, polymer, ceramics, composites as well as modified natural materials autogra fts, allog rafts and xenografts [3] An essential property for biomaterials i s biocompatibility, which is the ability to exist in contact with tissues of the human body without causing an unacceptable degree of harm to the body [4] Although, most implantable materials are non immunogenic and non toxic, implants made of such materials have been shown to trigger various degrees of host immune response which limits their performance and lifetime in vivo. The duration and inten sity of the immune response varies depending on t he size, shape, chemical and physical properties of the biomaterial as well as the site of the implant [5] The host immune response plays a role in various clinical problems associated with implants such as aseptic loosening of artificial joints, [6,7] degradation and surface cracking of pacemaker leads, [8] fibrous encapsulation around breast implants [9,10] and drug

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20 delivery systems [11] As these complications eventuall y lead to implant failure it requires surgical removal and replacement, increasin g both the risk to patients as well as health care costs [6] These host responses that ensue as soon as a bi omaterial is implanted in the body and described in the order that they occur are injury, blood c apsule development [5,12] T success or failure of an implanted device and hence, a number of studies have been conducted trying to elucidate the various steps of the host immune response a nd identify therapeutic targets to mitigate t his response. Injury, Blood material Interactions and Provisional Matrix Formation The process of implantation, regardless of the location of the implant, causes an injury due to the surgical procedure of implantation as well as disruption of tissue integr ity [13] This injury results in the implant coming in contact with blood, the fibrinogen in the blood is a blood clot is formed that promotes platelet adhesion and aggregation [14] Activated platelets release interleukin 8 (IL8), RANTES ( Regulated upon Activation, Normal T cell Expressed, and Secreted ) and macrophage inflammatory protein 1 alp phagocytes, [15] leading to the initiation of a non specific inflammatory response. The injury to vascularized connective tissue activates a number of wound healing systems such as extrinsic and intrin sic coagulation systems, the complement system, the fibrinolytic system, the kinin g enerating system and platelets [5] Thrombus for mation leads to the development of a blood based transient provisi onal matrix [5] The provisional matrix formed provides a structu ral framework composed of fibrin cross

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21 linked with factor XIIIa [16] and several biochemical and cellular components which participate in the processes of wound healing and foreign body reaction [5] This matrix contains adhesive molecules such as fibronectin and thrombospondin bound to fibrin as well as platelet granule components released during pla telet aggregation [16] Platelet granule components include thrombospondin released from granule s and cytokines include TGF Platelet derived growth factor ( PDGF ) platelet factor 4 and platelet derived endothe lial cell growth factor [16] Thus, t he provisional matrix comprises of naturally derived components from the body itself, i s biodegradable and functions as a sustained release system for bioactive agents that control the sub sequent phases of wound healing [5] The cells that infiltrate the implant site as a result of injury and release bioactive agen ts include neutrophils, mast cells, monocytes, macrophages fibroblasts and endothelial cells l isted in the temporal order as they arrive at the site of injury [5] Pro tein Adsorption on Biomaterial Surface Immediately following exposure to physiologic fluids, implanted materials spontaneously adsorb a layer of host proteins which form the mediating layer between the recruited leukocy tes and the implant surfaces [5,17,18] Thus, eve n before the cells come in contact with the implant, the biomaterial surface is coated with various proteins and the interaction of these proteins with adhesion receptors on inflammatory cells leads to the recognition of the foreign body. The adsorbed prot eins are not foreign to the body however they still help in recognition of foreign objects because the confirmation and density of proteins on the biomaterial surface is different than its native state and concentration in the body. Proteins such as fibrin ogen, fibronectin, vitronectin, albumin, immunoglobul ins and complement factor C3 have been shown to a dsorb onto

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22 biomaterial surfaces [19 21] The type and surface density of adsorbed proteins depend on surfac e properties of the biomaterial as well the solution concentration of proteins [18] I nterestingly, the Vroman effect dictates that the type and amount of protein adsorbed on the biomaterial surface is dynamic, as adsorbed proteins may desorb due to other proteins having highe r affinity for the surface [22] Upon adsorption, proteins can undergo conformational changes to reduce surface energy and thus expose different ligand binding sites and create bioactive sites for the interac tion of cells with biomaterials [5,17,21,23] For example when fibrinogen comes in contact with hydrophobic biomaterial surfaces it adopts an energetically more favorable con 202) and P2 395), [24] which are binding sites for 2 integrins present on macrophage surface [25,26] When surface receptors such as integrins bind to different ac tive sites of adsorbed proteins they modulate cellular responses such as adhesion, morphology, growth, differentiation, and activation [27] Based on different surface chemistries and thus differential p rotein adsorption, studies have demonstrated differences in cell behaviors including adhesion, [21,28,29] differentiation, [30,31] activation, [24,32 34] and apoptosis [35 37] on biomaterial surfaces. Thus adsor bed proteins lead to recognition of foreign substances in the body, macrophage adhesion to biomaterial surface and promote macrophage activation [5] Acute Inflammation The first cells to arrive at the implant site are neutrophils and m ast cells the main [5,38] The activated neutrophils secrete a number of chemokin es and cytokines such as IL 8 and MIP inflammatory response that further contribute to the development of the inflammatory

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23 response release causes phago facilitates phagocyte adhesion [5,17,38,39] During the degranulation process, mast cells also release interleukin 4 (IL 4) and interleukin 13 (IL 13) which have been shown to play a role in macrophage fusion to form foreign body giant cell (FBGC) [40,41] and thus determine the extent and degree of the subsequent development of the foreign body reaction. This acute inflammation around biomaterials usually resolves in less than one week, depending on the extent of injury at the implant site, [5,39] following which phase begins. Chronic Inflammation The chronic inflammation phase is characterized by the presence of mononucle ar cells, i.e. monocytes, macrophages, plasma cells and lymphocytes at the implant site [42] Unlike the acute inflammation phase which last only couple of days, this phase can extend from few days to even years depending on the type and persistence of inflammatory stimulus [5] In case of biocompatible materials chronic inflammatory response resolves in less than two weeks. I f it lasts longer it could indicate that there is some infection associated with the implant [39] Among the cells that arrive at the implant site during chronic inflammation, macrophages are most important as they secrete a large varie ty of biologically active products that participate in the sub sequent phases of wound healing [5] The biologically active products secreted by macrophages include neutral proteases, chemotactic factors, arachidonic acid metabolites, reactive oxygen metabolites, complement components, coagulation factors, growth p romoting factors, and cytokines [5]

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24 Granulation Tissue and Foreign Body Reaction After chronic inflammation, the next phase in the sequence of events following biomaterial implantation is formation of gra nulation tissue which marks the beginning of the wound healing phase. The granulation tissue is composed of macrophages, fibroblasts and vascular endothelial cells. The fibroblasts proliferate and synthesize collagen and proteoglycans which form the fibrou s capsule around implants. The endothelial cells proliferate, maturate and organize themselves into capillaries thus initiating neovascularization. The macrophages may fuse together to form FBGC, which form a part of the foreign body capsule along with mac rophages and collagen from fibroblasts [5] FBGCs release reactive oxygen intermediates (ROIs), degradative enzymes, and acid into t he space between the cell membrane and biomaterial surface which has been shown to mediate degradation of biomaterial surfaces [5,8] Approaches to Modulate F oreign Body Response Since protein adso rption on the surface is the first step in the cascade of events that occur upon biomaterial implantation, several studies have investigated non fouling surface which significantly reduce protein adsorption to modulat e foreign body response [43,44] A number of studies have also demonstrated that different surface chemistries result in differential protein adsorption on the surface, bot h in terms of type and protein confirmation, exposing different active sites for cell binding and ultimately influencing cellular response [21,45] For example hydrophilic and anionic surfaces have been shown to pro mote anti inflammatory cytokine secretion by monocytes and macro phages upon adhering to the surface [46] This can be attributed to differential protein adsorption on these surfaces as compared to hydrophobic and cationic surfaces which results in the availability of different set of binding sites for integrins. Mac rophage

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25 activation is controlled by binding and clustering of integrins on the cell surface that triggers the signaling reactions that are propagated into the cell (outside in signaling) leading to a functional response [47] The next interaction investigated for modulating macrophage r esponse is the binding of macrophages to the adsorbed protein layer that is mediated through surface receptors such as integrins. Numerous studies have examined the role of macrophage integrins in the various steps of the foreign body response. For example the binding of neutrophil and macrophage integrin, Mac 1 (CD11b/CD18), to fibrinogen adsorbed on biomaterial surface is shown to mediate adhesion of phagocytes to biomaterial implants [17,48] Different types of m acrophages are beneficial to the wound healing response as they participate in tissue repair and angiogenesis. Studies have been conduct ed to explore activation of macrophages towards the alternatively activated macrophages which have an anti inflammatory phenotype as compared to classically activated macrophages [49,50] Approaches that have been investigated to modulate inflammatory response to biomaterials involve ( 1) surface modification of biomaterials physical and chemical [51,52] ( 2) surface treatment of biomaterial surface in order to release bioactive molecules that have been shown to mod ulate the foreign body response [53] (3) t argeting receptors or signaling pathways in macrophages tha t participate in the inflammatory response to biomaterials [54] Macrophage Response to Biomaterials Macrophages are sentinels of the body, they participate in clearance of tissue debris and apoptotic cells and wound healing [55] They respond to foreign invasion in the body ranging from pathogens to biomaterial implants [5,55] As reviewed in the earlier section s, macrophages form maj ority of the cell type recruited to site of

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26 biomaterial implantation and they play a significant role in the various phases of foreign body response [5] Macrophages recruited to site of implant secrete cytokines, chemokines, and growth factors that signal the recruitment and activation of other cells such as lymphoc participate in the various stages of the foreign body response [5] Macrophages may undergo frustrated phagocytosis when trying to phagocytose bulk biomaterials; they fuse to form FBGCs and encapsulate the implanted material [5] The foreign body response and fibrous encapsulation severely limits the functional performance of the implanted biomaterial in vivo such as pacemaker leads, [8] drug delivery [11] and recording electrodes [56] S ince macrophages play such a central role in the response to implanted materials various studies have explored modulating macrophage response using different approaches such as surface chemistry and surface roughness to modulate macrophage adhesion to biom aterials [57 61] Some of these techniques reduce macrophage adhesion to biomaterial surface by making anti fouling surfaces which resist protein adsorption thus abrogating m acrophage surface interact ion [60] Nanot opograp hic surfaces which have features in the nanometer size range have also been explored to modulate m acrophage adhesion and function [62,63] Since features of nanotopographic surfaces are in the biological size range, they have been explored for var ious cell surface interactions [64 67] Another link between the macrophages and biomaterial surface are receptors cal led integrins present on macrophage surface that bind to the adsorbed protein. Binding of integrins to their ligands leads to integrin clustering and downstream signaling that may result in alteration in cell growth, differentiation, migration, attachment and spreading [68] Macrophages interact with

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27 adhesion proteins via integrins, which is evident from the decreas e in cell attachment observed in the presence of anti integrin antib odies [69] Integrins Integrins are a large family of cell adhesion receptors that play an important role in cell ECM and cell cell interaction [70,71] They are heterodimeric transmembrane r covalently associated, where each subunit contributes and is require d for ligand binding [70,71] integrin receptors [70] M 2 M 2 (Mac 1). Integrins have a long extracellular domain that forms the site for ligand binding and recognizes specific amino acid sequences [70] T hey have a short intracellular domain that directs intracellular signaling by providing binding sites for signaling molecules such as protein kinases, calcium binding proteins, focal adhesion kinases (FAK), tyrosine and MAP kinases [72] Ligand binding of integrins causes clustering of integrins to form focal contacts which contain structural proteins such as vinculin, talin [73] actinin as well as signaling molecules such as FAK, Src and paxillin. These structural proteins link the cytoskeleton to the ECM and thus modulate cell adhesion and signaling [72] Thus i ntegrins through this outside in signalling, enables a cell to sense its location, local environment, adhesive state and surrounding matrix [71] Integrins are normally present in an inactive state and when bound to a ligand, conformational changes t ake place These changes lead to affinity modulation for the ligand resulting in stronger binding [71] Th is is termed as inside out signaling and it enables cells to modulate adhesive behavior without changing the number of receptors on its surface [74] Integrins have binding sites for ligand interaction for example, majority of integrins that

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28 2 200 amino acids that acts as the major liga nd binding site [75] Integrins control various cell functions such as cell survival, proliferation, migration and differentiation [76] Thus they serve as therap eutic targets for directing or disrupting intracellular cell signaling. Cells interact with adhesive proteins adsorbed on biomaterial surface through these adhesion receptors integrins [5,14,77] Role of Integrins in Host Immune Response to Biomaterials Integrins are shown to play a role in the various stages of the host immune response. Blood material interaction: Platelet integrin IIb 3 mediates initial adhesion of platelets to biomaterial surface adsorbed fibrinogen [78] Acute inflammation: Integrins on neutrophils surface control the migration of neutrophils through ECM during their extravasation to the implant site [79] Neutrophils have several integrins such as M 2 V 3 and 1 integrins ( 4 5 6 ) that contribute to the adhesion and motility of neutrophils towards the site of implant [79] Upon binding of neutrophils integrins to ECM proteins, a signaling cascade is initiated within the cell t hat leads to change in shape, proliferation and survival [79] Chronic i nflammation: Upon chemokine signaling from the wound site by the neutrophils and mast cells, monocytes in circulation adhere to the endothelium and extravasate through th e blood vessels to the site of injury. The 1 and 2 integrin families have been shown to play a role in this process of adhesion and extravasation of mon o cytes [80] Upon reaching the implant site, 2 integrin family and particularly integrin has been shown to mediate adhesion to biomaterial surface [81]

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29 Granulation t issue and foreign body r eaction : 1 and 2 integrin have been shown to play a role in fusion of macrophages to form foreign body giant cells. [82] Macrophage Integrins 1 2 3 5 f amily. M acrophages and monocytes have multiple i ntegrins some of which include M 2 X 2 V 2 and M 2 [83] Integrins present on macrophages direc t various cell functions as explained here: 1. Macrophage adhesion to extracellular matrix(ECM) proteins: This interaction is important fr om the point of view of phagocytosis as interaction with different ECM proteins stimulates phagocytosis via F [83] 2. Macrophage adhesion to other cells: Macrophages are a component of the innate immune system which provides signaling for the adaptive immune system. This signaling requires cell cell int eraction between macrophages, T cells and B cell s [83] Addi tionally macrophages are scavengers of the body and are responsible for the clearance of apoptotic cells This requires the macrophage interaction and recognition of apoptotic cells. Recognition of apoptotic cells is mainly mediated by V int egrin and inge stion of these cells elicits an anti inflammatory response [83] 3. Macrophage migration and spreading: Integrins act as scaffolds between the cell cytoskeleton and the external environment thus directing cell migration and spreading. Cell migration is critical for the p rocess of extravasations of cells to sites of inflammation, spreading and activation [83] 4. P hagocytosis: The pathways involved in integrin m ediated spreading and migration are distinct from those involved in integrin mediated phagocytosis H ence it is possible to disr upt phagocytosis without disrupting migration. Various M 2 V 3 V 5 5 1 [83] Since integrins present on macrophages direct various inflammatory processes, they serve as ideal therapeutic targets for modul ating the macrophage inflammatory response. Mac 1 integrin Mac 1 is an important leukocyte receptor that plays a role in recruitment and activation of immune cells so as to mount an inflammatory response [84] It is

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30 expressed on monocytes, neut rophils and certain lymphocytes and it quantitatively up regulated and transformed to an active state by inflammatory mediators [84] Mac 1 integrin is kn M 2 and CD11b/CD18. It binds to a myriad of ligands such as ECM proteins (fibrinogen, [85] fibronectin, [86] collagen, [87] vitronectin [88] ), counter receptors such as ICAM 1,2,3, [89] products of coagulation and complement such as iC3b, [90] factor X, [91] complement factor H [92] as well as other non protein substances such as heparin [93] The integrin Mac 1 mediates cell adhesion to a number of proteins that adsorb out of physiologic fluids onto synthetic materials including complement factor fragment C3bi, album in, vitronectin, and fibrinogen [5,70,71] Mac 1 plays a role in phagocytosi s of C3bi complement coated targets such as pathogens, apoptotic cells, fat and oil droplets, [94,95] as well as in adhesion dependent respiratory burst and degranulation [94,96] When Mac 1 binds to its ligands it alters leukocyte adhesion and activation [97] For example d uring recruitment to an inflammation site, Mac 1 along with other integrins such as LFA 1 and VLA 4 modul ate leukocyte rolling, adhesion and extravasation through the endothelium and m igration through tissues by interaction with ECM proteins [97] Binding of Mac 1 to its ligands triggers numerous signaling cascades such as the NF chemokines which help amplify the inflammatory response [97] Notably, Mac 1 also mediates adhesion to denatured proteins [42,98] This is important because proteins that adsorb onto extremely hydrophobic surfaces of some biomaterials such as UHMWPE, underg o extensive denaturation/unfolding [5,14,77] Mac 1 has been shown to be the major macrophage recept or directing phagocytosis o f titanium alloy wear

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31 particles [99] For these reasons, Mac 1 is a primary candidate for mediating macrophage molecular recognition and re sponse to particulate as well as bulk biomaterials [33,98] Thus therapies that target the Ma c 1 receptor are expected to block macrophage activation and mitigate the inflammatory response to implanted biomaterials. RGD binding integrins The peptide sequence arginine glycine aspartic acid (RGD) was first discovered in the protein fibronectin as th e recognition site for fibronectin receptor ( 5 1 ) [100] How ever now it is known that it is the binding site within several proteins such as vitronectin, fibronectin, collagen [101] Of the known twenty integrins, definitely eight and possibly up to twelve of them recognize the RGD sequence in their ligands [101] The chain of the known RGD binding integrins 5 8 V and IIb form an integrin sub family as they show more sequence similarities as compared to other [102] Short peptides containing RGD sequence have been shown to have an adhesion promotin g as well as adhesion blocking effect. When immobilized on a surface it promotes cell adhesion [103,10 4] whereas in solution it prevents cell adhesion in a concentration dependant manner [105] As RGD peptides ca n bind to a number of integrin receptors and can be used to mimic several protein s, use of RGD peptides enables targeting a wide range of ligand receptor interactions. Integrin Targeted Therapies Anti adhesion therapies using antibodies, peptides, and pep tidomimetic inhibitors against adhesion receptors have shown to be effective in various animal disease models [106 108] as well as in clinical trials [109 ,110]

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32 Mac 1 Targeted Therapies 1, CD11b contains a 200 [75] Antibodies to this domain are shown to block binding to ICAM 1, iC3b and fibrinogen [75] Mutat ions within the I domain of Mac 1 have been shown to prevent binding of ICAM 1 and iC3b [111,112] Antibody blocking Antibodies to Mac 1 have been shown to interrupt the adhesive and migratory capability of leukocytes and reduce tissue i njury in models of inflammation [113,114] M1/70 is a rat 1 (CD11b) [115] with broad species cross reactivity. It is shown to block adhesion, homotypic aggregation, and complement dependent binding and phagocytosis of co mplement opsonized erythrocytes [116 118] Blocking adhesion and hence phagocytosis of wear particles can be an approach to modulate macrophage inflammatory response and reduce peri implant osteolysis. Neutrophil inhibitory factor (NIF) NIF is a 41 kDa glycoprotein isolated from canine hookworm, Ancylosto ma caninum [119] It binds to the I domain of Mac 1 integrin with high affinity and blocks the Mac 1 binding to several ligands such as C3bi, ICAM 1, and fibrinogen [119] It is shown to block neutrophils adhesion to protein coated surfaces and inhibit neutrophil functions [120] In sev eral animal models of disease it has been shown to be effective in attenuating the deleterious effects resulting from excessive activation of polymorphonucle ar cell in inflammatory disease [121]

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33 Targeted Therapies for RG D B indi ng Integrin Peptides containing the RGD sequence have been shown to inhibit cell adhesion F or example, RGD peptide is shown to inhibit adhesion of Human K562 erythroleukemia cells on fibronectin coated dishes in a concentration dependent manner [105] V 3 has been reported to be the primary integrin that mediates adhesion to RGD peptides for numerous cell types [122,123] Hence RGD mimetics t hat target integrins other than Mac 1 on the macrophage surface may compe te with protein coated biomaterial surface and arrest the macrophage inflammatory response at the very first step of adhesion. Disintegrins are a group of low molecular weight, cystei ne rich polypeptides isolated from snake venom and contain an Arg Gly Asp (RGD) loop maintaine d by specific disulfide bridges [124] They are shown to bind and interfere with various integrin mediated process such as inhibit ion of platelet aggregation via the blockade of IIb 3 integrin [124] Echistatin, a 49 residue protein purif ied from the venom of the saw scaled viper Echis carinatus Echistatin contains the Arg Gly Asp (RGD) sequence which is much more potent than the tetrapeptide Arg Gly Asp Phe [125] sequence and binds to integrins such as V 3 through this RGD binding site. Its binding to integrin V 3 is irreversible and has a high affinity. [126] Since V 3 is a receptor predominant ly expressed on osteoclasts e chistatin is shown to inhibit osteoclastic bone resorption in V 3 which is known to modulate osteoclast function [127] It is also shown to inhibit fibrinogen dependent platele t aggregation and cell adhesion [125] Thus echistatin through its RGD binding site can serve as an effec tive integrin targeted therapy.

