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The effect of titanium surface topography on macrophage behaviour in vitro Refai, Ali K. 2009

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 THE EFFECT OF TITANIUM SURFACE TOPOGRAPHY ON MACROPHAGE BEHAVIOUR IN VITRO by Ali K. Refai  BDS, King Saud University, Riyadh, SA 1995 Certificate in General Residency Program (GRP), University of Missouri, USA 1999. Certificate in Oral Medicine and Diagnostic Sciences, University of Missouri, USA 2001 MSc (MSOB), University of Missouri, USA 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Dental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2009  ©Ali K. Refai, 2009 ABSTRACT  Macrophages are pivotal to the survival of endosseous implants. Researchers have shown that macrophage-like cells attached to rough SLA surfaces and such rough surfaces were associated with a greater percentage of bone formation than smooth surfaces. Therefore, it is the hypotheses of this study to investigate the effect of surface topography on macrophage morphology, activation, and secretion of selected cytokines and chemokines, and the effect of the secretions on early events that take place during bone formation.  RAW 264.7 cells were cultured on replicas of four titanium topographies initially produced by polishing (PO), large-grit sandblasting (CB), acid-etching (AE), and sandblasting plus acid etching (SLA) with and without lipopolysaccharides (LPS). Morphology and motility were observed via scanning electron microscopy (SEM) and time-lapse video microscopy respectively. Cytokines and chemokines were measured using an enzyme-linked immunosorbent assay (ELISA) and cells counted by nuclear staining. Factorial design and two-way analysis of variance were employed to assess statistical significance and interaction.  SEM demonstrated that macrophages increased in number and exhibited diverse morphologies on the four tested surfaces. Although no differences in motility were found macrophages exhibited either an oscillatory movement or remained stationary. Unstimulated macrophages on AE and SLA increased secretion of tumor necrosis factor- α compared to those cultured on the PO surface. Cells cultured with suboptimal doses of LPS on SLA produced higher levels of interleukin-1β, interleukin-6, and TNF-α at 24  ii and 48 hours.  Relative to cells on PO surfaces unstimulated macrophages on SLA surfaces down-regulated their production of chemokines CCL2and CCL3, but increased their relative production when stimulated by LPS.  Using a Transwell co-culture system to determine the effects of macrophage secretions on rat osteoblasts we found that ALP activity was increased significantly after 1 week of culture for co-cultures on SLA surfaces compared to co-cultures on PO surfaces or control surfaces.  However, the number of bone-like nodules was unaffected by the presence or duration of macrophages in the co-culture model.  This in vitro study demonstrated that surface topography modulates production of cytokines and chemokines in a time-dependent manner. The success of these approaches in examining cytokines typical of classically activated macrophages, suggests that other activation patterns such as immunosuppression and wound healing should be similarly studied.   iii TABLE OF CONTENTS  ABSTRACT .................................................................................................................................. ii TABLE OF CONTENTS ............................................................................................................. iv LIST OF TABLES......................................................................................................................... x LIST OF FIGURES .......................................................................................................................xi LIST OF ABBREVIATIONS.......................................................................................................xv ACKNOWLEDGEMENTS.......................................................................................................xviii DEDICATION.............................................................................................................................xxi 1 CHAPTER 1: Introduction .......................................................................................................... 1 1.1 Overview........................................................................................................................ 2 1.2 The use of implants in dentistry ..................................................................................... 3 1.2.1 Biocompatibility and biofunctionality of implanted materials ............................... 3 1.2.2 Characteristics of titanium and titanium-based alloys ............................................ 4 1.2.3 Definition of osseointegration................................................................................. 6 1.3 Implant surfaces ............................................................................................................. 7 1.3.1 Blasted surfaces....................................................................................................... 8 1.3.2 Acid–etched surfaces ............................................................................................ 10 1.3.3 Blasted and acid-etched surfaces ..........................................................................10 1.3.4 Coated surfaces .....................................................................................................11 1.3.5 Oxidized surfaces.................................................................................................. 12 1.4 Comparisons of clinical effectiveness among implant systems ................................... 13 1.5 Biologic responses to materials.................................................................................... 15 1.5.1 Effect of surface topography on cell behavior ...................................................... 15 1.6 The macrophage........................................................................................................... 17 1.6.1 Origin .................................................................................................................... 17  iv 1.6.2 Distribution ........................................................................................................... 18 1.6.3 Biologic functions .................................................................................................18 1.6.4 Macrophage identification and markers................................................................21 1.6.5 Macrophage phenotypic differentiation ................................................................ 21 1.6.6 Macrophage activation and regulation ..................................................................24 1.6.7 Production of effector molecules by macrophages ............................................... 25 1.6.7.1 General ...........................................................................................................25 1.6.7.2 Cytokines .......................................................................................................27 1.6.7.3 Inflammatory cytokines secreted by macrophages ........................................28 1.6.7.3.1 Tumor necrosis factors............................................................................ 28 1.6.7.3.2 Interleukin-1 (IL-1) .................................................................................31 1.6.7.3.3 Interleukin-6 (IL-6) .................................................................................33 1.6.7.4 Cytokine control of repair ..............................................................................35 1.6.7.4.1 Positive regulation................................................................................... 35 1.6.7.4.2 Negative regulation .................................................................................35 1.6.7.5 Chemokines....................................................................................................36 1.6.7.6 Chemokines secreted by macrophages...........................................................37 1.6.7.6.1 CCL2 (MCP-1) .......................................................................................37 1.6.7.6.2 CCL3 (MIP-1α) ......................................................................................39 1.6.8 Macrophage involvement in inflammation and wound repair ..............................40 1.6.9 Macrophage cytokines modulate inflammation and the wound-healing process..42 1.6.10 Macrophage chemokines modulate inflammation and wound healing ...............44 1.6.11 Osteogenesis and macrophages...........................................................................45 1.6.11.1 Osteogenesis ................................................................................................45 1.6.12 Macrophage-biomaterial interactions and cytokine secretion.............................46 1.6.13 Macrophage participation in the integration of implants ....................................48  v 2 CHAPTER 2: Statement of problem and objectives ................................................................. 52 2.1 Background .................................................................................................................. 53 2.2 Problem ........................................................................................................................ 54 2.3 Hypotheses ................................................................................................................... 54 2.4 Rationale ...................................................................................................................... 55 2.5 Objectives..................................................................................................................... 55 2.6 Significance.................................................................................................................. 56 3 CHAPTER 3: Materials and methods........................................................................................ 57 3.1 Substrata....................................................................................................................... 58 3.1.1 Surface design .......................................................................................................58 3.1.2 Replica preparation ............................................................................................... 59 3.1.3 Surface cleaning....................................................................................................61 3.1.3.1 Ultrasonication ...............................................................................................61 3.1.3.2 Washing .........................................................................................................61 3.1.3.3 Glow-discharge cleaning................................................................................63 3.1.3.4 Titanium coating ............................................................................................ 63 3.1.3.5 Surface characterization .................................................................................63 3.2 Rats and cellular activators .......................................................................................... 67 3.3 Cell populations ........................................................................................................... 67 3.3.1 Macrophages and culture conditions..................................................................... 67 3.3.2 Osteoblast strains and culture conditions.............................................................. 68 3.3.3 Media supplements ............................................................................................... 69 3.4 Macrophage adhesion .................................................................................................. 69 3.4.1 DAPI staining........................................................................................................70 3.4.2 SEM investigation.................................................................................................70 3.5 Time-lapse cinemicrography........................................................................................ 72  vi 3.6 Production of cytokines and chemokines..................................................................... 73 3.7 Sandwich ELISA assay................................................................................................ 74 3.7.1 Cytokine and chemokine determinations ..............................................................74 3.8 Bone formation ............................................................................................................ 75 3.8.1 Transwell co-culture .............................................................................................75 3.8.2 Macrophage secretion and mineralization ............................................................77 3.8.3 Alkaline phosphatase activity assay......................................................................79 3.9 Bone-like nodule formation ......................................................................................... 80 3.9.1 Epifluorescence microscopy ................................................................................. 80 3.9.2 NIH image protocol and photoshop editing ..........................................................80 3.10 Phosphotyrosine immunostaining ................................................................................ 81 3.11 Statistics ....................................................................................................................... 82 4 CHAPTER 4: Morphologic studies of macrophage cell line RAW 264.7 on surface topographies of varying roughness ............................................................................................ 83 4.1 Scanning electron microscopy of RAW 264.7 on different surface topographies ....... 84 4.2 Time-lapse microscopy ................................................................................................ 91 4.3 Macrophage activation ................................................................................................. 93 4.4 Conclusion ................................................................................................................... 95 5 CHAPTER 5: Effect of titanium surface topography on macrophage activation and secretion of pro-inflammatory cytokines and chemokines ............................................................................ 96 5.1 Cytokine responses to surface topography................................................................... 98 5.1.1 Production of tumor necrosis factor-α ..................................................................98 5.1.1.1 Unstimulated macrophages ............................................................................98 5.1.1.2 Stimulated macrophage..................................................................................98 5.1.2 Production of interleukin-1β ...............................................................................102 5.1.2.1 Unstimulated macrophages ..........................................................................102  vii 5.1.2.2 Stimulated macrophages ..............................................................................102 5.1.3 Production of interleukin-6 .................................................................................104 5.1.3.1 Unstimulated macrophages ..........................................................................104 5.1.3.2 Stimulated macrophages ..............................................................................104 5.2 Chemokine responses to surface topography .............................................................106 5.2.1 Monocyte chemoattractant protein-1 production ................................................106 5.2.1.1 Unstimulated macrophages ..........................................................................106 5.2.1.2 Stimulated macrophages ..............................................................................106 5.2.2 Production of macrophage inflammatory protein-1α .........................................110 5.2.2.1 Unstimulated macrophages ..........................................................................110 5.2.2.2 Stimulated macrophages ..............................................................................110 5.3 Synergism: surface topography and LPS ...................................................................113 5.4 Interaction between sandblasted and acid-etched components of the SLA surface...113 6 CHAPTER 6: Measurement of the effects of secretions by macrophage plated on varying surface topographies on osteoblasts in a co-culture model......................................................118 6.1 Alkaline phosphatase activity ....................................................................................119 6.2 Bone-like nodules ......................................................................................................121 7 CHAPTER 7: Discussion and future directions ......................................................................128 7.1 Cellular morphology, adhesion, locomotion, and activation .....................................130 7.2 Induction of inflammatory cytokines and chemokines ..............................................138 7.3 Surface roughness ......................................................................................................143 7.3.1 Increase in surface area .......................................................................................145 7.3.2 Cofactor modification of test surfaces ................................................................145 7.3.3 Quorum sensing .................................................................................................. 146 7.4 Determination of the effect of macrophage cytokines produced on the test surfaces in the induction of osteogenesis................................................................................................151  viii 7.5 Future directions .........................................................................................................157 8 References ................................................................................................................................159  ix LIST OF TABLES  Table 3.1: Quantitative surface composition (atom %), derived from XPS data, of titanium-coated epoxy resin replicas and polished commercially pure titanium surfaces (Adapted from Weiland M 2002). ................................................................................................................. 65 Table 3.2: Roughness parameters Ra and Rq of smooth surfaces, typically used in cell culture assays, measured with atomic force microscopy on a 10  10 µm2 area (Schuler M et al 2009). .................................................................................................................................... 65 Table 3.3: Roughness values (Ra, Rq, Rz, and Sm) of an SLA commercially pure titanium disk and its 8 TiO2-coated epoxy replicas measured with confocal white light microscopy on a 770  770 µm2 area (Schuler M et al 2009)......................................................................... 66 Table 3.4: XPS elemental concentrations of TiO2-coated epoxy replica, TiO2-Coated Si wafer, and commercially pure titanium SLA disk measured on a 6 mm2 area (Schuler M et al 2009). .................................................................................................................................... 66  x LIST OF FIGURES  Figure 1–1: Dental implants share with natural teeth the need to interface with epithelium and connective tissue, and anchorage of the natural or artificial crown must be sufficiently secure to support mastication. A major difference is that titanium dental implants are typically integrated with bone, whereas natural teeth are supported by a periodontal ligament. (Courtesy of Shine Dental.) .................................................................................... 5 Figure 1–2: Examples of dental implants made by various manufacturers (www.dentistkerala.com, www.suindental.co.kr, www.astratechdental.dk, www.ec21.com, www.bangkokdental.com, www.dentalcompare.com, and www.coastalimplant.com).......... 9 Figure 1–3: Differentiation and distribution of macrophages in vivo. (Adapted from Gordon S, 2003.) Copied under licence from Access Copywrite. Further reproduction prohibited...... 20 Figure 1–4: Two hypotheses advanced to explain macrophage functional diversity. (A) Function- specific monocyte precursors are viewed as giving rise to function-specific....................... 23 Figure 1–5: Inducers and selected functional properties of different polarized macrophage populations. Macrophages polarize and acquire different functional ................................... 26 Figure 3–1: SEM view of the four surfaces used in this study: PO = polished; AE = acid etched; CB = coarsely blasted; and SLA = sandblasted, large-grit, acid etched. .............................. 60 Figure 3–2: Schematic diagram of the co-culture model. Macrophages were plated in the upper compartments (inserts) onto surfaces and/or directly onto the membrane and into the lower compartments, where osteoblasts were plated onto tested surfaces. SLA = sandblasted and acid etched; PO = polished; Mac = macrophages; Osteo = osteoblasts; Ti-GCS = titanium glass-covered slip; L comp = lower compartment................................................................ 76 Figure 3–3: Schematic diagram of the three period sequences used to investigate mineralization. CE = continuous effect; LE = late effect; EE = early effect. ................................................ 78  xi Figure 4–1: a and 4-1b. SEM images show macrophage morphology on the four titanium-coated epoxy replica used in this study (a) after 24 hours and (b) after 48 hours............................ 86 Figure 4–2 Number of attached macrophages, based on SEM observations of the four titanium- coated epoxy replica used in this study, at 24 & 48 hours. Asterisk denotes p .................... 87 Figure 4–3: Number of attached macrophages, as determined by the DAPI assay, on the four titanium-coated epoxy replicas used in the study after 24 and 48 hrs. Asterisk denotes p < 0.05. ...................................................................................................................................... 88 Figure 4–4 A & B: Number of spread and round macrophages on the four titanium-coated epoxy replicas used in the study after 24 and 48 hours. Asterisk denotes p < 0.05. ....................... 89 Figure 4–5: Macrophages expressed varied morphology on the SLA surface. They were capable of stretching into deep pores and establishing multiple points of attachment that anchored fine cell processes within the microstructure of the SLA surface (D). ................................. 90 Figure 4–6 a and 4-6b: show a series of images of macrophages stained with the fluorescent dye Cell Tracker Orange from the (a) SLA and (b) titanium GCS time-lapse studies. Circles reveal no movement, whereas arrows reveal slight movement. ........................................... 92 Figure 4–7: Macrophage staining for phosphotyrosine after 24 hours. Phosphotyrosine was positively stained and present throughout the macrophages associated with both SLA and PO surfaces and was also present at structures typical of focal adhesion. Bar = 20 µm. ..... 94 Figure 5–1: TNF-α production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. At 24 hours, TNF-α ................... 100 Figure 5–2: TNF-α production by lipopolysaccharide- (LPS) stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24,.......................... 101 Figure 5–3: Interleukin-1β (IL-1β) production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24,.......................... 103 Figure 5–4: Interleukin-6 (IL-6) production by LPS-stimulated macrophages (6.4 ng/mL) cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 ............................ 105  xii Figure 5–5: Monocyte chemoattractant protein-1 (MCP-1) production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24,.... 108 Figure 5–6: MCP-1 production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours................................ 109 Figure 5–7: Macrophage inflammatory protein 1α (MIP-1α) production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24,.... 111 Figure 5–8: MIP-1α production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours................................ 112 Figure 5–9: Interaction between topography and LPS stimulation at 48 hours. Cytokine and chemokine profiles of macrophages (stimulated vs. unstimulated) cultured on all tested surfaces. After 48 hours, stimulated macrophages cultured on an SLA surface secreted significantly more cytokines and chemokines than TCP-positive controls (p ≤ 0.05). (A) IL- 1β, (B) IL-6, (C) TNF-α, and (D) MCP-1. ......................................................................... 115 Figure 5–10: Interaction effects between the features produced by blasting and etching on the secretion of cytokines and chemokines by stimulated macrophages at 48 hours. .............. 