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34 Polymers for Sustain ed Release of Integrin targeted Therapies The therapies discussed above need to be delivered to the site of implant in a controlled time release fashion in order for it to appropriately intervene and dis rupt the different stages of the foreign body response. Depending on the specific application, a number of polymers have been explored in biomaterials application for achieving desired release characteristics as well ensuring that the therapeutic molecule is released in the biologically active state [128] For example, for tissue engineering as well as temporary drug delivery application, degradable polymers such as Poly(lactide co glycolide) are de sired as they are biocompatible and degrade into safe by products in the body obviating the need for polymer removal [129] Controlled release systems for drug delivery are useful in applications requiring a continuous, long term sustained release of the drug at certain specified concentrations [130,131] These controlled release systems include polymer matrices that encapsulate the drug, can be implanted at site of action thus preventing systemic effect and can be designed to achieve the desired release profile [130] ethylene vinyl acetate copolymer is non inflammatory, non biodegradable and has been greatly explored for drug delivery to the brain since its early reports by Langer and Folkman in the 1970s [131] It has been invest igated for delivery of various compounds in a slow release, sustained fashion to the brain [132,133] as well as for orthodontic applications [134] can be molded into desired shapes for placement into specific tissue regions, can be designed to release the drug over extended periods of time [135] and macromolecules have been shown to retain their biological activity after release from [136]

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35 Clinical Significance Arthritis and Total Joint Replacement The term arthritis is used to describe a milieu of conditions resulting from inflamed or damaged joints and is a leading cause of disabili ty amongst Americans [137] It is a chronic condition accompanied with pain and immobility leading to severe economic loss both in terms of decrease in work productivity as well as health care costs amounting t o $128 billion annually in 2003 [138] Some of the common medical conditions that lead to arthritic joints are osteoarthritis, rheumat oid arthritis, juvenile arthritis, gout, fibro myalgia and Lupus erythematosus [137] In 2006, 46 million Americans (1 in 5 adults) suffered from arthritis or chronic joint symptom s making arthritis second only to heart disease as the l eading cause of work disability [137] For arthritic patients, joint replacement is considered only when physical therapy a nd pain management have failed [139] Hip joint replacement is a successful therapy f or patients suffering from debilitating arthritis (osteoarthritis and rheumatoid arthritis), congenital hip dysplasia (CHD) and joint fracture to return mobility and relieve pain by replacing the damaged hip joint. Total hip replacement system replaces the head of the femur as well as the acetabular cup with a pair of material surfac es articulating with each other [140] Over the years, different combinations of surfaces have been investigated as apposing joint surfaces such as metal on polyethylene, ceramic on polyethylene, metal on metal and ceramic on ceramic [141] From these, the most popular combination has been the combination of an ultra high molecular weight polyethylene (UHMWPE) acetabular component and a metal femoral component [141]

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36 Failure of Total Hip Replacement: Aseptic loosening Hip replacement surgery is one of the most successful surgeri es of the 20th century [142] In 2004 over 230,000 hip replacement surgeries were performed in the U.S alone each costing an average of $35,000 [143] However the replaced joint has a functional life of about 10 15 years with a failure rate of 10% at 10 years post s urgery [144] As younger an d younger people are electing for this surgery, having a longer functional life of the joint is necessary. Additionally, a report published by the American joint replacement registry predicts savings of $65.2 million annually through a mere reduction of 2% in U.S rates for revision surgeries that are performed to replace the malfunctioning implant [145] Aseptic loosening is a major reason for the failure of the artificial joint accounting for greater than 70% of the revision surgeries [146] This loosening occurs due to osteolysis around the joint known as pe riprosthetic osteolysis. As a result of osteolysis (bone degradation), the tight fit between the artificial joint and bone is lost, resulting in micro motion causing joint instability, pain and ultimately need for revision surgery [6] Revision surgeries are more complicated and less reliable than primary joint replacement surgeries due t o the osteolysis around the implant and hence require special bone grafting techniques [6] In 2004, about 46,000 revision surgeries were performed in the US each costing an avera ge of $45,000 [143] The majority of hip jo ints that are replaced in the US have a metallic or ceramic femoral stem and a metal ball articulating with a UHMWPE acetabular cup liner [147 149] UHMWPE is widely used in orthopedic implants because of its higher impact strength and abrasion properties compared to other polymeric materials [150] In spite of UHMWPE having better wear resistance and mechanical characteristics compared to other materials, due to the constant motion/cycl ic mechanical load at the joint results i n

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37 the UHMWPE liner wearing out and generation of micron size d wear debris particles [148] The wear debris consists of particles derived from the v arious components of the implant including UHMWPE, bone cement in the form of PMMA, or metal, with predominant proportion of UHMWP E particles [147,149] The size range of the generated wear part icles is 0.5 range [151] The wear particles settle in the space between the joint and the bone where macropha ges phagocytose these microparticles and become activated [152] Activated macrophages release prostaglandins and cytokines such a s TNF 6 and PGE 2 which lead to activation of bone resorbing pathways via formation of osteoclasts leading to periprosthetic osteolysis [152] Extensive research is focused towards development of bearing surfaces with better frictional and wear properties in order to reduce wear debris generation H owever friction and wear cannot be completely eliminated. I n order to mitigate the problem of peri prosthetic osteolysis various pharmacological approaches targeting cytokines such as TNF [153 155] as well as use of biophosphanates [156,157] have been investigated. Since activated macrophages and the cytokines they secrete ultimately lead to the problem of aseptic loosening, they ar e potential targets for therapeutic intervention. Role of Macrophages in Wear Debris Induced Periprosthetic Osteolysis Microparticles settle in the space between the implant shaft and the bone. Macrophages phagocytose these microparticles and become acti vated giving rise to an inflammatory response [152] An analysis of tissue explanted from patients undergoing joint r evision surgeries indicates that the pseudosynovial memb rane formed around the artific ial joint is rich in macrophages and foreign body giant cells (FBGC) associated with UHMWPE partic les [147 ,158 161] Since the UHMWPE particles are not

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38 biodegradable, they are not cleared from the body when macrophages phagocytose them. When the amount of wear particles generated exceed the clearance capacity of macrophages, the particles persist in the body, form granulomatous tissue and lead to chronic inflammation [162] The activated macrophages release prostaglandins, cytokines, metalloproteinases and lysosomal enzymes such as TNF 6, and PGE 2 which lead to activation of bone resorbing pathways [7,152,163,164] IL 1, IL 6 and TNF precursors thus participating in osteoclasts mediated bone resorption [163,165,166] Thus there is osteocl ast formation, osteolysis of bone around the impl ant and ultimately implant loosening [167] A schematic of the process of aseptic loosening is depicted in the Figure 1 1. Macrophage s have integrin receptors on their surface through which they interact with the layer of proteins adsorbed on the wear particle surface result ing in macrophage activation. When cultured in serum free media with parti cles free of adsorbed proteins, macrophages exhibit only basal levels of activation, as measured by secretion of IL IL 6 and TNF [42] Due to their hydrophobic nature p olyethylene wear particles have been shown to activate complement pathway leading to opsonization with iC3b which is a ligand for Mac 1 receptor on macrophages [168] UHMWPE surfaces have also been shown to adsorb other proteins such as albumin, fibronectin and IgG [169] Thus disrupting this integrin protein interaction will down regulate contact, adhesion and resulting phagocytosis of wear particles by macrophages and open up avenues for mitigating macroph age inflammatory response.

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39 Role of Osteoclasts and Integrins on O steoclasts Osteoclasts are the primary cells respons ible for bone resorption that leads to peri prosthetic osteolysis resulting in aseptic loosening of joint implants [170] Cytokines such as I L 1, IL 6 and TNF released by activated macrophages have been reported to stimulate differentiation and matur ation of osteoclast precursors to mature osteoclasts which participate in the actual process of bone resorption [163,165,166] The inflammatory cytokines also cause up regulation of RANKL on stromal cells [167] The RANKL on osteoblasts binds to RANK on osteoclast precursor s causing them to mature. Osteoclasts actively migrate on the surface of bone s and undergo alte rnating cycles of migration and resorption [171] Osteoclasts migrate to the site of resorption bind to bone surface creating a sealing zone between the osteoclasts and bone [172] The sealing zone provides the perfect microenvironment isolated from the surrounding for the products secreted from the osteoclasts such as acids and proteolytic enzymes that degrade the mineralized bone mat rix [173] The integrins present on oste oclasts surface are V 3 [174,175] 2 1 [174,175] V 5 [175] an d V 1 [174,175] Integrins mediate the migration and attachment of the osteoclasts to the bone surface which involves integrin interaction with extracellular matrix (ECM) For example int V 3 has been shown to play a role in osteoclast migration and formation of the sealing zone [176] Integrin 2 1 mediates osteoclasts adhesion to native collagen type I [177] Thus, integrins are key players in the osteoclast resorption process [122] Surface Modification Approaches to Modulate FBR Implantation of biomaterials into the body results in initial tissue injury as well as long term inf lammatory responses [5] Macrophages are recruited to the site of

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40 biomaterial implantation and are the primary mediator of the forei gn body response [5,14] Macrophages and foreign body giant cells (FBGC), formed by fusion of macrophages, secrete reactive oxygen species, enzymes and acidic products which degrade the biomaterial [39] Furthermore, macrophages secrete inflammatory cytokines which recruit more macrophages and other cell types to the impl ant site such as fibroblasts, which secrete a collagenous m atrix to form a fibrous capsule [39] All together, this foreign body response is responsible for the isolation and degradation of the imp lant [178,179] This limits the function of numerous implanted devices such as cardiac pacemaker leads, ele ctrodes and orthopedic implants [149,180] Due to the primary role that various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces in order to mitigate inflammatory responses [52,181] Notably, the extent of the inflammatory response mounted by the body has been shown to be influenced by the implant mate rial and its surface properties [5,39] Because of their unique surface properties, there is great interest in exploring nanostruct ured materials for potential biomaterial applications. Nanostructured Materials Nanostructured materials, whose structural elements have dimensions in the range of 1 100 nm, exhibit u nique properties compared to bulk material s due to small dimensions and l arge surface area relative to volume [182] These nanostructured materi als are being investigated for use in an increasing number of applications such as microelectronics, sensor technology, semiconductors and cosmetics as well as medical applications such as biosensors, tissue engineering and drug delivery vehicles [183] .Sinc e biological systems operate in the nanometer size range, nanostructured

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41 materials present possibilities for unique biological interactions. For example, increased osteoblast adhesion and mineralization has been demonstrated on nanostructured surfaces of both titanium dioxide and zinc oxide (ZnO), as compared to mi cron sized surface topographies [65] Interestingly, di fferent cell types have been demonstrated to elicit differential responses to a given nanostructured material. For example, carbon nanotubes have been shown to promote adhesion of osteoblasts [66] whereas they inhibit adhesion of other cells such as fibroblasts, [184] chondrocytes, [18 4] smooth muscle cells [184] as well as macrophages [63] In particular, altered cell adhesion and viab ility of fibroblasts, umbilical vein endothelial cells and capillary endothelial cells has been reported on ZnO nanorods as compared to ZnO flat substrates [185] Zinc Oxide (ZnO) Nanorod Surface Zinc oxide has unique optical, semiconducting, piezoelectric and magnetic properties hence, it is used for different applications in fields such as semiconductors biosensors and piez oelectrics [186] Furthermore, ZnO is used in a number of both exploratory and well established biomedical applications. For example, ZnO nanorods grown on high electron mobility transistors devices have been shown to be highly sensitive for glucose detection, [187] while ZnO has long been used as a component in various biomedica l applications such as dental filling materials (e.g., temporary fillings) [188] and sunscreens [189] Additionally, ZnO has been investigated as a c omponent in topical wound healing ointments [190 192] and is used in commercially available products for the treatment of venous ulcers [193] and acne [194] Nanoparticles of ZnO are also known for thei r anti bacterial activity against both gram negative and gram positive bacteria [65,195,196]

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42 Due to various biological applications of ZnO, especially as nanoparticl es, a number of research groups have investigated the cytotoxicity of ZnO. Cell type specific results have be en reported. Zinc oxide i s reported as non toxic to cultured human dermal fibroblasts [197] and T cells [196] whereas it exhibited toxicity to neuroblastoma cells [198] and vascular endothelial cells [199] Implant Infection Medical implants are being extensively used in every organ of the human bo dy, with success in replacing or repairing physiologic functions. However, a major impediment is implant associated infections caused by bacterial adhesion to biomaterials, which necessitate implant removal, extended care and prolonged antibiotic treatment [200 202] This additional care significantly contributes to health care costs. For example, of the 2.6 million orthopedic devices implanted annually in the US, approximately 112,000 (4.3%) beco me infected [203] The primary cause of revision surgeries aseptic loosening has been discussed in detail above however implant associated infections happen to rank second in the list for reasons for revision surgery especially for knee arthroplasties [204] The most common cause of infection is the generally non pathogenic and ubiquitous bacteria S. epidermidis which is normally found on human skin and under normal circumstances is well tolerated by the immune system [205,206] However, when adherent to implanted surfaces, bacteria develop a protective biofilm resista nt to immune and antibiotic attack and can develop multiple resistance to antibiotics [207,208] Many implant associated infections therefore require surgical removal of the implant. Estimated costs of implant associated infectio ns exceeds $3 billion annually in the US [203] Although Staphylococcus species account for majority (45 to 55%) of orthopedic implants associated infections [209]

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43 Pseudomon as aeruginosa is also seen to be the cause of infections in 4 to 6% of infected orthopedic devices [210] In order to address this problem, implant coating strategies have been developed with the goal to elim inate initial bacterial adhesion and/or kill adherent bacteria. Various strategies have been investigated. For example, surface coatings which support low levels of protein adsorption, termed non fouling, including surfaces modified with polyethylene glyco l, polyethylene oxide brushes and hydrophilic polyurethanes, demonstrate resistance to bacterial adhesion [211,212] However, the effectiveness of these coatings toward resisting biofilm formation is limited and results vary depen ding on bacterial species [213] provide continuous release of bactericidal agents [214 217] Thesis Outline This thesis addresses the common issues associated with implanted biomaterials and suggests two separat e approached to modulate this response. The first approach is based on macrophage integrins that have been shown to modulate various steps of the macrophage inflammatory response. The second approach is a surface modification approach studying the effect o f nanostructured biomaterial surface on macrophage adhesion and viability. Macrophage response to two classes of biomaterials particulate and bulk has been investigated and the role of integrins in response to these has been quantified. Chapter 1 provi des background of the field and the significance of this project. Chapter 2 details the role of integrins in macrophage response to particulate biomaterials that mimic the wear particles generated from wear of joint implants. Mac 1 and RGD binding integri ns have been investigated for their role in in vivo osteolysis resulting from the

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44 particulate biomaterial. The steps and mechanisms involved in this osteolysis process are further elucidated by studying macrophage response to these microparticles in vitro. Chapter 3 explores the role of the Mac 1 and TGD binding integrins in the in vivo response to subcutaneously implanted bulk biomaterials and explores them as therapeutic targets for modulating the foreign body response. It further explores a drug delivery coating for biomaterials for the delivery of anti integrin peptides. Chapter 4 present results demonstrating the role of ZnO nanorod surface on adhesion and viability of macrophages and the in vivo effect of coating biomaterials with the nanostructured co ating. Based on the results of Chapter 4, ZnO nanorod surface was investigated as an anti bacterial surface for common implant associated infections in Chapter 5. Finally, Chapter 6 gives overall conclusions and recommendations for future work.

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45 Figure 1 1. Schematic of the process of aseptic loosening resulting from activation of macrophages upon phagocytosis of wear particles

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46 CHAPTER 2 ROLE OF INTEGRINS IN MACROPHAGE RESPONSE TO PARTICULATE BIOMATERIALS Background Aseptic loosening due to peri impl ant osteolysis is one of the major reasons for failure of joint implants [218] This osteolysis results from biological response to wear debris particles generated during mechanical l oading of the artif icial joints [158] Joint implan ts have components made of metals, polymers and ceramics hence micron size particles of these materials resulting from wear are found in the space between the implant and bone where they medi ate the bone resorptive process [147,149,151,158,219] An analysis o f tissue explanted from patients undergoing joint revision surgeries indicates that the pseudosynovial membrane formed around the artificial joint is rich in macrophages and foreign body giant cells (FBGC) associated with wear particles [147,159 161,220] Macrophages are the scavengers of the body and they attempt to clear the particles by the process of phagocytosis [152] When the amount of wear particles generated exceed the clearance capacity of macrophages, the particles persist in the body, form granulomatous tissue and lead to chronic inflammation [162] The activated m acrophages release prostaglandins, cytokines, metalloproteinases and lysosomal enzymes suc h as TNF 6, and PGE 2 which lead to activation of bone resorbing pathways [7,152,163,164] Due to osteolysis the tight fit between the bone and implanted joint established during joint surgery is lost; there is joint instability, pain and need of revision surg ery [7] Revision surgeries are more complicated and less reliable than primary joint replacement surgeries due to the osteolysis around the implant which requires s pecial bone grafting techniques [6] As

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47 younger and younger people are electing for joint implant surgery, having a longer functional life of the artificial joint is necessary. W ear debris particles generated in the body are coated with adhesive proteins, which spontaneously ads orb onto the biomaterial surface from the physiologic fluids [5,18] Macrophages interact with wear particles through the interface of the adsorbed proteins and cell surface receptors called integrins [5,14,77] Integrins present on macrophages direct various cell functions such as adhesion to extracellular matrix (ECM) proteins, adhesio n and signaling to other cell types, cell migration and sp reading as well as phagocytosis [83] As integrins such as Mac 1 present on macrophages direct various inflammatory processes, they serve as ideal therapeutic targets for modulating the macrophage inflammatory response [94 96,221] Fibrinogen is one of the primary components of plasma deposited on biomaterial surface mediating the acute inflammatory response through phagocyte recruitment t o implanted material [17] Mac 1, a leukocyte integrin present on macrophages and neutrophils functions as a fibrinogen receptor and this receptor mediated interaction between Mac 1 and fibrinogen has been shown to direct macrophage adhesion and activation [222] Additionally albumin has been shown to b e adsorbed from human serum onto wear particles of various materials (titanium alloy, poly ( methyl methacrylate ) and critically, polyethylene ) and enhance particl e induced macrophage activation [42] The leukocyte integrin Mac M 2 CD11b/CD18) mediates macrophage adhesion to adsorbed proteins and is shown to direct phagocytosis of titanium al loy wear particles [99] Among the mileu of proteins that adsorb onto the biomaterial surface, majority of them such as fibrinogen, fibronectin and vitronectin, cont ain a tripeptide, Arg Gly Asp

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48 (RGD), that serves as the recogn ition sequence for integrins [101] Hence, RGD peptide upon binding to integrins on the macrophage surface may compete with protein coated wear particles and arrest the macrophage inflammatory response a t the very first step of adhesion. Peptides containing the RGD sequence have been shown to inhibit cell adhesion F or example, RGD peptide is shown to inhibit adhesion of human K562 erythroleukemia cells on fibronectin coated dishes in a concentration depe nd ent manner [105] In this chapter, we have investigated the role of integrin Mac 1 and RGD binding integrins in macrophage response to particulate biomaterials such as those generated as wear particles from total joint replacements. To study the role of integrins in particle induced osteolysis in vivo, polyethylene (PE) MPs were implanted on mouse calvaria and t he resulting osteolysis was quantified. For delivering the RGD peptide for receptor blocking to the site of MP implant, discs prepared from polymer loaded with the RGD peptide were placed over the implanted MPs on the calvarial surface. is a non inflammatory and non biodegradable polymer that has been investigated for slow and sustained release of compounds into the brain [132] as well as tooth space [134] For the particulate biomaterial system we have used commercially available spherical polystyrene (PS) microp articles (MPs) of 1 m diameter as well as spherical and oblong PE MPs of size 0.5 5 m. Macrophage inflammato ry response depends on size and shape of phagocytosed particle. Sizes 0.5 5 m are shown to be most reactive for macrophages [223,224] hence, we have selected MPs in this size range To study the role of integrin Mac 1 in macrophage response to particulate biomaterials, we

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49 q uan tify phagocytosis of PS MPs and PE MPs and the subsequent inflammatory cytokine secretion by macrophages harvested from Mac 1 knockou t (KO) mice and compare it to wild type (WT) controls. Similar studies were performed to study the role of RGD binding i ntegrins using RGD peptide for blocking the integrin receptors on macrophages The RGD peptide binds to the integrins on macrophage surface, blocking the site of binding and thus competing with the protein coated MPs preventing their binding. Additionally, when integrins are bound by a soluble ligand, the soluble ligand cannot generate sufficient force required for integrin signaling which is dependant on a physical resistance force [225] Integrin signaling upon binding to soluble ligands is incomplete as the full range of signaling proteins found i n substrate immobilized integrin ligand interaction are not recruited due to the lack of mechanical resistance [226] This results in cells receiving a negative or unproductive signal regarding its environment and alters cell functioning [227] Various studies have reported a lack of inflammatory cyt okine secretion in the absence of detectable endotoxin levels on the MPs used for macrop hage phagocytosis [7,228 230] Endotoxin refers to the lipopolysaccharide (LPS) compl ex associated with the outer membrane of Gram negative pathogens such as Escherichia coli [231] In order to further investigate the effect of endotoxin in this work, we quantify macrophage particle uptake and subsequent cytokine secretion upon exposure to particles with known amount of endotoxin and compare it to particles with undetectable endotoxin levels (< 0.02EU/ml). We also investigate different receptor ligand interactions involved in MP uptake and inflammatory cytokine secretion by coating MPs with different opsonizing proteins such as bovine serum albumin (BSA), fibrinogen (Fg), fibronectin

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50 (FN), vitronectin (VN) and serum (Ser) and c omparing responses across different proteins. By studying the phagocytosis, inflammatory cytokine secretion and in vivo osteolytic response of macrophages from receptor knockout mice as well as using integrin blocking studies, we aim to identify and block receptors responsible for macrophage adhesion, phagocytosis and activation in response to biomaterials. Another aspect of macrophage material interaction is binding of macrophage integrins to proteins adsorbed onto biomaterials which trigger down stream s ignaling in the macro phage leading to its activation. S tudying this ligand receptor interaction further increases our understanding of macrophage biomaterial interactions. Once the role of receptor s that mediates foreign body response is delineated, they can serve as a therapeutic target for integrin targeted therapies. Experimental Procedure Disc Preparation for Controlled Release of RGD In order to deliver RGD peptide to the site of MP implantation, discs loaded with RGD peptide were pre pared by solvent extraction method. Briefly, a 10% (w/v) solution of the polymer was prepared by dissolving it in methylene chloride. Polymer/drug discs were prepared by mixing the polymer solution and RGD dissolved in phosphate buffered sali ne (PB S) (10mg in 50 L ) in a ratio of 6.5:3.5 (v/v) in sealed glass vessels. This solution was agitated vigorously for 15 min followed by sonication in a water bath at 25 C for 15 min. Each disc was made using 3 5 L of the resulting dispersion by placing a drop on a treated coverslip, followed by quick freezing on dry ice and then dried under vacuum to remove any solvent by evaporation. The coverslip was treated with Rain X to allow easy peeling of the film

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51 from the coverslip surface. The resulting films w ere 7 mm in diameter and 0.2 mm in thickness. Contro l discs prepared from polymer were prepared similarly. Determination of Loading and Release Kinetics of RGD from Discs To study the loading efficiency of the RGD peptide into the polymer, the discs were dissolved in methylene chloride followed by addition of PBS in order to extract the peptide in the aqueous phase. This emulsion was agitated vigorously for 15 min followed by sonication in a water bath at 25 C for 15 min. The emulsi on was then centrifuged at 10000 g for 10 min to enable separation of the oil and water phase of the emulsion. RGD dissolved in PBS was collected and spectrophotometric analysis was used to determine the concentration of peptide encapsulated in the disc. T o study the release kinetics of the encapsulated RGD peptide from the prepared discs, the discs were placed on a shaker in PBS (pH=7.4) at 37 C. The supernatant was collected and replenished with fresh PBS every 3 days. Using spectrophotometric analysis, t he concentration of RGD peptide in the supernatant was determined and the release was plotted as a percentage of loaded RGD released over 3 weeks. Mouse Calvarial Osteolysis Model To study the role of different integrins in wear particle induced osteolysis we used an established mouse calvarial osteolysis model [165,232,233] UHMWPE microparticles were implanted directly on the surface of the calvarial bone and the extent of osteolysis was quantified using histomorphometry. Briefly, mice were anesthetized with 70 80 mg/kg ketamine and 5 7 mg/kg xylazi ne. The head of the mouse was shaved and the c alvaria were exposed with a one centimeter incision in the frontal plane between the two ears 6 mg of UHMWPE MPs in 30 L of FBS was spread on the calvarial surface and the incision was closed using wound clip s. Mice designated

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52 without particles spread on the calvarium. Mice L of FBS without particles spread on the calvarium. Mice were placed in different groups based on the hypothes is to be tested. Hypothesis 1: Mac 1 integrin plays a role in wear particle induced osteolysis Group 1: Mac 1 KO mice with 0.1% Serum coated MPs Group 2 : WT mice with 0.1% Serum coated MPs Control 1: Vehicle WT mice with 0.1% Serum Control 2 : Sham surger y on WT mice Hypothesis 2 : RGD binding integrins play a role in wear particle induced osteolysis Group 1 : WT mice with 0.1% Serum coated MPs with RGD peptide loaded polymer film Group 2 : WT mice with 0.1% Serum coated MPs with blank polymer film Control 1: Vehicle WT mice with 0.1% Serum Control 2 : Sham surgery on WT mice All the surgeries were performed at the same time. C ommon groups across differen t hypothesis tests were not r epeated but were used for analysis and comparison. Seven days post surgery, mice were euthanized by CO 2 asphyxiation and the calvaria removed for histological processing. The calvaria were fixed in 10% formal in and decalcified using 10% Ethylenediaminetetraacetic acid (EDTA) at 4 C for 7 days. The calvaria were embedded in paraffin and five micron sections were taken in the frontal plane around the area where the MPs were spread and osteolysis was expected. The sections were sta ined with hemotoxylin and eosin for nuclei (dark blue) and

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53 collagen (pink) and imaged using phase contrast microscopy ( Axiovert 200M Carl Zeiss inverted fluorescent microscope) Area of bone resorption was measured by tracing the area of bone a round the mi dline suture Macrophage Generation Bone marrow derived macrophages were generated from 7 10 week old C57BL6/J mice u sing a 10 day culture protocol [234,235] Animals were handled in accordance with protocol approved by the In stitutional Animal Care and Use Committee (IACUC) at University of Florida. Briefly, mice were euthanized by CO 2 asphyxiation followed by cervical dislocation and tibias and femurs were harvested for isolating marrow cells. Marrow cells were obtained by fl ushing the shaft of the bones with a 25 G needle using wash media comprising of media (Hyclone Laboratories Inc, Logan, Utah) containing 1% fetal bovine serum (FBS) (Hyclone Laboratories Inc, Logan, Utah) and 1% penicillin streptomycin neomycin anti biotic mixture (Hyclone Laboratories Inc Logan, Utah). The macrophage we re cultured in a growth media comprising of Dulbecco's Modified Eagle's Medium (DMEM)/F12(1:1) (Cellgro, Herndon,VA) medium containing 1% penicillin streptomycin, 1% L glutamine (L onz a, Walkersville, MD), 1% non essential amino acids (Lonza, Walkersville, MD), 1% sodium pyruvate (Lonza, Walkersville, MD), 10% fetal bovine serum (FBS) and 10% L 929 cell conditioned media (LCCM). The LCCM serves as an established source of macrophage col ony stimulating factor, which pushes the differentiation of marrow cells towards the macrophage phenotype [236] To produce LCCM, L 929 cells were grown to a confluent monolayer in 150 cm 2 tissue culture flasks. 50 mL media was added to each flask for 7 days after which all the media in the flask was replaced with fresh media for 7 additional days. The media collected at day 7 and 14 was pooled, sterile filtered and stored at 20 C. From

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54 the isolated marrow cells, the red blood cells were lysed using AC K (Ammonium Chloride Potassium ) lysis buffer. (Lonza, Walkersville, MD) The precursor cells were i solated using centrifugation, resuspended in macrophage growth medium and then seeded in a tissue culture treated T flask (day 0) to remove fibroblasts and mature macrophages as they would adhere to the flask. After 48 hours (day 2), the floating cells wer e collected, resuspended in fresh media and seeded on low attachment plates for 4 additional days. The cells in the low attachment plates were supplemented with 1 ng/mL IL 3 (Peprotech, Rocky Hill, NJ) for expansion of the macrophage precursor cells. Half of the media in the low attachment wells was exchanged on day 4 with fresh growth media. At the end of 6 days, cells were lifted from the low attachment wells by gentle pipetting, re suspended and seeded on tissue culture treated polystyrene plates for 2 m ore days to allow macrophage adhesion and maturation. On day 8, all the media in the wells was replaced with fresh media and a t day 10 of culture, the cells we re ready for experiment. The purity of the macrophage culture was verified by staining for CD11b 1) and F4/80 murine macrophage markers and analyzed using flow cytometry. Macr ophages isolated from at least 4 separate mice were used for each type of experiment. Polystyrene(PS) Microparticle Preparation For the particulate biomaterial system, we used commercially available fluorescent polystyrene particles Fluoresbrite YG Microspheres (Polysciences Inc, Warrington, Pennsylvania). Macrophage inflammatory response depends on particle size and shape. Sizes 0.5 5 m are shown to be most reactive, [223,224] hence we have used commercially available spherical microparticles (MPs) of 1 m diameter. According to previously published studies, detectable level of endotoxin on MPs has been shown to

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55 be a prerequisite for inflammatory cytokine secretion [228 230] Hence we have compared phagocytosis and subsequent cytokine secretion of MPs coated w ith known level of endotoxin to MPs with undetectable endotoxin levels. In order for the particles to have a known amount of endotoxin, the particles were coated with LP S by incubating in 0.7 1.2 g/mL (depending on the protein to be adsorbed later) LPS so lution for 3 hours to obtain a final endo toxin concentration of 100 EU/mL The LPS coated MPs were then protein coated by incubating with various ECM proteins such as human plasma derived fibronectin (FN) (BD Bioscience), human plasma derived vitronectin ( VN) (BD Bioscience) and bovine plasma fibrinogen (FG) (Mp Bi omedicals) as well as with FBS and Bovine serum albumin (BSA) (Fisher Bioreagents) Species specific protein sequence homologies, as compared to murine, are as follows: FN 92%, COL 89%, VN 76%, FG 81% and BSA 70%; determined by HomoloGene, an online resource made available through the National Center for Biotechnology Information. The concentration of protein soluti on used for coating is 200 g/mL and the particles were incubated overnig ht in the protein solution. The particles are vortexed into the protein solution and allowed to incubate with the protein overnight at 4 C. After overnight incubation, the particles are separated from protein solution by filtration (pore size 0.22 ) and resuspended in PBS. Analysis of endotoxin on MPs was performed in duplicate with the Chromo limulus amoebocyte lysate ( Chromo LAL ) assa y (Associates of Cape Cod Incorporated, Falmouth, MA). Endotoxin Testing A high sensitivity Limulus Amebocyte Lysate (LAL) test was performed to measure the endotoxin level on the MPs coated with or without LPS. 50 uL of the Chromo LAL substrate (Limulus Amebocyte Lysate colyophilized with chromogenic substrate) is

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56 plate. The 96 well plate is then read in a microplate reader that has been set to read absorbance at 405 nm ev ery 2 min for 2 h ours A threshold value of 0.2 for absorbance was selected and the time point that the sample crossed the threshold value was used for calculating the endotoxin concentration with the help of a standard curve plotted with the control stand ard endotoxin (CSE) provided by the manufacturer. The detection limit of the assay at 2 h is 0.04 EU/mL. Quantification of Macrophage Phagocytosis of PS MPs 1 x 10 5 macrophages were plated in 96 well tissue culture plates. A predetermined number of fluor escent polystyrene MPs are added to each well in order to feed a fixed number (10, 20 and 40) of particles per cell and incubated at 37 C, 5% CO 2 to allow phagocytosis. Phagocytosis was evaluated at the end of 2, 3.5, 5 and 7.5 h ours after two PBS washes t o remove MPs not phagocytosed by cells and read in a fluorescent plate reader. The number of MPs phagocytosed was determined from a standard curve obtained by plotting relative fluorescence intensity versus MP number. The results are reported as MPs/cell a nd normalized for cell numbers by staining cell nuclei with DAPI and measuring fluorescence intensity per well. For the RGD blocking experiments, macrop hages were incubated with 1 mM and 10 m M RGD in macrophage media for 1 h our prior to feeding the MPs for quantifying MP uptake at 2 and 24 h ours respectively. MP uptake at 2 h ours and 24 h ours after feeding 20 MPs per cell was quantified as described above. Quantification of Macrophage Cytokine Production upon Phagocytosis of PS MPs For quantification of mac rophage cytokine production, 1 x 10 6 macrophages were plated in 12 well tissue culture plates. MPs were added to each well in order to get a cell

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57 to MP ratio of 1:25. The well plates are incubated at 37 C, 5% CO 2 for 24 h ours to allow phagocytosis and secr etion of cytokines. A negative control of cells incubated with no MPs and positive contro l of cells incubated with 1g/mL LPS was also set up to study baseline cytokine secretion and activation by a strong inflammatory signal (LPS). After 24 h ours the supe rnatant is collected and frozen at 20 C for cytokine analysis using sandwich ELISA. The supernatant was assayed for cytokines TNF 6 using sandwich ELISA kits (R & D S ystems) according to manufacturer's directions. For the RGD blocking experiment s, 1 x 10 5 macrophages were incubated with 2.5 M RGD in macrophage media for 1 h prior to feeding 20 MPs per cell. After 24 h, the supernatant was collected and frozen at 20 C for cytokine analysis using sandwich ELISA. Polyethylene (PE) Microparticle Pr eparation Polyethylene microparticles form the majority of the wear particles isolated from the periprosthetic tissue harvested from patie nts undergoing revision surgery [147,149] The retrieved polye thylene particles are rounded or elongated and in the size range of 0.1 5 m with a high proportion of submicron size particles [219,237,238] For mimicking the wear debris particles generated in the body, we have used commercially available UHMWPE particles (Shamrock Technologies, Newark, NJ) in the size range of 0.5 5 m. M acrophage inflammatory response to wear particles depends on the size and shape of the particles with a size range of 0.5 5 shown to be most reactive [223,224] PE MP preparation for phagocytosis experiments In order to visualize and quantify the number of PE MPs phagocytosed by the macrophages the proteins used to coat the particles are fluorescently labeled using

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58 Alexa Fluor succinimidyl ester fluo rescent dyes. Succinimidyl esters provide an efficient and convenient way to selectively link the Alexa Fluor dyes to primary amines (R NH2) located on proteins. For protein conjugation the Al exa Fluor dye was mixed with protein (5 mg/mL ) according to the desired labeling efficiency and allowed to react by stirring for one h our at room temperature. After one hour, the unreacted dye was separated from the protein solution by centrifugation through spin filters with a pore size of 3 kDa. The particles are vo rtexed into the protein solution and allowed to incubate with the protein overnight at 4 C. After overnight incubation, the particles are separated from labeled s onication into unlabelled protein solution PE MP preparation for cytokine experiments According to previously published studies, detectable level of endotoxin on MPs has been shown to be a prerequisite for inflammatory cytokine secretion [228 230] Hence we have quantified cytokine secretion upon phagocytosis of MPs coated with known level of endotoxin to MPs with undetectable endotoxin levels. In order to remove any adherent endotoxin on t he UHMWPE MPs, they were washed in 70% ethanol for 72 h ours by continuous shaking and ethanol change every 24 h ours To coat the clean MPs with a known amount of endotoxin, the particles were incubated with 1 00% ethanol containing 3 5 g/mL (depending on t he protein to be adsorbed later) LPS for 1 h our The clean MPs to be used as endotoxin free controls were also incubated in 100% ethanol without LPS. After 1 h our the MPs are separated from ethanol by spin filtration (pore size 0.22 m) and resuspended in solutions of various ECM proteins such as human plasma derived fibronectin (FN) (BD Bioscience) and bovine plasma fibrinogen (FG) (Mp Bi omedicals) as well as with FBS and Bovine serum albumin (BSA) (Fisher

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59 Bioreagents). The concentration of FN,Fg and BSA used for coating was 250 g/m L and serum was 5% in PBS. MPs with or without LPS coating were incubated with protein solutions for 2 h ours after which the endotoxin levels on the MPs was measured. The size distribution of the particles with different adsor bed proteins was determined using LS1320 Coulter counter (PERC, University of Florida) to ensure different proteins do not change the size distribution of MPs due to aggregation. The size and surface properties of MPs were also visualized using Scanning El ectron Microscopy (SEM). (MAIC facility, University of Florida). Inverted Cell Culture Technique for Phagocytosis of UHMWPE MPs The density of UHMWPE is 0.94g/cm 3 which is lesser than water (1g/cm 3 ) hence when suspended in media, the MPs float to the surfa ce contact with macrophages adherent on well bottom. A num ber of studies have used various techniques to confine the MPs to the bottom of well plates. For example, Harada et al have embedded polyethylene particles in agarose solution coated on the bottom of the wells and cultured macrophages on the agarose layer to ensure cell MPs contact [239] Shanbag et al suspended the particles in serum and coated well bottoms followed by macrophage seeding [240,241] The disadvantage of such techniques is the limited interaction of the macrophages with the confined particles. Rao et al have used well plates sealed with silicone sheets and invert ed to bring macrophages in contact with particles [242] I created an inverted culture system comprising of a glass coverslip inverted over Viton O rings which keep the coverslip at some height fr om the bottom of the well as shown in the Figure 2 4 The UHMWPE MPs float up to the surface of the media which is adjusted to be at the level of the coverslip allowing free interaction