116 Figure 6–1: ALP expression as determined by relative area stained in osteoblast cultures on Ti- coated glass cover slips when exposed by means of a transwell mode for 1, 2, and .......... 122 Figure 6–2: ALP staining of osteoblasts at the end of week 1in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the transwell membrane) and Mem (no macrophages).................................................. 123 Figure 6–3: ALP staining of osteoblasts at the end of week 2 in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the transwell membrane) and Mem (no macrophages).................................................. 124  xiii Figure 6–4: ALP staining of osteoblasts at the end of week 4 in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the Transwell membrane) and Mem (no macrophages). ............................................... 125 Figure 6–5: Bone-like nodule formation by percentage surface area at various time periods (means ± SD). EE =  early effect (osteoblasts exposed to macrophage secretions............. 126 Figure 6–6: Examples of Bone-like nodules demonstrated by alizerone complexone stained formed after 3 weeks by osteoblasts cultured on Ti-coated coverslips under .................... 127 Figure 7–1: Diagram of macrophage responses to biomaterials. Macrophages respond to small particles (<10 µm) by phagocytosis (I). For large particles, macrophages fuse ................. 131   xiv LIST OF ABBREVIATIONS  AC assay : Alizarin complexone assay AE  : Acid-etched surface. Al2O3  : Aluminum oxide. ALP  : Alkaline phosphatase. ANOVA : Analysis of variance. APC  : Antigen presenting cell. ATCC : American Type Culture Collection. bFGF  : Basic fibroblast growth factor. BMP-2 : Bone morphogenetic protein-2. BSA  : Bovine serum albumin. C°  : Degree centigrade. CCD  : Charge-coupled device. CCL2  : Cysteine:cysteine ligand 2 chemokine CCL3  : Cysteine:cysteine ligand 3 chemokine CXC  : Cysteine:one amino acid:cysteine chemokine family. CX3C : Cysteine:3 amino acids:cysteine chemokine family. C  : Cysteine chemokine family. CD  : Cytochalasin D. CD14  : Cluster of differentiation-14. CD68  : Cluster of differentiation 68. CD64  : Cluster of differentiation 64. CFU-GM : Colony-forming unit-granulotcyte-macrophage. CpTi  : Commercially pure titanium. CB  : Sand-blasted surface DAPI  : 4’,6-diamidino-2-phenylindole. DES  : Discontinuous edged surfaces ECM  : Extracellular matrix. ED1  : Lysosomal rat macrophage ED1 antigen. ED2  : Surface membrane rat macrophage ED2 antigen. ELISA : Enzyme-linked immunosorbent assay. ERK1/2 : Extracellular signal-regulated kinases1/2. FAK  : Focal adhesion kinase. FAs  : Focal adhesions. FBGC : Foreign body giant cells. Fc-g  : Fc-gamma receptor. GM-CSF : Granulocyte-macrophage colony stimulating factor. HA  : Hydroxyapatite. IFN-g  : Interferon-g. IFN-α : Interferon-α. IGF-1  : Insulin-like growth factor. IL-1a  : Interleukin-1alpha. IL-1b  : Interleukin-1beta. IL-1RI/II : Interleukin-1 receptor I/II.  xv IL-1 RAcP : Interleukin -1 receptor accessory protein. IL-3  : Interleukin-3. IL-4  : Interleukin-4. IL-6  : Interleukin-6. IL-6Ra : Interleukin-6 binding receptor IL-10  : Interleukin-10. IL-12  : Interleukin-12. IL-13  : Interleukin-13 kDa  : Kilodalton. LAK  : Lymphokine activated killer cell. LPM  : Non-contact laser profilometer. LPS  : Lipopolysaccharide. α-MEM : Alpha minimal essential media M1  : Classically activated macrophages. M2  : Alternatively activated macrophages. mAb  : Monoclonal antibody MAPK : Mitogen-activated protein kinase. MCP-1 : Monocyte chemotactic protein-1. M-CSF : Macrophage-colony stimulating factor. MIP-1 a : Macrophage inflammatory protein 1 alpha. ml  : Milliliter. mm  : Millimeter. mM  : Millimoler. MPS  : Mononuclear phagocyte system. NBS  : Newborn bovine serum. NF-Kb : Nuclear factor-kappa beta. NK cells : Natural killer cells nm  : Nanometer. OPN  : Osteopontin OsO4  : Osmium tetroxide. PO  : Polished surface. P388D1 : Macrophage cell line. PBS  : Phosphate buffer saline. PDGF : Platelet derived growth factor. pH  : A measure of the degree of the acidity or the alkalinity of a solution. PI3 K  : Phosphatidylinositol 3-kinase. Pyk2  : protein tyrosine kinase 2 beta PP2  : 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine. QS  : Quorum sensing. Ra,q,z,m : Average roughness value. RCOs  : Rat calvarian osteoblasts. RPM  : Revolutions per minute. RPMI 1640 : Roswell Park Memorial Institute 1640 medium. SEM  : Scanning electron microscope. SLA  : Sand-blasted large-grit acid-etched. Src  : Proto-oncogenic tyrosine kinase.  xvi TCP  : Tissue culture plastic. TGF-a : Transforming growth factor alpha. TGF-b1 : Transforming growth factor-beta1. Th1  : T helper cell 1. Th2  : T helper cell 2. Ti  : Titanium. Ti GSC : Titanium glass cover slips Ti-6AL-4V : Grade 5 titanium alloy. TiO2  : Titanium oxide. TLRs  : Toll-like receptors. TNF-α : Tumor necrosis factor alpha. TNF-β : Tumor necrosis factor beta. TNFRI/II : Tumor necrosis factor receptor I/II. TPS  : Titanium plasma sprayed. ml  : Microliter. mm  : Micrometer. W  : Watts XPS  : X-ray photoelectron spectrometry.  xvii ACKNOWLEDGEMENTS  First And Foremost I Would Like To Thank ALLAH My Almighty God For All His Blessings.  I would like to express my deep gratitude and appreciation to my supervisors, Dr. Doug Waterfield and Dr. Don Brunette. You both are extremely helpful and understanding professors. You both guided me through this entire process. I would imagine that without your incredible support this would have been a very different experience and difficult task and cannot be finished without your both continuous efforts and sincere support. I admire and respect both of you greatly, as a teachers and a persons. You both helped me to publish a great article out of this thesis accepted and cited by many scientist in the field. Indeed, it is both of you who helped me to become as small person with little experience in the field known among big scientists. THANK YOU VERY MUCH.  I would like to thank my committee members, Dr. Clive Rebort and Dr. Ed Putnins for their generously given time and expertise to improve my work. I thank them for their contribution and their good-natured support. My special thanks for my internal and external examiners for their sincere advise and valid corrections.I would like to thank Dr Marcus Textor , his Lab and ITI foundation. My particular thanks goes to my colleague and good friend Salem Ghrebi who spent precious long hours helping and aiding me and have a tearful and hearty support throughout the program. I would like to thank Dr. Babak Chehroudi for his assistance and emotional support and Mr. Andre Wong for his technical assistance and the time he spent to teach me in microscopic field. I would also like to thank all my colleagues in Dr Brunette Lab.  xviii Special thanks go to Dr. Marco Wieland for his support and guidance during my beginning of the program. As for all members of the oral biology department and faculty of dentistry, you made this journey a bearable one. The years, we spent together working our way through the good and bad times will never be forgotten. Thank you for being there, I wish you all the best. Vicky Koulouris, what can I say, you are one of the reasons I was able to survive and get through some very difficult times. I personally and my wife remember your kind and sympathetic feeling toward us during my medical problems and your memorized visit when my wife delivered our angel son, Khalid. Your motherly love and care is beyond what word scan describe, thank you.  And finally, the closest people to my heart, my family. I cannot express my love or give enough credit to my mother and father’s soul. You supported me morally, intellectually, financially and above all showered me with your unconditional love throughout my whole life. God bless both of you. As for my wife Amnah and as partner in life, she was the one who had to go through all the good and bad circumstances with me, tolerate my mood swings, take care of the kids and be the shoulder I could lean on. This achievement is every part yours as it is mine; I love you and I cannot live without you, you are a gift from Gad. My gorgeous kids, Raneem and Rand, Khalid, you are my life and the love I have for you in my heart is unparalleled.    xix The chapters presented in this thesis have been prepared by the candidate with guidance from the graduate supervisors, Dr. Douglas Waterfield and Dr. Don Brunette. The data in this thesis was acquired and prepared by the candidate.   xx DEDICATION  This thesis is dedicated to :  My Father’s Soul and my Mother  My Wife AMNAH, my kids ( Raneem, Rand and Khalid)  My Brothers and my Sisters  My Colleagues and close Friends whom stand beside me during my medical problems and encouraging me to finish my PhD Degree.     xxi  1 CHAPTER 1: Introduction  1 1.1 Overview  All biomaterials, including medical devices, dental implants, and prostheses, evoke a host response when implanted into living human tissue. When a dental implant is placed into the oral cavity, its surface is conditioned by tissue fluids that contain macromolecules, which influence the behavior of cells when they encounter the surface. What follows is a series of cell-material interactions leading to the release of growth factors, cytokines, and chemotactic factors that modulate cellular activity in the surrounding tissue (Anderson JM et al 2008). Also influencing the interaction between implant materials and the host tissue are the physical properties of the implant, such as surface chemistry and topography. For example, an increase in surface roughness of titanium implants has been associated with an increase in the amount of bone formed at the interface as well as increased osteoconduction  (Cooper LF 1998).  Such findings suggest that surface topography may directly affect the differentiation and migration of osteogenic cells such as osteoblasts. The surface migration and subsequent proliferation and differentiation of osteoblasts and other mesenchymal cells involved in endosseous wound healing is controlled by cytokines, chemokines, and growth factors secreted by other cell types dominating the wound site. Among those cell types are the macrophages, which arrive in the vicinity of the implant within the first 24 hours of implant insertion.  The macrophage plays a central role in directing host inflammatory and immune processes. Thus, establishing its response to biomaterials is critical to understanding material-mediated host responses. The macrophage also plays a pivotal role in  2 modulating the repair process, initiating phagocytosis, and producing myriad cytokines to control wound healing, cell recruitment and proliferation, and possible bone formation. This monograph will elucidate the role of the macrophage in each of these events.  1.2 The use of implants in dentistry 1.2.1 Biocompatibility and biofunctionality of implanted materials  The interaction between any biomedical device and its host depends on both the biofunctionality and the biocompatibility of the device. Biofunctionality refers to the ability of a device to perform its designated function (Williams DF1991; Cook SD 1992), while biocompatibility refers to its ability to elicit an appropriate host response in a specific application  (Gotman I 1997; Williams DF 1987). Figure 1-1 illustrates the biofunctional and biocompatible qualities of a dental implant. The implant is biofunctional in its capacity to replace the missing tooth. Its biocompatibility allows it to become successfully osseointegrated.  The placement of a dental implant into the human body triggers a sequence of healing processes and host responses involving vascular, humoral, and cellular systems, known collectively as inflammation (Anderson JM 1993; Thomsen P and Gretzer C 2001). Because they are biocompatible, the materials in implants generally set off an inflammatory response that progresses from a high-intensity, acute phase to a low- activity, quasi-equilibrium state known as the foreign body reaction (Jenney CR, 1998).   3 1.2.2 Characteristics of titanium and titanium-based alloys  Titanium and titanium alloys are the materials of choice for oral implantology and other dental and medical devices (Wang RR and Fenton A 1996; Meffert RM, 1992). Titanium has been alloyed with aluminum (usually 6%) and vanadium (4%) for common medical purposes such as hip and knee prostheses. Aluminum and vanadium were alloyed with titanium to increase strength and elasticity while maintaining corrosion resistance when implants contact body fluids (Meffert RM 1997). Titanium’s suitability for medical and dental applications can be attributed to its mechanical, physico-chemical, and biologic properties, including immunotolerance and cytocompatability (Donley TG and Gillette WB 1991). Titanium has a high corrosion resistance because it has an oxide surface layer that is 2 to 5 nm thick and chemically nonreactive to any surrounding tissues (Kasemo B and Lausmaa J 1988; Stanford CM and Keller JC 1991). It has a modulus of elasticity of approximately 110 GPa, which is half that of cobalt-based alloys but at least five times greater than that of bone. Due to their low densities, titanium and titanium-based alloys have a specific strength (force per unit area at failure /density) superior to other metals. However, titanium has poor shear strength and wear resistance, making it unsuitable for articulating surfaces and bone screw applications (Cook SD 1992).  4  Figure 1–1: Dental implants share with natural teeth the need to interface with epithelium and connective tissue, and anchorage of the natural or artificial crown must be sufficiently secure to support mastication. A major difference is that titanium dental implants are typically integrated with bone, whereas natural teeth are supported by a periodontal ligament. (Courtesy of Shine Dental.)  5 1.2.3 Definition of osseointegration  Titanium implants, as they are used in clinical dentistry and orthopedics, become intimately associated with bone, a process resulting in osseointegration (Brånemark PI et al 1977; Branemark R et al  2001). The term osseointegration was originally defined as a direct structural and functional connection between ordered living bone and the surface of a load-bearing implant (Brånemark PI et al 1977). This definition was later revised, and today an implant is regarded as osseointegrated when there is no progressive relative movement between the implant and the bone with which it is in direct contact (Brånemark PI, et al 1983). Osseointegration thus constitutes an anchorage mechanism by which nonvital components can be reliably and predictably incorporated into living bone, and this anchorage can persist under all normal conditions of loading (Worthington P 1997).  Osseointegrated implants are used for the replacement of missing single teeth, for the restoration of the partially edentulous segment of the mouth, and for the reconstruction of the completely edentulous patient by means of implant-supported fixed bridges or removable overdentures (Worthington P 1991). In the pioneering stages of their development, dental implants were most commonly placed by oral-maxillofacial surgeons in carefully selected patients and restored by prosthodontists. Currently, dental specialists and nonspecialists alike place dental implants. Moreover, implants are now placed into more challenging sites with less bone support and even in medically compromised patients. These challenges have led to the development of new implant  6 systems, and a key factor in their design is the surfaces at the macro, micro, and nano scales (Mendonca G, et al 2008).  1.3 Implant surfaces  Among the commercially available implants most frequently described in the dental literature and used clinically, two basic types of surface can be identified: (1) roughened surfaces obtained by subtractive processes such as sand blasting, acid etching, or a combination of the two processes; electropolishing; or mechanical polishing; and (2) roughened surfaces obtained by additive processes such as coating with titanium plasma- spray (TPS) or hydroxyapatite or through ion deposition. Surfaces are further subdivided as either isotropic, meaning they have no orientation of their features, or anisotropic, meaning they contain features such as fine lines or grooves produced by turning on a lathe (machining).  Moreover, the surfaces are integrated into a larger structure with visible, so-called macro geometric properties, such as screw threads and vents, which are designed to facilitate mechanical stability. The macro shape varies among manufacturers; some examples are shown in Fig. 1-2. (In reviewing the approaches used to modify implant surfaces, I have selected examples from several implant manufacturers. No attempt has been made to be comprehensive because of the large number of implant systems available and the numerous, sometimes subtle variations in production methods. The emphasis is on those implants that are widely used or illustrate particular principles of surface modification.)  7 1.3.1 Blasted surfaces  Blasted surfaces can be produced using a variety of blasting particles, including aluminum oxide, TiO2, and hydroxyapatite. Manufacturers attempt to gain commercial advantage by introducing proprietary processes that claim to optimize the surface. For example, some elements of the blasting particles—such as aluminum in the alumina- blasted implants—can become embedded in the implant surface (Sittig C, et al 1999). To obviate possible problems caused by incorporated elements such as aluminum, Astratech developed the TioblastTM surface (Astratech, Molndal, Sweden), which is produced by blasting with TiO2 particles. Like other blasting processes, this one produces an isotropic surface. The topographic characterization of these surfaces can be somewhat complex; the most sophisticated and comprehensive approach is the wavelength-dependent method of Wieland et al  (2002). More typically, implant topography parameters, if they are given at all, are reported only summarily. For example, the Tioblast implants had an average height deviation (Sa) of 1.07 µm, an average wavelength (Scx) of 10.11 µm, and an increased surface area (Sdr) of 29% (Albrektsson T and Wennerberg A 2004).  8  (A) IMZ                   (B) Sterioss                    (C) Tioblast                (D) Astra   (E) Nobel Biocare   (F) TiUnite   (G) TiUnite   (H) Osseotite   (I) SLA  Figure 1–2: Examples of dental implants made by various manufacturers (www.dentistkerala.com, www.suindental.co.kr, www.astratechdental.dk, www.ec21.comT, www.bangkokdental.com, www.dentalcompare.com, and www.coastalimplant.com).  9 Topographic characterization of the surface features used in this thesis, originally produced by blasting and etching, are given in chapter 4. It should be noted, however, that comparison of parameters such as Sa is difficult because the particular values obtained depend on the characterization method being used (e.g., values obtained by contact profilometry).  1.3.2 Acid–etched surfaces  The OsseotiteTM (Implant Innovations, Palm Beach Gardens, FL) is etched in a two-step procedure that employs HCl and HsSO4, resulting in an isotropic surface with a high frequency of irregularities. The measured surfaces had an average height deviation of 0.94 µm, an average wavelength of 11.68 µm, and an increased surface area of 20% (Albrektsson T  and Wennerberg A 2004). However, acid oxidation treatment, such as hydrofluoric acid, can be used to create topographies in the nanoscale (Nanci A, et al 1998), conventionally defined as structures having at least one of their dimensions in the range of 1 to 100 nm. An important application of this principle is the surface developed by Ellingsen and coworkers (2006) in which the Tioblast surface is treated with hydrofluoric acid and marketed as Osseospeed owing to its ability to induce rapid bone formation at the implant surface.  1.3.3 Blasted and acid-etched surfaces  The SLATM (sandblasted, large grit, acid etched) titanium surface was developed by Straumann (Waldenburg, Switzerland) (Scacchi M et al  2000) (Fig. 1-3). The SLA  10 surface was created by sandblasting with large-grit particles of Al2O3 followed by acid etching with HCL/H2SO4 at an elevated temperature for 5 minutes (Wong M et al  1995). The SLA implants had average height deviations of 1.42 µm, average wavelengths of 16.60 µm, and an increased surface area of 33% (Albrektsson T and Wennerberg A 2004). SLA has compared favorably in histometric and biomechanical testing with some other implant surfaces such as TPS (Li D et al  2002). SLA has also been successfully used in the clinic (Cochran DL 2000) and has increased removal torque values, compared to the machined and acid-etched surfaces, by 75% to 125% at each healing period (Li D et al  2002). More characterization of this surface is provided in chapter 4.  1.3.4 Coated surfaces  An example of a coated surfaces is the Steri-OssTM family of implants, which are available with a hydroxyapatite (HA) or a TPS coating. The HA coating obviously alters the surface chemistry as well as the topography. This dual alteration has at least the potential to combine beneficial effects synergistically. In vitro experiments comparing bone formation on microfabricated grooves of different depths with surfaces consisting of titanium or HA coating found that the latter produced consistently more bone nodules at all depths with a statistically significant interaction term, indicating synergy between the effects of coating and topography. The Steri-Oss HA-coated implants had an average height deviation of 1.68 µm, an average wavelength of 13.74 µm, and an increased surface area of 55% (Albrektsson T and Wennerberg A 2004). The Steri-Oss TPS surface was rougher than its HA-coated counterpart, with an average height deviation of 3.86  11 µm, an average wavelength of 19.55 µm, and an increased surface area of 134%. No long-term follow up studies have been published (Albrektsson T and Wennerberg A 2004).  1.3.5 Oxidized surfaces  The electrochemically-oxidized surface of the TiUniteTM (Nobel Biocare, Goteborg, Sweden) is a highly crystalline and phosphate-enriched titanium oxide characterized by a microstructured surface with open pores in the low micrometer range (Hall J, et al  2005). It is an isotropic surface that features increased thickness of the oxidized layer in the apical direction with craterous structures. The implants had an average height deviation of 1.08 µm, an average wavelength of 10.98 µm, and an increased surface area of 37% (Albrektsson T  and Wennerberg A 2004). As might be expected, the surface chemistry of this implant differs from commercially pure (cp) titanium due to incorporation of anions of the electrolyte used in oxidization process. X-ray photoelectron spectroscopy (XPS) analysis indicated that the TiUnite implants contained more than 7 at% of P in the oxide layer and higher amounts of hydroxides compared to titanium surfaces that were not electrochemically treated (Kang BS, et al  2009).  From this brief review it can be appreciated that the diverse processes used in production of various implant systems result in surfaces that differ from one another both in chemistry and topography. Moreover, a particular problem in interpreting the effects of topography has been exacerbated as a result of the current trend to introduce nanoscale  12 features. As noted by Mendonca et al  (2008), it is extremely difficult to isolate topographic effects from chemistry or charge effects induced by nanotopography because at the atomic level of material assembly surface properties are influenced by quantum phenomena. Nevertheless, they state that it is important to distinguish distinct topographic effects from changes in surface energy or chemical reactivity. Because the intent of this thesis is to explore the effects of particular topographies, the strategy used was to replicate the surfaces in epoxy and then coat them with commercially pure titanium. Thus, the effects of topography could be investigated independent of chemistry because the chemical properties were those of commercially pure titanium for all surfaces.  1.4 Comparisons of clinical effectiveness among implant systems  Regardless of the procedures used to produce them, many types of roughened surface have proved successful in practice. The clinically and commercially important question arises: which implant system is best? The answer to this question is not clear at present because of the complexities and problems involved in clinical trials of dental implants: 1. It should be noted that many implant systems have high success rates for certain patient populations; thus, distinguishing small relative effects requires large samples. 2. There is heterogeneity among patients with respect to medical conditions, compliance with oral hygiene instructions, and habits such as smoking or clenching. These factors when not dealt with by exclusion from the patient pool or methods to distribute their effects, can lead to increased variance in the data.  13 3. Local sites can vary markedly in aspects such as quality of bone within the same patient. 4. Implant systems are developed at a faster rate than the time required to get long-term clinical data. 5. To some extent, implant placement can be technique sensitive, so the skill of the surgical team contributes to the overall success rate, and studies vary in the experience of the surgeons used to place the implants. 6. Investigators are frequently associated with and sponsored by manufactures of particular implant systems. When such investigators perform comparative studies, the system with which they are most familiar would appear to have an advantage in that the investigators would encounter fewer technical problems than they would face with unfamiliar systems. 7. There is ample evidence in pharmaceutical research that systematic bias exists favoring products made by the company that sponsors the research. Explanations offered to explain this well-established phenomenon include the selection of inappropriate comparison products or conditions and publication bias (studies that show the company’s product to be inferior are simply not published. Although there is no evidence that this phenomenon applies to implant research, it must be counted as a possibility given that implant systems cannot be placed “blind” (in the experimental design sense of the investigator not knowing which implant system he/she is using). If different teams associated with different manufacturers get contradictory results, meta-analysis of the studies will be hard pressed to find differences.  14 Considering these problems, it is not surprising that the most recent Cochrane systematic review (Esposito M, et al  2007) concluded there was no evidence showing that any particular type of dental implant has superior long-term success. The authors noted that their conclusions were based on a few randomized clinical trials, often at high risk of bias, with few participants and relatively short follow-up periods.  1.5 Biologic responses to materials  Fundamental aspects of tissue responses to materials can be traced along the so-called tissue response continuum, which is characterized by injury, blood-material interactions, provisional matrix formation, acute inflammation, chronic inflammation, granulation tissue, foreign body reaction, and ultimately fibrous encapsulation of the biomaterial, medical device, or prosthesis (Anderson JM 2001).  1.5.1 Effect of surface topography on cell behavior  The main goal of any biomaterial is to obtain an appropriate host tissue response (Hunt JA and Shoichet M 2001; Soskolne WA et al  2002; Kieswetter K et al  1996). The clinical success of dental implants depends in part on their mechanical properties, surface chemistry, and surface topography (Albrektsson T et al  1981). One of the major determinants of implant performance in vivo, surface topography influences cell adhesion, cell shape, cell selection, cell orientation, mechanical interlocking, topographic (contact) guidance, tissue organization, production of local microenvironments  15 (Albrektsson T et al  1981; Brunette DM 1996), and production of growth factors and cytokines (Kieswetter K et al  1996).  The response to surface topography (roughness) and micromachined surfaces by fibroblasts, epithelia, and osteoblasts has been extensively studied (Kieswetter K 1996 et al ; Brunette DM 1996; Brunette DM and Chehroudi B 1999; Brunette DM 1999; Wieland M et al  2002). Oakley et al demonstrated that the cytoskeleton of fibroblasts was found to be the primary structure involved in topographic guidance in vitro (Oakley C et al 1997). Cell differentiation was also found to be influenced by surface topography. Perizzolo et al  reported that bone-like nodule production was increased on a titanium surface with deep grooves. This increase was enhanced when the surface topography was coated with hydroxyapatite (Perizzolo D 2001). Furthermore, microfabricated substrata have also been found to affect both cell adhesion (Chehroudi B et al 1989) and extracellular matrix organization of collagen (Qu J et al  1996). Many studies have also demonstrated the effect of surface topography on the capacity of cells to attach, proliferate, differentiate, and secrete extracellular matrix and proteins (e.g., TGF-β1 and BMP-2) (Kieswetter K 1996 et al ; Takebe J et al  2003; Martin JY et al  1995). Thus, the surface topography seems to affect gene expression. Moreover, increasing the surface roughness of titanium implants generally results in an increased resistance to compressive, tensile, and shear forces as well as increased bone formation at the implant surface (Buser D et al  1991; Carlsson L et al  1988; Gotfredsen K et al  1995; Feighan JE et al  1995).   16 1.6 The macrophage 1.6.1 Origin  Macrophages are part of the mononuclear phagocyte system (MPS) (van Furth R et al 1972) and are produced in the bone marrow (Fig. 1-5). Differentiation of bone marrow stem cells into monocytes/macrophages is a process whereby progenitor cells, known as colony-forming unit–granulocyte-macrophages (CFU-GM), develop from pluripotent hemopoietic stem cells (Metcalf D 1971; Ogawa M 1983); these progenitor cells then give rise to monocyte precursors, monoblasts, and promonocytes (Goud TJ and van Furth R 1975; Goud TJ et al  1975). The promonocytes then differentiate and enter the blood to form the blood monocyte (van Furth R 1982; van Furth R and Dulk MM 1970). Thereafter, the blood monocytes circulate in the bloodstream until they enter tissue and fully differentiate to become resident tissue macrophages without endogenous inflammatory stimulus (van Furth R and Cohn ZA 1968; Hume DA et al  2002). The process of differentiation and proliferation of macrophages is controlled by cytokines and growth factors such as macrophage–colony stimulating factor (M-CSF), granulocyte- macrophage–colony stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-1 (IL- 1), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) (Takahashi K, et al  1996; Kazuo M 1998; Valledor AF, et al  1998; Riches DW, et al  1996; Clarke S and Gordon S 1998).     17 1.6.2 Distribution  When macrophages migrate to different tissues, they acquire the characteristics and functional heterogeneity of their tissues of residence (Gordon S et al  1988; Gordon S 1995). The terms used to describe macrophages also designate the specific organ or connective tissue in which they reside. For instance, in the central nervous system, macrophages are known as microglia cells; in the liver, they are kupffer cells; in the lung, alveolar macrophages; in the connective tissue, histiocytes; in the spleen and lymph nodes, fixed and free macrophages, respectively; in the serous cavities, pleural and peritoneal macrophages; in the bone, osteoclasts; in the skin, langerhans cells; and in the lymphoid tissue, dendritic cells. At implant sites, macrophages are known as inflammatory macrophages (Gordon S 1995; Abbas AK 1997).  1.6.3 Biologic functions  Because they are widely distributed and participate in both specific and innate immunity, macrophages comprise a range of functional and morphologic phenotypes. For example, they regulate hemopoiesis, contribute to tissue homeostasis, and engulf large particles via a process known as phagocytosis (Gordon S 1995; Abbas AK 1997, 58–59). Macrophages can function as antigen-presenting cells, introducing processed foreign antigens to naïve and primed T-lymphocytes and allowing the induction, enhancement, or inhibition of specific immune responses. In addition, macrophages can be activated by immune stimuli to display enhanced antimicrobial resistance and more efficient microbicidal functions (Takahashi K, et al  1996; Kazuo M 1998; Riches DW, et al  18 1996; Gordon S 1995; Abbas AK 1997; Laskin DL, et al 2001). Macrophages are also potent secretory cells that release cytokines, chemokines, and growth factors; these secretions have been found to orchestrate the immune response (Takahashi K, et al  1996; Kazuo M 1998; Gordon S 1995).  19   Figure 1–3: Differentiation and distribution of macrophages in vivo. (Adapted from Gordon S, 2003.) Copied under licence from Access Copywrite. Further reproduction prohibited.  20 1.6.4 Macrophage identification and markers  Specific monoclonal antibodies (mAbs) have been developed to allow detection of tissue- resident macrophages as well as those recruited to sites of inflammation and infection. Antibody staining reveals the heterogeneity in macrophage populations and serves to emphasize the diverse physiologic roles of macrophages in normal tissues and at inflammatory sites (Martinez-Pomares L, et al  1996). Some mAbs (e.g., CD68) recognize all macrophages (Pulford KA 1990), while others (e.g., F4/80) recognize only a subpopulation of tissue macrophages (Hume DA, et al  2002; Austyn JM and Gordon S 1981). Studies have shown that the monoclonal antibodies ED1 and ED2 can be used to identify subpopulations of macrophages in rats (Dijkstra CD, et al  1985). The ED1 antibody identifies membrane markers associated primarily with newly recruited blood monocytes (acute stage of inflammation), while the ED2 antibody identifies mature tissue macrophages (chronic stage of inflammation) (Dijkstra CD, et al  1985; Rosengren A, et al  1997). Recent studies (Chehroudi B, et al  in press) have shown that the macrophages attaching to the SLA surface in a rat model are ED1 positive—that is, recruited macrophages.  1.6.5 Macrophage phenotypic differentiation  In addition to its well-defined role in the uptake and degradation of foreign agents and tissue debris, the macrophage is now recognized for its diverse contributions to host defense as well, especially its vital roles in the degradative and reparative phases of the  21 inflammatory response. Two general hypotheses have been advanced to explain the heterogeneity exhibited by macrophages (Fig. 1-4). The first postulates that diversity is produced by functionally distinct and committed subpopulations of macrophages. Despite its conceptual attractiveness, this hypothesis has received little experimental support (David W and Riches H 1996). The second hypothesis argues that macrophages are pleuripotential cells that adapt to the prevailing stimuli or conditions at the site to which they have been attracted. Functional heterogeneity in this hypothesis is viewed as an adaptive response by a relatively homogeneous population of precursor macrophages. Significant data have been published in support of this hypothesis (David W and Riches H 1996).  22  Macrophage Functional Diversity  Homogenous population of cells respond to stimuli Function specific precursor population   Figure 1–4: Two hypotheses advanced to explain macrophage functional diversity. (A) Function-specific monocyte precursors are viewed as giving rise to function-specific subpopulations of monocytes and ultimately to function-specific subpopulations of macrophages. (B) Monocytes are viewed as a relatively homogeneous population of cells that respond to the stimuli and conditions that prevail at the site to which they have been attracted. In this hypothesis, macrophage functional diversity is viewed as being induced in an adaptive fashion. Abundant data support this hypothesis (B). (Adapted from David W and Riches H 1996). Copied under licence from Access Copywrite. Further reproduction prohibited.  23 1.6.6 Macrophage activation and regulation  The complex mechanisms of macrophage activation and regulation are integrated to accomplish the intended host response. Three pathways with three distinct biologic functions have been identified in macrophage activation (Mosser DM 2003). The first pathway produces the classically activated macrophage, which is involved in the Th1 cellular immune responses. The macrophage requires two signals to be classically activated: (a) obligatory cytokine IFN-γ, which primes macrophages for activation but does not itself activate macrophages; and (b) tumor necrosis factor (TNF) or an inducer of TNF such as lipopolysaccharide (LPS) (Nathan C 1991). The second pathway produces alternatively activated macrophages that have been implicated in immunosuppression, tissue repair, and extracellular matrix (ECM) deposition. It has been demonstrated that macrophages treated with either IL-4 or IL-13 assume an alternative activation phenotype (Stein M et al 1992).  The third pathway is type II–activated macrophage, which inhibits inflammation and induces Th2-type humoral immune responses. The type II-activated macrophage was initially identified during an examination of conditions under which IL- 12 transcription was terminated. It was observed that the ligation of Fcγ Rs on activated macrophages unexpectedly turned off IL-12 synthesis (Sutterwala FS, et al  1985) and induced the secretion of large amounts of IL-10 (Sutterwala FS, et al  1998). Type II– activated macrophages are clearly distinct from alternatively activated macrophages described above in two ways: they do not induce arginase; and, with the exception of IL- 12 and IL-10, the production of many of the other cytokines produced by classically activated macrophages (such as TNF, IL-1, and IL-6) remains intact (Gerber JS and  24 Mosser DM 2001). In conclusion, these three types of macrophage activation may form their own regulatory network to prevent a well-intentioned immune response from progressing to immunopathology (Mosser DM 2003).  In 2004, Mantovani et al further refined the functions and nomenclature of different polarized macrophage populations (Fig. 1-5). Macrophages polarize and acquire different functional properties in response to environment-derived signals. Macrophage exposure to IFN-α and LPS drives M1 polarization, with potentiated cytotoxic and antitumoral properties, whereas M2 macrophages are in general more prone to immunoregulatory and protumoral activities. In particular, M2a (induced by exposure to IL-4 and IL-13) and M2b (induced by combined exposure to immune complexes and TLR or IL-1R agonists) exert immunoregulatory functions and drive type II responses, whereas M2c macrophages (induced by IL-10) are related more to suppression of immune responses and tissue remodeling.  1.6.7 Production of effector molecules by macrophages 1.6.7.1 General  Macrophages release myriad substances, including pro-inflammatory and cytotoxic cytokines, growth factors, bioactive lipids, hydrolytic enzymes, reactive oxygen intermediates, and nitric oxide. These secretions orchestrate the immune response, play a central role in acute and chronic inflammation, and are implicated in tissue remodeling and wound repair after injury.  25  Figure 1–5: Inducers and selected functional properties of different polarized macrophage populations. Macrophages polarize and acquire different functional properties in response to environment-derived signals. (Abbreviations: DTH, delayed-type hypersensitivity; IC, immune complexes; IFN-g, interferon-g; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MR, mannose receptor; PTX3, the long pentraxin; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; SLAM, signaling lymphocytic activation molecule; SR, scavenger receptors; TLR, toll-like receptor.) (Adapted from Mantovani A et al , 2004.) Copied under licence from Access Copywrite. Further reproduction prohibited.  26 1.6.7.2 Cytokines  Cytokines encompass a broad group of signaling proteins that are produced transiently after cellular activation. They modulate the functions of individual cells and regulate processes taking place under normal, developmental, and pathologic conditions (Dinarello CA, et al  1990; Meager A and Meager T 1998). Most cytokines act either locally on their own (autocrine) or on other cell types (paracrine), or systemically (endocrine). Their action, which is short-lived, is initiated via specific receptors expressed primarily on the cell membranes of their target cells (Miyajima A, et al  1992). A close examination of the physiologic and pathologic effects of regulated and deregulated expression of cytokines has shown that they are involved in the regulation of virtually all general systemic reactions of organisms, including immune responses, inflammation, hematopoiesis, and wound healing (Foster JR 2001). Some of the largest groups of cytokines are interleukins, which are named and categorized according to their function (e.g., lymphokin and monokin); interferons, which were initially thought to be involved in antiviral responses; colony stimulating factors, which support the growth of cells and differentiation; and tumor necrosis factors, which have anti-tumor, anti- proliferative, and cytotoxic effects (Norman S, et al  1993).  The cytokine network is highly complex, but two general principles are important to the present discussion. First, the cytokine system demonstrates great redundancy, which, in this context, means that similar functions can be performed by many different cytokines. For example, many different cytokines can act to stimulate cell division in activated T  27 cells.  Second, through pleiotropism, the action of a single cytokine can have many different functional effects on different cell types and sometimes even on the same cell (Abbas AK, et al  1997).  1.6.7.3 Inflammatory cytokines secreted by macrophages  As noted earlier, the macrophage is a major source of cytokines and growth factors. Only cytokines of relevance to this project will be discussed.  1.6.7.3.1 Tumor necrosis factors  Tumor necrosis factor-α  (TNF-α), also known as cachectin (Carswell EA, et al  1975; Beutler B, et al  1985), and tumor necrosis factor-β (TNF-β), also known as lymphotoxin (Williams TW and Granger GA 1968; Ruddle NH and Waksman BH 1968), are closely related proteins that bind to the same cell surface receptor and produce a vast range of similar, though not identical, effects. Despite the similarity of their biologic activities and functions, however, the regulation of the expression and processing of the two factors is quite different (Vilcek J and Lee TH 1991; Ruddle NH 1992). TNF-α is produced by activated macrophages and other cell types, including neutrophils, activated T and B lymphocytes, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells, and some transformed cells (Vilcek J and Lee TH 1991; Ruddle NH 1992). TNF-β, conversely, is produced only by lymphocytes (Vilcek J and Lee TH 1991; Ruddle NH 1992).  28 Mature human TNF-α is a polypeptide of 157 aa residues. (For purposes of comparison, it is useful to know that mouse, rat, and rabbit TNF-α are each one aa shorter [Vilcek J and Lee TH 1991].) The apparent molecular weight of human TNF-α under denaturing conditions is approximately 17 kDa (Davis JM, et al 1987), and, in contrast to TNF-β, it shows no N-glycosylation (Vilcek J and Lee TH 1991).  TNF-α does not possess a typical signal peptide sequence but is initially synthesized as a larger protein, of which the mature 17 kDa factor comprises the C-terminal portion. The N-terminal sequence of the precursor contains both hydrophilic and hydrophobic domains, and its presence results in the occurrence of TNF-α as a membrane-bound form from which the mature factor is released by proteolytic cleavage (Kriegler M, et al  1988; Luettig B, et al  1989; Perez C, et al  1990). Evidence suggests that the membrane- anchored form of TNF-α on the surface of macrophages and/or monocytes, serving as a reservoir for release of soluble TNF-α, exhibits lytic activity and may play an important role in intercellular communication (Kriegler M, et al  1988; Luettig B, et al  1989; Perez C, et al  1990).  Researchers have identified two distinct receptor types that specifically bind TNF-α and TNF-β, and virtually all cell types express one or both of these receptor types. TNFR-II (Type A) is a transmembrane glycoprotein with an apparent molecular weight of 75 kDa (Dembic Z, et al  1990); TNFR-I (Type B) is a transmembrane glycoprotein with an apparent molecular weight of 55 kDa (Schall TJ, et al  1990; Loetscher H, et al  1990). The intracellular domains of the two TNF receptors are apparently unrelated, suggesting  29 that the two types employ different signal transduction pathways (Dembic Z, et al  1990). Each receptor type can bind TNF-α or TNF-β with high affinity, and there is no evidence that interaction between them is necessary for signal transduction (Loetscher H, et al 1990; Smith CA, et al  1990; Engelmann H, et al  1990). Soluble forms of both types of receptors have been found in human serum and urine (Seckinger P, et al  1989; Olsson I, et al  1989; Engelmann H, et al  1990). These soluble receptors are capable of neutralizing the biologic activities of both TNF-α and TNF-β and may serve to modulate and localize the activities of the TNFs or act as a reservoir for their controlled release (Seckinger P ,et al  1989; Olsson I ,et al  1989; Engelmann H, et al  1990).  The two TNFs are extremely pleiotropic due to the ubiquity of their receptors, their ability to activate multiple signal transduction pathways, and their capacity to induce or suppress the expression of a number of major genes, including those for growth factors and cytokines, transcription factors, receptors, inflammatory mediators, and acute phase proteins (Vilcek J and Lee TH 1991; Kronke M, et al  1991). TNFs also play a critical role in normal host resistance to infections and to the growth of malignant tumors, serving as immunostimulants and as mediators of the inflammatory response (Vilcek J and Lee TH 1991; Kronke M, et al  1991). Many of the effects produced by the TNFs are functionally similar to those produced by IL-1 (Kronke M, et al  1991).      30 1.6.7.3.2 Interleukin-1 (IL-1)  Interleukin 1 (IL-1) is a family of proteins that includes IL-1α, IL-1β, and the IL-1 receptor antagonist. IL-1α and IL-1β bind to the same cell surface receptors and perform the same biologic functions. In general, IL-1 is produced by macrophages and various other cell types in response to stimuli such as inflammatory agents, infections, or microbial endotoxins (LPS) (Dinarello CA 1991; Oppenheim JJ, et al  1986; Dinarello CA and Wolff SM 1993; Dinarello CA 1994).  IL-1α and IL-1β are structurally related polypeptides that show approximately 25% homology at the amino acid level (Oppenheim JJ, et al  1986). They are synthesized as 31 kDa precursors and are then cleaved into proteins with molecular weights of approximately 17.5 kDa (Giri JG, et al  1985; Hazuda DJ, et al  1988). Both exert their effects by binding to specific receptors. Two distinct receptor types that bind both forms of IL-1 have been isolated. IL-1 RI (an 80 kDa membrane-bound receptor protein) has been isolated from T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, and hepatocytes (Dinarello CA, 1991; Dinarello CA and Wolff SM 1993; Urdal DL, et al  1988; Sims JE, et al  1988). IL-1 RII (a 68 kDa membrane-bound receptor protein) has been isolated from B cells, neutrophils, and bone marrow cells (Dinarello CA 1991; Dinarello CA and Wolff SM 1993). The two IL-1 receptor types exhibit approximately 28% homology in their extracellular domains; however, the type II receptor has a cytoplasmic domain of only 29 amino acid residues, whereas the type I  31 receptor has a cytoplasmic domain of 213 amino acid residues (Dinarello CA 1991; Sims JE, et al  1988). Recently, a third type of IL-1 receptor, referred to as IL-1 receptor accessory protein (IL- 1 RAcP), has been cloned from murine cells (Greenfeder SA, et al  1995). While IL-1 RAcP does not appear to bind IL-1 directly, it is required for transduction of biologic responses and forms a complex that binds IL-1 with higher affinity than IL-1 RI alone. IL-1 RII does not appear to be involved in IL-1 signaling and may function as a “decoy” receptor that attenuates IL-1 function (Slack J, et al  1993).  IL-1 conducts a wide variety of biologic activities and plays a central role in mediating immune, normal host, and inflammatory responses (Dinarello CA 1991; Dinarello CA and Wolff SM 1993; Dinarello CA 1994). IL-1 can be released into a local environment following bacterial or immunoglobulin ligation of monocyte/macrophage CD14, the LPS- Toll receptor (Nockher WA and Scherberich JE 1997) or CD64, the IgG receptor (van de Winkel JG and Capel PJ 1993). Within this environment, IL-1 affects a variety of cells. For example, capillary endothelial cells are stimulated by IL-1 to secrete chemokines such as CCL2 (Parry GC, et al  1998) and to upregulate the expression of vascular adhesion molecules such as E-Selectin, ICAM-1, and VCAM-1 (Kupper TS and Groves RW 1995). CCL2 acts as a stimulus for chemotaxis and activates mononuclear cell integrins (Rollins BJ 1997), thus facilitating mononuclear infiltration into an area of early inflammation. IL-1 also induces expression of “itself” in newly arriving monocytes and macrophages, thus amplifying the overall process (Dinarello CA 1987). In terms of other pro-inflammatory molecules, IL-1 apparently is needed for the efficient production of  32 IFN-γ. On resident NK cells, IL-1 appears to work in conjunction with macrophage- derived IL-12 to induce IFN-γ secretion (Hunter CA 1995), resulting in an IFN-γ– induced activation of macrophages (Billiau A 1996). Finally, IL-1 also stimulates release of MMPs from resident fibroblasts. MMP expression has at least two in situ effects: first, extracellular matrix degradation can facilitate monocyte migration; and second, MMPs are known to degrade IL-1β and a number of chemokines, thus down-modulating the local inflammatory response initiated by IL-1 (Ito A, et al  1996; McQuibban GA, et al 2002).  