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60 between the cells and MPs. Such a free interaction mimics the kind of interaction between macrophages and wear particles in the joint space around the implant. Quantification of Macrophage Phagocytosis of PE MPs 1 x 10 5 macrophages were plated in 96 well tissue culture plates. A predetermined n umber of fluorescently labeled protein coated PE MPs are added to each well in order to feed a fixed number ( 20 and 40) of particles per cell The wells are filled to the brim, sealed with a sealing tape, inverted and incubated at 37 C, 5% CO 2 to allow phagocytosis. Phagocytosis was eva luated at the end of 2, 3.5, 5 and 7.5 h ours after two PBS washes to remove MPs not phagocytosed by cells and read in a fluorescent plate reader. The number of MPs phagocytosed was determined from a standard curve obtained by plotting relative fluorescence intensity versus MP number. The results are reported as MPs/cell and normalized for cell numbers by staining cell nuclei with DAPI and measuring fluorescence intensity per well. Quantification of Macrophage Cytokine Production For quantification of macro phage cytokine production, 1 x 10 6 macrophages were grown on 22 x 22 mm glass coverslips.(Fisherbrand, Fisher Scientific) To allow macrophage contact with MPs for phagocytosis, the inverted culture system as described in the previous section was utilized. Protein coated MPs with or without adsorbed LPS were added to each well in order to get a cell : MPs ratio of 1:10. The well plates are incubated at 37 C, 5% CO2 for 24 h ours to allow phagocytosis and secretion of cytokines. A negative control of cells inc ubated with no MPs and positive contro l of cells incubated with 1g/mL LPS was also set up to study baseline cytokine secretion and activation by a strong inflammatory signal (LPS). After 24 h ours the supernatant is collected and frozen at 20 C for cytoki ne analysis using sandwich

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61 ELISA. The supernatant was assayed for cytokines TNF 6 using sandwich ELISA kits (R & D systems) according to manufacturer's directions. Statistical Analysis Statistical analyses were performed using general linear neste d model ANOVA, using Systat (Version 12, Systat Software, Inc., San Jose, CA). Pair wise comparisons were made between the different groups separately for each protein using Tukey's Honestly Significant Difference Test with p values of less than or equal t o 0.05 considered to be significant. Results Loading and Release Kinetics of RGD from Discs In order to determine the loading efficiency of RGD into the discs and determine the exact amount of RGD loaded per disc, the discs were dissolved in methylene chloride and the RGD was extracted into water phase of the emulsion. The l oading efficiency of RGD into the disc was 92%, with 2.3 mg RGD encapsulated per disc. The release of RGD from the disc was characterized over 3 weeks to estimate the peptide that may be released into the implant site. There is burst of RGD from th e discs in the 1 st 4 days, releasing about 50% of the encapsulated peptide (Figure 2 1). This is followed by a gradual increase to 65 % of the encapsulated peptide in the next 4 days followed by a plateau phase (Figure 2 1). During the plateau phase there is negligible release of 0.5% of the encapsulated peptide per week for the next 2 weeks of testing (Figure 2 1). Role of RGD binding Integrins in MP induced Osteolysis In order to determine the role of RGD binding integrins in particle induced osteolysis, the resulting osteolysis upon implantation of PE MPs along with

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62 polymer discs releasing RGD peptide was quantified. The area of osteolysis was compared to the calvaria which had PE MPs along with blank polymer discs implanted upon it. Cont rol groups of sham, vehicle only and vehicle + blank were included to study osteolysis resulting from surgery alone, 0.1% Serum used to resuspend the MPs and the blank disc respectively. The area of osteolysis between the control groups veh icle only and vehicle + blank was not significantly different hence, they were combined under the vehicle control group. The area of osteolysis in the mice releasing RGD peptide was 50% lower as compared to the mice with the blank discs (Fi gure 2 2 A), indicating a major role played by RGD binding integrins in the osteolytic process. Hematoxyline and Eosin stained sections of the mouse calvaria from the different groups (Figure 2 2 B,C,D,E) depicting the area of osteolysis(Figure 2 2 D & E, yellow arrows) enable visualization of the difference in area of osteolysis. The area of osteolysis for the MPs and RGD releasing samples as well as MPs and blank samples was significantly higher than vehicle and sham control groups (Figure 2 2 A). The area of osteolysis between the two control groups sham and vehicle only was not significantly different (Figure 2 2 A ), indicating that the 0.1% FBS used as a vehicle to resuspend the MPs does not significantly contribute to the osteolytic process. Role of Mac 1 Integrins in MP induced Osteolysis Using an established model to quantify osteolysis, the role of Mac 1 inte grin in wear particle induced osteolysis was quantified. PE MPs mimicking the wear particles generated from total joint replacements in vivo was implanted on mouse calvarium and the resulting osteolysis in 1 week was quantified using histomorphometry. Cont rol

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63 groups of sham and vehicle only were common between the RGD binding integrin and Mac 1 integrin studies. The area of osteolysis in the Mac 1 KO mouse was 60% lower as compared to WT controls (Figure 2 3 A). Hematoxyline and eosin stained sections of th e mouse calvaria from the different groups (Figure 2 3 B,C,D,E) depicting the area of osteolysis (yellow arrows) enable visualization of the difference in area of osteolysis between Mac 1 KO and WT mice. When compared to the vehicle control group, the area of osteolysis in Mac 1 KO mice was not significantly higher (Figure 2 3 A) indicating that the absence of the integrin significantly reverses the osteolytic effect resulting from the MPs. Purity of Macrophage Culture and Mac 1 KO Macrophages The percen tage of WT macrophages co expressing CD11b and F4/80 was ~90%, indicating a relatively pure WT macrophage population (Figure 2 4 A ) The percentage of macrophages from Mac 1 KO mice expressing CD11b was ~1%, thus confirming absence of the receptor on the m acrophage surface (Figure 2 4 B) Role of Mac 1 and RGD binding Integrins in Macrophage MP Uptake of PS MPs To explore the role of different ligand receptor interaction as well as endotoxins in macrophage particle uptake, MPs were coated by adsorption with different opsonizing proteins and LPS respectively. There was no significant difference in the macrophage MP uptake between PS MPs pre coated with and without LPS (Figure 2 5 A) Macrophages phagocytosed differential number of particles depending on the p rotein coated on the PS MPs with the highest uptake of MPs for fibrinogen coated MPs and lowest uptake of BSA coated MPs for most ratios and time points tested (Figure 2 6, 2 7 & 2 8 )

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64 In order to determine the role of integrin Mac 1 in macrophage MP upta ke, we quantified the MP uptake at different cell to MP ratios and different time points for Mac 1 KO macrophages and compared it to macrophages from WT mice. The absence of Mac 1 integrin on macrophage surface down regulates phagocytic uptake of fibrinoge n, serum, vitronectin and fibronectin opsonized PS MPs with MP uptake down to 60 85% as compared to WT controls (Figure 2 6, 2 7, 2 8 ) Uptake of MPs coated with BSA was higher for WT macrophages as compared to Mac 1 KO at lower cell:MP ratio of 1:10 (Figu re 2 6 ) However for higher ratios of 1:20 and 1:40 this relationship is reversed (Figure 2 7 & 2 8 ) To determine the role of RGD binding integrins in macrophage MP uptake we quantified the MP uptake at cell : MP r atio of 1:20 in a 1 mM (for 2 h ours ) an d 10 mM (for 24 h ours) RGD peptide solution in the macrophage culture media At the end of 2 and 24 h ours the number of MPs taken up by the cells was quantified and compared to unblocked samples. At 2 h ours the MP uptake of RGD blocked samples decrease t o 30 50% for FN, Fg, Ser and VN coated MPs and 77% for BSA as compared to unblocked samples (Figure 2 9 A ) This difference in MP uptake became further amplified at 24 h ours when the MP uptake was reduced to 10 30% as compared to unblocked samples for all the protein coated MPs (Figure 2 9 B) Role of Mac 1 and RGD binding Integrins in Macrophage Inflammatory Cytokine Secretion in Response to PS MPs To determine the role of endotoxins adsorbed on MPs in macrophage activation and inflammatory cytokine secre tion, macrophages were fed MPs coated with LPS and without LPS. There was a significant difference in the macrophage secretion of TNF and IL 6 between MPs pre coated wi th LPS and without LPS as MPs without LPS had

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65 undetectable levels (< 30pg/mL ) of cytokine secretion (Figure 2 5 B) The role of different ligand receptor interaction in macrophage inflammatory cytokine secretion was explored by feeding macrophages with MPs coated with different opsonizing proteins and quantifying the resulting cytokine secretion. For Mac 1 KO macrophages, BSA coated MPs had the highest IL 6 and TNF as compared to other protein coatings (Fig ure 2 10 A & B) Additionally, for Mac 1 KO macrophages, FN coated MPs had the higher TNF as compared to Fg and Serum coated MPs. For WT macrophages, Ser and F N coated MPs had significan tly higher IL 6 secretion as compared to Fg and BSA and significantly higher TN F only (Figure 2 10 A & B) In order to determine the role of integrin Mac 1 in macrophage inflammatory cytokine secretion, we quantified the secr etion of TNF 6 upon MP uptake by Mac 1 KO macrophages and compared it to macrophages from WT mice. The absence of Mac 1 integrin on macrophage surface down regulates IL 6 secretion upon uptake of Fg, FN, Serum and VN opsonized PS MPs with a reduct ion of 30 50 % as compared to WT control (Figure 2 10 A) A similar reduction is observed in TNF uptake of Serum FN and VN opsonized PS MPs with a reduction of 20 40 % as compared to WT control (Figure 2 10 B) There is significant decreas e in the TNF IL 6 secretion by Mac 1 KO macrophages for the positive controls incubated with soluble LPS, the secretion was reduced to 20 40% as compared to WT control s Interestingly, TNF 6 secretion for the Mac 1 KO macrophages was 3.4x an d 1.3x higher than the WT controls for the BSA coated MPs. All the MP fed samples for Mac 1 KO and WT macrophages were significantly higher than their respective no MP

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66 negative control For the WT macrophage samples with MPs were significantly lower than the LPS positive control (Figure 2 10 ) However for the Mac 1 macrophage samples the protein coated MP samples and LPS positive control group were not significantly different (Figure 2 10 ) In order to determine the role of RGD binding integrins in macroph age inflammatory cytokine secretion we quantified the secretion of TNF 6 from WT macrophages upon MP exposure for 24 h ours in the presence of 1 0 mM RGD dissolved in cell media and compared it to unblocked samples. There was a significant decrease in the production of both TNF 6 at 24 h ours for samples incubated with RGD peptide for all the protein coatings (Figure 2 11 A & B) The decrease in IL 6 secretion was 90% for BSA coated MPs and greater than 98% for all the remaining proteins fo r RGD blocked samples (Figure 2 11 A) whereas TNF below detecti on limits of the assay (30 pg/mL ) (Figure 2 11 B) UHMWPE MPs Size Distribution The size distribution of UHMWPE MPs was determined after coating particles wit h different protei ns to verify different protein coated MPs are still in the same size range. It is possible for MPs coated with proteins to form aggregates due to MPs clumping together H ence care was taken to ensure that this does not happen to the part icle solutions. The MPs coated with different proteins were in the size range of 0.5 5 m with over 35% of them in the submicron size range (Figure 2 12 A D) SEM images of the protein coated PE MPs depicts that the MPs are oblong shape and have a rough su rface texture (Figure 2 12 E).

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67 Role of Mac 1 Integrins in Macrophage MP Uptake of PE MPs To explore the role of different ligand receptor interaction in macrophage particle uptake, PE MPs were coated by adsorption wit h different opsonizing proteins. Macrop hages phagocytosed differential number of particles depending on the protein coated on the MPs with the highest uptake of MPs for fibrinogen coated MPs and lowest uptake of BSA coated MPs for most ratios and time points tested (Figure 2 13 and 2 14 ). In o rder to determine the role of integrin Mac 1 in macrophage MP uptake, we quantified the MP uptake at different cell to MP ratios and different time points for Mac 1 KO macrophages and compared it to macrophages from WT mice. The absence of Mac 1 integrin o n macrophage surface down regulates pha gocytic uptake of fibrinogen, fibronectin and BSA opsonized PE MPs with MP uptake down to 50 80 % as compared to WT controls (Figure 2 13 and 2 14). Role of Mac 1 Integrins in Macrophage Inflammatory Cytokine Secretion in Response to PE MPs In order to determine the role of integrin Mac 1 in macrophage inflammatory cytokine secretion, we quantified the secretion of IL 6 and TNF Mac 1 KO macrophages and compared it to macrophages from WT mice. The inv erted culture setup as depicted in the Figure 2 15 was utilized to ensure macrophage contact with the PE MPs as PE MPs are lighter than media and float to the surface. The lack of Mac 1 integrin on macrophage surface down regulates IL 6 secretion upon upta ke of BSA opsonized PE MPs with a reduction of 15% as compared to WT control (Figure 2 16 A). The absence of Mac 1 integrin on macrophage surface also down regulates TNF modest

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6 8 reduction to 80 90% as compared to WT control (Figure 2 16 B ). There is significant decrease in the secretion of TNF and IL 6 by Mac 1 KO macrophages for the positive controls incubated with LPS with secretion reduced to 35% and 75% for TNF 6 respectively as compared to WT control (Figure 2 16 A & B ) All the MP fed samples for Mac 1 KO and WT macrophages were significantly higher than their re spective no MP negative control (Figure 2 16 A & B ) Impact of the Study Macrophages form the sentinels of the body in response to implant ed biomaterials as well as particles generated over time du e to wear of these biomaterials [5,152,243] Macrophages interact with implanted biomaterials through the layer of physiologic proteins adsorbed on the biomaterial surface and integrin receptors present on macrophage surface [5,14,18,42] Binding of the inte grin receptors initiates a down stream signaling mechanism leading to macrophage activation and release of inflammatory cytokines such as TNF 6, IL E [152,163,244,245] and chemotactic factors such as MIP 8 [246] For non particulate biomaterials which cannot be phagocytosed by macrophages, macrophages fuse togethe r to form foreig n body giant cells which is also an integrin dependent process [82] Because in tegrins present on macrophages are involved at the initial stages of material macrophage inter ac tions, they serve as potential therapeutic targets for modulating the macrophage inflammatory responses to implanted materials. To identify target integrins, we have quantified the role of different macrophage integ rins Mac 1 and RGD binding integrins in i nflamma tory response to particulate biomaterials using in tegrin knockout mice and integrin blocking studies.

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69 For the particulate biomaterial system we first quantified the in vivo osteolysis resulting from implanting polyethylene microparticles on the sur face of mouse calvaria. These polyethylene microparticle mimic the wear particles generated from wear of polyethylene liners, one of the apposing components of artificial joint replacements and a major source of wear debris [147,149] To study the role of RGD binding integrins in the osteolysis response, the particle induced osteolysis was quantified in the presence and absence of RGD peptide eluted from polymer discs. The properties of the ELV polymer solution and the polymer drug emulsion were adjusted to achieve release of 70% of the encapsulated peptide within the 1 week of implantation. The area of osteolysis in mice with RGD releasing discs was 50% as compared to mice with blank discs. Integrin V 3 is expressed on activated macrophages [247] Blocking the macrophage integrin receptors such as 5 1 V 3 and V 5 that have an RGD binding site with soluble RGD peptide, prevents interaction and binding of the protein coated MPs (Figure 2 9). When the integrin is bound with a soluble ligand rather than surface adsorbed ligand, the mechanical resis tance required for integrin signaling is insufficient [225] The cellular signaling molecules that are normally recruited during surface bound ligand binding are not recruited which affect s the strength of the adhesions as well as initiation of other inflammatory signaling pathways [226,248] This may play a role in the reduction in MP uptake as well as subsequent cytokine secretion upon blocking integrins with RGD peptide (Figure 2 9 & 2 11). Additionally, RGD peptide binds to several integrins present on osteoclasts surface such as V 3 [174] 2 1 [174] and V 1 [174] Osteoclasts actively migrate on the surface of bone s and undergo alte rnating cycles of migration and resorption [171]

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70 Both of these processes involve interaction with extracellular matrix (ECM) which occurs through integrins thus, integrins are key players in the osteoclast resorption process [122] RGD peptide binding to the integrin receptors is kn own to disrupts osteoclasts ECM interaction, prevents binding of osteoclasts to bone surface and prevent formation of a sealing zone which is required for the resorption process [249] Besides the role of RGD binding integrins in osteoclast funct ioning, our in vitro data also suggested a major role that these integrins play in macrophage inflammatory response to particulate biomaterials. The inflammatory response of macrophages has been shown to depend on the size, shape, surface area and texture of the wear particles [223,224] and sizes 0.5 5 m are shown to be most reactive, [223,224] in the peri prosthetic osteolysis process. In order to better understand the integrin protein micropart icle interaction without having to account for changes in size, shape and texture we investigated MPs of a standard size and shape. Hence, the initial experiments were conducted with commercially available fluorescent polystyrene particles Fluoresbrite Y G Microspheres which are spherical and 1 m in diameter. The PS MPs were coated with different opsonizing proteins in order to study receptor ligand interactions involved in MP uptake and cytokine secretion. Integrins have a binding specificity to its liga nds hence; coating MPs with different opsonizing proteins helps target specific integrin ligand interactions. We observed ligand specific variations in the uptake of PS MPs with highest uptake for fibrinogen coated MPs and lowest uptake of BSA coated MPs f or most ratios and time points tested. F ibrinogen is one of the primary components of plasma deposited on biomaterial surface [17,23] and a ligand for Mac 1 [85] an integrin shown to play a role

PAGE 71

71 in macrophage phagocytosis [94,95] Upon adso rption onto a hydrophobic biomaterial surfac e, Fg adopts an energetically favorable conformation thereby exposing the hidden 395) which are known to be binding sites for integrin Mac 1 [24] There have been contradicting report s in the field regarding the role of adsorbed endotoxin on the immunogenicity of the wear particles. Some groups have demonstrated an inflammatory cytokine response with undetectable levels of endotoxin [250,251] where as other have reported a lack of inflammatory cytokine secretion in the absence of detectable endotoxin levels on the MPs used for macrop hage phagocytosis [228 230] Thus, we conducted studies to understand the r ole of adsorbed endotoxin in our experimental setup. We found that MP uptake does not depend on the level of endotoxin adsorbed on the MPs. However, macrophages that phagocytosed MPs with undetectable levels of endotoxin had undetectable levels of cytokine secretion. Thus we used a normalized amount (# EU/mL/mg) of LPS adsorbed onto MPs for all the in vitro experiments. For studying the role of RGD binding integrins in MP uptake, we blocked RGD receptors using soluble RGD peptide dissolved in cell cultur e media. At 2 h ours after blocking integrin receptors with RGD peptide the MP uptake was significantly reduced for MPs coated with proteins fibronectin and fibrinogen. At 24 h ours there was significant decrease in MP uptake for all the protein coatings f or samples blocked with RGD peptide. A remarkable decrease in the cytokine production at 24 h matching the decreased MP uptake results demonstrates a role of RGD binding integrins in the macrophage inflammatory process. Synthetic peptides containing the RG D sequence