1.6.7.3.3 Interleukin-6 (IL-6)  Interleukin 6 (IL-6) is a multifunctional cytokine that plays important roles in host defense, acute phase reactions, immune responses, and hematopoiesis (Akira S, et al 1993; Hirano T 1994; Van Snick J 1990; Kishimoto T, et al  1992). It is produced by a variety of cells, including T cells, B cells, monocytes/macrophages, fibroblasts, keratinocytes, vascular endothelial cells, osteoblasts, and tumor cells (Akira S, et al 1993). The production of IL-6 is upregulated by mitogenic or antigenic stimulation, cytokines, and numerous other signals (Hirano T 1994). IL-6 expression in monocytes is inhibited by IL-4, IL-10, and IL-13 (Akira S, et al  1993; Hirano T 1994; Van Snick J 1990; Kishimoto T, et al  1992).  Mouse IL-6 cDNA encodes a 211 aa residue precursor polypeptide with a hydrophobic signal peptide that is cleaved to generate the 187 aa residue mature protein. At the protein  33 sequence level, there is approximately 39% identity between mouse and human, 39% identity between rat and human, and 87% identity between mouse and rat IL-6 (Northemann W, et al  1989). Although human and mouse IL-6 are equally active on mouse cells, mouse IL-6 is not active on human cells (Tanabe O, et al  1988; Van Snick J, et al  1988).  IL-6 produces its effects by binding to a high-affinity receptor complex consisting of two membrane glycoproteins: an 80 kDa IL-6 binding receptor (IL-6Ra) and a 130 kDa signal-transducing protein (gp130). The IL-6Ra binds IL-6 with low affinity without transducing an IL-6 signal. In the absence of IL-6Ra, IL-6 does not bind to gp130. However, the presence of IL-6Ra together with gp130 will result in high-affinity IL-6 binding and subsequent signal transduction (Akira S, et al  1993; Hirano T 1994; Van Snick J 1990; Kishimoto T, et al  1992).  It is well established that IL-6 is a pro-inflammatory cytokine, and yet it has also been described as an anti-inflammatory molecule (Barton BE 1997; Tilg H, et al  1997). Furthermore, IL-6 has been shown to influence IL-4 production. It has been suggested that antigen-driven and APC-derived IL-6 may influence naïve CD4+ T cells to produce IL-4 and express the IL-4 receptor. Thus, given the close approximation of the APC and T cell in induction of an immune response, transient APC-derived IL-4 could initiate a T cell autocrine loop whereby naïve T cells direct their own phenotype commitment (O'Garra A 1998; Rincon M, et al  1997).   34 1.6.7.4 Cytokine control of repair  Macrophage growth factors and cytokines stimulate mesenchymal cell proliferation, are implicated in angiogenesis, and contribute to extracellular connective tissue matrix synthesis. Consequently, it is important to identify the mechanisms that both positively and negatively control the expression of these mediators.  1.6.7.4.1 Positive regulation  The means by which TNF-α functions as a growth factor for fibroblasts in angiogenesis (Leibovich SJ and Wiseman DM 1988; Leibovich SJ, et al  1987) are indirect and require the expression of other growth factors and cytokines (Riches DWH 1996). TNF-α also induces the expression of PDGF by fibroblasts and of IGF-I by macrophages (Battegay EJ, et al  1995; Kirstein M, et al  1992; Fournier T, et al  1995). In addition, TNF-α induces the expression of IL-6, which in turn induces the expression of platelet-derived growth factor (PDGF) (Ikeda U 1991). Thus, it has been concluded that TNF-α more accurately serves as a mediator of growth factor production (Riches DWH 1996).  1.6.7.4.2 Negative regulation  The negative mechanisms in the repair process are unclear. However, some studies have shown that IFNs inhibit formation of collagen (Hyde DM, et al  1988) and abrogate the  35 stimulation of IGF-I synthesis by macrophages in response to both hyaluronic acid and TNF-α (Lake FR et al  1994).  1.6.7.5 Chemokines  Chemotactic cytokines, or chemokines, belong to a superfamily of small proteins that perform a crucial role in immune responses. At present, about 50 chemokines have been identified in humans (Matsushima K 2000; Locati M, et al 2001). Based on the relative position of cysteine residues, these chemokines have been classified into four families: CXC, CC, C, and CX3C (Matsushima K 2000; Locati M, et al 2001). Chemokines play a role in the induction of leukocyte migration, angiogenesis, collagen production, and the proliferation of hematopoietic precursors (Locati M 2001:Luster AD 1998). These biologic activities are mediated by seven transmembrane domains, all of them G-protein– coupled receptors. At present, five receptors for CXC (CXCR1-5), nine receptors for CC (CCR1-9), one for C (XCR1), and one for CX3C (CX3CR1) chemokines have been identified (Locati M, et al  2001; Luster AD 1998). The complexity of the cytokines and their receptors enables close control of immune responses but poses a formidable problem for investigators because the possibilities for alterations are so numerous.  36 1.6.7.6 Chemokines secreted by macrophages  Like cytokines, macrophages are also a major source of chemokine production; examples include CCL2 (monocyte chemotactic protein 1 [MCP-1]) (Proost P, et al  1996) and CCL3 (macrophage inflammatory protein-1α [MIP-1α]) (Cook DN 1996). CCL2 and CCL3, both members of the CC subfamily of chemokines (Proost P, et al  1996; Cook DN 1996), have monocyte chemotactic activity and play a key role in immunoregulatory and inflammatory processes (Proost P, et al  1996; Cook DN 1996; Gu L, et al  1999; Gillitzer R and Goebeler M 2001). More specifically, each plays a critical role in macrophage recruitment into wounds, upon which appropriate tissue repair depends (Gillitzer R and Goebeler M 2001; DiPietro LA, et al 1998).  1.6.7.6.1 CCL2 (MCP-1)  CCL2 is a member of the β or CC subfamily of chemokines (Ernst CA, et al 1994). Human CCL2 is highly homologous to the mouse JE gene product (Yoshimura T, et al 1989). Originally, the JE gene was identified as a PDGF-inducible gene in murine fibroblasts. Today, however, it is considered the mouse homologue of human CCL2 (Yoshimura T, et al  1989). Functionally and structurally, JE is most closely related to the MCP/eotaxin subfamily of proteins, which includes five human proteins (CCL2, 7, 8, 13, and CCL11) and four mouse proteins (JE/CCL2, MARC/ CCL7, CCL12, and CCL11) (Ernst CA, et al  1994; Rollins BJ, et al  1988; Luster AD and Rothenberg ME 1997). At the amino acid–sequence level, mature human CCL2 shows 55%, 59%, and 66% identity  37 with the analogous regions of mouse JE, MARC, and CCL12, respectively (Ernst CA, et al 1994; Rollins BJ, et al  1988; Luster AD and Rothenberg ME 1997). Mouse JE/CCL2 cDNA encodes a 148 aa residue precursor protein with a predicted 23 aa residue signal peptide that is cleaved to generate a putative mature protein of 125 aa residues. Compared to mature human CCL2, mouse JE/CCL2 has a 49 aa residue carboxy-terminal extension that is rich in serine and threonine residues (Ernst CA, et al  1994; Rollins BJ, et al  1988; Luster AD and Rothenberg ME 1997).  CCL2 and/or JE are produced by many cells, including fibroblasts, epithelial cells, macrophages, mast cells, endothelial cells, osteoblasts, and ameloblasts (Luster AD and Rothenberg ME 1997; Luo Y, et al  1994; Sarafi MN, et al  1997; Frazier-Jessen MR and Kovacs EJ 1995; Volejnikova S, et al  1997; Van Damme J, et al 1991). The expression of mouse JE/ CCL2 is induced by inflammatory stimuli such as viruses, LPS, and cytokines such as TNF-α, IL-1, IFN-γ, and PDGF (Rollins BJ, et al 1988; Luster AD and Rothenberg ME 1997; Luo Y, et al  1994; Van Damme J,et al  1991; Gu L, et al  1997). JE/CCL2 is a potent chemoattractant for monocytes/macrophages and lymphocytes (Luster AD and Rothenberg ME 1997; Luo Y, et al 1994; Gu ,L et al 1997). JE/CCL2 has also been shown to play a role in the regulation of Th1/Th2 lymphocyte differentiation, enhancing Th2 development by increasing IL-4 production and inhibiting IL-12 production (Karpus WJ, et al  1997; Lukacs NW, et al  1997). The activities of JE and/or CCL2 are mediated by the mouse CC chemokine receptor CCR2, which has been classified as a G protein–coupled, seven transmembrane domain receptor. CCR2 receptor  38 exists in the two isoforms CCL2RA and CCL2RB (Gao JL and Murphy PM 1995; Kurihara T and Bravo R 1996).  1.6.7.6.2 CCL3 (MIP-1α)  Macrophage inflammatory proteins 1α and 1β (CCL3 and CCL4) are closely related but distinct proteins that were originally co-purified from medium conditioned by an LPS- stimulated mouse macrophage cell line (Sherry B, et al  1988). The CCL3 proteins are also members of the β or CC subfamily of chemokines, a large superfamily of small, inducible, secreted, pro-inflammatory cytokines involved in a variety of immune and inflammatory responses (Miller MD and Krangel MS 1992; Kelner GS and Zlotnik A 1995). Most chemokines have been shown to act primarily as chemoattractants and activators of specific types of leukocytes (Miller MD and Krangel MS 1992; Thomas JS 1994; Kelner GS and Zlotnik A 1995). Human and mouse CCL3 cDNA encode a 92 aa residue precursor protein with a 22 aa (for human) and 23 aa (for mouse) residue signal peptide that is cleaved to generate the secreted mature protein (Thomas JS 1994; Davatelis G, et al  1988). Mature mouse CCL3 shares approximately 70% amino acid identity with mouse CCL3.  CCL3 expression can be induced in a variety of cell types, including T cells, B cells, monocytes, mast cells, neutrophils, Langerhans cells, astrocytes, endothelial cells, fibroblasts, and smooth muscle cells (Thomas JS 1994,; Kelner GS and Zlotnik A 1995; Davatelis G, et al  1988; Kasama T, et al  1994; Koch AE, et al  1994; Lukacs NW 1994;  39 Parkinson EK, et al  1993). The human CCL3 gene has been mapped to chromosome 17 along with all other human β chemokine genes (Thomas JS 1994). Similarly, the mouse CCL3 gene has been mapped to mouse chromosome 11 along with all other mouse β chemokine genes (Thomas JS 1994). The biologic activities of CCL3 overlap with those of other β chemokines. CCL3 acts as a monocyte and macrophage chemoattractant (Miller MD and Krangel MS 1992) and has been reported to have differential chemoattractant and pro-adhesive effects on T-lymphocytes (Tanaka Y, et al  1993, Taub DD, et al  1993).  The biologic activities of CCL3 and CCL4 are mediated by CCR5, a receptor that binds both factors. CCR5 receptor is a seven transmembrane–spanning G-protein–coupled receptor (Samson M, et al  1996) that is expressed in lymphoid organs such as thymus and spleen and on monocytes, macrophages, T-cells, and B-cells. The CCR5 receptor binds the chemokines CCL3, CCL4, and CCL5 with high affinity (Raport CJ, et al  1996) and generates inositol phosphates in response.  1.6.8 Macrophage involvement in inflammation and wound repair  Wound repair is an interactive process that involves soluble mediators, extracellular matrix components, resident cells (keratinocytes, fibroblasts, endothelial cells, nerve cells), and infiltrating leukocyte subtypes (neutrophils/macrophages) all participating differentially in the classically defined three phases of wound healing: inflammation, tissue formation, and tissue remodeling (Clark RAF 1996; Singer AJ and Clark RAF  40 1999). The macrophage plays a pivotal role in the transition between inflammation and repair and in modulating the repair process. Within wounds, macrophages phagocytose debris, become activated, and secrete a large number of bioactive substances, including cytokines and growth factors (Leibovich SJ and Wiseman DM 1988, Gretzer C et al 2006).  The first direct evidence that explained the role of macrophages in a wound was presented in 1975 by Leibovich and Ross, who showed that depletion of monocytes and macrophages in guinea pigs led to a remarkable delay in wound healing. Following this initial observation, several studies demonstrated the multifunctional roles of macrophages and their secretions within wounds. For example, activated macrophages and their conditioned media were found to promote angiogenesis (Polverini PJ, et al  1977; Koch AE, et al  1986), and wound macrophages were shown to release factors that stimulate both angiogenesis and collagen synthesis (Clark RA, et al  1976, Hunt TK, et al  1984). Rappolee et al  (1988) reported that macrophages isolated from subepidermally implanted wound cylinders in mice contained the mRNA transcripts for PDGF, TGF-β, basic fibroblast growth factor (bFGF), IGF-I, TGF-α, and IL-1β. Thus, macrophages are known to be an important source of multiple cytokines and growth factors at wound sites. Macrophages have also been shown to affect the extracellular matrix composition after injury via the production of extracellular matrix molecules, proteases, and protease inhibitors (Fukasawa M, et al  1989; Porras-Reyes BH 1991 ; DiPietro LA, et al  1996). Macrophages also affect tissue repair through the production of inflammatory cytokines and growth factors that influence the recruitment, proliferation, and differentiation of  41 endothelial cells, fibroblasts, keratinocytes, and other cell types (Polverini PJ, et al  1977; Clark RA, et al  1976; Kovacs EJ and DiPietro LA 1995; Sunderkötter C, et al  1994). The capability of the macrophage to enhance wound healing has been demonstrated by in vivo studies showing that the application of macrophage-activating substances accelerates the wound-repair response (Mustoe TA, et al  1987, Leibovich SJ and Danon D 1980). The injection of additional macrophages into healing wounds also augments the repair response in both normal and impaired wound-healing models (Danon D, et al 1989).  Interestingly, alternatively activated macrophages have shown a potential to mediate wound healing, angiogenesis, and ECM deposition (reviewed by Mosser DM 2003). For example, alternatively activated macrophages produce high levels of fibronectin and a matrix-associated protein, beta IG-H3 (Gratchev A, et al  2001), and they promote fibrogenesis from fibroblastoid cells (Song E, et al  2000). The induction of arginase in these cells may lead to polyamine and proline biosynthesis, promoting cell growth, collagen formation, and tissue repair (Hesse M, et al  2001). Thus, the alternatively activated macrophage may serve as the cell involved in regulation and recovery function (Mosser DM 2003; Goerdt S, et al 1999).     42 1.6.9 Macrophage cytokines modulate inflammation and the wound-healing process  Following implantation of a biomaterial, immune cells migrate to the site in response to cytokine and chemokine production. The adherent monocyte/macrophage population is one of the main sources of cytokines at the tissue-implant interface. Based on their effects on an immune response, a recent classification of proinflammatory cytokines places them into Th1 and Th2 subgroups, promoting either a cellular or humoral-mediated response, respectively (Abbas AK 1997). Th1 cytokines include IL-1β, TNF-α, and interleukin-8 (IL-8), while Th2 cytokines include IL-6, IL-10, and IL-1RA (Brodbeck WG, et al  2002, Brodbeck WG, et al  2003).  In terms of biocompatibility, cytokines are categorized based on their role in influencing the foreign body response to the implanted material, promoting either chronic inflammation or wound healing. IL-2, IL-8. IL-6 and TNF-α are generally categorized as proinflammatory/anti–wound healing cytokines because of their collective ability to promote inflammation by cellular activation and chemotaxis. Cytokines that inhibit the inflammatory response and promote wound healing such as IL-1 RA, IL-4, IL13 and TGF-β, are classified as anti-inflammatory/pro-wound healing. IL-1β and IL-10 are unique in this classification scheme because they represent the extremes of the responses. IL-1β is considered a proinflammatory/pro-wound healing cytokine because of its ability to activate both inflammatory cells (lymphocytes and monocytes) and wound-healing cells (fibroblasts). IL-10 is classified as an anti-inflammatory/anti-wound healing  43 cytokine because it counteracts IL-1 β proinflammatory effects. IL-10 downregulates the activity of the inflammatory cells and suppresses further cytokine production, resulting in an anti-inflammatory/anti–wound healing effect  (Brodbeck WG, et al  2002; Brodbeck WG, et al  2003). Macrophages adherent to endosseous dental implant surfaces in vitro also produce TGF-β and BMP-2 (Takebe J et al 2003), will be discussed in more detail later.  1.6.10 Macrophage chemokines modulate inflammation and wound healing  Several studies have investigated the expression and function of CC chemokines in healing wounds (DiPietro LA, et al  1995, DiPietro LA, et al  1998, Fahey TJ, et al 1990). Within wounds, the tissue macrophage is considered the major cellular source for the CC chemokines (DiPietro LA, et al  1995; DiPietro LA, et al  1998). Both resident and recruited macrophages are instrumental in the recruitment of additional macrophages to the wound site (Fahey TJ, et al  1992). This concept has been elucidated in a murine model system where antibody neutralization of CCL3 resulted in both a decrease in the number of wound macrophages and a delay in the wound-healing response (DiPietro LA, et al  1998; Fahey TJ, et al  1992).Two key CC chemokines, CCL2  and CCL3, showed distinct patterns of expression during wound healing (DiPietro LA, et al  1995; DiPietro LA, et al  1998). CCL2 protein levels peaked at day 1, whereas CCL3 levels peaked at day 3 simultaneous with the number of macrophage cells present (DiPietro LA, et al 1995, DiPietro LA, et al  1998).  Although the major function of CC chemokines is macrophage recruitment during wound repair, the sheer redundancy of these cytokines  44 and their ability to recruit different subsets of leukocytes suggest that the interplay of chemokines in wound healing may be quite complex ( Luttikhuizen DT et al 2007).  1.6.11 Osteogenesis and macrophages 1.6.11.1 Osteogenesis  Osteoblasts and osteoclasts are specialized cells responsible for bone formation and bone remodeling, respectively. The mechanisms of osteoblast and osteoclast differentiation and proliferation are highly regulated by growth factors (e.g., bone morphogenetic proteins), cytokines (e.g., TNF-α, IL-1), and hormones (e.g., parathyroid hormone). Macrophages are a major sources of such cytokines and growth factors (Chwartz Z and Boyan BD 1994; Katagiri T and Takahashi N 2002). Generally, bone formation on an implant surface requires recruitment of osteoblast precursor cells, their differentiation into secretory osteoblasts, production of nonmineralized extracelluar matrix (osteoid), and calcification of the extracelluar matrix (Katagiri T and Takahashi N 2002).  The process by which endosseous implants become integrated in bone can be divided into three distinct stages. The first stage, osteoconduction, relies on the migration of differentiating osteogenic cells to the implant surface. The second, de novo bone formation, involves the laying down of a mineralized interfacial matrix (similar to that observed in cement lines in natural bone tissue) either on the implant surface (Davies JE 1998; Davies JE and Baldan N 1997) or near it (Clokie CM and Warshawsky H 1995).  45 The third stage, bone remodeling, results in the development of a bone-implant interface comprising de novo bone at discrete sites (Davies JE 1998).  Osborn and Newesley (1980) describe two mechanisms, distance osteogenesis and contact osteogenesis, by which bone can become juxtaposed to an implant surface. In distance osteogenesis, new bone forms not on the implant surface but rather on the surface of old bone at the peri-implant site. The bone surfaces provide a population of osteogenic cells that lay down new matrix, which encroaches on the implant. In this way the implant becomes surrounded by bone.  In contact osteogenesis, the implant surface must be colonized by bone cells before bone matrix is formed. In this case new bone forms first on the implant surface as a result of the presence of differentiating osteogenic cells  (Osborn JF and Newesely H 1980).  1.6.12 Macrophage-biomaterial interactions and cytokine secretion Although macrophages and foreign body giant cells have long been associated with implanted devices, more recent studies have explored the molecular and cell biological processes taking place at the macrophage/biomaterial interface. Several in vitro studies have demonstrated a rapid upregulation of IL-1 mRNA (Haskill S, et al  1988), TNF-α, and IL-6 (Blaine TA, et al  1996) following the adhesion of monocytes to material surfaces. Specifically, /macrophage secretion of IL-1α (Shanbhag AS, et al  1995; Voronov I 1998), IL-1β (Ingham E and Fisher J 2000; Green TR, et al 1998), IL-6 (Ingham E and Fisher J 2000; Pollice PF, et al  1998), IL-10 (Pollice PF, et al 1998), and TNF-α (Pollice PF, et al  1998; Cardona MA, et al  1992; Boynton EL, et al  46 2000) was stimulated in response to adhesion to polysterine (PS) (Miller KM, et al  1989; Hogquist K, et al  1991; Hurme M and Serkola E 1991; Bonfield TL, et al  1992; Benahmed M, et al  19971), polydimethylsiloxane (PDMS) and silicone (Anderson JM, 1995);  PE, Dacron, expanded polytetrafluoroethylene (ePTFE), and PDMS (Cardona MA, et al  1992; Miller KM, et al  1988; Miller KM and Anderson JM 1989); and titanium surfaces (Gretzer C,  et al  1996 and 2006). In general, relatively low levels of secreted proteins are detected unless exogenous stimuli are added (Gretzer C, et al  1996; De Fife KM, et al  1995; Yun JK, et al  1995; Gonzales O, et al  1996; Harada Y, et al 1996; Gretzer C and Thomsen P 2000). LPS is a major stimulus used to induce increased release of IL-1α, IL-1β, IL-6, TNF-α, and prostaglandin E2 (PGE2) (Miller KM, et al 1989; Miller KM and Anderson JM 1989; Gonzales O, et al  1996). Using macrophage J744A.1, cell line cultured for 24-72 h on cpTitanium disks prepared with smooth and grit-blasted/acid rough surface topographies. pro-inflammatory cytokines (IL-1beta) and (IL-6) and the anti-inflammatory cytokine (IL-10) mRNA were measured with PCR in the presence and absence of LPS. In the absence of LPS, IL-1beta levels were increased on grit-blasted/acid rough surfaces during the first 48 h. In contrast, IL-6 levels were reduced on the grit-blasted/acid rough surfaces. When cultures were treated with LPS, high levels of IL-1beta and IL-6 expression were measured, irrespective of surface topography (Tan KS et al 2007).  Macrophage production of IL-10, as noted earlier (Moore KW, et al  1993), is stimulated by pro-inflammatory cytokines (Pollice PF, et al  1998). IL-10 also is implicated in regulation of inflammation at material surfaces. It has been shown that 1- to 3-µm  47 particles of titanium triggered secretion of IL-10 by human monocytes, and this inhibited secretion of TNF-α and IL-6 (Pollice PF, et al  1998), therby damping down the inflammatory response.  Very few in vivo studies in soft tissues have elucidated the presences of TNF-α protein and TNF-α mRNA in association with macrophages around poly(ether)urethane implants using immunohistochemistry and in situ hybridization (Hunt JA, et al  1996). Studies also have shown IL-1β and TNF-α expression around PDMS implants in mice. Maximum immunostaining was observed at 14 days and remained elevated throughout a 70-day observation period (Dalu A, et al  2000). IL-1β and TGF-β immunostaining were distributed adjacent to titanium implants 3 to 30 days after implantation in rats but did not correlate to the number of inflammatory cells present (Rosengren A, et al  1997). Recent study showed that differential material-dependent cytokine profiles are produced by surface adherent macrophages and foreign body gaint cells in vivo. The authors concluded that IL6 and TNF alpha levels did not decrease with time indicating that cytokine production from adherent macrophages may have a prolonged influence on the normal inflammatory and wound healing response to injury (Rodriguez A et al 2009)  In conclusion, the peri-implant macrophages express different types of cytokines, and the expression of such cytokines is not correlated to implant type. The types of cytokine- secreting cells and the cytokine levels obtained at the interface around different types of materials have not been established. Further, the precise modulatory effects of cytokines  48 on the inflammatory and reparative processes around implants are not known (Thomsen P and Gretzer C 2001).  1.6.13 Macrophage participation in the integration of implants  The placement of endosseous implants results in extravasation of blood, hemostasis, and fibrin clot formation at the bone-implant interface. Among the cells residing in this fibrin network are monocytes, which ultimately differentiate as macrophages (Davies JE 1998; Park JY and Davies JE 2000; Thomsen P and Gretzer C 2001). It is likely that the monocytes and macrophages residing in this fibrin clot contribute to the inductive and chemotactic signals that support osseointegration (Cooper LF 2003). These monocytes arrive at the surgical site in response to locally produced chemoattractant factors.  Macrophages are detected on different types of material surfaces and in the peri-implant tissue after implant placement in soft tissue (Anderson JM and Ziats NP 1991, Thomsen P and Ericson LE 1991). A number of studies have shown that these macrophages have different phenotypes (Johansson CB, et al  1992) and express different surface antigens (Hunt JA, et al  1996) depending on time after implantation (Dijkstra CD, et al  1985; Rosengren A, et al  1997, Rodriguez A et al 2009). Implant-adherent macrophages and multinuclear cells are also observed prior to bone formation at the surfaces of different materials inserted in bone (Larsson C, et al  2001). In our laboratory, implant surfaces of varying roughness were implanted subcutaneously in rats. The results revealed that macrophage-like cells, as assessed by morphology, were  49 observed on rough SLA surfaces in greater numbers than on either smooth surfaces or grooved microfabricated surfaces in which each facet of the groove was smooth. More interesting, the SLA surfaces were associated with a greater degree of bone formation than the smooth or grooved surfaces (Brunette DM, et al  2003, Chehroudi et al. 2009). Therefore, the authors suggested that the presence of macrophages on an implant surface is not necessarily detrimental and that possibly the secretions of macrophages on the rough surfaces may exert positive effects on bone formation.  Control of bone formation and maintenance at the endosseous implant interface is considered an essential factor in maintaining the immediate and long-term survival of the dental implant (Cooper LF 1998; Stanford CM and Brand RA 1999). Bone formation depends on the regulation of osteoblastic cells derived from host tissues and influenced by osteoinductive signals, osteogenetic stimuli, and osteoconductive mechanisms (Einhorn TA 1999). It has been postulated that osteoblast differentiation and proliferation are controlled by cytokine signals from nonmesenchymal cells (Frost A, et al  1997; Gerstenfeld LC, et al  2001). Moreover, bone formation is stimulated through the induction of bone marrow-derived mesenchymal stem cells along the osteoblastic lineage. This inductive process may require or may be modulated by nonmesenchymal cells, including circulating cells of the immune system. Therefore, it has been proposed that osteoinduction is a function of the implant–resident cell population rather than osteoblastic adhesion to an endosseous implant surface alone (Cooper LF 2003). As noted earlier, macrophages play a role in endosseous wound healing (Thomsen P and Ericson LE 1991; Sennerby L, et al  1993; DiPietro LA 1995). Recent studies have  50 shown that macrophage cell lines are able to produce osteoinductive signals, including BMPs and the osteogenic cytokine TGF-β (Champagne CM, et al  2002; Takebe J et al 2003 and Tan KS et al 2007), and their production was topographically dependent (Takebe J, et al  2003). The previous studies (Champagne CM, et al  2002; Takebe J, et al  2003) are the first to demonstrate that BMPs are produced by cells other than osteoblasts.  It is important to investigate whether macrophage cytokines stimulated by implant surfaces of varying degree of roughness have a positive effect on osteoblast differentiation and subsequent bone mineralization  51  2 CHAPTER 2: Statement of problem and objectives  52 2.1 Background  The success or failure of implanted biomaterials depends in large part on the cellular interactions occurring at the bone-implant interface. A successful outcome requires not only successful wound healing but also successful bone formation. These processes are highly regulated and involve a complex interplay between a number of different cell types. At the early stages of endosseous wound healing, macrophages are among the first cells to come into contact with the implant surface and hence play a pivotal role in the events that follow. They have the potential to secrete myriad cytokines and growth factors that initiate tissue destruction as well as the healing or reparative response.  All cells implicated in bone formation are affected in significant ways by the surface characteristics of implanted devices. For this reason, surface topography is considered an important factor in the successful osseointegration of implants.  Today, most dental implants are made of either commercially pure titanium or titanium alloys because of the biocompatibility of these materials both in vitro and in vivo. To improve the bone integration of these titanium implants, surfaces of varying degrees of roughness have been developed through sandblasting, acid-etching, and a combination of the two. Straumann’s SLA surface is an example of a surface topography created by sandblasting and acid-etching that has been clinically successful.  53 2.2 Problem  The involvement of macrophages in tissue responses to implants is well documented. Since surface topography has the capacity to influence cell behavior, it is important to understand the responses of macrophages to the various types of surface topography currently in use. Thus, in vitro testing procedures are required to quantify the degree to which implants of different topographies elicit inflammatory and/or healing responses by macrophages. Such tests would enable the screening of topographies for their effects on macrophages before the surfaces were tested in animal models or clinical applications. Moreover, the in vitro approach provides an efficient method for designing topographies to produce desired responses. In this thesis, macrophage interactions with surface topography (SLA and its components) were investigated for their effects on cytokine and chemokine secretion using an in vitro model. Furthermore, the role of macrophage/surface topography in the generation of mineralized tissue by osteoblasts was studied using a transwell coculture model.  2.3 Hypotheses  1. Surface topography differentially affects macrophage attachment, proliferation, and morphology. 2. Attached macrophages selectively (depending on topography) secrete cytokines, chemokines, and growth factors that influence the local inflammatory response, cellular recruitment, and early events that take place during bone formation.  54 2.4 Rationale  Surface topography is considered an important factor in implant success. In our laboratory, implant surfaces of varying degrees of roughness have been implanted subcutaneously in rats (Chehroudi et al. 2009). Our observations revealed that macrophage-like cells attached to rough SLA surfaces in greater numbers than to either smooth or to grooved microfabricated surfaces in which each facet of the groove was smooth. Moreover, the SLA surfaces were associated with a greater percentage of bone formation than either the smooth or grooved surfaces. It has been suggested that increased macrophage activity and their secreted cytokines, chemokines, and growth factors contribute to the inflammatory and reparative changes that occur during endosseous wound healing. However, there is little direct evidence for the functional importance of macrophages or their secretions in bone formation.  2.5 Objectives  1. To quantify, using scanning electron microscopy (SEM), RAW 264.7 macrophage cell line adhesion to the four types of surface topography used in this study: polished (PO), coarsely blasted (CB),  acid etched (AE), coarsely blasted and acid etched (SLA) 2. To determine, using SEM, whether cell morphology of RAW 264.7 macrophages varies with surface roughness 3. To characterize the RAW 264.7 macrophage locomotion on SLA and related topographies in terms of velocity and persistence  55 4. To examine the effect of topography on RAW 264.7 macrophage activation using immunostaining of phosphotyrosine 5. To examine the effect of surface topography on modulation of RAW 264.7 macrophage secretion of pro-inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) and CC chemokines (CCL2 and CCL3) with and without a substimulatory dose of lipopolysaccharide in a factorial design. This design enables, for any response, the determination of the statistical significance of an interaction between the CB and AE topographic features. 6. To investigate the effects of RAW 264.7 macrophage secretions as results of surface topography on osteogenesis using a transwell coculture model  2.6 Significance  The results of the studies will help elucidate the effects of surface roughness on the macrophage contribution to tissue remodeling and bone formation at tissue-implant interfaces as well as possibly guide the future design and development of implant surfaces.  56  3 CHAPTER 3: Materials and methods  57 3.1 Substrata 3.1.1 Surface design  Titanium disks (15-mm diameter X 1-mm thick) of four surface topographies were used (Fig. 3-1). The surfaces were produced via (1) mechanical polishing (PO), (2) coarse sandblasting (CB), (3) acid etching (AE), and (4) a combination of sandblasting and acid etching (SLA) and were provided by Institute Straumann (Waldenburg, Switzerland). Macrophage activation and secretion of cytokines were investigated using an enzyme- linked immunosorbent assay (ELISA), the four topographies, and a tissue culture polystyrene (TCP) to serve as a control. One disk containing the four experimental surfaces in equal dimensions (5 mm per surface, total surface area = 1 cm2) was produced to assess macrophage morphology using scanning electron microscopy (SEM). Macrophage motility on 6 surfaces, including the four topographies (PO, CB, AE and SLA) as well as (5) glass cover slips and (6) titanium-coated glass cover slips, was assessed by means of time-lapse cinemicrography. Within a Transwell co-culture model, cytokines produced by macrophages on the various surfaces in the upper chamber could diffuse to influence osteoblasts growing on titanium-coated glass cover slips in the lower chamber.  PO, SLA, and titanium glass cover slips were used to investigate this cytokine-induced bone formation as indicated by alkaline phosphatase activity.    58 3.1.2 Replica preparation  Impressions of the four surface topographies (PO, CB, AE, and SLA) were made with poly(vinyl siloxane) impression material (Provil Light; Dormagen, HK, Germany). These poly(vinyl siloxane) negative replicas of the four surfaces were then used to cast positive replicas out of epoxy-resin (EPO-TEK 302-3; Epoxy Technology, Bellerica, MA), which were then placed in a fume hood for 24 hours for initial polymerization. To complete polymerization, the positive replicas were baked at 58°C for 3 days.  59   Figure 3–1: SEM view of the four surfaces used in this study: PO = polished; AE = acid etched; CB = coarsely blasted; and SLA = sandblasted, large-grit, acid etched.  60 3.1.3 Surface cleaning  The epoxy replicas were processed to ensure cleanliness and sterility for cell culture experiments. The three-step cleaning and sterilization process comprised: 1. Ultrasonication 2. Washing 3. Glow-discharge cleaning Steps 1 and 2 were completed prior to coating of the replicas in titanium; step 3 was completed afterward.  3.1.3.1 Ultrasonication  The epoxy surfaces were placed in a 250-mL plastic beaker filled with equal concentrations of water and 7x O-Matic Laboratory Detergent (MP Biomedicals, Irvine, CA). To remove any remaining particulate or organic debris adhering to the surfaces, the beaker was transferred to the ultrasonicator for 30 minutes.  3.1.3.2 Washing  After ultrasonication, the epoxy surfaces were washed 10 times with tap water, then washed 10 times again with distilled water. Such thorough washing was required to remove all traces of the highly concentrated detergent used in the ultrasonication process. Throughout these washings, the epoxy surfaces were handled with sterilized titanium  61 forceps to avoid potential contamination from organic matter or extraneous metals such as those found in stainless steel instruments, and care was taken to avoid scratching the surfaces. The epoxy surfaces were then dried overnight in a Petri dish under a tissue- culture laminar-flow fume hood.  62 3.1.3.3 Glow-discharge cleaning  This step of sterilization was completed after the epoxy surfaces were coated with titanium. Titanium-coated replicas were treated for 3 minutes in an argon glow-discharge chamber (Aebi and Pollard, 1987) to clean the surfaces and increase their surface energy, which is necessary for cell attachment and spreading (Meenaghan MA, et al 1979; Baier RE, et al 1984; Baier RE, et al 1982).  3.1.3.4 Titanium coating  After ultrasonication, washing, and drying, epoxy surfaces were sent to the electrical engineering department to be coated with 50 nm of titanium using a sputter-coating machine (Randex 3140 Sputtering System, Palo Alto, CA). Afterward, the titanium- coated replicas were glow-discharged for cell culture experiments as described above.  3.1.3.5 Surface characterization  Surface characterization and comparison between titanium-coated epoxy-resin replica surfaces and their originals has been described in detail (Wieland M, et al 2002; Schuler M, et al 2009). Briefly, for this process X-ray photoelectron spectroscopy (XPS) is used to determine the chemical composition of the oxide layer of the titanium-coated epoxy- resin replica surfaces (Table 3-1). Surface topographies of replicas prepared in this manner have been characterized using a noncontact laser profilometer (LPM) (UBM  63 Messtechnik, Ettlingen, Germany) equipped with a microfocus sensor (based on an autofocusing system) as well as a scanning electron microscope (Cambridge Stereoscan 260, Cambridge, UK) and the stereo SEM application. The quantitative topographic comparisons of the original surfaces and their replicas found that the replicas faithfully model the original surfaces. Tables 3-2 to 3-4 show the integral roughness parameter values (i.e., Ra, Rq, Rz) calculated from stereo SEM and noncontact LPM for both the original surfaces and their replicas (Schuler M, et al 2009).  64 Table 3.1: Quantitative surface composition (atom %), derived from XPS data, of titanium-coated epoxy resin replicas and polished commercially pure titanium surfaces (Adapted from Weiland M 2002).  Element  Ti-coated replicas (atom %) Glow-discharged Ti-coated replicas (atom %) Polished cp Ti surfaces (atom %)a  Titanium  17.3 ± 2.7  23.5 ± 2.6  26.1 ± 0.9  Oxygen  49.6 ± 2.9  54.5 ± 3.9  54.4 ± 2.0  Carbon  32.1 ± 4.7  20.9 ± 0.8  19.0 ± 2.9  Nitrogen  1 ± 0.2  1.1 ± 0.3  0.5 ± 0.1 n = 5; mean value ± SD aCleaned with organic solvents and passivated in 30 vol % nitric acid (HNO3)   Table 3.2: Roughness parameters Ra and Rq of smooth surfaces, typically used in cell culture assays, measured with atomic force microscopy on a 10  10 µm2 area (Schuler M et al 2009). Surface Ra (nm) Rq (nm) Tissue culture polystyrene (TCP)a  2.30 ± 0.20 2.80 ± 0.20 Epoxy replica coated with 60-nm TiO2b  2.66 ± 0.31 3.33 ± 0.42 Si wafer coated with 60-nm TiO2  0.44 ± 0.02 0.60 ± 0.03 Polished CP titanium disk  2.50 ± 0.57 3.35 ± 0.96 aMaster surface for replicating the smooth topography; mean values of 3 samples bMean values of 8 consecutive casts using the same negative replica Ra: Arithmetic average of absolute values of all points of the profile Rq: Root mean square of values of all points of the profile  65 Table 3.3: Roughness values (Ra, Rq, Rz, and Sm) of an SLA commercially pure titanium disk and its 8 TiO2-coated epoxy replicas measured with confocal white light microscopy on a 770  770 µm2 area (Schuler M et al 2009). Surface Ra (µm) Rq (µm) Rz (µm) Sm (µm) SLA CP Ti diska  3.90 ± 0.08 4.93 ± 0.06 18.33 ± 0.15 32.76 ± 0.63 Epoxy replica coated with 60-nm TiO2b 4.08 ± 0.04 5.10 ± 0.05 19.30 ± 0.18 28.65 ± 0.46 aMean values of 3 samples bMean values of 8 consecutive casts using the same replica Ra, Rq: see Table 3-2 for explanations Rz: arithmetic average of maximum peak-to-valley height Sm: Arithmetic average spacing between fallings flanks of peaks on mean line   Table 3.4: XPS elemental concentrations of TiO2-coated epoxy replica, TiO2-Coated Si wafer, and commercially pure titanium SLA disk measured on a 6 mm2 area (Schuler M et al 2009). Smooth surfaces SLA surfaces Elementa (at%)  TiO2-coated epoxy replica TiO2-coated Si wafer TiO2-coated epoxy replica CP Ti disk Ti  23.0 ± 0.1 25.5 ± 1.3 22.7 ± 1.1 26.8 ± 1.3 O  68.2 ± 0.9 70.8 ± 0.9 66.9 ± 0.9 65.8 ± 0.9 C  8.8 ± 0.2 3.7 ± 0.1 10.4 ± 0.2 7.3 ± 0.1 aMean values of three samples.  66 3.2 Rats and cellular activators  Sprague-Dawley rats were purchased from the UBC Animal Care Facility. The rats were maintained on a standard diet with access to water ad libitum. Rat experiments were approved by the Animal Care Committee, University of British Columbia. Lipopolysaccharides (LPS) from E. coli were purchased from Sigma, St. Louis, MO.  3.3 Cell populations  The RAW 264.7 cell line used in this thesis was obtained from American Type Culture Collection (ATCC, Manassas, VA). The osteoblast-strain were derived from neonatal rat calvaria.  3.3.1 Macrophages and culture conditions  The RAW 264.7 macrophages were cultured on TCP in 75-cm2 flasks (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) and grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Rockville, MD) and an antibiotic mixture consisting of 0.5 mg penicillin G (Sigma), 5 mL gentamycin sulfate (Sigma), and 60 mL amphotericin B (Fungizone; Gibco, Grand Island, NY). They were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells were routinely passaged by scraping with a disposable cell scraper (Fisher Scientific, Edmonton, Canada) and divided 1:5 for subculture. For induction of cytokines,  67 the cell lines were counted electronically with a cell counter (Coulter Electronics, Luton, England) and then plated onto the titanium-coated epoxy-resin replica surfaces at a population density of 5 5 105 cells per well.  3.3.2 Osteoblast strain and culture conditions  Osteogenic cells were enzymatically isolated from newborn Sprague-Dawley rat calvaria and cultured as previously described (Aubin JE 1998 andWieland M, et al 2002). In brief, osteoblast cells were isolated from the frontal, parietal, and occipital bones of newborn (24- to 48-hour) Sprague-Dawley rats by sequential digestion in a mixture containing 180 U/mL clostridial collagenase, type 1a (Sigma), and 5 mg/mL trypsin (Gibco, Burlington, Ontario, Canada). Osteoblasts were then cultured on TCP in 75 cm2 flasks (Becton Dickinson Labware) in alpha minimal essential medium (α-MEM) (STEMCELL Technologies), 15% newborn bovine serum (NBS) (Cansera; Rexdale, Ontario, Canada), and an antibiotic mixture consisting of 100 µg/mL penicillin G (Sigma), 50 µg/mL gentamycin (Sigma), and 3 µg/mL amphotericin B (Fungizone), and then incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The primary culture was designated subculture zero.  Once the osteoblasts strain reached confluence, the medium was removed and the cells were rinsed with 10 mL of a 0.25% trypsin solution consisting of 0.5 g trypsin (Gibco) in 200 mL citrate saline (pH 7.8) and 10% glucose (Sigma). The washing trypsin was then removed, and an additional 10 mL of the trypsin solution were added to the cells. The  68 cells were incubated at 37°C for 10 minutes until they detached from the floor of the flask. Afterward, another 10 mL of medium was added, and the entire contents of the flask were transferred to a sterile test tube and centrifuged at 1,500 rpm for 5 minutes. Thereafter, the supernatant was discarded and the pellet was resuspended in 10 mL of warm α-MEM supplemented with 15% fetal calf serum and antibiotics.  For osteoblast strain cultures, the medium was changed 3 times weekly and divided 1:2 for subculture. Osteoblasts strain from subcultures 1, 2, and 3 were used in the co-culture model to assist mineralization resulting from macrophage secretions.  3.3.3 Media supplements  When macrophages/osteoblasts strain were used in the co-culture model, the culture medium was supplemented with 58 µg/mL L-ascorbic acid phosphate magnesium salt n- hydrate (Wako Pure Chemical Industries, Richmond, VA) and 3.15 mg/mL Na-β- glycerol-phosphate (Sigma) from day 1 of the experiment. The medium was changed twice a week. The supplements were added to promote mineralization.  3.4 Macrophage adhesion  Macrophage adhesion was quantified by DAPI staining  of DNA as well as by SEM.   69 3.4.1 DAPI staining  After the supernatants were harvested, the macrophage monolayers attached to the surfaces were covered with 2 mL of Tris buffer and sonicated using an ultrasonic processor with a microtip attachment (Branson Sonifier 250; VWR Scientific, Danbury, CT). Macrophage disruption was checked using phase-contrast microscopy. To assess DNA content, 4’,6-diamidino-2-phenylindole (DAPI only fluoresces when bound to DNA) was added to each well to a final concentration of 0.5 µg/mL. The contents of the wells were then transferred to a cuvette and read in a fluorescence spectrofluorometer at 365 nm excitation and 460 nm emission. The concentration of DNA was calculated from a standard curve obtained using known concentrations of DNA (Shapira L,et al 1993). Finally, all of the results obtained from the assays were standardized (ng/µg DNA/mL).  3.4.2 SEM investigation  Macrophage morphology and enumeration on each of the four titanium surface topographies was assessed via SEM.  To assess macrophage morphology, one each of the four experimental surfaces (SLA, AE, SB, and PO) was assembled in equal dimensions (5 mm per surface). The RAW 264.7 cells (2 X 104/mL) were cultured on these surfaces for periods of 24 and 48 hours. At the end of these periods, the surfaces were washed in warm (37оC) RPMI-1640  70 medium (STEMCELL Technologies), fixed for 1 hour in 2.5% glutaraldehyde (Sigma) at 4оC, and rinsed 3 times in 0.1 M phosphate-buffered saline (PBS).  The next treatment steps were performed using a microwave technique (3470 Hornet Microwave System; Pelco International, Redding, CA). Macrophages were irradiated in the cold spot area of the microwave at 37°C at 750 W of power for post-fixation and at 45°C at 750 W of power for dehydration. The cells were further fixed in 2% osmium tetroxide (OsO4) in PBS for 40 seconds, then rinsed twice in 0.1 M PBS for 30 seconds. This was followed by a 2% tannic acid treatment (JBS-Chem, Dorval, Quebec, Canada) for 40 seconds to enhance the osmium fixation. The cells were again rinsed two times in 0.1 M PBS for 30 seconds each. The macrophages were then treated again with 2% OsO4 for 40 seconds, followed by two rinses in 0.1 M PBS. After that, the macrophages were dehydrated with graded ethanol (two times each with 50%, 70%, and 90% ethanol for 30 seconds each time, and three times with 100% ethanol for 40 seconds each time). They were then critical point dried with CO2 (Samdri-795 Critical Point Dryer; Tousimis, Rockville, MD), and sputter-coated (Hummer VI Sputtering System; Technics, Alexandria, VA) with 10 nm of gold.  The cells were observed under SEM (Cambridge Stereoscan 260) at an accelerating voltage of 10 to 12 keV, and images were captured using Orion 10 software. The number of cells were averaged and counted at 500X magnification to allow macrophages located within the pits of the SLA structure to be counted. Five random fields per surface and three surfaces with the same topography were selected for enumeration. Cellular  71 spreading was assessed based on the presence of cell processes and the elongation of the central cytoplasmic region. The numbers of spread and round macrophages were also counted in five fields per surface. Mean numbers were calculated for each titanium surface used in the study.  3.5 Time-lapse cinemicrography  Time-lapse cinemicrography was used to monitor macrophage motility on the different surface topographies. Macrophages were harvested, adjusted to 1 X 106/10 mL, and centrifuged for 5 minutes at 1,500 rpm. Then the macrophage pellet was diluted in 3 mL of RPMI-1640 medium containing 5 µL fluorescent stain (CellTracker Orange CMTMR Molecular Probes; Invitrogen, Carlsbad, CA) and incubated for 30 minutes at 37ºC. Afterward, cells were centrifuged for 5 minutes at 1,500 rpm, and the pellet was again washed with 10 mL RPMI-1640 medium and again incubated for 30 minutes at 37ºC. Thereafter, cells were centrifuged for 5 minutes at 1,500 rpm and the pellet was diluted in medium containing serum and antibiotics. Cells were plated on the surfaces (PO, CB, AE, and SLA) at a final concentration of 1 X 104 cells/300 µL and incubated for 30 minutes. Then the surfaces were mounted on glass slides and placed into a Pentz chamber (Bachofer, Reutlingen, Germany); the chamber was injected with medium containing serum and antibiotics using a sterile needle. The chamber was then placed in a stage incubator (Bachofer) that controls temperature (37°C), humidity, and CO2 concentration inside the chamber. Cell motility was viewed with epifluorescence microscopy equipped with a video camera (imaging-Q) and Northern Eclipse software (Empix Imaging,  72 Mississauga, Ontario, Canada). Images of cells were captured every 10 minutes for 5 hours. The images were edited using Adobe Photoshop software.  3.6 Production of cytokines and chemokines  The macrophage cell lines RAW 264.7 was adjusted to 5 X 105 cells/mL in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and the antibiotic mixture described above. The cells were plated in quantities of 1 mL onto the titanium-coated epoxy resin replica surfaces (PO, CB, AE, and SLA) in 24-well tissue culture plates (Falcon; Becton Dickinson Labware). All samples were set up in triplicate.  For production of pro-inflammatory cytokines and chemokines, LPS was added at the suboptimal dose of 6.4 ng/mL to one set of the surfaces (PO, CB, AE, and SLA), while a second set of surfaces contained only macrophages. This suboptimal dose was established based on the response of macrophages to serial dilutions of LPS; 6.4 ng/mL was the concentration that failed to produce cytokines on TCP. The purpose of using it was to determine whether surface topography acted synergistically with LPS.  