PAGE 72

72 have been shown to compete with adhesive proteins for binding to these int egrin receptors and thus, inhibit integrin related functions in different cell systems. Along with the MP samples, there was also a complete down regulation in the cytoki ne secretion from the soluble LPS positive controls when soluble RGD was present. This finding is corroborated by literature showing that integrin v is shown to be involved in LPS induced TLR4 si gnaling pathways leading to macrophage adhesion and cytokine release [252] Thus, blocking RGD binding integrins can provide an inflammatory blockade at multiple levels. RGD is the integrin binding site present in several proteins such as fibr onectin, fibrinogen, vitronectin and laminin [101] known to adsorb on to biomaterial surfaces. The RGD peptide binds to multiple integrin receptors on macrophage surface such as 5 1 V 3 and V 5 Integrin binding by a soluble nature ligand results in an attenuat ed integrin signal and this may play a role in the reduction in MP uptake and cytokine secretion. To study the role of Mac 1 integrin in the osteolysis response, the particle induced osteolysis in Mac 1 KO mice was compared to WT controls. The area of oste olysis in Mac 1 KO mice was 60% lower than WT and not significantly higher than the vehicle control group. The role of Mac 1 integrin in this osteolysis process may be at least two fold; first through the modulation of macrophage response to wear particles and second through its role in osteoclasts maturation and activation. The integrin Mac 1 has been shown to mediates macrophage adhesion to adsorbed proteins and direct phagocytosis of titanium alloy wear particles [99] It has also been reported that blocking the Mac 1 receptor using antibodies against CD11b ( M ) and CD18 ( 2 ) results in inhibition of osteoclast differentiation in both RAW264.7 cells and bone marrow d erived

PAGE 73

73 macrophages upon addition of RANKL [253] To understand the role of Mac 1 integrin in macrophage response to microparticles, we have further investigated MP uptake as well as subsequent cytokine secretion from macrophages harvested from Mac 1 KO mice and compared it to WT macrophages. The MP uptake and subsequent cytokine secretion upon phagocytosis of PS MPs was initially investigated. The study was then extended to PE MPs as they better mimic the size, shape and material of the wear particles generated in the body from joint implants. We quantified particle uptake and inflammatory cytokine secretion in response to PS MPs coated with different adhesive proteins and LPS MP uptake for decreasing cell : MP ratios at various time points starting from 2 h ours to 7.5 h ours for Mac 1 KO macrophages was compared to WT control. F or Fg coated MPs, the MP uptak e of Mac 1 KO macrophages is significantly lower than WT (Figure 2 6,2 7 and 2 8) Since fibrinogen is one of the primary components of plasma deposited on biomaterial surface [17] in the absence of integrin Mac 1, a Fg receptor [85] macrophage adhesion is disrupted leading to reduced particle uptake. MPs coated with fibronectin and v itronectin other Mac 1 ligands [86,88] showed decreased particle uptake for certain ratios and time po ints tested (Figure 2 6,2 7 and 2 8) Along with Fg, Fn and VN serum coated MPs also showed decreased particle uptake for Mac 1 KO macrophages as compared to WT for ratio 1:20 and 1:40 beyond 3.5 h ours as well as for ratio 1:10 at 7.5 h ours similar to FN a nd VN (Figure 2 6,2 7 and 2 8) Fibronectin and vitronectin present in the serum adsorb on to MPs and in the absence of Mac 1 which binds to both fibronectin and vitronectin MP uptake is reduced. BSA coated MPs did exhibit reduced MP uptake for Mac 1 KO ma crophages as compared to WT for ratio 1:10 however for

PAGE 74

74 higher ratios this relationship was unexpectedly inversed although the differences were small (Figure 2 7). The differences in the Mac 1 and WT macrophage particle uptake although small, are indicative of differences in the kinetics of macrophage phagocytosis in the absence of Mac 1 integrin. These differences in kinetics may play a role in the recruitment of signaling molecules and contribute to differences in cell signaling and functioning. We studied the role of integrin Mac 1 in inflammatory cytokine s ecretion in response to PS MPs at 24 h ours upon particle exposure. Similar to the trends seen in MP uptake, TNF and IL 6 secretion from Mac 1 KO macrophages is lower than WT for FN, serum and VN coate d MPs. IL 6 secretion from Mac 1 KO macrophages is l ower than WT for Fg coated MPs. Additionally we see that TNF and IL 6 secretion from Mac 1 KO macrophages is lower than WT for the positive control of soluble LPS. This may be explained by the finding that integrin Mac 1 is shown to form a receptor complex with CD 14 and TLR 4 in response to LPS which is required for expression of the full spectrum of genes [254] CD 14 does not have a transmembrane component [255] and requires a signaling co receptor for intracellular signaling. We conducted experiments similar to PS MPs with PE MPs as PE better represent the wear particles generated in vivo. However, we see that the results of the PS and PE MPs experiments do not follow the same trend for the all the opsonizing proteins. The biggest difference between the results of PS and PE MPs experiments was seen for BSA coated MPs. For PS MPs, the MP uptake and subsequent cytokine secretion was higher for Mac 1 KO macrophages as compared to WT. However, for PE MP this inequality was reversed and MP uptake as well as cytokine secretion from Mac 1 KO

PAGE 75

75 macrophages was lower than WT controls. The uptake of fibrinogen and fibronectin coated MPs was lower than WT for some of the conditions tested however the reduction was not as effective as seen with PS MPs. Cytokine secretion from Mac 1 KO macrophages was reduced as compared to WT fo r some protein coatings however similar to uptake experiments the reduction was a modest 10 15%. These differences in the response to PS and PE MPs can be attributed to the differences in the material properties, size and shape of the two MP types. The con centration and confirmation of proteins adsorbed on biomaterial surface depend on the surface properties [18] Upon adsorption proteins can undergo conformational changes to reduce surface energy and thus expose different ligand binding sites thus creating bioactive sites for the interactio n of cells with biomaterials [18] The PE MPs are extremely hydrophobic as compared to PS MPs and it has been shown that p roteins that adsorb onto extremely hydrophobic surfaces such as UHMWPE, undergo extensive de naturation/unfolding [14,39,77] This denturation may result in differences binding sites being exposed on the PE MPs as compared to PS MPs. Integrin Mac 1 is shown to mediate adh esion to denatured proteins [42] Thus we observe a reduction in the PE MP uptake as well as cytokine secretion for Mac 1 KO macrophages as compared to WT macro phages for BSA opsonized MPs. Thus we have identified integrins that play a role in the in vivo osteolytic response which is mediated by osteoclasts but is initiated by macrophage response to particulate wear debris particles. We have further elucidated t he mechanisms and ligand receptor interaction involved in this process by studying MP uptake and subsequent cytokine secretion from macrophages with the integrin knocked out or blocked using peptides.

PAGE 76

76 Thus Mac 1 and RGD binding integrins can serve as thera peutic target for design of anti integrin therapies to mitigate peri implant osteolysis.

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77 Figure 2 1. Release kinetics of RGD from polymer disc. Release kinetics were studied at 37 C in PBS (pH = 7.4) with continuous sha king. Each point represents the mean of 3 samples. The standard deviation was <10% of the respective values.

PAGE 78

78 Figure 2 2. RGD binding integrins modulates osteolysis in response to particulate biomaterials. A) The area of osteolysis around the midline suture (1.5 mm) was quantified at 7 days. The average area of osteolysis was qu antified by pooling data from 10 sample points per mouse and 6 mice per group Plotted are mean and standard error. (* indicates statis tically significant difference (p < 0.05) from all other groups. Decalcified calvaria were sectioned and stained with Hematoxylin and Eosin for osteolysis measurement. Representative images of calvaria from B) Sham and C) Vehicle D) MP + Blank and E ) MP + RGD loaded are shown to depict the difference in area of osteolysis. A rea of osteolysis is shown with blue arrows Vehicle only Area of Osteolysis(m 2 ) Sham MPs + RGD loaded ELVAX MPs + Blank ELVAX 0 10 000 20000 30000 40000 50000 60000 70000 80000 Not Significant Significant Significant A

PAGE 79

79 Figure 2 3. Mac 1 integrin modulates osteolysis in response to particulate biomaterials. A) The area of ost eolysis around the midline suture (1.5 mm) was quantified at 7 days. The average area of osteolysis was qu antified by pooling data from 10 sample points per mouse and 6 mice per group Plotted are mean and standard error. (* indicates statistically signifi cant difference (p < 0.05) from all other groups. Decalcified calvaria were sectioned and stained with Hematoxylin and Eosin for osteolysis measurement. Representative images of calvaria from B) Sham and C) Vehicle D) WT and E) Mac 1 KO are shown to depict the difference in area of osteolysis. A rea of osteolysis is shown with yellow arrows A Sham Vehicl e WT Mac 1 KO B C E D

PAGE 80

80 Figure 2 4 A) Purity of the macrophage culture was determined to be ~90% as determined by immunofluorescent quantification of F4/80 and CD11b murine macrophage markers by flow cytometric analysis. B) The deficiency of Mac 1 receptor on macrophages from the Mac 1 KO mouse was verified by staining 1). The percentage of cells expressing CD11b was determined to be ~1% Figure 2 5 Presence of endotoxin plays a role in cytokine production upon phagocytosis of LPS coated PS MPs however it does not play a role in MP uptake. A) After feeding PS MPs at cell:MP ratio of 1:20, the MP uptake at 7.5 h ours was quantified for MPs coated with or wit hout LPS(100EU/20 million MPs/mL ). The average number of MPs taken up by macrophages was quantified by pooling data from at least 9 samples from 3 separate runs. Plotted are mean and standard error. B) Quantificatio n of TNF from WT macrophages upon exposure to MPs coated with or without LPS and different opsonizing proteins for 24 h ours The cytokine concentration was quantified by pooling data from 4 samples. Plotted are mean and standard error. (* indic ates statistically significant difference (p < 0.05) between MPs coated with or without LPS for the same protein coating). A B F4/80 CD11b CD11b F4/80

PAGE 81

81 Figure 2 6 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages. After fee ding PS MPs at cell:MP ratio of 1:10, the MP uptake at A) 2 hours B) 3.5 h ours C) 5 h ours D) 7.5 h ours was quantified in Mac 1 KO macrophages and compared to WT control. The average number of MPs taken up by macrophages was quantified by pooling data from at least 15 samples from 5 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between Mac 1 KO and WT control samples for the protein coating) t = 2 h A t = 3.5 h B t = 5 h C t = 7.5 h D

PAGE 82

82 Figure 2 7 Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages. After feed ing LPS (100EU/20 million MPs/mL ) and different protein coated PS MPs at cell : MP ratio of 1:20, the MP uptake at A) 2 h ours B) 3.5 h ours C) 5 h ours D) 7.5 h ours was quanti fied in Mac 1 KO macrophages and compared to WT control, The average number of MPs taken up by macrophages was quantified by pooling data from at least 15 samples from 5 separate runs. Plotted are mean and standard error. (* indicates statistically signifi cant difference (p < 0.05) between Mac 1 KO and WT control samples for the protein coating) t = 2 h A t = 3.5 h B t = 5 h C t = 7.5 h D

PAGE 83

83 Figure 2 8. Integrin Mac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages. After feed ing LPS (100EU/20 million MPs /mL ) and different protein coated PS MPs at cell : MP ratio o f 1:40, the MP uptake for A) Fg B) FN C) Ser D ) VN was quantified in Mac 1 KO macrophages and compared to WT control, The average number of MPs taken up by macrophages from 2 7.5 hours was quanti fied by pooling data from at least 15 samples from 5 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between Mac 1 KO and WT control samples for the protein coating) VN D C Serum Fg A * B FN

PAGE 84

84 Figure 2 9. Phagocytosis of protein opsonized PS MPs by macrophage is modulated by blocking RGD binding integrins with soluble RGD peptide. A) MP uptake of LPS (100EU/20 mil lion MPs/mL ) and different protein coated MPs at 2 hours after blocking with 1 mM RGD peptide. The average number of MPs taken up by WT macrophages was quantified by pooling data from 18 samples from 6 separate runs. B) MP uptake at 24 hours after blocking with 10 mM RGD peptide. The average number of MPs taken up by WT macrophages was quantified by pooling data from 8 samples from 2 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between samples incubated with RGD peptide and control samples for the same protein coati ng) Figure 2 10. Integrin Mac 1 modulates inflammatory cytokine secretion from macrophages upon exposure to protein and LPS coated PS MPs. A) Quantification of IL 6 secretion from Mac 1 KO and WT macrophages upon exposur e to LP S(100EU/20 million MPs/mL ) and protein coated PS MPs for 24 hours. B) Quantification of TNF 1 KO and WT macrophages upon exposure to protein coated PS MPs for 24 hours. The cytokine concentration was quantified by pooling data from 30 samples from 5 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between Mac 1 KO and WT control samples for the same protein coating) A B A B A B

PAGE 85

85 Figure 2 11 Macrophage cytok ine secretion upon exposure to protein coated PS MPs is modulated by blocking RGD binding integrins. A) Quantification of IL 6 secretion from WT macrophages upon blocking RGD receptors by soluble RGD (10 mM) and exposur e to LPS(100EU/20 million MPs/mL ) and different protein coated PS MPs for 24 h ours B) Quantification of TNF from WT macrophages upon blocking RGD receptors by soluble RGD (10 nM) and exposure to LPS and protein coated PS MPs for 24 h ours The cytokine concentration was quantified by pooling data from 6 samples from 2 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between RGD peptide and control samples for the protein coating) A B A B

PAGE 86

86 E Figure 2 12 Particle size distribution of protein coated UHMWPE MPs. MPs coated with A) BSA B) Fg C) FN and D)Serum were analyzed for size distribution using Coulter counter. E) SEM image of BSA coated PE MP ( scale bar = 10 m) showing the size distri bution, shape and surface texture of the PE MPs. C B A D

PAGE 87

87 Figure 2 1 3 Integrin Mac 1 modulates phag ocytosis of protein opsonized PE MPs by macroph ages. After feeding LPS (100EU/2 0 million MPs/mL ) and different protein coated PE MP s at cell : MP ratio of 1:20, the MP uptake at A) 2 h ours B) 3.5 h ours C) 5 h ours D) 7.5 h ours was quantified in Mac 1 KO macrophages and compared to WT control, The average number of MPs taken up by macrophages was quantified by pooling data from at least 24 samples from 4 separate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between Mac 1 KO and WT control samples for the protein coating) * t = 2 h t = 3.5 h t = 5 h t = 7.5 h A B C D

PAGE 88

88 Figure 2 1 4 Integrin M ac 1 modulates phagocytosis of protein opsonized PS MPs by macrophages. After feed ing LPS (100EU/20 million MPs/mL ) a nd different fluorescently labeled protein coated PE MPs at cell : MP ratio of 1:40, the MP uptake for A) BSA B) Fg C) FN was quantified in Mac 1 KO macrophages and compared to WT control, The average number of MPs taken up by macrophages from 2 7.5 h ours was quantified by pooling d ata from at least 24 samples from 4 separate runs. Plotted are mean and standard error. (* indicates statistical ly significant difference (p < 0.05) between Mac 1 KO and WT control samples for the protein coating) BSA FN Fg * * A B C

PAGE 89

89 Figure 2 15 Detail of a single well for inverted culture phagocytosis assay. The less dense UHMWPE particles float to the surface of the media where they establish contact with macrophages on the coverslip inverted on Viton O rings. Such a setup ensures free interaction between cells and particles similar to the interaction in the joint space in the body. Figure 2 16 Integrin Mac 1 modulates inflammatory cytokine secretion from macrophages upon exposure to protein and LPS coated UHMWPE MPs. A) Quantification of IL 6 secretion from Mac 1 KO and WT macrophages upon exposur e to LPS(100EU/20 million MPs/mL ) and protein coated P E MPs for 24 h ours B) Quantification of TNF 1 KO and WT macrophages up on exposure to protein coated PE MPs for 24 h ours The cytokine concentration was qu antified by pooling data from 24 samples from 6 sepa rate runs. Plotted are mean and standard error. (* indicates statistically significant difference (p < 0.05) between Mac 1 KO and WT control samples for the same protein coating) Coverslip Cell monolayer Rubber spacer Media level Floating UHMWPE particles A B

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90 CHAPTER 3 INTEGRIN DIRECTED MODULATION OF MACROPHAGE RESPONSE TO BULK BI OMATERIALS Background Following the implantation of a biomaterial within the body a series of host immune responses terme d as foreign body response ant it which attempt to eliminate or isolate the foreign body [39] Macrophages play a major role in this foreign body response as they form majority of the cell type recruited to the site of biomaterial implantation and they secrete various c ytokines and signaling molecules that lead to material degrad ation [39] If the implant is too big for the macrophages to phagocytose mac rophages fuse t o form foreign body giant cells ( FBGCs ) which release reactive oxygen intermediates (ROIs), degradative enzymes, and acid into the space between the cell membrane and biomaterial surface and is shown to mediate degradation of biomaterial sur faces [5,8] The macrophages and FBGCs along with recruited fibroblast s lead to fibro us encapsulation of the implant [5] This fibrous encapsulation severely limits the functional performance of several implanted biomaterial such as pacemaker leads, glucose sensors [256] electrodes in vivo [257] and drug delivery devices [11] For these applications, it is particularly important to have uninterrupted exchange of nutrients and cellular byproducts with the surrounding medium and the fibrous capsule prevents this free exchange. Hence various research groups have been investigating several surface modification approaches for reducing fibrous capsule formation. These techniques focus on reducing protein ad sorption on the biomaterial surface, one of the primary and critical steps of the foreign body response. It is critical because m acrophages interact with biomaterials through the interface of the adsorbed proteins and cell surface receptors called integrin s [5,14,77] The modification approaches to reduce protein adsorption

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91 include self=assembled monolay ers [258] polymer brushes [259] and nanotopographic structures [260] It is also possible to reduce the fibrous capsule formation by modulating the macrophage response as macrophages play a central role in this process. Macrophages have integrin receptors on its surface which direct various cell functions such as adhesion to extracellular matrix (ECM) proteins, adhesion and signaling to other cell types, cell migration an d sp reading as well as phagocytosis [83] Since integrins present on macropha ges direct various processes that are involved in the inflammatory response they serve as ideal therapeutic targets for modulating the macrophage inflammatory response. Fibrinogen is one of the primary components of plasma deposited on biomaterial surface mediating the acute inflammatory response through phagocyte re cruitment to implanted material [17,23] Mac 1 a leukocyte integrin present on macrophages and neutrophils functions as a fibrinogen receptor and this receptor mediated interaction between Mac 1 and fibrinogen has been shown to direct mac rophage adhesion and activation [222] Additionally integrin Mac 1 mediates cell adh esion to a number of other proteins that adsorb out of physiologic fluids onto synthetic materials including complement factor fragment C3bi, albumin, vitronectin, and fibronectin [5,48,261] As integrin Mac 1 present on macrophages direct various inflammatory processes, it may serve as ideal therapeutic target for modulating the macrophage inflammatory response [94 96,221] Among the mileu of proteins that adsorb onto the biomaterial surface, majority of them such as fibrinogen, fibronectin and vitronectin, contain a tripeptide, Arg Gly Asp (RGD), that s erves as the recognition sequence for integrins [101] Hence, RGD

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92 mimetics that target integrins on the macrophage surface may competitively bind to the integrins thus disrupting its binding to the protein adsorbed on biomaterial surfaces. This may arrest the macro phage inflammatory response at the very first step of adhesion. Echistatin, a 49 residue protein purified from the venom of the saw scaled viper Echis carinatus contains the RGD sequence which is much more potent than the tetrapeptide Arg Gly Asp Phe [125] and binds to integrins such V 3 through this RGD binding site. Echistatin is shown to inhibit osteoclastic bone resorption in vitro and in vivo by V 3 which is known to modulate osteoclast function [127] Through its RGD binding site, echistatin can serve as an effective integrin targeted therapy. In this paper, we have studied the role of the integrins in foreign body response to subcutaneously implanted biomaterials. For subcutaneous implantation of bulk biomaterials we have used a common biomaterial polyethylene terephthalate which is used in several medical applications such as vascular grafts [262] surgical mesh [263] and sutures. To investigate the role of integrin Mac 1 in the foreign body r esponse (FBR) to implanted biomaterials we implanted PET discs in Mac 1 KO mice and WT controls. The thickness of the collagenous capsule formed around the disc seven days after implantation was used as a measure to quantify FBR. Similarly, to investigate RGD bind ing integrins for their role in FBR, we coat the PET discs with ethylene vinyl acetate ( ) polymer loaded with echistatin which is slowly released by diffusion from the polymer matrix. is a non inflammatory and non biodegradable polymer that ha s been investigated for slow and sustained release of compounds into the brain [132] a s well as tooth space [134] Once the role of these integrin receptor s in

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93 the foreign body response is delineated, they can serve as a therapeutic target for integrin targeted therapies. Experimental Procedure Biomaterial Implantation and Analysis Discs (7 mm diameter, 0.5 mm thick) were cut from PET sheets, washed, and sterilized by washing in 70% ethanol for 48 h ours At the end of 48 hours the endotoxin level on the discs was determined us ing Chromo assay and the endotoxin levels on the disc were determined to be below the recommend ed maximum FDA level. (0.5 EU/mL 5 samples/group) in accordance with protocol approved by the U niversity of Florida IACUC committee. The wounds were closed with wound clips which were removed at day 7 after implantation. PET discs were explanted at 14 days, formalin fixed and paraffin h ematoxylin e osin stain for nuclei (dark blue) and collagen (pink) and the thickness of the capsule surrounding the disc was determined by bright field microscopy. For studying the role of Mac 1 integrin, discs were implanted in Mac 1 KO mice and WT control mice. For the RGD blocking experiments, echistatin a nonpeptide RGD mimetics was load ed into polymer. Polymer / drug coatings were prepared from an emulsion of copolymer beads (5% by weight) and ec histatin (final conc. = 50 g/mL ) in a ratio of 9:1 ( : drug) in dichloromethane in sealed glass vessels. This solution was agitated v igorously for 15 min followed by sonication in a water bath at 25 C for 15 min. Approximately 2 0 L of solution was then pipetted onto each PET disc and discs were allowed to dry overnight under mild vacuum to remove solvent by evaporation. Control discs w ith only coatings were prepared similarly.