An additional reason for testing the effect of surface topography in the presence of LPSs is the likelihood that all implanted materials would be exposed to LPSs in vivo. There is evidence that LPSs are present to some degree even under aseptic conditions. For example, LPSs are thought to be found at the surgical site and because of bacterial colonization (Fürst MM et al 2007)  73 The cultures were incubated for various time periods as established by the design of the experiments. Supernatants were then collected and stored at –80ºC for use in further assays. TCP wells and TCP wells plus LPS (E.coli, Sigma) were employed as negative and positive controls, respectively. Pro-inflammatory cytokines (interleukin-β [IL-β], interleukin 6 [IL6], and tumor necrosis factor-α [TNF-α]) and CC chemokines CCL 2 [(monocyte chemoattractant protein-1 [MCP-1]] and CCL 3 [macrophage inflammatory protein -1α [MCP-1α] were assayed using ELISA kits.  3.7 Sandwich ELISA assay 3.7.1 Cytokine and chemokine determinations  Levels of cytokines (TNF-α, IL-1β, and IL-6) and chemokines (MCP-1 and MIP-1α) were measured with sandwich enzyme-linked immunosorbent assays (ELISA) following manufacturer instructions (Quantikine; R&D Systems, Minneapolis, MN). Briefly, 50 µL of standard, control, or sample supernatants were added per well. The test samples were then incubated for 2 hours at room temperature. After unbound substances were washed away, 100 µL of an enzyme-linked polyclonal antibody were added per well, and the samples were incubated for 2 hours at room temperature. Following a second washing to remove any unbound antibody-enzyme reagent, 100 µL of substrate solution were added to each well, and the samples were incubated in darkness for 30 minutes. At this point a stop solution was added to each well and its optical density was determined using a microplate reader (Flow Laboratories, Titertek Multiskan, Mississauga, Ontario, Canada)  74 set to 450 nm and corrected at 570 nm. The sample values were determined by comparison with a standard curve. All samples were assayed in triplicate.  3.8 Bone formation 3.8.1 Transwell co-culture  Osteoblasts strain (2 X 105 per well) were plated on the titanium glass cover slip (tested surfaces) in the lower compartment of a 6-Transwell plate that was 24 mm in diameter and had a 0.4-µm pore size (Costar; Corning, Corning, NY). Macrophages (5 X 105 per well) were seeded on SLA and PO titanium surfaces in the upper compartments (Fig. 3- 2). The Transwell plate allows exchange of soluble components of  the medium  such as cytokines, but not cells. That is, direct cell-to-cell interactions between osteoblasts and macrophages were inhibited by polycarbonate membranes with 0.4-µm pores. Both cell types were fed with α-MEM, 15% NBS, and an antibiotic mixture (as described above) and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Further, to promote mineralization, osteoblasts were treated with 58 µg/mL L-ascorbic acid phosphate magnesium salt n-hydrate and 3.15 mg/mL Na-β-glycerol-phosphate from day 1 of the experiment. The medium was changed twice a week. Inserts (i.e., upper compartments) with feeding medium free of cells and macrophages were employed as a control.  75   Figure 3–2: Schematic diagram of the co-culture model. Macrophages were plated in the upper compartments (inserts) onto surfaces and/or directly onto the membrane and into the lower compartments, where osteoblasts strain were plated onto tested surfaces. SLA = sandblasted and acid etched; PO = polished; Mac = macrophages; Osteo = osteoblasts; Ti-GCS = titanium glass-covered slip; L comp = lower compartment.   76 3.8.2 Macrophage secretion and mineralization  The effect of macrophage secretions on osteoblasts (i.e., increased alkaline phosphatase activity and formation of bone-like nodules) was assessed over three periods to investigate its change over time. First, to investigate an early effect, inserts with macrophages plated on SLA and PO titanium surfaces were placed for 1 week and then removed. Second, to investigate a later effect, inserts with macrophages plated onto SLA and PO titanium surfaces were added after 1 week. Third, to investigate the effect of continuous macrophage secretion on osteoblasts, inserts with macrophages plated onto SLA and PO titanium surfaces were added from day 1. Osteoblasts were plated on the titanium glass cover slip (tested surface) on the lower compartment of a 6-Transwell plate from day 1 and fed with α-MEM, 15% NBS, and antibiotics supplemented with 58 µg/mL L-ascorbic acid phosphate magnesium salt n-hydrate, 3.15 mg/mL Na-β-glycerol- phosphate, and 5 µg/mL alizarin complexone over the next 4 weeks of the experiment. Toward the end of the experiment (4 weeks), the osteoblasts plated onto titanium glass cover-slips (tested surfaces) were examined for bone-like nodule formation.  77              Figure 3–3: Schematic diagram of the three period sequences used to investigate mineralization. CE = continuous effect; LE = late effect; EE = early effect. Upper compartments (inserts) where macrophages were plated onto surfaces and/or directly on the membrane, and lower compartments where osteoblasts were plated onto tested surfaces.   78 3.8.3 Alkaline phosphatase activity assay  Measurement of alkaline phosphatase (ALP) activity, an early osteoblast marker method, is based on the hydrolysis of p-nitrophenolphosphate into p-nitrophenol under alkaline conditions. ALP activity was measured at 1, 2, and 4 weeks using a commercial kit (Sigma). Osteoblasts cultured on titanium glass cover-slips were fixed in 60% citrate- buffered acetone for 30 seconds and rinsed in deionized water for 45 seconds. Then the samples were stained using a diazonium-coupling reaction (Fast Blue RR salt and naphthol AS-MX phosphate alkaline solution) for 30 minutes. Afterward, samples were rinsed thoroughly in deionized water for 2 minutes. All staining procedures were carried out at room temperature. Surfaces stained for alkaline phosphatase were observed using bright-field microscopy and a 205 objective lens (Jena-Pol; C. Zeiss, Jena, Germany) equipped with a digital camera (EOS D60; Canon, Tokyo, Japan) and controlled by remote capture program. The amount of alkaline phosphatase activity (ALP) was assessed using NIH image software by measuring the area of the surface that stained positive with the dye (purple-blue color). The scale was calibrated based on an object of known size using the same magnification (205). Ten randomly selected images were captured per surface, and the images were imported into NIH Image (National Institutes of Health, Bethesda, MD). Contrast between the staining and the surfaceswas maximized, and the area of staining was highlighted using the NIH Image software. The threshold for each image was then set and the area of staining measured. The measurements were imported into a Microsoft Excel spreadsheet and the mean percentage surface area of staining was calculated for each surface.  79 3.9 Bone-like nodule formation  Bone nodule formation was assessed by means of an alizarin complexone (AC) assay, which relies on the chelation of the alizarin red S dye with a calcium molecule (red- orange color). After 4 weeks, samples were washed in warm α-MEM, fixed in 10% buffered formalin phosphate (Fisher Scientific) for 1 hour at room temperature, and rinsed 3 times in deionized water.  3.9.1 Epifluorescence microscopy  Nodules incorporating alizarin complexone were identified with an Axioskop 2 epifluorescence microscope (C. Zeiss, Oberkochen, Germany) using ultraviolet light (λmax = 488 nm) with a 205 objective. Images were captured with a low light level charge-coupled device (CCD) camera (PentaMax 12 bit CCD; Princeton Instrument, Trenton, NJ) using Northern Eclipse 6.0 software. All images were saved in TIF format. The percentages of surface area comprising bone-like nodules were estimated using NIH Image software.  3.9.2 NIH image protocol and photoshop editing  In the beginning, all of the frames were edited in Photoshop, grayscaled, auto-leveled, and size adjusted. NIH image was then calibrated to a known distance, and the surface was measured under the same magnification used for the picture (20X). The known  80 distance was 175 µm and number of pixels was 197; hence, the resulted scale was 1.26 pixels/µm. The total surface area of the frame was calculated under the same magnification and scale. After that, the surface area occupied by ALP staining and bone- like nodules was estimated based on the scale and threshold as calculated. Finally, the percentage of the surface area was calculated.  3.10 Phosphotyrosine immunostaining  Macrophages were plated on SLA and PO surfaces at a density of 1.5 X 105 cell/mL for 1 hour. Samples were washed in 0.1M PBS for 2 minutes, fixed in 10% formaldehyde for 5 minutes, and washed in PBS 3 more times for 5 minutes each. Permeabilization was performed in 0.5% Triton X-100 (Sigma) for 5 minutes, and the cells were washed 1 time in PBS for 2 minutes. Blocking of nonspecific binding was performed using 1% bovine serum albumin (BSA, Sigma), and samples were incubated with primary monoclonal IgG2b antibody anti-mouse p-tyr (PY 99; Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 1:100 for 1 hour. The samples were then washed 3 times in 0.1% BSA in PBS for 10 minutes each and incubated with the secondary antibody Alexa Fluor goat anti-mouse IgG conjugated to Texas red (Molecular Probes, Oregon, WA) at a concentration of 1:200 for 1 hour. All antibodies were diluted with PBS containing 0.1% BSA. The samples were then washed in PBS 5 times for 5 minutes each.  Labeling for phosphotyrosine was visualized on a Nikon confocal microscope (Eclipse E600) using magnifications up to 63X objectives. Images were captured using EZ-C1 software (EZ C12.3 Nikon). Images were enhanced with Photoshop 8.0 using the level feature.  81 3.11 Statistics  All data were analyzed using the software program SPSS Version 10 for Windows XP (Chicago, IL). Statistical comparison between the surfaces and the controls was performed using the 2-way analysis of variance (ANOVA) including interaction effect between surface and time. Post hoc analysis was performed using the Bonferroni and Tukey multiple comparison adjustment testing. P < 0.05 was the level adopted to reject the null hypothesis and thus establish significance.  82  4 CHAPTER 4: Morphologic studies of macrophage cell line RAW 264.7 on surface topographies of varying roughness  83 For long-term survival of dental implants, osseointegration must be established and maintained. Along with infection and mechanical factors, host immunologic response is a potential cause of implant failure. While immunologic responses to biomaterials vary, it has been shown that failed implants can be associated with a chronic inflammatory response, and when this occurs, macrophages are preferentially located near the tissue- biomaterial interface.  Given the plasticity of macrophage behavior, their activity at the tissue-biomaterial interface of failed implants is not fully understood. It is clear that macrophages can interact with a variety of surface topographies. Indeed, the roughness of a topography influences the level of attachment, the morphology, and the secretory profile of macrophages, as described in chapter 1. This chapter investigates the morphology of RAW 264.7 cells when interacting with four surface topographies (polished, acid etched, sand blasted, and sand blasted–acid etched).  4.1 Scanning electron microscopy of RAW 264.7 on different surface topographies  The morphology of macrophages associated with the four surface topographies at 24 and 48 hours is shown in Fig. 4-1. In general, the SEM investigation revealed that the number of macrophages increased with time of incubation; further, the SLA surface was found to contain the greatest number of macrophages at 24 and 48 hours (Fig. 4-2). This time- related increase reflects either increased initial adherence, or later enhanced proliferation, or both in relation to the SLA surface compared to the other surfaces. On PO, CB, and  84 AE surfaces, no significant change in macrophage number was discerned. These SEM observations of macrophage adhesion/proliferation were confirmed by the DAPI assay (Fig. 4-3).  While SEM investigations demonstrated the capability of macrophages to exhibit diverse shapes and structures, including spread, flattened, ruffled, and elongated forms, on each of the titanium-coated epoxy replicas, an increased spreading was found on the roughest surface (SLA). Similar to the observations regarding macrophage adhesion, the amount of spreading on the PO, SB, and AE surfaces did not differ significantly. Rounded macrophages were also found on all titanium-coated epoxy replicas (Fig. 4-4A).However, this findings were not significant when the cells spreading were calculated in percentage. This might be explained by indulging of macrophages within the fine features of SLA surface and their ability to build bridging layers, masking their correct number calculation (Fig. 4-4B). Macrophages grown on rougher surfaces (i.e., CB and SLA) displayed more extended processes that stretched over the coarse pores and developed a more complex three-dimensional shape, reflecting the underlying surface. For example, macrophages grown on the SLA surface expressed varying morphologic characteristics, including significantly elongated and more fully developed layers of cells that bridged over each other. Moreover, macrophage processes reached into deep pores and provided multiple points of attachment that anchored fine cell processes within the microstructure of the SLA surface (Fig. 4-5).  85  Figure 4–1: a and 4-1b. SEM images show macrophage morphology on the four titanium- coated epoxy replica used in this study (a) after 24 hours and (b) after 48 hours.  86   Figure 4–2 Number of attached macrophages, based on SEM observations of the four titanium-coated epoxy replica used in this study, at 24 & 48 hours. Asterisk denotes p < 0.05. Only on the SLA surface did the number of macrophages increase significantly between 24 and 48 hours.  87   Figure 4–3: Number of attached macrophages, as determined by the DAPI assay, on the four titanium-coated epoxy replicas used in the study after 24 and 48 hrs. Asterisk denotes p < 0.05.  88    Fi ep W tes co  A B gure 4–4:  Number of spread and round macrophages on the four titanium-coated oxy replicas used in the study after 24 and 48 hours. Asterisk denotes p < 0.05. (A). hen the number recalculated in percentage, no significant was found between all the ted surfaces, however, the percentage of cell spreading at SLA is still higher mparing to PO at 24 and 48 hours(B). 89   Figure 4–5: Macrophages expressed varied morphology on the SLA surface. They were capable of stretching into deep pores and establishing multiple points of attachment that anchored fine cell processes within the microstructure of the SLA surface (D).  90 4.2 Time-lapse microscopy  Cell movement on the different surface topographies was assessed by means of time- lapse microscopy. In general, macrophages either remained stationary or exhibited an oscillatory and stationary type of movement (i.e., limited movement with no overall change in position), and no significant differences were found between any of the experimental surfaces (PO, CB, AE, SLA) and the titanium glass cover slip (GCS). Figure 4-6 presents a series of images of macrophages on SLA and Ti GCS surfaces stained with fluorescent dye. Almost all of the macrophages on the SLA surfaces maintained their positions over the entire 5-hour length of the time-lapse microscopy study, whereas a few cells on the Ti GCS surface expressed a slight movement and the majority of cells exhibited an oscillatory type of motion. Although macrophages showed no signficant net movement, some 70% to 80% of  the cells on all experimental surfaces exhibited oscillations around their initial location.In general, cells were alive and tightly adhere to rough surfaces, however they exhibited no preferred direction of migration, because they was no consistent cue specifying a direction of migration.  91  Figure 4–6 a and 4-6b: show a series of images of macrophages stained with the fluorescent dye Cell Tracker Orange from the (a) SLA and (b) titanium GCS time-lapse studies. Circles reveal that no movement, whereas arrows reveal slight oscilation.    92 4.3  Macrophage activation  To examine the effect of surface topography on macrophage activation, immunostaining of phosphotyrosine was carried out 24 hours after the samples were plated. Phosphotyrosine was positively stained in the macrophages associated with both SLA and PO surfaces (Fig. 4-7). On the SLA surface, the macrophages were spread apart to a greater degree and had more intense staining in the structures resembling focal adhesions might be present. The spreading of the macrophages on the SLA surface, including their attachments within the pits, has the consequence that not all parts of the cell are in the same focal plane, and any one focus level may not demonstrate the total number of focal adhesions present. Thus the number of focal adhesions of macrophages on SLA surfaces will tend to be underestimated unless three-dimensional reconstructions are performed.  93   20 µm20 µm PO SLA  Figure 4–7: Macrophage staining for phosphotyrosine after 24 hours. Phosphotyrosine was positively stained and present throughout the macrophages associated with both SLA and PO surfaces and was also present at structures typical of focal adhesion.  94 4.3 Conclusion  The results described in this section demonstrate that macrophages can attach to rough surfaces and that the types of rough surface to which they attach affect their proliferation and shape. Macrophages proliferated on the SLA surface between 24 and 48 hours, but significant increases in cell numbers were not observed on the other surfaces. The SLA surface in particular allowed for greater spreading of the macrophages, and the multiple attachment points provided by the rough complex surface were often associated with phosphorylated tyrosine indicative of activated cell signaling pathways, possibly related to signaling molecules at those sites. Activation of signaling pathways carries the implication that cell functions such as secretion might possibly be activated as well, and this possibility is tested in Chapter 5.  95  5 CHAPTER 5: Effect of titanium surface topography on macrophage activation and secretion of pro-inflammatory cytokines and chemokines The results shown in this section was published in J Biomed Mater Res 70A: 194–205, 2004 and cited more than 35 times (Refai AK et al, 2004)   96 The potential role of macrophages in the performance and longevity of an implant is not well understood. The macrophage is one of the first cells to arrive at the tissue-implant interface. Macrophage interaction with implanted materials is believed to involve adhesion, activation, and secretion of cytokines at the implant site. Typically, macrophages prefer rough surfaces to smooth ones (a phenomenon called rugophilia) and elongate significantly on grooved substrata. This preference was confirmed by the results reported in chapter 4, where RAW 264.7 cells  demonstrated increased adherence/proliferation and spreading on the SLA surface after 48 hours compared with other surfaces.  Macrophages are a major source of cytokines, chemokines, and growth factors. Cytokines and chemokines are involved in both the destructive and reparative stages of early wound healing following insertion of the implant. Pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and chemokines such as CCL2 (monocyte chemoattractant protein-1) and CCL3 (macrophage inflammatory protein-1α) are widely expressed at these early stages as well. Their presence at the wound-healing site offers evidence of the important role they perform in directing host responses such as cellular recruitment and possibly bone formation. In this chapter, the topographies of the four titanium surfaces were investigated for their capacity to induce production of these pro-inflammatory cytokines in RAW macrophages.    97 5.1 Cytokine responses to surface topography 5.1.1 Production of tumor necrosis factor-α 5.1.1.1 Unstimulated macrophages  At 6 hours, TNF-α secreted by unstimulated macrophages cultured on the acid-etched (AE) surface increased significantly compared to the amounts secreted on the tissue culture polystyrene (TCP), mechanically polished (PO), coarsely sandblasted (CB), or sandblasted and acid-etched (SLA) titanium surfaces (Fig 5-1 p ≤ 0.05). At 24 hours, the amount of TNF-α secreted by the macrophages cultured on SLA and AE surfaces was significantly greater than that secreted on all other titanium surfaces, and the response to the AE surface was the greatest of all (p ≤ 0.05). By 48 hours, TNF-α secreted by the macrophages cultured on the AE surface was significantly greater than that on all other titanium surfaces (p ≤ 0.05).  5.1.1.2 Stimulated macrophage  After 6 hours of incubation, there was no difference in the amount of TNF-α secretion when lipopolysaccharide- (LPS) stimulated macrophages were cultured on all titanium surfaces; the levels were significantly increased only as compared to the TCP surface (Fig. 5-2 p ≤ 0.05). After 24 and 48 hours, the macrophages cultured on SLA surfaces secreted ninefold and fourteenfold more TNF-α, respectively, than the amounts secreted by unstimulated macrophages cultured on TCP over the same periods. The increased production of TNF-α by the macrophages on the SLA surface was statistically significant  98 when compared to all other surfaces, including the positive control (TCP + LPS), as well (p ≤ 0.05). TNF-α levels secreted on the other titanium surfaces, including PO, CB, and AE, did not vary significantly.  99  Figure 5–1: TNF-α production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. At 24 hours, TNF-α secreted by the macrophages cultured on SLA and AE surfaces increased significantly compared to that of all other titanium surfaces (p ≤ 0.05). The TNF-α content was measured by enzyme-linked immunosorbent assay (ELISA) and quantified by 4',6- diamidino-2-phenylindole (DAPI) staining. Data are presented as mean ± SEM, and the studies were performed in triplicate. Two-way analysis of variance (ANOVA) and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisk denotes p ≤ 0.05 compared to all titanium surfaces and to TCP at each period tested. Reproduced with permission from (Refai AK et al , 2004)  100   Figure 5–2: TNF-α production by lipopolysaccharide- (LPS) stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. The macrophages cultured on SLA surfaces secreted significant amounts of TNF-α compared to all other tested surfaces at 24 hours and 48 hours (p ≤ 0.05). The TNF-α content was measured using ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP + LPS at each time tested. Reproduced with permission from (Refai AK et al , 2004)  101 5.1.2 Production of interleukin-1β 5.1.2.1 Unstimulated macrophages  No IL-1β secretion was detected in unstimulated macrophage cultures on any surface at any of the times tested.  5.1.2.2 Stimulated macrophages  Treatment of macrophages with LPS resulted in significant IL-1β production (Fig 5-3). For example, IL-1β levels produced by stimulated macrophages cultured on SLA surfaces increased approximately sixfold at 24 hours and sixteenfold at 48 hours compared to unstimulated macrophages cultured on TCP (data not shown). At 6 hours, IL-1β production by the macrophages cultured on the PO surfaces increased significantly compared to that on the SLA surfaces. IL-1β production by the macrophages cultured on the positive control (TCP + LPS) also increased significantly at 6 hours compared to that on all other surfaces (p ≤ 0.05). At 24 hours, the level of IL-1β secretion by the macrophages cultured on the SLA surface increased significantly compared to that on the PO and AE surfaces (p ≤ 0.05). At 48 hours, the level of IL-1β secretion by the macrophages increased on the SLA surface compared to all other surfaces, including the positive control (TCP + LPS) (p ≤ 0.05).   102  Figure 5–3: Interleukin-1β (IL-1β) production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours.Treatment of macrophages with LPS resulted in significant IL-1β production; IL1-β was not detected in unstimulated cultures. The IL1-β content was measured by ELISA and quantified by DAPI. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP + LPS at each time tested. Reproduced with permission from (Refai AK et al , 2004)  103 5.1.3 Production of interleukin-6 5.1.3.1 Unstimulated macrophages  Like IL-1β, IL-6 was undetectable in the unstimulated macrophage cultures on any surface at any of the times tested.  5.1.3.2 Stimulated macrophages  Similar to IL-1β, IL-6 production increased significantly in the presence of LPS (Fig 5- 4). For example, IL-6 levels produced by stimulated macrophages cultured on SLA surfaces increased approximately sevenfold at 24 hours and thirteenfold at 48 hours compared to unstimulated macrophages cultured on TCP (data not shown). At 6 hours, IL-6 production by the macrophages cultured on SLA surfaces increased significantly compared to that on TCP (p ≤ 0.05). At 24 hours, the level of IL-6 produced by the macrophages cultured on the SLA surface was greater than that observed on the TCP, TCP + LPS, PO, or AE surfaces (p ≤ 0.05). At 48 hours, IL-6 levels produced by the stimulated macrophages on the SLA and AE surfaces differed significantly and were greater than those on all other surfaces, including the positive control  (p ≤ 0.05).   