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94 Foreign Body Giant Cell (FBGC) Formation Fusion was induced in expanded macrophages obtained at the end of 10 day culture protocol described in Chapter 2 The macrophages were lifted and replated onto non tissue cu lture treated 24 well plates with 10E6 cells per well. Fusion was induced by addition of 10 ng/mL recombinant mouse (rm) G M CSF (R&D Systems) and 10 ng/mL rm IL 4 (R&D Systems) to macrophage growth medium depleted of LCCM. Complete media in the wells was r eplaced on day 3 and 5 and fusion was analyzed on day 7. Cells with 3 or more nuclei were counted as FBGC as cells with 2 nuclei could be a dividing cell. Determination of Loading and Release Kin etics of Echistatin from C oating around PET Discs To study the loading efficiency of the e chistatin into the polymer, the coating around the PET discs was dissolved in methylene chloride. Water was added in order to extract the e chistatin in the aqueous phase and t hi s solution was agitated vigorously for 15 min followed by sonication in a water bath at 25 C for 15 min. The solution was then centrifuged at 10000 g for 10 min in order to separate out the oil and water phase of the emulsion. The water was collected and s pectrophotometric analysis was used to determine the concentration of echistatin encapsulated around each disc. To study the releas e kinetics of the encapsulated e chistatin from the prepared discs, the discs were placed on a shaker in PBS (pH=7.4) at 37 C. The supernatant was collected and replenished with fresh water every alternate day. Using spectrophotometric analysis, the concentration of e chistatin in the supernatant was determined and the release was plo tted as a percentage of loaded e chistatin over a span of 3 weeks.

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95 Statistical Analysis Statistical analyses were performed using general linear nested model ANOVA, using Systat (Version 12, Systat Software, Inc., San Jose, CA). Pair wise comparisons were made between the different groups separately for each protein using Tukey's Honestly Significant Difference Test with p values of less than or equal to 0.0 5 were considered to be significant. Results Role of Mac 1 in Foreign Body Response to Implanted Biomaterial In order to study the role of Mac 1 in foreign body response to implanted biomaterial we performed subcutaneous implantation of PET discs in Mac 1 KO and WT mice. One of the standard methods to evaluate chronic inflammation to synthetic materials is measurement of fibrous capsule thickness fol lowing subcutaneous implantation. PET discs were implanted subcutaneously for 14 days after which a smooth acellular fibrous capsule was formed around the implant as part of the foreign body response that the body mounts against implanted foreign materials The thickness of the capsule formed around the PET discs implanted in Mac 1 KO mice was significantly thinner as compared to WT controls for the body wall side of the disc as well as the average of both sides (Figure 3 1 ) The capsule thickness was 40% t hinner on the body wall side and 15% thinner on the side facing the skin in Mac 1 KO mice as compared to WT controls (Figure 3 1 A) Overall the capsule thickness in Mac 1 KO mice was 27% thinner as compared to WT controls (Figure 3 1 A) Hematoxylin and e o sin stained sections of the disc with the fibrous capsule are depicted for the purpose of visualizing and understanding the difference in the capsule formed around the two groups (Figure 3 1 B )

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96 Role of Mac 1 in Foreign Body Giant Cell Formation Since th e fibrous capsule is composed of macrophages, FBGCs and fibroblast, we investigated the role of M ac 1 integrin in FBGC formation by studying fusion of macrophages t o form FBGC and comparing the p ercentage of fusion to WT controls There was no significant difference in the percentage of fusion between Mac 1 and WT controls (Figure 3 2 ) Loading and Release Kinetics of Echistatin from Discs In order to deter mine the loading efficiency of e chistatin into the coating on the P ET discs and determine the amount of e chistatin loaded per disc, the polymer coating on PET discs was dissolved in methylene chloride Echistatin was th en extracted into water phase of the emulsion by sonication. The loading efficiency of e chisatin into the disc was 95%, with 12 g e chistatin encapsulated in the polymer around e ach disc. Next, the release of e chistatin from the disc was characteriz e d over 3 weeks to estimate the e chistatin that may be released into the i mplant site. There is burst of e chistatin from the discs in the first 2 days, releasing about 20% of the encapsulated e chistatin (Figure 3 3). This is followed by a gradual release o f 7 10 % of the encapsulated e chistatin every 2 days in the next 10 days. After 10 days, there is a plateau phase during the next 7 days when the release e chistatin falls to 3 5% every 2 days (Figure 3 3). Role of RGD binding Integrins in Foreign Body Res ponse to Implanted Biomaterial In order to study the role of RGD binding integrins in foreign body response to implanted biomaterial we performed subcutaneous implantation of PET discs coated with polymer loaded with RGD mimetic echistatin. The thic kness of the

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97 capsule formed around the echistatin coated PET discs was 15% thinner as compared to only coated control discs (Figure 3 4 ) Impact of the Study Biomaterial adherent macrophages are central mediat ors of the foreign body response which in some cases results in the isolation and degradation of implanted materials. For many biomedical devices such as sensors, drug delivery devices and implanted electrodes, the foreign body reaction limits implant efficacy [11,256,257] Since macrophages play such a central role in the response to implanted materials various studies have explored modulating macrophage response using different approaches such as surface chemistry and surface roughness to modulate macrophage adhesion to biomateri als [57 61] Some of these techniques reduce macrophage adhesion by making anti fouling surfaces which resist protein adsorption which results in abrogating macrophage intera ction with the surface [60] Nanotopographic sur faces which have features in the nanometer size range have also been explored to modulate m acrophage adhesion and function [62,63] Another link between the macrophages and biomaterial surface are the integrin receptors that bind to the adsorbed p rotein. Binding of integrins to their ligands leads to integrin clustering and downstream signaling that may result in alteration cell growth, differentiation, migration, attachment and spreading [68] Macrophages interact with adhesion proteins via integrins, which is evident from the decrease in cell attachment observed in the presence of anti integrin antib odies [69] Since integrins play such an important role in macrophage adhesion and activation, we have investigated the role of Mac 1 and RGD binding integrins in the in vivo FBR to subcutaneously implanted bioamaterials. The thickness of the foreign body capsule formed around the implant was used as a metric for comparing the FBR. We

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98 saw a 30% reduction in the thickness of capsule formed around discs implanted in Mac 1 KO mice as com pared to that implanted in WT mice. F ibrinogen is one of the primary components of plasma deposited on biomaterial surface [17] Hence, in the absence of integrin Mac 1, a Fg receptor [85] mac rophage adhesion to the biomaterial surface may be abrogated. Reduced macrophage adhesion may be one of the reasons for the reduced capsule thickness in the Mac 1 KO mice. Our results from the previous chapter have also demonstrated that integrin Mac 1 pla ys a role in macrophage activation and cytokine secretion. These cytokines secreted by m acro phages at the site of implant endothelial cells, and smooth muscle cells that participate in the various stages of the foreign body response [5] The reduced cytokine secretion from Mac 1 KO macrophages may result in stunted signaling to other cells that form the fibrous capsule that results in a thinner capsule. Another cell type that participates in the fibrous capsule formation is FBGC, which is formed by the fusion of macrophages [5] We have investigated the role of Mac 1 integrin in FBGC formation and there was no significant difference in the percentage of fusion between the Mac 1 and WT macrophages. Thus FBGC formation may be ruled out as a factor contributing to the difference in the capsule thickness. We observed a 15% reduction in the thickness of fibrous capsule formed around discs secreting e chistatin which contains the RGD sequence [125] Echistatin binds to integrins such a V 3 through this RGD binding site Blocking the macrophage integrin receptors such as 5 1 V 3 and V 5 that have an RGD binding site with e chistatin can disrupt macrophage binding to the biomaterial surface In the previous chapter, we have

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99 reported that RGD binding plays a major role in the macrophage response to particulate biomaterials. Blocking the RGD binding integrins with soluble RGD peptide resulted in a 98% decrease in cytokine production by macrophages upon phagocytosis of microparticles. A similar effect can be expected in the macrophage response to bulk biomaterials. The cytokines secreted by m acro phages at the site of implant play a major role in recruitment of other cell types that play a role in the foreign body response [5] The reduced cytokine secretion from macrophages upon blocking RGD binding integrins with Echistatin may result in altered signaling to other ce lls that form the fibrous capsule and results in a thinner capsule. We have identified integrins that play a role in the foreign body response and fibrous capsule formation to subcutaneously implanted biomaterials. Thus Mac 1 and RGD binding integrins can serve as therapeutic target for design of anti integrin therapies to reduce fibrous capsule formation.

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100 Figure 3 1 Integrin Mac 1 modulates foreign body response to subcutaneously implanted biomaterials. A) The capsule thic kness around the implanted PET discs was quantified at 2 weeks for both the dermis side and body wall side of the disc. B and C) Hematoxylin and Eosin stained sections (collagen pink;cell nuclei:dark blue) of tissue response to implanted PET discs. Discs i mplanted in Mac 1 KO mice (C) had a thinner capsule (indicated by arrow) as compared to WT control(B). B B C A B

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101 Figure 3 2 Integrin Mac 1 does not play a role in fusion of macrophages to form foreign body giant cells. Macrophage fusion was quantified as the percentage of giant cell nuclei relative to the total number of nuclei. The fusion percentage was quantified by pooling data from 12 samples from 3 separate runs. Plotted are mean and standard error. (* indicates statistically signifi cant difference (p < 0.05) between Mac 1 KO and WT control samples Fig ure 3 3. Release kinetics of Echistatin from polymer coating on PET disc s Release kinetics were studied at 37C in PBS (pH = 7.4) with continuous shak ing. Each point represents the mean of 3 samples. The standard deviation was <10% of the respective values.

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102 Figure 3 4 RGD binding integrins modulates foreign body response to subcutaneously implanted biomaterials. A) The ca psule thickness around the implanted PET discs was quantified at 2 weeks. Echistatin coated discs had a thinner capsule (indicated by arrow) as compared to control. B and C) Hematoxylin and Eosin stained sections (collagen pink;cell nuclei:dark blue) of tissue response to implanted PET discs. Discs coated with Echistatin loaded (B) had a thinner capsule (indicated by arrow) as compared to only E coated controls (C). 50 m 50 m A B C

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103 CHAPTER 4 CONTRIBUTIONS OF SURFACE TOPOGRAPHY AND CYTOTOXICITY TO THE MACROPHAGE RESPONSE TO ZINC OXIDE NANORODS Background Nanostructured materials, whose structural elements have dimensions in the range of 1 100 nm, exhibi t unique properties compared to the bulk material due to small dimensions and large surface area relative to volume [182] These nanostructured materials are being investigated for use in an increasing number of applications such as microelectronics, sensor technology, semiconductors and cosmetics as well as medical applicati ons such as biosensors, tissue enginee ring and drug delivery vehicles [183] Because biological systems operate in the nanmeter size range, nanostructured materials present possibilities for unique biological interactions. For example, increased osteoblast adhesion and mineralization has been demonstrated on nanostructured surfaces of both titanium dioxide and zinc oxide (ZnO), as compared to mi cron sized su rface topographies [65] Interestingly, different cell types have been demonstrated to elicit differential responses to a given nanostructured material. For example, carbon nanotubes have been shown to promote adhesio n of osteoblasts [66] whereas they inhibit adhesion of other cells such as fibroblasts, [184] chondrocytes, [184] smooth muscle cells [184] as well as macrophages [63] In particular, altered cell adhesion and viability of fibroblasts, umbilical vein endothelial cells and capillary endothelial cells has been reported on ZnO nanorods as co mpared to ZnO flat substrates [185] Zinc oxide has unique optical, semiconducting, piezoelectric and magnetic properties hence, it is used for different applications in fi elds such as semiconductors biosensors and piezoelectrics [186] Furthermore, ZnO is also used in a number of both exploratory and well established biomedical applications. For example, ZnO nanorods

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104 grown on high electron mobility transistors devices have been shown to be highly sensitive for glucose detection, [187] while ZnO h as long been used as a component in various biomedical applications such as dental filling materials (e.g., temporary fillings) [188] and sunscreens [ 189] Additionally, ZnO has been investigated as a component in topical wound healing ointments [190 192] and is used in commercially available products for the treatment of venous ulcers [193] and acne [194] Nanoparticles of ZnO are also known for their anti bacterial activity against both gram negative and gram positive bacteria [65,195,196] Due to various biol ogical applications of ZnO, especially as nanoparticles, a number of groups have investigated the cytotoxicity of ZnO. Cell type specific results have been reported. Zinc oxide was reported to not be toxic to cultured human dermal fibroblasts [197] and T cells [196] whereas it exhibited toxicity to neuroblastoma cells [198] and vascular endothelial cells [199] In this work, we evaluate the response of m acrophages to ZnO nanorod surfaces which have previously been shown to modulate adhesion and viability of fibroblasts and endothelial cells [185] We investigate macrophage adhesion and viability on ZnO nanorod surfaces compared to sputtered ZnO as a relatively flat substrate in order to gauge effects due to surface topography and those intrinsic to the material. Our goal is to explore the potential for nano structured surfaces to modulate macrophage responses. As macrophage adhesion to biomaterial surface is one of the early steps of the inflammatory response to an implanted material, [5,178,179] a surface which modulates macrophage adhesion may serve to direct the foreign body response.

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105 Experimental Procedure Fabrication of ZnO Nanorods ZnO nanorods were made by a solution based hydrothermal growth method [185,264] First, ZnO nanoparticles were prepared by mixing 10 mM zinc acetate dehydrate (Sigma Aldrich, St. Louis, MO) w ith 30 mM of NaOH (Sigma Aldrich, St. Louis, MO) at 58 C for 2 h ours Next, ZnO nanoparticles were spin coated onto the substrate several times and then post baked on a hot plate at 150C to promote adhesion. Seeded substrates were then suspended face down in a Pyrex glass dish 100 mL aqueous solution containing 20 mM zinc nitrate hexahydrate and 20 mM hexamethylenetriamine (Sigma Aldrich, St. Louis, MO). To arrest the n anorod growth, the substrates were removed from solution, rinsed with de ionized water and dried in air Kurt Lesker CMS 18 Multi Target Sputter Deposition system. In orde r to compare topographical features, scanning electron microscopy and atomic force microscopy images of substrates were obtained using Raith 150 E Beam writer and AFM Dimension 3100. Macrophage Generation Bone marrow derived macrophages were generated fro m 7 10 week old C57BL6/J mice u sing a 10 day culture protocol [234,235] as described in Chapter 2. Substrate Preparation and Macrophage Culture The two ZnO substrates were grown on 22 mm square glass coverslips (Fisherbrand, F isher Scientific) which were also was used as glass reference substrates. Prior to cell seeding, the ZnO substrates were sterilized by ethanol wash for

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106 15 min followed by UV treatment for 15 min. The glass coverslips were O 2 plasma etched using a Plasma Pr een II 862 plasma etcher followed by ethanol wash and UV treatment. In order to examine dynamic behavior of macrophages on nanorods using time lapse video microscopy, macrophages were cultured overnight on ZnO nanorods. Macrophages were seeded on the nano rod substrate and allowed to adhere for 1 h our after which a time lapse video of individual cells was taken in order to study the change in spread area of the cell over time. Phase contrast imaging of single cells was performed overnight using a Nikon TE 2 000 microscope. Images were collected every 1 min for 13 h, using a 40x objective. Cells were stained for actin using rhodamine phalloidin (Molecular Probes, Inc., Eugene, OR) [265] Adhesion and viability studies were performed using macrophages pre loaded with calcein [266] AM (AnaSpec Inc, San Jose,CA) in 2 mM dextrose solution by incubating it at 37 C for 20 min, pelleted and resuspended in mac rophage culture media. Macrophages in this cell suspension were counted by hemocytometer and 500,000 cells in 3 mL media were seeded onto substrates for 16 h ours The number of cells adherent on the surfaces was quantified at 3 h ours and 16 h ours post seed ing. At the time of quantification, media was aspirated from the wells and 7 AAD (Beckman Coulter, Fullerton, CA) in 1% BSA calcein and did not stain with 7 AAD were cou nted as live while 7 AAD positively stained cells were counted as dead. Three non overlapping fields were imaged per sample for the purpose of quantifying the total adherent, live and dead cells. Data was

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107 averaged from 6 8 replicates obtained from 3 separa te runs. In order to quantify the amount of zinc dissolved in the media due to dissolution of ZnO from the substrates, supernatants were collected at 3 h ours and 16 h ours of cell culture. Supernatants were analyzed for Zn content using Perkin Elmer Plasma 3200 Inductively Coupled Plasma Mass Spectroscopy (ICP) system. Data was averaged from three separate samples. In order to verify whether the cell death was due to apoptosis or necrosis, cells were immunofluorescently stained using antibodies specific to a ctive caspase 3, a marker for cells undergoing apoptosis [267] The cells were first fixed with 3.7% formaldehyde for 15 min at 4 C. The cells then were permeabalized with 0.5% Triton X in PBS for 15 min at room temperature. C ells were blocked with 1% goat serum for 1 h our at room temperature followed by incubation with rabbit polyclonal to active caspase 3 (Abcam Inc, MA) for 1 h at room temperature. Cells were then incubated with alkaline phosphatase conjugated goat anti rabb it antibody for 45 min at room temperature, washed and then incubated for 20 min with Vector Red substrate. Vector Red substrate is cleaved by alkaline phosphatase to produce a red reaction product, visible in bright field microscopy. The number of cells stained red was quantified from images taken from 3 non overlapping fields to determine the percentage of cell population which was apoptotic at 3 h ours and 16 h ours In order to test the non contact based toxicity of ZnO substrates, 2 million macrophages per well, in 6 well plates, were exposed to the three substrates placed face down on top of sterilized Viton O rings used as spacers to separate the substrates from the cells (Figure 2.5 A ). The substrates were maintained in the cultures in this configura tion for 7 days, with media change every alternate day. At day 7, the

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108 substrates were removed and 7 AAD was added to the wells in order to stain the dead cells. Phase contrast and fluorescence images of 3 non overlapping fields per well were taken in order to quantify the number of viable cells adherent on the wells. The data was collected and averaged from 8 replicates obtained from 2 separate runs. In vivo Response to ZnO Nanorod Coating In order to determine the foreign body response mounted against ZnO nanorod coated biomaterials, polyethylene terephthalate (PET) discs coated with ZnO nanorods and sputtered ZnO were implanted subcutaneously in mice on the dorsal side of the thorax in accordance with a protocol approved by the University of Florida IACUC committee. Uncoated PET discs served as a reference. Two discs were implanted per mouse and there were two animals in each experimental group. Discs (7 mm diameter, 0.5 mm thick) were cut from PET sheets and ZnO nanorods were grown on its surface as descr ibed in fabrication of ZnO nanorod section. Prior to implantation, the nanorod coated and uncoated discs were sterilized by washing in 70% ethanol. The wounds were closed with wound clips which were removed at day 7 after implantation. PET discs were expla nted at 14 days, formalin fixed and paraffin embedded. Histological blue) and collagen (pink) and examined by phase contrast microscopy. Statistical Analysis Statistical analyses were performed using general linear nested model ANOVA Inc., San Jose, CA). Pair wise comparisons were made using Tukey's Honestly Significant Difference Test wi th p values of less than or equal to 0.05 considered to be

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109 concentration in the cell media was calculated using zinc concentration values at 16 h, for the purpose of simplif ying the analysis as media was changed every alternate day during the 7 day culture period. Results ZnO Substrate Characterization Surface topography of the two ZnO substrates was characterized by scanning electron microscopy (Figure 4 1) The nanorods wer e found to be approximately 50 nm in diameter and 500 nm in height. Furthermore, atomic force microscopy of the sputtered ZnO revealed root mean squared roughness value of 31 nm, while glass substrate had a mean squared roughness value of 0.3 nm. Comparing the length scale of topographical features normal to the surface for the ZnO substrates, the sputtered ZnO length scale is an order of magnitude less than the nanorod length scale (nanorod height), indicating that sputtered ZnO substrates can be used for comparison as a relatively smooth ZnO surface. Macrophage Adhesion, Spreading and Viability on ZnO Nanorods In order to qualitatively determine the influence of surface topography on macrophage spreading and adhesion, macrophage spreading on ZnO nanorods w as examined by time lapse video microscopy. Macrophages were allowed to adhere for 1 h our on the nanorod substrate following which, a time lapse videos were taken to observe cell spreading over time. Images of a representative cell from the 13 hour time in terval are shown (Figure 4 2) At 1.5 h ours the macrophage was well spread however, at 2 h ours there was retraction of lamellopodia and at 3 h ours the cell was completely rounded. At 6.5 h ours protrusions from the well rounded cell became evident. These protrusions grew in size up to 9 h ours after which they remain constant

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110 in size and cellular movement ceased. Further investigation revealed that these protrusions contain actin (data not shown) suggesting cytoplasmic leakage. Compared to the ZnO nanorods, macrophages on the glass substrate remained well spread over the 13 h ours time span (data not shown). In order to quantify the extent to which ZnO nanorod substrates were able to modulate macrophage adhesion, the adherent number and viability of macrop hages on ZnO nanorod surface were compared to that on sputtered ZnO substrates (as a relatively flat ZnO surface) and glass substrates as a reference. The sum of the live and dead cells was computed for total adherent cell number. The most salient feature is that for both 3 h ours and 16 h ours of culture, the number of viable cells adherent on both nanorod and sputtered ZnO substrates was reduced compared to glass, with the ZnO nanorod substrates supporting the lowest cell numbers (Figure 4 3) In more deta il, at 3 h ours there was no difference between the total number of adherent cells on sputtered ZnO and glass; in contrast, the total number of adherent cells on ZnO nanorods was 50% of the total adherent number on glass substrates (Figure 4 3) Furthermore at 3 h ours the number of live cells adherent on sputtered ZnO and ZnO nanorods was 75% and 50% of the number of live cells adherent on glass, respectively. Lastly at 3 h ours the number of adherent dead cells on the sputtered ZnO substrate was 3 fold hig her compared to glass while dead cell numbers on the ZnO nanorods and glass were comparable. At 16 h ours the total number of adherent cells on sputtered ZnO was 50% compared to glass, while the total number of adherent cells on ZnO nanorod was 30% compare d to glass (Figure 4 3) The number of live cells adherent on sputtered ZnO

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111 and ZnO nanorods was 52% and 12%, respectively, compared to the number of live cells adherent on glass at 16 h ours Finally, at 16 h ours the number of dead cells adherent on the sp uttered ZnO substrate was 1.3 fold greater than the number of dead cells on glass, while the dead cell number on the ZnO nanorods was 1.6 fold greater than on glass. In order to investigate the mechanism of macrophage death, cells were immunostained agai nst activated caspase 3, which is expresse d by cells undergoing apoptosis [267] The percentage of total cells positively stained for activated caspase 3 was <1%, indicating the lack of apoptosis and attributing cell death to necrosis. All together, these data suggest that although substrates presenting nanorod topography demonstrated a dramatic reduction in the number of adherent macrophages compared to the control substrates, which could indicate a role for nanotopography th e sputtered ZnO substrates, lacking such nanotopographical features, also exhibited a significant reduction in macrophage adhesion, which strongly suggests that material toxicity of ZnO is a factor in this response. Dissolved Levels of Zn and Non contact B ased Toxicity of ZnO A mechanism by which ZnO substrates could induce cell toxicity is through dissolution of zinc into cell culture media followed by cellular internalization. In fact, dissolved zinc has been shown to induce cell toxicity in a cell type d ependent manner via production of reactive oxygen species and disruption of energy metabolism [268,269] In order to further explore this pr ospect, we quantified the amount of dissolved Zn present in cell culture media when macrophages were cultured on the substrates for 3 h ours and 16 h ours using Inductively Coupled Plasma Mass spectroscopy (ICP) (Figure 4 4) The amount of dissolved Zn in the cell culture media

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112 ours and 16 h ours for the sputtered ZnO at 3 h ours ours for the ZnO nanorod substrate. N otably, the level of dissolved Zn in the culture media presents the trend ZnO nanorod > ZnO sputtered > glass, which is the inverse trend of adherent cell viability; glass > ZnO sputtered > ZnO nanorod. In order to ascertain if ZnO substrates can modulate macrophage numbers through substrate dissolution in the absence of direct contact between cells and substrates, the substrates were incubated 7 d ays in shared media with macrophages cultured on tissue culture plastic. Substrates were inverted above macrop hage cultures by placing substrates on top of O rings serving as spacers and media was exchanged with fresh media every other day (Figure 4 5 A) The number of viable cells after 7 d ays of culture in media shared with both sputtered ZnO and nanorod ZnO sub strates was approximately 50% the number of viable cells cultured with glass substrates (Figure 2 5 B) Significant differences in viable cell numbers were not detected between sputtered ZnO and nanorod ZnO substrates. These data demonstrate that ZnO subst rate dissolution occurs and those dissolution products modulate viable macrophage numbers. Foreign Body Response to Zinc Nanorod Coated PET Although in vitro analysis provides valuable insight into adhesion, viability and toxicity to ZnO substrates, it can implanted material. Furthermore, the use of ZnO in current medical applications motivated additional in vivo analysis. Histological analysis of fibrous capsule formation subsequent to subcutaneous implanta tion was performed as an established means to

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113 evaluate chronic inflammation to implanted materials [5,270] Polyethylene terephthalate (PET) discs were coated with ZnO by sputtering or through growth of ZnO nanorods (Figure 4 6 A) and uncoated PET discs were utilized as reference. Coated and uncoated PET discs were implanted subcu taneously for 14 d ays Histological analysis of explanted discs demonstrated that the uncoated PET discs implanted subcutaneously had a smooth acellular fibrous capsule (Figure 4 6 B) In contrast, surrounding the discs coated both with sputtered ZnO and Z nO nanorods there was an elevated number of accumulated leukocytes and a lack of a continuous fibrous capsule (Figure 4 6 C & D) representing unresolved inflammation, likely a result of cell necrosis indicated from in vitro studies. Impact of the Study Na notopography has previously been shown to modulate cell adhesion and function [62,65,66,184,185,198,271 273] Depending on the cell type, the effect of nanoparticles or nanostructures s uch as nanoposts, nanopits, nanotubes and nanoislands varies from enhanced cell function [65,66] to toxicity [198,272] In particular, ZnO nanorods, nanowires, and nanotubes are attractive for biosensing applications, given their chemical stability, high specific surface area, and electrochemical activity [186,274 277] Furthermore, engineering ZnO n anorods with well controlled aspect ratio and spacing has been demonstrated [278] Our interest in ZnO nanorods was to investigate their potential as a well controlled nanotopography model system to modulate macrophage adhesion and subsequent responses. Biomaterial adherent macrophages are central mediators of the foreign body reaction, which results in th e isolation and degradation of implanted materials. For many biomedical devices such as sensors, drug delivery devices and implanted electrodes,

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114 the foreign body reaction limits implant efficacy. Biomaterial surface engineering approaches to modulate cell adhesive responses have precedent in general [30,31,266,279 281] and for macrophages in particular [282] Furthermore, ZnO nanorods have pr eviously been reported to modulate cell adhesion and anchorage dependent viability in a cell type dependent fashion [185] O ur investigation of macrophage adhesion demonstrated the ability of ZnO nanorods to dissuade macrophage adhesion. At the earlier time point of 3 h ours the number of adherent macrophages on ZnO nanorods was half that of glass and this reduction in adhesion furthered by 16 h ours Additionally, at 3 h ours the number of dead cells on the ZnO nanorod was low, equivalent to that of glass, although this number increased 30% by 16 h ours Taken in isolation, these data could be viewed optimistically as a feasible means to modulate macrophage adhesion with ZnO nanorod coatings. In fact, if macrophage death were to occur through apoptotic mechanisms, then even this cell death could represent a reasonable approach for biomaterial coating to limit foreign body reaction as has been suggested previously [282] However, reviewing the cell adhesion and viability results from the flat ZnO (sputtered) control clearly indicates the limitation of substrates formed from ZnO the material itself possesses toxicity resulting in non apoptotic (necrotic) cell dea th. Furthermore, given the reduced adherent cell numbers on sputtered ZnO compared to glass, this toxicity itself may provide some inhibition of cellular adhesion. Unfortunately, this confounds the ability to attribute modulation of macrophage adhesion to nanotopography as opposed to toxicity, and clearly indicates that investigation in nanotopography directed modulation of macrophages will require an alternative base material. Furthermore,

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115 these data indicate that ZnO is not an ideal biomaterial coating, i n particular for applications where long term macrophage biomaterial contact is expected such as soft tissue residing implanted devices. This notion is underscored by the observed unresolved inflammation when ZnO coated substrates were implanted subcutaneo usly in mice. concentration of solubilized Zn (measured via ICP mass spectroscopy) revealed moderate negative correlations of 0.4 and 0.5 for both 3 h ours and 16 h ours respectively (Figure 4 7) numbers when not in contact with substrates versus Zn concentration in the shared media revealed a moderate negative correlation ( 0.4) as well (Figure 4 7) This indicates correlation between an increase in zinc concentration and a decrease in the number of live cells, for which there is clear precedent. Zinc has been shown to induce cellular toxicity via production of reactive oxygen species [268] and disruption of energy metabolism [269] [283,284] However, there are still informative points to consider. It is noteworthy that the association between viable cell number and amount of soluble Zn, allowing for a possible role of nanotopography influencing the observed decrease in cell ad hesion and viability. In fact, there are a number of reasons to suspect such a contribution of nanotopography driven modulation of cell adhesion and function. For example, a recent study that conformally coated ZnO nanorods with SiO2 to prevent the leachin g of ZnO into the solution demonstrated reduced adhesion and survival of endothelial cells and

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116 fibroblasts, which was attributed to a lack of sustainable adhesive complexes on nanorod substrates [273] Since macrophages are also adhesion dependent cells, they may be driven to u ndergo cell death due to a lack of adhesive cues, termed anoikis, [2 85] a form of programmed cell death, or apoptosis. However, for macrophages seeded on the bare ZnO substrate system examined here, this is not likely the case, given the fact that the macrophages adherent to the ZnO nanorods did not express activated casp ase 3. Another possibility is cell penetration by nanorods. Given the dimensions (50 nm in diameter, 500 nm in height), it is possible that a large number of these ZnO nanorods may pierce the cell membrane and lead to a loss of membrane integrity, which co uld drive cell death. Thus, while we originally set out to investigate the role of nanotopography on modulating macrophage adhesive responses, this work indicates that use of ZnO nanorods for this goal does not represent a tenable approach for macrophages. Although our results cannot rule out the effects of nanotopography, interpretation is intractably confounded by the dissolution and inherent toxicity of Zn from these substrates. Future efforts in this vein will need to focus on use of nontoxic approaches

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117 Figure 4 1. Surface topography of sputtered ZnO and ZnO nanorods. A.) SEM image of sputtered ZnO. Scale bar is 200 nm. B.) SEM images of ZnO nanorods indicating upright growth of nanorods. Nanorod diameter of is ~50 nm and height is ~500 nm. Scale bar is 200 nm. C.) AFM image of sputtered ZnO substrate. The surface roughness is approximately 31 nm, which is an order of magnitude less than the height of nanorods, validating selection as a relatively smooth surface compared to ZnO nanorods.

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118 Figure 4 2. Time lapse images of adherent macrophage seeded on ZnO nanorods demonstrating initial adhesion followed by contraction and apparent cytoplasm leakage. Macrophages on ZnO nanorod surface were imaged for 13 h ours in culture and i mages from a representative cell are shown. Scale bar is 20 um. 5 h 1.5 h 2 h 2.5 h 3 h 6.5 h 7.5 h 9 h 11 h 13 h 1 h 4 h

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119 Figure 4 3. Macrophage adhesion and viability on ZnO substrates. Macrophage number live, dead and total on the 3 substrates glass, sputtered ZnO and ZnO nanor ords was quantified at A) 3 h ours and B) 16 h ours post seeding. Average numbers of live and dead macrophages on substrates were quantified by pooling data from 6 samples from 3 separate runs. Three images were taken per sample. Plotted a re mean and standar d error. (* indicates statistically significant difference (p < 0.05) from all other conditions in the same category (i.e. live, dead, total)). A B

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120 Figure 4 4. Dissolved levels of zinc in culture media when macrophages are cultured on zinc oxide substrates. Zinc concentration in the cell culture media was quantified at 3 h ours and 16 h ours with macrophages cultured on the glass, sputtered ZnO and ZnO nanorod substrates using Inductively Coupled Plasma mass spectroscopy. (* indicates statistic ally significant difference (p < 0.05) from all other conditions in the same category (i.e., 3 h ours and 16 h ours )).

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121 Figure 4 5. A) Setup to determine cytotoxicity of ZnO when cells are not present in contact with th e substrates. B) Viability of macrophages when ZnO substrates are present in the same media but not in direct contact with cells. Cells are cultured at the bottom of a 6 well plate and substrates are inverted in the well as shown with the help of Viton O r ings in order to provide shared culture media. The average number of live adherent cells on glass, sputtered ZnO and ZnO nanorod substrates was quantified by pooling data from 8 replicates from 2 separate runs. Three images were taken per replicate. Plotte d are mean and standard error. (* indicates statistically significant difference (p < 0.05) from all other conditions.) Coverslip Cell monol ayer Rubber spacer Media level A B

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122 Figure 4 6. Foreign body response to zinc oxide coated PET discs implanted subcutaneously in mice. Zinc ox ide coatings on implanted discs prevent formation of acellular fibrous capsule around discs, indicative of unresolved inflammation. A.) SEM image of ZnO nanorods coated on the PET discs. (Scale bar is 200 nm ) H & E section of tissue response to implanted b iomaterials B) Uncoated PET C) ZnO nanorod coated PET D) Sputtered ZnO coated PET A B C D Uncoated PET disc ZnO nanorod coated disc Sputtered ZnO coated disc B C D

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123 Figure 4 7. Correlation between dissolved zinc levels in media and macrophage viability. Macrophage viability has a moderate negative correlati on with dissolved Zn concentration in the media. The number of live cells adherent on substrates at both 3 and 16 hours and the number of live cells adherent on the well bottom at 7 days with inverted substrates negatively correlates with zinc concentratio live substrate adherent macrophages and zinc concentration at 3 h is 0.40, between live substrate adherent macrophages and zinc concentration at 16 h ours is 0.50, demonstrating moderate correlation. This indicates that as Zn correlation coefficient for live macrophages at 7 days with inverted substrate s and zinc concentration was 0.44, demonstrating moderate correlation. # Live A dherent C ells ( per coverslip in thousands) # live cells p er well (in millions) 0.44 Live cells at 7 days C 0.40 Live cells at 3 hrs A 0.50 Live cells at 16 hrs B Macrophages Cultured in Shared Media with Substrates Zinc concen

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124 CHAPTER 5 ANTIBACTERIAL EFFECT S OF ZINC OXIDE NANO ROD SURFACES Background The US medical device industry is a $30 billion industry and medical implant demand is predicted to rise 9.3 percent annually through 2011 [286] Medical implants are being extensively used in every organ of the human body, with success in replacing or repairing physiologic functions. However, a major impediment is implant associated infections caused by bacterial adhesion to biomaterials, which necessitate implant removal, extended care and prolonged antibiotic treatment [200 202] This additional care significantly contributes to health care costs. For example, of the 2.6 million orthopedic devices implanted annually in the US, approximately 112,000 (4.3%) become i nfected [203] The mos t common cause of infection is the generally non pathogenic and ubiquitous bacteria S. epidermidis which is normally found on human skin and under normal circumstances is well tolerated by the immune system [205,206] However, when adherent to implanted surface s, bacteria develop a protective biofilm resistant to immune and antibiotic attack and can develop multiple resistance to antibiotics [207,208] Many implant associated infections therefore require surgical removal of the implant Estimated costs of implant associated infections exceeds $3 billion annually in the US [203] In order to address this problem, implant coating strategies have been developed with the goal to eliminate initial bacterial adhesion and/or kill adherent bacteria. Various strategi es have been investigated. For example, surface coatings which support low levels of protein adsorption, termed non fouling, including surfaces modified with polyethylene glycol, polyethylene oxide brushes and hydrophilic polyurethanes,

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125 demonstrate resista nce to bacterial adhesion [211,212] However, the effectiveness of these coatings toward resisting biofilm formation is limited and results vary depending on bacterial species [213] provide continuous release of bactericidal agents [214 217] Complimentarily, nanotechnologies can provide orthogonal approaches which have the potential to be combined with both non fouling and active release strategies. Nanostructured materials, whose struc tural elements have dimensions in the range of 1 100 nm, exhibit unique properties compared to the bulk material due to small dimensions and large surface area relative to volume [182] These nanomaterials are used in an over broadening array of applications such as microelectronics, sensor technology, semiconductors, cosmeti cs as well as medical applications such as biosensors, tissue engineering and drug delivery vehicles [183] Formation of nano scale rod investigated to coat complex implant surfaces [185,260,264] These nano scale coatings approach provides increased stability compared to nanoscale powders and avoids health and stability issues asso ciated with nanoparticulates [287] Notably, nanoparticles of zinc oxide (ZnO) have demonstrated bactericidal effectiveness for various bacterial strains such as B. atrophaeus [288] E. coli [288,289] S. agalactiae [195] S. aureus [195] and S. epidermidis [65] suggesting ZnO as a promising material for use in antimicrobial coati ngs. Furthermore, nanotopographic surfaces have been reported to modulate bacterial adhesion [290] These facts motivated us to investigate the potential of ZnO nanorod surfaces to resist to bacterial adhesion and possess bactericidal activity.