104   Figure 5–4: Interleukin-6 (IL-6) production by LPS-stimulated macrophages (6.4 ng/mL) cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. IL-6 production was not detected in unstimulated macrophages. The IL-6 content was measured by ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP + LPS at all times tested. Reproduced with permission from (Refai AK et al , 2004)  105 5.2 Chemokine responses to surface topography  The surface topography on which the unstimulated and stimulated macrophages were cultured also influenced the secretion of chemokines.  5.2.1 Monocyte chemoattractant protein-1 production 5.2.1.1 Unstimulated macrophages  Generally, the SLA surface was found to downregulate secretion of monocyte chemoattractant protein-1 (MCP-1) in unstimulated macrophages at 6 hours, 24 hours, and 48 hours compared to the effect of the other surfaces (Fig 5-5). This decrease in secretion of MCP-1 was statistically significant compared to the AE surface at 6 and 24 hours and the PO surface at 24 hours (p ≤ 0.05).  5.2.1.2 Stimulated macrophages  LPS treatment of macrophages cultured on the SLA surface resulted in a fourfold, eightfold, and fourteenfold increase in the secretion of MCP-1 compared to unstimulated macrophages cultured on an SLA surface at 6, 24, and 48 hours, respectively (Fig. 5-6). At 6 hours, MCP-1 production by the macrophages cultured on all titanium surfaces— including the positive control (TCP + LPS)—was significantly greater than that observed on the TCP surface (p ≤ 0.05). At 24 hours, the level of MCP-1 produced by the macrophages cultured on the SLA surface increased significantly compared to that  106 produced on all other surfaces except CB and the positive control (TCP + LPS) (p ≤ 0.05). At 48 hours, the level of MCP-1 produced by the macrophages cultured on the SLA and AE surfaces was significantly greater than that observed in cultures prepared on all other surfaces, including the positive control  (p ≤ 0.05).  107   Figure 5–5: Monocyte chemoattractant protein-1 (MCP-1) production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. The SLA surface downregulated MCP-1 production in unstimulated macrophages at 6, 24, and 48 hours compared to the effect of all other surfaces (p ≤ 0.05). The MCP-1 content was measured by ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two- way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP at each time tested. Reproduced with permission from (Refai AK et al , 2004)  108  Figure 5–6: MCP-1 production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. LPS treatment resulted in an approximately fourfold, eightfold, and fourteenfold increased secretion of MCP-1 by macrophages cultured on the SLA surface compared to the secretion of MCP- 1 by unstimulated macrophages cultured on the SLA surface at 6, 24, and 48 hours, respectively (p ≤ 0.05). The MCP-1 content was measured by ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP + LPS at each time tested. Reproduced with permission from (Refai AK et al , 2004)  109 5.2.2 Production of macrophage inflammatory protein-1α 5.2.2.1 Unstimulated macrophages  The SLA surface was also found to downregulate macrophage inflammatory protein-1α (MIP-1α) secretion in unstimulated macrophages at 6 hours, 24 hours, and 48 hours compared to the effect of the other surfaces (Fig 5-7). This downregulation effect was statistically significant compared to the effect of the AE surface at 6 hours, all of the other surfaces at 24 hours, and the AE surface at 48 hours (p ≤ 0.05).  5.2.2.2 Stimulated macrophages  In contrast, LPS treatment of macrophages cultured on the SLA surface resulted in a nearly twofold, a threefold, and an eightfold increase in the secretion of MIP-1α compared to unstimulated macrophages cultured on an SLA surface at 6, 24, and 48 hours, respectively (Fig. 5-8). At 6 and 24 hours, MIP-1α secretion did not differ significantly between cultures on any of the titanium surfaces or the positive control (TCP + LPS). At 48 hours, the level of MIP-1α increased significantly on the SLA surface compared to that on all other surfaces and the positive control (p ≤ 0.05).  110  Figure 5–7: Macrophage inflammatory protein 1α (MIP-1α) production by unstimulated macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours. Similar to its effect on MCP-1, the SLA surface downregulated MIP-1α secretion in unstimulated macrophages at 6, 24, and 48 hours when compared to other surfaces (p ≤ 0.05). The MIP-1α content was measured by ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP at each time tested. Reproduced with permission from (Refai AK et al , 2004).  111  Figure 5–8: MIP-1α production by LPS-stimulated (6.4 ng/mL) macrophages cultured on TCP and titanium surfaces of varying roughness after 6, 24, and 48 hours.MIP-1α production by macrophages cultured on the SLA surface was upregulated nearly twofold, threefold, and eightfold compared to unstimulated macrophages cultured on the SLA surface at 6, 24, and 48 hours, respectively (p ≤ 0.05). The MIP-1α content was measured by ELISA and quantified by DAPI staining. Data are presented as means ± SEM, and the studies were performed in triplicate. Two-way ANOVA and Tukey post hoc analysis were performed to test significance (p ≤ 0.05). Asterisks denote p ≤ 0.05 compared to all titanium surfaces and to TCP + LPS at each period tested. Reproduced with permission from (Refai AK et al , 2004).  112 5.3 Synergism: surface topography and LPS  Surface topography was observed to act synergistically with the LPS. This synergistic effect was time dependent and did not reach statistical significance until the 24 and 48 hour periods. Generally, when macrophages were stimulated with LPS, all of the titanium surfaces increased their production of pro-inflammatory cytokines and chemokines. Interestingly, the stimulated macrophages cultured on the SLA surfaces secreted significantly more pro-inflammatory cytokines and chemokines at 48 hours than they did on all other titanium surfaces or the positive control (TCP + LPS) (Fig. 5-9).  5.4 Interaction between sandblasted and acid-etched components of the SLA surface  The data obtained by our study was analyzed using the two-way analysis of variance (ANOVA), which allows testing for interaction between the components of the surfaces on cytokine and chemokine secretion. Briefly, the surfaces can be considered as two factors: acid etching at two levels (present and absent) and sandblasting at two levels (present and absent). Thus, for example, the SLA surface comprises both the acid etching– and sandblasting-produced features, whereas the PO surface has neither. The combination of the sandblasted and acid etched features on the SLA surface resulted in stimulated macrophages releasing greater amounts of pro-inflammatory cytokines and chemokines than that predicted by combining the amounts produced by blasting and acid etching. This interaction was statistically significant for TNF-α and MCP-1 at 24 hours and for all of the cytokines and chemokines tested at 48 hours (p ≤ 0.05; Fig. 5-10).  113 The synergistic relationship demonstrated by LPS stimulation can be illustrated by an interaction graph in which the values for each response on each surface are plotted against the presence or absence of LPS (Fig 5-9). Parallel lines indicate no interaction. For IL-1ß, IL-6, TNF-α, MCP-1 and MIP1α, the SLA line is not parallel to those of other surfaces, and the responses to the SLA surface are clearly greater than the sum of the AE and CB surfaces. Similarly, the surface processes (blasting and acid etching) show a significant interaction (Fig. 5-10).  114     E Figure 5–9: Interaction between topography and LPS stimulation at 48 hours. Cytokine and chemokine profiles of macrophages (stimulated vs. unstimulated) cultured on all tested surfaces. After 48 hours, stimulated macrophages cultured on an SLA surface secreted significantly more cytokines and chemokines than TCP-positive controls (p ≤ 0.05). (A) IL-1β, (B) IL-6, (C) TNF-α, (D) MCP-1 and (E) MIP1α.Modefied from Refai AK et al 2004.    115    E Figure 5–10: Interaction effects between the features produced by blasting and etching on the secretion of cytokines and chemokines by stimulated macrophages at 48 hours. The combination of CB and AE resulted in a significant production of all cytokines and chemokines tested in this study at 48 hours (p ≤ 0.05). Presence of both coarse sandblasting (CB) and acid etching (AE) features is characteristics of the SLA surface, whereas the mechanically polished (PO) surface has neither. Two-way ANOVA was used to test for significance and the interaction effects between the two components of the SLA surface. (A) IL-1β (B) IL-6 (C) TNF-α, (D) MCP-1 and (E) MIP1α.Modefied from Refai AK et al 2004.  116 The results presented in this chapter demonstrate that the surfaces on which macrophages are cultured can dramatically alter their secretion of chemokines and cytokines. In the complex milieu surrounding a recently inserted implant, it would be expected that these cytokines and chemokines would exert biologic effects. For dental implants where osseointegration is desired, a primary concern is how such a complex mixture of secretions would affect bone. The next chapter introduces a co-culture model to investigate this question.  117  6 CHAPTER 6: Measurement of the effects of secretions by macrophage plated on varying surface topographies on osteoblasts in a co-culture model  118 Bone formation around dental implants depends on the regulation of osteoblastic cells derived from host tissues (Einhorn TA 1999). Osteoblast differentiation and proliferation are controlled by cytokine signals from non-mesenchymal resident cells, including circulating cells of the immune system (Frost A, et al 1997; Gerstenfeld LC, et al 2001; Cooper LF 2003). As detailed in previous chapters, macrophages perform a critical role in endosseous wound healing (Thomsen P and Ericson LE 1991; Sennerby L, et al 1993; DiPietro LA 1995). Recent studies showed that macrophage cell lines can produce osteoinductive signals in the form of bone morphogenetic protein (BMPs) and the osteogenic cytokine (TGF-β) (Champagne CM, et al 2002; Takebe J et al 2003), and the production of these signals was controlled in part by surface topography on which the macrophages were cultured (Takebe J, et al 2003).  This chapter investigates whether cytokines from the RAW 264.7 macrophage cell line stimulated by implant surface topographies of varying degrees of roughness affect osteoblasts’ production of mineralized tissue and alkaline phosphatase, a characteristic enzyme produced by osteogenic cells forming bone in a co-culture model.  6.1 Alkaline phosphatase activity  In this series of experiments, the responding cells, osteoblasts, were plated on a titanium glass cover slip in the lower compartment of a 6-Transwell plate, as described in the Material and Methods section (Chapter 3). Macrophages, the cells whose secretions would be assessed by the responding osteoblasts were then seeded on sandblasted and  119 acid-etched (SLA) and mechanically polished (PO) titanium-coated surfaces in the upper compartments of the co-culture model (see Fig. 3-2). The transwell allows the exchange of only the soluble medium components. Alkaline phosphatase (ALP) isoenzyme, which detects the earliest stages of mineralization initiated by osteogenic cells, is one of the most widely markers for identifying bone differentiation  (Ballanti P 1995).  By the end of week 1, ALP activity increased significantly (p < 0.05) in transwells containing macrophages plated on SLA surfaces, compared to ALP activity in transwells containing macrophages plated on both PO surfaces and the two control surfaces (i.e., membrane only and macrophages only) (Fig. 6-1). At the end of week 2, surfaces in transwells containing macrophages plated on SLA surfaces similarly showed higher expression of ALP activity compared with those plated on PO surfaces and controls (membrane only and macrophages only). However, these differences were significant only in comparison with the macrophages-only control and were not significant in comparison with the membrane-only control. By the end of week 4, ALP activity increased significantly (p < 0.05) in transwells containing the membrane-only control when compared to macrophages plated on SLA surfaces, PO surfaces, and the macrophages-only control surface. However, ALP activity increased in transwells containing SLA surfaces when compared to macrophages plated on PO surfaces and the macrophages-only control surface. Figure 6-2 shows examples of ALP staining of osteoblasts in transwells containing macrophages plated on the various surface topographies at 1, 2, and 4 weeks .   120 6.2 Bone-like nodules  A second series of studies was performed to assess the formation of bone nodules in transwells containing macrophages plated on the various surfaces. Alizarin complexone (AC), a marker of later bone-like nodule formation, is easily observed by epifluorescence microscopy. Figures 6-2 to 6-5 show bone nodule formation (means ± SD) by osteoblasts on the test surfaces (titanium-coated glass cover slips) after 3 weeks in the presence of macrophages on SLA and PO surfaces in the Transwell co-culture system. The number of bone-like nodules was not affected by the presence of macrophages in the co-culture model for all testing protocols (early effect, late effect, continuous effect). Figure 6-6 shows some examples of AC staining in Transwells containing macrophages plated on the various surface topographies in groups from each test protocol.  121   Figure 6–1: ALP expression as determined by relative area stained in osteoblast cultures on Ti-coated glass cover slips when exposed by means of a transwell mode for 1, 2, and 4 weeks to the secretions of macrophages cultured on SLA,  and PO surfaces as well as controls comprising macrophages cultured on the membrane (Mac) and the absence of macrophages (i.e. membrane alone (Mem) , and the control surface topographies on osteoblasts using a co-culture model. Macrophages cultured on SLA had an early effect (week 1 W1) on ALP expression but no effect was seen relative to the Mem control at week 2  (W2) or week 4 (W4)  122  Figure 6–2: ALP staining of osteoblasts at the end of week 1in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the transwell membrane) and Mem (no macrophages).  123  Figure 6–3: ALP staining of osteoblasts at the end of week 2 in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the transwell membrane) and Mem (no macrophages).  124  Figure 6–4: ALP staining of osteoblasts at the end of week 4 in transwells containing either macrophages plated on the SLA and PO surface topographies or controls Mac (macrophages on the Transwell membrane) and Mem (no macrophages).  125  Figure 6–5: Bone-like nodule formation by percentage surface area for various periods of exposure to macrophage secretion (means ± SD). EE =  early effect (osteoblasts strain exposed to macrophage secretions for first week only), LE = late effect (osteoblasts exposed to macrophage secretions during weeks 2 and 4 only and CE = continuous effect (osteoblasts strain exposed to macrophage secretions for all four weeks).Cont= control.  126  Figure 6–6: Examples of Bone-like nodules demonstrated by alizerone complexone stained formed after 3 weeks by osteoblasts cultured on Ti-coated coverslips under various testing protocols: (a,b)  macrophages on SLA (a) and PO (b) under the EE protocol ; (c,d) macrophages on SLA (c) and PO (d)  under the LE protocol; (e,f)) macrophages on SLA (e) and PO (f)  under the CE protocol.   127  7 CHAPTER 7: Discussion and future directions  128 The implantation of a biomaterial into soft and/or hard tissue induces a number of host responses. This thesis was undertaken to study in vitro the topographically influenced effects of implant surfaces on the macrophage, a member of the mononuclear leukocyte population.  The placement of endosseous implants results in leakage of blood from damaged blood vessels and the formation of a fibrin clot at the bone-implant interface. The clot functions as a reservoir of cytokines and growth factors to recruit circulating inflammatory cells, consisting mainly of monocytes/macrophages and neutrophils. Among the cells residing in this fibrin network are newly arrived monocytes, which ultimately differentiate into macrophages (Davies JE 1998; Park JY and Davies JE 2000; Thomsen P and Gretzer C 2001). An early function of these macrophages is the destruction of bacteria in the wound. Moreover, it is likely that these cells contribute to the early inductive and chemotactic signals that support osseointegration (Cooper LF 2003). Macrophages can also be found on the implant surface and in the peri-implant tissues after implant insertion (Anderson JM and Ziats NP 1991; Thomsen P and Ericson LE 1991). A number of studies have shown that these recruited macrophages have different phenotypes (Johansson CB, et al 1992) and express specific cell surface markers (Hunt JA, et al 1996) at various times following implantation (Dijkstra CD, et al 1985; Rosengren A, et al 1997). Implant-adherent macrophages and multinuclear cells are observed prior to bone formation as well. Further evidence of the role of mononuclear cells in bone formation is the presence of foreign body giant cells (FBGCs) on the surface of implanted biomaterials (Vogel M, et al 2001).  129 Given that macrophages appear to be highly influenced by the surface characteristics of implanted devices, the purpose of this thesis was to investigate the effect of the surface topography on the response of macrophages in an in vitro model. Three approaches were taken in this study: 1. Evaluation of cellular morphology, adhesion, locomotion, and  activation of RAW 264.7 cells on the test surfaces. 2. Induction of inflammatory cytokines and chemokines in response to the test surfaces. 3. Determination of the effect of macrophage cytokines produced on the test surfaces in the induction of osteogenesis using a Transwell co-culture model. Each of these studies is discussed in turn.  7.1 Cellular morphology, adhesion, locomotion, and activation  Several processes direct macrophage interaction with biomaterials. First, the macrophage response is constrained by the size of the material (Xia Z, et al 2006). Material particle sizes smaller than a single-nucleated macrophage (typically around 10 µm in diameter) may be phagocytosed by macrophages. Large particles that cannot be phagocytosed (between 10 µmand 300+ µm) may be engulfed by multinucleated giant cells formed by the fusion of macrophages. In response to a medical device, such as an implant, pin, plate, suture, or membranes, macrophages and/or macrophage-derived FBGCs form and attach to its surface, where they may remain for the lifetime of the device (Fig 7-1).  130   Figure 7–1: Diagram of macrophage responses to biomaterials. Macrophages respond to small particles (<10 µm) by phagocytosis (I). For large particles, macrophages fuse to form foreign body giant cells (FBGCs), either to engulf the particles (II) or to remain on the surface of bulk materials (III). (Adapted from Xia Z 2006.) Copied under licence from Access Copywrite. Further reproduction prohibited.   131 Second, the macrophage response is affected by the nature of the materials: that is, whether it is degradable or non-degradable. Biodegradable materials will be degraded within phagosomes after phagocytosis or eroded via extracellular resorption, with or without the involvement of FBGCs. Any associated inflammation will be resolved after total resorption of the biodegradable materials. Materials that cannot be degraded, either within the macrophage phagosome or by extracellular resorption, may pose a different challenge. Macrophages will infiltrate continuously to phagocytose undigested particles or to fuse into FBGCs and remain on the surface of the implanted materials. Macrophages and FBGCs have been found at the implant site for up to 150 days in vivo and may possibly remain for the lifespan of the implant (Anderson JM, 2001).  Therefore, for non-degradable biomaterials, it would appear desirable to choose those that induce fewer macrophage responses for long-term implantation.  For many cell types, interaction with the ECM involves specialized, elongated structures of the plasma membrane, which interact with the substrate (leaving a gap of approximately 10–15 nm). These sites, termed focal complexes, have a dash-like appearance, measure approximately 0.5 µm in length, and constitute some of the earliest matrix contacts formed by a protruding cell. They are integrin-based, enriched in talin, paxillin, and vinculin, and are associated with actin microfilaments at their cytoplasmic aspects (Zamir E and Geiger B 2001). Focal complexes can mature into focal adhesions through the application of force, such as occurs during the retraction of cellular protrusions, for example. At present, there are at least 50 proteins associated with focal contacts and their targeted extracellular matrix adhesions, and the most fundamental of  132 these are the integrin molecules. By acting as mechanosensors, integrins serve as an interface between structural cues in the extracellular environment and biochemical signals in the cytosol. Thus, the capacity of integrins to respond to mechanical force is central to the role of integrins in multiple biologic processes.  Highly motile cells such as osteoclasts and macrophages differ from most cell types in their mechanism of attachment, which involves the formation of podosomes. Podosomes are specialized adhesion sites that are structurally and functionally distinct from focal adhesions (Marchisio PC, et al, 1984, 1987; Tarone G, et al, 1985; Nermut MV, et al, 1991). Although podosomes and focal adhesions have many of the same proteins, focal adhesions are relatively stable, while podosomes are dynamic attachment structures that undergo assembly and disassembly every 2 to 12 minutes  (Stickel SK and Wang YL 1987). Their most distinguishing feature is their dual component architecture, which consists of a core of F-actin and actin-associated proteins surrounded by a ring of proteins such as talin or vinculin. Consequently, actin regulatory pathways exert a major additional influence on podosome-type contacts.  In our study, we investigated the morphology, adherence, and locomotion of RAW 264.7 cells on our test surfaces. While macrophages spread, flattened, ruffled, and elongated on all titanium-coated epoxy replica surfaces, increased spreading was found on the roughest surface (SLA) at 24h and 48hr. Rounded macrophages were also found on all titanium- coated epoxy replicas. Macrophages grown on the SLA surface displayed podosomes that were more extended, stretching into deep pores and forming multiple points of  133 attachment that anchored fine cell processes within the microstructure of SLA. Moreover, these cells were significantly elongated and developed layers of cells that bridged over each other. In general, the number of macrophages increased in correlation with time of incubation; more macrophages were adherent to the SLA surface at 24 and 48 hours than on the PO surface. This increase reflects either increased adherence at early time points or enhanced proliferation at later time points or both for the SLA surface. On the PO, CB, and AE surfaces, macrophages showed no significant differences in cell number. These scanning electron microscopy (SEM) results were confirmed by means of 4'-6- Diamidino-2-phenylindole (DAPI) staining. Thus, surface topography markedly affected both proliferation and morphology.  Macrophages are highly mobile cells that can traverse endothelial barriers and connective tissue with ease. Such movement through the extracellular matrix involves cyclic attachment and detachment of individual adhesive structures such as podosomes and focal adhesions, each adhesive structure establishing  a localized anchorage at the leading edge of the cell, stabilizing the sites of cell protrusion and in turn eliciting directional movement. Because this mechanism is a dynamic process, it has been suggested that integrins in the focal adhesions, for example, serve as bidirectional transducers of force, directing cellular contractile forces to the extracellular environment while also detecting forces in that environment and translating associated messages to the cytoplasm. The attachment of cells to the ECM via integrins leads to activation of several protein tyrosine kinases, of which Pyk2, Src, and Cbl play pivotal roles. It has been suggested that activation of Src is associated with a shift from stable focal adhesions to the more  134 dynamic podosome assemblies, thereby regulating cell motility (Kaplan D, et al 1995; Meng F and Lowell CA, 1998; Felsenfeld DP, et al 1999).  