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126 Experimental Procedure Fabrication of ZnO Nanorods ZnO nanorods were made by a solution based hydrothermal growth method as described in Chapter 4. Bacterial Culture Bacterial adhesion and viability experiments were performed using P. aeruginosa and S. epidermidis. These represent the most common strains in device re lated infections, exhibit increased resistance to antibiotics when associated with device related infections and have structural differences in their cell walls (being Gram negative and Gram positive, respectively). Bacteria were cultured in Luria Broth co nsisting of 10 g Bacto tryptone, 5 g yeast extract and 5 g sodium chloride per liter of sterile water. This blend has been shown to inhibit extracellular polysaccharide production by bacteria and therefore biofilm production [291] By scraping the surface of the frozen ba cteria stock with a sterile 1 L loop a small amount of bacteria were transferred into the Er lenmeyer flask containing 50 mL of Luria Broth. The mixture was agitated for approximately 16h in an inc ubator shaker at 37 C and 225 rpm. The bacterial optical density (OD) was assessed in a Nanodrop Spectrophotometer ND 1000. The optical density (OD) of the culture was converted to bacteria concentration based on standard curve generated by plotting OD ve rsus bacteria number, obtained by direct bacterial enumeration. The culture was diluted in Luria Broth to obtain 500,000 cells/mL Substrate Preparation and Bacterial Adhesion S tudies Before seeding bacteria, substrates were cleaned and sterilized by washi ng in 95% ethanol for 1 min and then rinsing with phosphate buffered saline (PBS). This

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127 procedure was repeated three times with a final ethanol rinse. The substrates were then left to dry. Substrates were then placed in 35 mm x 10 mm cell culture dishes ( Corning Inc. Lowell, MA) and 1x10 6 bacteria in suspension were seeded. Bacteria were allowed to adhere for 2 h in a bacterial incubator at 37 C. After incubation all substrates were rinsed twice with PBS to remove non adherent bacteria and placed in new 3 5 mm x 10 mm cell culture dishes. Next, 2 mL of PBS were added to each dish and they were then incubated at 37 C for another 22 h. Fluorescence Staining and Imaging After 22 h of culture, non adherent cells were removed by two gentle washes with PBS. Total adherent bacteria were then stained with Hoechst 33258, (Invitrogen) a membrane permeable DNA binding dye, by incubating for 1 h followed by PBS wash. Dead bacteria were incubated for 30 min with 7 Aminoactinomycin D (7 AAD) (BD Pharmingen) a membrane imp ermeable DNA binding dye, which stains bacteria only when the bacterial cell wall is compromised. Images were obtained with a fluorescence microscope (Nikon TE 2000) and analyzed using Axiovision software 4.7.2 (Carl Zeiss Imaging Solution) to determine to tal adherent and dead bacteria cell counts. At least three images were taken per sample; samples were prepared at least in triplicate and repeated at least three times. The average number and standard error of adherent and dead bacterial cells on the subst rates were computed. Statistics Statistical analyses were performed using ANOVA, using Systat (Version 12, Systat Software, Inc., San Jose, CA). Pair Honestly Significant Difference, with p values of less than or equal to 0.05 considered to be significant.

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128 Results In order to quantify the extent to which ZnO nanorod substrates were able to modulate bacterial adhesion, the adherent number and viability of bacteria on ZnO nanorod substrates were compared to that on s puttered ZnO substrates and glass as a reference. Overall, the most salient feature is that for both bacterial strains cultured, the number of dead bacteria on ZnO nanorod substrates was much higher compared to glass, and for S. epidermidis higher compared to sputtered ZnO substrates (Figures 5 1 B and 5 3 B) For P. aeruginosa, ZnO substrates demonstrated some ability to resist adhesion, where total numbers of adherent bacteria on ZnO substrates was reduced compared to glass. Representative images of stain ed P. aeruginosa on the three substrates are shown in the Figure 5 2 which is an overlay of fluorescence images with DNA stained blue for all adherent bacteria and red for the dead bacteria. The images clearly demonstrate the reduction of adherent bacter ia and the increased number of dead bacteria on the ZnO surfaces. Pooled data was analyzed by ANOVA and the mean and standard error plotted for each substrate. Overall, groups were found to be significant by ANOVA (p value < 0.04), and the total adherent b acteria number on both ZnO substrates was found to be significantly less than on glass substrates (50% of the total adherent number on ZnO nanorods, and 65% of the total adherent number on sputtered ZnO, compared to glass) (Figure 5 1 A) Total adherent num bers were not significantly different between the two ZnO substrates, however (p = 0.16). In order to determine the antimicrobial potential of substrates for P. aeruginosa, the numbers of killed bacteria were compared. Overall, groups were found to be sig nificant by ANOVA (p value < 1x10 3). Notably, ZnO nanorod substrates

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129 demonstrated enhanced antimicrobial efficiency compared to sputtered ZnO and glass, with the number of killed P. aeruginosa on ZnO nanorods (19% killed) being 2.5 fold higher than glass (7.5% killed) and 1.5 fold higher than ZnO sputtered (13% killed) (Figure 5 1 B). The numbers of killed P. aeruginosa were not significantly different between the sputtered ZnO and glass substrates, however (p = 0.1). In order to determine the effects of Z nO substrates on bacteria with a different cell wall structure, adhesion and viability experiments were repeated using S. epidermidis. In contrast, to the Gram negative P. aeruginosa, the Gram positive S. epidermidis demonstrated a number of adherent bacte ria on the ZnO nanorod substrate that was equivalent to glass. Furthermore, the number of adherent S. epidermidis on sputtered ZnO was higher than the other substrates. Although adherent bacteria numbers did not follow the same trend, the ratio of killed S epidermidis on the substrates followed the same general trend as P. aeruginosa. Representative images of stained S. epidermidis bacteria on the substrates are shown in the Figure 5 4, which is an overlay fluorescence images with DNA stained blue for all adherent bacteria and red for the dead bacteria. The images show a slightly increased adhesion on sputtered ZnO and a higher number of dead bacteria on the ZnO substrates. Pooled data was analyzed by ANOVA and the mean and standard error plotted for each substrate. Overall, groups were found to be significant by ANOVA (p value < 0.05). The total numbers of adherent S. epidermidis on ZnO nanorod and glass substrates were equivalent, while the number of adherent S. epidermidis on sputtered ZnO was 15% highe r than the ZnO nanorod and glass substrates (Figure 5 3 A)

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130 Next, to determine the antimicrobial potential of substrates for S. epidermidis, the numbers of killed bacteria were compared. Overall, groups were found to be significant by ANOVA (p value < 1x1 0 10). Notably, ZnO nanorod substrates demonstrated enhanced antimicrobial efficiency compared to sputtered ZnO and glass substrates (Figure 5 3 B), with the number of killed bacteria on ZnO nanorods (16%) being 30 fold higher than glass (0.5%). The number of killed bacteria on ZnO nanorods was 1.5 fold higher than on sputtered ZnO, while the number of killed bacteria on sputtered ZnO (11%) was 20 fold higher than glass. Impact of the Study ZnO nanorods, nanowires, and nanotubes have been explored for var ious biomedical applications because of their attractive properties such as electrochemical activity, high specific surface area and chemical stability [183 ] We have recently reported that ZnO nanorod surfaces are capable of modulating adhesion and viability of multiple mammalian cell types [185,260] Relevant work with ZnO nanoparticles has reported a bactericidal effect on numerous gram positive and gram negative bact erial strains [65,195,288,289] Proposed mechanisms for the anti bacterial activity of ZnO nanoparticles include the induction of H 2 O 2 a strong oxidizing agent [292 295] disruption of cell membrane and leakage of its cytoplasmic contents [288,289] as well as internalization of the nanoparticles [289] Hence our in terest in investigating ZnO nanorod surface, a well controlled nanotopography model system to evaluate bacterial adhesion and viability and explore its potential as an antibacterial implants coating. In this study the antibacterial activity of ZnO nanorods and sputtered ZnO substrates for bacterial strains P. aeruginosa and S. epidermidis has been investigated. Antibacterial surface coatings should possess activity against a wide range of bacteria as the type of

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131 bacteria found in different implants varies g reatly [201] We selected both gram positive as well as gram negative strains of bacteria in order to study the effect of nanorods on bacteria with different cell wall structures. Importantly, S. epidermidis is the s train that is most associated with device related infection, while the nocosomial associated P. aeruginosa is also involved in device related infection, with the ability to develop antibiotic resistance [296] Fewer P. aeruginosa adhered on ZnO subs trates, with ZnO nanorods and sputtered ZnO supporting 65% and 50% of the total number adherent on the reference glass substrate. The ZnO nanorod substrate killed the most P. aeruginosa, with a 1.5 fold and 2.5 fold increase compared to sputtered ZnO and g lass, respectively. These anti adhesive and cytotoxic effect are in line with previously published data on effect of ZnO nanorod surfaces on mammalian cells [185,260] as well as other bacterial strains [195,288] Notably, the anti adhesive effect of the ZnO substrates on the gram positive S. epidermidis bacteria was not as profound a s the gram negative P. aeruginosa bacteria. In fact, the sputtered ZnO had the highest adherent bacteria numbers with approximately 15% more adherent bacteria as compared to glass and ZnO nanorod substrates. The decrease in bacteria viability followed the same trend for both strains with the highest number of killed bacteria on ZnO nanorod substrates. However, while there was a 30 fold increase over the number of dead bacteria on glass for S. epidermidis, there was only a 2.5 fold increase for P. aeruginosa The difference in activity against the two types of bacteria could be attributed to differences in their cell walls. Specifically, gram positive bacteria have a thick layer of peptidoglycans and a cytoplasmic membrane, whereas the gram negative bacterial wall consists of a thin

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132 layer of peptidoglycan between the cytoplasmic membrane and the outer membrane [297,298] The structural differences in the bacterial cell wall of these strains may differentially interact with the ZnO nanorods through strain specific differences in non specific electrostatic and van der Waals interactions. Recent work by Tam et al. also reported antimicrobial activity of ZnO nanorod surfaces using the bacterial strains E. coli and Bacillus atrophaeus [288] They determined that for these strains, the antibacterial activity of the ZnO nanorods was attributed to damage of the cell membranes, which caused leakage of cell contents and cell death [288] Although the exact cause of the membrane damage in the current work requires additional investigation, the release of Zn2+ ions is a plausible reason for the antibacterial activity of ZnO substrates. ZnO is known to be unstable in solution and breaks down to produce Zn2+ ions. Zinc ions can pene trate cell membranes, disrupt the [299] Previously we have shown that the amount of Zn dissolved i n cell culture media is significantly higher for ZnO nanorod substrates (450 M) compared to sputtered ZnO substrates (150 M) [260] Another possibility is direct penetration of the bacterial cell wall by nanorods and this loss of membrane integrity can result in cell death. Even though bacteria are relatively small, a large number of ZnO nanorods are estimat ed to be in contact with a single bacteria (estimated ~250 500 nanorods; with a nanorod surface density of 126 nanorods per m 2 for our substrates [185] ). Although in this work we used ZnO nanorods of one set length, these antimicrobial mechanisms could potentially be differentially modulated by different nanorod lengths and optimized in the future. Although approaches using ti med release antibiotic coatings reportedly achieve

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133 higher levels of bactericidal efficacy in earlier time points, low levels of antibiotic release at later time points raise concerns of developing antibiotic resistance for chronic or recurring infections [300] All together these findings support the continued investigation of ZnO nanorod coatings as complimentary, orthogonal antimicrobial coating approaches with potential to both reduce bac terial adhesion and viability.

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134 Figure 5 1. Pseudomonas aeruginosa adhesion and viability is reduced on ZnO nanorod substrates. Bacterial cell number s: total (A), and dead (B) on glass sputtered ZnO and ZnO nanorod substrates were quantified at 24 h post seeding. Mean and standard error are plotted for the number of adherent and dead bacteria on subst rates, with a sample number, n = 22, from 3 separate runs. ( A) indicates significant difference from glass (B) indicates si gnificant difference from all other surfaces. Adherent bacteria per field 1050 0 150 300 450 600 750 900 Glass ZnO nanorod A Sputtered ZnO Dead bacteria (%) Glass 0 5 15 ZnO nanorod B Sputtered ZnO 10 20 25

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135 Figure 5 2. Pseudomonas aeruginosa demonstrate decreased adhesion and viability of bacteria on ZnO nanorod. Bacteria on substrates: glass (A), ZnO nanorods (B) and sputtered ZnO (C ) were stained after 24 h of culture. Fluorescence images of Hoechst staining (blue) represent total adherent bacteria and staining with 7 AAD (red) represent dead bacteria. A B C

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136 Figure 5 3. Staphylococcus epidermidis viabili t y is reduced on ZnO substrates. Bacterial cell number s: total (A), and dead (B) on glass sputtered ZnO and ZnO nanorod substrates were quantified at 24 h post seeding. Mean and standard error are plotted for the number of adherent and dead bacteria on su bst rates, with a sample number, n = 12, from 4 separate runs. indicates significant difference from all other surfaces. Glass 0 25 50 75 100 125 150 ZnO n anorod A Sputtered ZnO Sputtered ZnO Dead bacteria (%) 20 0 5 10 15 ZnO nanorod B Glass

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137 Figure 5 4. Staphylococcus epidermidis demonstrate decreased viability, but comparable adhesio n on ZnO nanorod. Bacteria on substrates: glass (A), ZnO nanorods (B) and sputtered ZnO (C) were stained after 24 h of culture. Fluorescence images of Hoechst staining (blue) represent total adherent bacteria and staining with 7 AAD (red) represent dead ba cteria. A B C

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138 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The goal of my research was to modulate macrophage response to biomaterials in order to mitigate the foreign body response, which has been shown to limit the functioning, performance and lifeti me of implanted biomaterials. I have explored two approaches to achieve this end goal integrin targeting as well as exploring nanotopographic surfaces as anti adhesion surface. My work focused mainly on understanding the role of the integrins in the vario us biomaterial related inflammatory processes such as peri prosthetic osteolysis, fibrous encapsulation of biomaterials and more specifically the role of integrins in macrophage response to particulate and bulk biomaterials. Through a series of experiments I demonstrated the role of integrin Mac 1 and RGD binding integrins in these inflammatory processes. The role of these integrins varied from a major player to some modulation depending on the process and integrin being studied. Recommendations for futur e experiments include many interesting avenues. During my research, I explored the role of integrins in the in vivo processes and then further investigated the mechanisms involved in these processes using in vitro experiments focusing on macrophages. How ev er other cell types are also involved in the host response to implanted materials and studying the role of integrins in osteoclasts and neutrophils functioning is a research topic worth exploring. In order to study the role of Mac 1 integrin, I used a Mac 1 KO mouse for all the experiments. However in order to move towards a therapeutic approach, functional antibody blocking experiments could be carried out by delivering the antibodies to the site of implant only. The Mac 1 KO mice have the integrin knocke d out from all the cell types such as neutrophils and osteoclasts which express this receptor. Thus the

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139 effects seen in the in vivo experiments are a compound effect of the absence on integrin on several ce ll types and thus we see an enhanced effect of a bsence of Mac 1 in vivo as compared to the in vitro responses. Mac 1 integrin play a role in different macrophage functions. Hence the functional blocking studies will have to focus on titrating the dosage as well as the temporal release of the antibodies into the implant site. Most in v ivo experiments conducted were based on established animal models however; they involved very small times of implantation ranging from 1 2 weeks. However implants are required to stay in the body for much longer periods of time in most applications. Hence experiments involving longer implantation time s need to be conducted to study long term effects of integrin blocking on response to implants. The properties of the controlled rele ase system and the dose of RGD/e chistatin will need to be modified to match the time of implantation. Other RGD antagonists such as Abciximab and tirofiban which have been approved for clinical use in the United States can also be exp lored as therapeutic approaches to mitigate peri implant osteolysis and fibrous capsule formation. Abcixima well as Mac 1 integrin. The ZnO nanorod surface exhibited toxicity towards macrophages due to the dissolved Z n making result interpretation regarding the role of nanotopography challenging. Nanorod surface made from other materials such as titanium shown to be non toxic and used in several orthopedic implants can be explored to study the role of nanotopography w ithout confounding toxicity issues. The length scale of the nanorods

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140 can also be varied to study the role of height of nanotopographic features in adhesion modulation. My research primarily focused on understanding the role of integrins and nanotopogrpahy in modulating macrophage response to biomaterials. Based on the insight gained through this work, future work in this direction can focus on translating this understa nding to a therapeutic approach.

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141 LIST OF REFERENCES [1] NIH Consens Statement. In Clinical Applications of Biomaterials.198; 4(5): 1 19. [2] Global Biomaterial Market (2009 2014). 2009. MarketsandMarkets. Accessed on 10 11 2010. Available at http://www.reportlinker.com/p0148007/Global Biomaterial Market.html [3] Ratner BD. Biomaterial Science : An Interdisciplinary endeavor. In Biomaterials Science: An introduction to materials in medicine. 2010. p 1 9. [4] Williams DF. On th e mechanisms of biocompatibility. Biomaterials 2008;29:2941 2953. [5] Anderson JM. Biological responses to materials. Annu Rev Mater Res 2001;31:81 110. [6] Archibeck MJ, Jacobs JJ, Roebuck KA, Glant TT. The basic science of periprosthetic osteolysis. Instr Course Lect 2001;50:185 195. [7] Bauer TW, Schils J. The pathology of total joint arthroplasty.II. Mechanisms of implant failure. Skeletal Radiol 1999;28:483 497. [8] Zhao Q, Topham N, Anderson JM, Hiltner A, Lodoen G, Payet CR. Foreign body gian t cells and polyurethane biostability: in vivo correlation of cell adhesion and surface cracking. J Biomed Mater Res 1991;25:177 183. [9] Gordon M, Bullough PG. Synovial and osseous inflammation in failed silicone rubber prostheses. J Bone Joint Surg Am 1982;64:574 580. [10] Smahel J. Foreign material in the capsules around breast prostheses and the cellular reaction to it. Br J Plast Surg 1979;32:35 42. [11] Anderson JM, Niven H, Pelagalli J, Olanoff LS, Jones RD. The role of the fibrous capsule in t he function of implanted drug polymer sustained release systems. J Biomed Mater Res 1981;15:889 902. [12] Luttikhuizen DT, Harmsen MC, van Luyn MJA. Cellular and molecular dynamics in the foreign body reaction. Tissue Eng 2006;12:1955 1970. [13] Anders on JM. Inflammatory response to implants. ASAIO Trans 1988;34:101 107.

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168 BIOGRAPHICAL SKETCH Toral Zaveri was born in 1983, in Mumbai, India. She completed her high school in 2001 from Mumbai. She received her Bachelors of Engi neering (BE) degree majoring in Biomedical Engineering from the Thadomal Shahani Engineering College, University of Mumbai, India in 2005. For the BE degree, her specialization was biomedical instrumentation and she interned at Breach Candy Hospital for pr actical training on biomedical instrument repair and maintenance. To further pursue her interest in biomedical engineering research she came to University of Florida for a MS degree in Fall 2005. She started working with Dr Benjamin Keselowsky in January 2 006 and her interest in her research motivated her to switch over to the PhD program under the mentorship of Dr Kesel owsky. She received her Master of Science degree in biomedical engineering from University of Florida in 2008 while continuing with her Do ctorate. friction property of endothelial cells and modulating macrophage response to biomaterials which formed her PhD research. Her doctoral research was in the area of in vestigating macrophage response to particulate biomaterials and identifying major integrins that can serve as therapeutic targets. She has also looked at surface modification techniques using nanotopography as an approach for modulating foreign body respon se. During her graduate studies she has acquired teaching and mentoring skills due to her participation in the Science training program for high school students. She hopes to join a research and development company so that she can use her acquired skills a nd knowledge for translational research and biomaterial product development.