With respect to the macrophage, only limited studies have investigated the effect of surface topography on cellular motility in vitro. Our analysis of cell movement on the different surface topographies was assessed using time-lapse microscopy. Our findings indicated that the macrophages did not exhibit net migration on any of the four surface topographies used in the study. In general, macrophages exhibited an oscillatory type of movement—that is, limited movement with no overall change in position—and no discernible differences were found among the five experimental surfaces (PO, CB, AE, SLA, and titanium glass cover slip). About 70% to 80% of the cells moved in an oscillatory fashion on all experimental surfaces. The stationary and oscillatory types of movement observed in this study correlated with the observations of others (Jenney CR 1998). In contrast to our findings, Wojciak-Stothard B, et al (1995) reported that macrophages were able to move faster on microfabricated grooved (patterned) surfaces than on plain substrata. Also, they found that persistence of macrophage movement was higher on patterned than on plain substrata. It should be noted, however, that the movement reported by this study did not exceed twice the diameter of the cell and that such grooved surfaces increase cell motility (Damji A, et al 1996). Thus, the differences in macrophage migratory behavior may be related to the differences between regular and irregular patterned substrata.   135 As noted above, the non-receptor tyrosine kinase Src is involved in integrin signaling, in the regulation of the cell cytoskeleton, and in cell migration (Sanjay A, et al 2001; Frame MC, 2004; Playford  MP and Schaller MD  2004; Alper O and Bowden ET 2005). Furthermore, Src activation is associated with the reorganization of actin in specific adhesion structures. Indeed, expression of v-Src, the oncogenic form of Src, induces a rearrangement of the actin cytoskeleton, characterized by a switch from stress fibers to podosomes in BHK cells (Tarone G, et al 1985).  To examine the effect of topography, specifically surface roughness, on general macrophage activation, we carried out immunostaining of phosphotyrosine, 24 hrs hours after plating. The macrophages stained positive for phosphotyrosine, which was found throughout the cells associated with both SLA and PO surfaces. On the SLA surfaces, the macrophages were spread out and exhibited intense staining within the cytoplasm. In contrast, the macrophages on the PO surfaces exhibited staining in both the cytoplasm and the podosomes. However, the spreading of macrophages on the SLA surface, combined with the attachment of cell processes within the pits of the surface, may have interfered with the observation of localized podosome staining.  The results of our study confirm that there is activation of signaling pathways within the podosome. The disks used in our study have irregular surfaces with features sometimes described as randomly distributed. Microfabricated surfaces, in contrast, are often designed with regular features such as grooves, boxes, or gap-cornered boxes described as discontinuous edged surfaces (DESs).  136 Regular microfabricated surfaces influenced the arrangement of F-actin, microtubules, and vinculin, and cells such as osteoblasts could be guided in their direction of migration. Microfabricated surfaces were also found to alter the staining intensity and localization pattern of phosphotyrosine and Src-activated focal adhesion kinase-1 (FAK) localization. Cell multilayering, matrix deposition, and mineralization were enhanced on DESs when compared with smooth controls. DES thus alters adhesion, migration, and proliferative responses from osteoblasts at early time points (<1 week) and promotes multilayering, matrix deposition, and mineral deposition at later times (2 to 6 weeks). Such topographic patterns could potentially be employed as effective surface features on bone-contacting implants or in membrane-based periodontal applications (Hamilton  DW, et al 2006).  Substratum surface topography is a powerful modulator of cell behavior, but how it influences intracellular signaling remains largely unknown. This area of research is now attracting more intense scrutiny. Our group investigated the influence of microfabricated topographies on the activation of nonreceptor tyrosine kinases Src, FAK, and extracellular signal-regulated kinases 1 and 2 (ERK 1/2) and on the transcription factor Runx2 in rat osteoblasts cultured on substrata that varied in their capacity to promote bone-like tissue formation. In summary, total tyrosine phosphorylation increased on grooves, tapered pits, and gap-cornered boxes relative to the levels found on smooth surfaces; the greatest activity occurred at 1 week. Src levels were higher on smooth surfaces than on any other surface, but FAK and ERK 1/2 phosphorylation were highest on groove and gap-cornered boxes for periods of up to 6 weeks. Inhibition of Src phosphorylation with PP2 inhibited FAK and ERK 1/2 phosphorylation on grooves but  137 had no detectable effect on either FAK or ERK 1/2 on smooth substratum. Osteoblast response to substrata with specific topographic features appeared to require FAK–Y397– Src–Y416 complexes for ERK 1/2 phosphorylation, whereas Src-independent methods of ERK 1/2 activation are present on smooth surfaces (Hamilton DW and Brunette DM 2007).  7.2 Induction of inflammatory cytokines and chemokines  The second part of this thesis study was an investigation of the effect of the topography of titanium surfaces on early macrophage activation and their secretory profile. In this study we evaluated the effects of surface topography on the induction of pro- inflammatory cytokines with and without LPS. Because it is a component of the gram- negative bacteria associated with external wound sites, LPS is considered to be a common influence in macrophage activation. It activates macrophages via Toll-like receptor 4 (TLR 4) that in turn connects to a signal pathway, activating the transcription factor complex NFκB.  NFκB initiates the transcription of a wide variety of genes involved in the production of acute inflammatory mediators, including pro-inflammatory cytokines and CC chemokines (Aderem A 2000). Thus, stimulation with LPS can not only increase all cytokine levels but can also influence the types of cytokines being expressed and consequently may lead to a possible misinterpretation of data (i.e., cross talk). Nevertheless, LPS is commonly found around implants in the form of endotoxins. Therefore, in this study we included conditions with a suboptimal dose of LPS (i.e., a dose that failed to produce cytokines in macrophages on tissue culture polystyrene  138 [TCP]) to determine whether LPS would interact with surface topography in directing macrophage responses.  Without the presence of LPS, macrophages increased their secretion of the pro- inflammatory cytokine TNF-α when attached to rough surfaces (AE and SLA). Compared to IL-1β and IL-6, which were not detected in this study without exogenous stimuli, this increase was detectable. In macrophages stimulated with a suboptimal dose of LPS, the SLA surface produced higher levels of IL-1β, IL-6, and TNF-α at 24 and 48 hours compared to that produced by all other surfaces. Surface topography also modulated macrophage secretion of the chemokines CCL2 (MCP-1) and CCL3 (MIP- 1α). Untreated macrophages cultured on SLA surfaces downregulated their production, while LPS-stimulated macrophages upregulated their production. These results are consistent with previous findings in which the increase in TNF-α secretion from LPS- stimulated macrophages was associated with surface roughness (Soskolne WA, et al 2002). This study involving measurement of inflammatory cytokine secretion in a cultured macrophage cell line RAW 264.7 on different surface topographies by ELISA differs from the methodololgy used by Tan et al, (2006). They used real time quantitative PCR to measure proinflammatory cytokines and chemokines at the mRNA level whereas I measured proinflammatory cytokines and chemokines at the protein level. They showed that IL1β and IL6 increased over the time using J744A.1 cell line in the absence of LPS stimulation, but TNF-α was not detected. This finding is the reverse of the present study where macrophage cell line RAW 264.7 secreted only very low amounts of TNF-α and failed to secret detectable levels of  IL1β and IL6 in the absence of LPS stimulation.The  139 differences between the two studies might be attributed to different gene expression (some upregulated and some downregulated), at the different times studied and/or differences between cell lines. However, the LPS stimulation effects studies agreed on the surface topography effects (Tan KS etal 2006).  More recently, macrophages were cultured on different surfaces in the presence and absence of  lipopolysaccharide (Paul NE et al 2008). The microstructured surface but not the nanotexured significantly affected the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. Gene array analysis was performed in order to relate gene to protein expression. They found that the release of several mediators was influenced by surface topography as well as by LPS stimulation. Further, they were able to show that proinflammatory cytokines and chemokines were upregualted and an anti-inflammatory cytokine was downregulated. Interstingly, genes were found to be regulated by topography; a total of 360 genes were upregulated by the regular microstructured surface of which 180 were not affected by LPS stimulation. Five hundred and eighty genes were downregulated. Among these, 50 genes were found to be specifically regulated by the microstructure.   This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Such findings may explain the different mode of chemokines secretions in absence and presence of LPS stimulation in this study. In general, we can conclude from this study that surface topography induced mediators responsible for both the activation and effector phases of innate and adaptive immunity.  140  It has become evident that these inflammatory mediators play a role in the latter stages of tissue repair as well. Both TNF-α and IL-1β contribute to the reparative phase either directly, by influencing endothelial and fibroblast functions, or indirectly, by inducing additional cytokines and growth factors (Clarke RAF 1996). TNF-α has been implicated in angiogenesis during wound healing (Clarke RAF 1996), stimulation of osteoclast activity (Mundy GR 1993), and regulation of osteoblast proliferation (Frost A, et al 1997; Gerstenfeld LC, et al 2001). It is possible that these functions have potential importance in wound regeneration at the implant-tissue interface. Gerstenfeld and coworkers (2001) suggest that the pro-inflammatory cytokine TNF-α influences the recruitment of mesenchymal cells into the osteoblastic lineage or, alternatively, influences the differentiation of the osteoprogenitor cells into osteoblasts in response to the injury. Furthermore, impaired intramembranous bone formation was reported following bone marrow ablation in the absence of TNF-α signaling (Gerstenfeld LC, et al 2001; Cho T et al 2001). IL-1 has been shown to participate in neovascularization (angiogenesis) during wound healing (Clarke RAF 1996) and to stimulate fibroblast and granulocyte proliferation (Lachman LB 1983; Gillis S 1983). IL-6 also plays a significant role in endosseous wound healing since it is evidently involved in the development of both osteoclasts and osteoblasts and consequently in the remodeling stage during bone formation. (Manolagas SC 1998; Jilka R 1992).  IL-6 has also been shown to be crucial to both epithelialization and and the formation of granulation tissue during wound healing (Gallucci RM, et al 2000). Studies show that CCL2 and CCL3 (MCP-1 and MIP-1α, respectively) play a critical role in macrophage recruitment into wounds, upon which  141 appropriate tissue repair depends. They are also implicated in orchestrating the immune response at wound sites (Gillitzer R 2001; DiPietro LA 1998); for example, it has been found that MCP-1 knockout mice exhibit a remarkable delay in wound healing (DiPietro LA, et al 1998).  The effect of material surfaces on macrophage activation and cytokine secretion has been predominantely studied in vitro. It has been found that macrophages secrete IL-1, IL-6, IL-10, and TNF-α after stimulation by different material surfaces (Thomsen P and Gretzer C 2001). The secretion of such cytokines was generally low without exogenous stimulation.  The addition of LPS elicited a marked increase in the production of pro- inflammatory cytokine by macrophages attached to titanium alloy particles and titanium disks. Gretzer  and coworkers (2003) reported that low levels of TNF-α were detected in untreated monocytes on titanium disks, while TNF-α levels increased fourfold in response to LPS stimulation. In another study, it was found that untreated macrophages did not induce IL-1β and TNF-α production when macrophages were challenged with titanium alloy particles unless LPS was added to the medium (Gretzer C, et al 2003). These results concur with the findings of the present study.  Only a few in vivo studies in soft tissue have reported finding the expression of TNF-α, IL1-β, and TGF-β around implant materials (Hunt JA 1996; Dalu A 2000; Rosengren A 1997). In a comprehensive review of macrophage and material surface interactions, Thomsen P (2001) concluded that the precise modulatory effects of cytokines on inflammatory and reparative processes around implants are not known. Two general  142 problems that make clear interpretation difficult are redundancy and pleiotropism of such cytokines. Redundancy exists because a given cellular function can be initiated by many different types of cytokines. For example, a variety of different cytokines can act to stimulate cell division in activated T cells. Pleiotropism exists because a single cytokine has many functional effects, not only on different cell types but even on the same cell type (Abbas AK 1997). Given our findings in this section, it is relevant to ask just how surface topography can affect activation of the macrophage. Such a discussion can be approached at both the macro and the micro levels. At the macro level, there are a number of possibilities.  7.3 Surface roughness  Surface roughness plays a critical role in both cell and tissue responses to biomaterials because it is the surface that forms the interface with the tissue (Brunette DM 1999). One way in which surface topography influences cell behavior is by altering cell shape (Brunette DM 1996). It has been postulated that alterations in cell shape due to surface topography affect many aspects of cell behavior, including adhesion, spreading, proliferation, and differentiation. It has been hypothesized that surface topography may produce a microenvironment in which cellular differentiation and activation can take place (Brunette DM 1996). This microenvironment hypothesis may also explain why, in our study, macrophages proliferate, differentiate, and secrete more cytokines and growth factors when they are plated on an SLA surface compared to other surfaces. In support of this hypothesis, Martin and coworkers (1995) found that surface roughness decreased  143 osteoblast proliferation while, at the same time, increasing cellular differentiation and matrix production. Further, surface roughness also altered osteoblast thickness and morphology but did not affect cell volume (Wieland M, et al 2002). The results presented in Chapter 4 demonstrated that surface topography altered macrophages shape. Macrophages were found to attach, spread, proliferate, and differentiate differently on the SLA surface compared to the other surfaces. Given that surface-directed cell shape alterations can affect gene expression, the conformational changes in macrophage shape on the test surfaces possibly directed differential gene expression, which resulted in increased proliferation, differentiation, and adhesion.  We also suggest that this alteration of cell shape in response to surface roughness affects induction of growth factor production as well. Recent studies in our laboratory investigated the effect of surface topography on the regulation of macrophage signaling cascades. Similar to this study, macrophages (RAW 264.7) were seeded onto titanium- coated epoxy replicas of polished, blasted, etched, and blasted and etched (SLA) surfaces. Immunocytochemistry and western blotting techniques were used to assess cell response. The results revealed that at day 1, macrophages on SLA surfaces exhibited a twofold increase in ERK1/2 phosphorylation compared to those on polished surfaces. Src activation also correlated with increased surface roughness. It was postulated that implant surface topography affects signaling pathways that control cytokine release and thus may modulate wound healing around the implants (Ghrebi S, thesis 2009).  144 7.3.1 Increase in surface area  The second possible mechanism whereby surface topography may affect macrophage function relates to an increase in the surface area of a rough surface relative to a smooth one. The SLA surface has a greater surface area (33%) than its simpler components, CB (29%) and AE (20%) (Wennerberg A 2003). This increase in surface area alters opportunities for attachment and spreading as well as the disposition of cell processes.For all the surfaces however the macrophages were able to spread but of course did not engulf the surface, instead gaint cell will form and attach to the surface and may stay for the lifetime of implant. In contrast when macrophages encounter particles of size range 10-100µm that they can phagocytose, the importance of surface area is increased and has been suggested by a study reporting that macrophages secreted more IL-1 when exposed to titanium particles at higher surface area ratios (Shanbhag AS 1994).  7.3.2 Cofactor modification of test surfaces  The third mechanism involves cofactor modification of the test surfaces. In this scenario, the interaction between a titanium surface and conditioned medium results in a thin layer of adsorbed macromolecules that modify cell behavior. There has been speculation that a rough surface absorbed more fibronectin than other surfaces (Pearson BS, et al 1988), leading to increased macrophage attachment through their RGD integrin receptor domains. This organization of attachment sites on titanium surfaces of varying roughness  145 might also affect the macrophages’ secretory profiles via signaling pathways (Gherbi S thesis 2009).  7.3.3 Quorum sensing  The fourth possible mechanism is quorum sensing, a phenomenon whereby gene expression responds to population density (Fuqua WC, et al 1994). Today, the term quorum sensing is used to describe the phenomenon whereby the accumulation of signaling molecules enables a single cell to sense the number of other cells in the vicinity (i.e., the cell population density). Although most studies of this phenomenon involve prokaryotic organisms (Fuqua WC, et al 1994), it is possible that such a mechanism could exist in cells of the monocytic lineage. If true, we speculate that a quorum sensing– like mechanism might explain the effect of SLA surfaces on CCL2 and CCL3 secretion, where these chemokines were downregulated on SLA surfaces but not stimulated relative to control values when LPS was present. It is evident that untreated macrophages proliferated more than stimulated macrophages on SLA surfaces. We speculate that under the condition of high cell population density, interactions occur between cells in the pits of the SLA surfaces. The untreated macrophages may sense that additional macrophages are not required and decrease chemokine production. In contrast, when macrophages were stimulated with LPS and cell number decreased, CCL2 and CCL3 production were elevated to recruit more macrophages to the surfaces.Another speculation is the upregualtion and downregulation of gene expression (Paul NE et al 2008).   146 The macro level effect(s) must surely translate into specific effects at the cell surface (i.e., micro level). Monocyte adherence induces the formation of podosomes, the sites of interaction between the biomaterial, the cytoskeletal proteins, and the intracellular signaling pathways. To understand the structure and function of podosomes, their complexity and dynamics must be considered.  As noted earlier, podosomes comprise a core of F-actin and actin-associated proteins embedded and linked to a ring structure of integrins and integrin-associated proteins. Many of the specialized podosome components are localized to either the core or the ring structure. Typical core components are actin; actin regulators, such as the Arp2/3 complex; gelsolin; and cortactin. The adhesion mediators, such as paxillin, vinculin, and talin, preferentially associate with the ring structure. Podosome signaling kinases, such as phosphoinositide 3-kinase (PI3-K), proline-rich tyrosine kinase 2/focal adhesion kinase (Pyk2/FAK), and Src, are also located in the ring structure (Linder S and Aepfelbacher M 2003; Buccione R, et al 2004). Podosomes are anchored in the ECM through a number of specific proteins, of which the integrin family plays an important role. Selective integrin isotypes are involved in the podosome, and their distribution is specifically localized.  Podosome adhesive sites are highly dynamic. They have a half-life of 2 to 12 minutes, with their inner core turning over 2 to 3 times during the lifespan of the podosome. The question of whether podosomes are heterogeneous in function has yet to be answered.   147 A model for podosome formation can be summarized as follows. Formation is initiated by attachment of the cell to the target substrate. Integrins then cluster and induce activation of a number of receptor tyrosine kinases, such as protein kinase C (PKC). Downstream of these initial events, a variety of pathways are initiated, most notably PI3K signaling (Chellaiah M, et al 1998), which in turn leads to the activation of FAK. This direction of signaling is also influenced by the Rho family of GTPases that are crucial to the initiation of actin filament formation, which gives rise to the formation of the actin-rich core of the podosome. It is unclear how core and ring formation are coordinated. An important subsequent switch at FAK is activation of Src in the ring, leading directly to activation of components of the mitogen-activated protein kinase (MAPK) pathway and subsequent transcription of pro-inflammatory cytokines. It is thus clear that Src is pivotal in regulating initial podosome formation, podosome lifespan, turnover of actin within the podosomes, and initiation of activation of the pro- inflammatory process.  Significant data support this model. Adherence augments human monocyte LPS-induced TNF-α production. This surface effect has been attributed to the actin cytoskeleton that is integral to adherence. Disrupting the actin cytoskeleton with cytochalasin D (CD) in adherent monocytes inhibited LPS-induced TNF-α production by 55%, thereby abrogating adherence-induced priming. Moreover, CD pretreatment abrogated adherence- induced activation of Pyk2, a major focal adhesion kinase, and ERK 1/2, a component of the MAPK signaling pathway. McGilvray and coworkers (1998) supported these findings, reporting that adherence induced ERK 1/2 phosphorylation and nuclear  148 translocation of NF kappa B and ultimately enhanced tissue factor expression. The conclusions of these studies were that the actin cytoskeleton is integral to the reprogramming of the monocyte for enhanced cytokine production and to maintenance of the cells in a ”primed“ state (Rosengart MR 2001; Haskill S, et al 1988; Yurochko AD, et al  1992; Shaw RJ, et al 1990; Lofquist AK, et al 1995; Bernardo J, et al 1997). As to the mechanism, it has been proposed that the cytoskeletal rearrangements enlist the actin architecture in the facilitation of signal transduction cascades of adherent cells (Rosengart MR 2001). Adherence is also a potent stimulus for monocyte differentiation. The adhesive interactions generated during monocyte recruitment are pivotal in induction of macrophage maturation, marked by enhanced phagocytosis and cytokine production.  It has been established that pro-inflammatory cytokines and CC chemokines are mediated through a well-known signaling pathway designated as NF-kappa B and are activated via toll-like receptor-4 (TLR-4). LPS is considered an archetypal factor in macrophage activation. It activates macrophages via TLR-4, which consequently activates the transcription factor complex NFkappa B. As noted above, NFkappa B has been shown to be a tightly regulated agent for initiating the transcription of a wide variety of genes involved in the production of acute inflammatory mediators, including pro-inflammatory cytokines and CC chemokines (Aderem A and Ulevitch RJ 2000). It has been speculated that biomaterials may activate macrophage responses in a manner similar to that of gram- negative bacteria or LPS, which activate macrophages via TLR4; however, the consequences of microbe invasion and biomaterial implantation are different. Microbes cause infections, which are resolved by adaptive immune responses via activation of B  149 and T lymphocytes, while implantation of most synthetic biomaterials causes inflammation and foreign body responses without the involvement of antibody-producing B cells (Xia Z 2006). More recently, members of our group also investigated the role of surface topography on activation of the NFκB signaling pathway in macrophages. They studied the effect of implant surface topography on activation of the NFκB signaling pathway in the RAW 264.7 macrophage cell line. They also examined, via SEM, the effect of surface roughness on the adhesion of the macrophages and on their morphology. They found that activation of the NFκB pathway was surface topography–dependent. The smooth surface showed the highest level of activation followed by the etched surface, and finally the SLA surface. The addition of suboptimal concentrations of LPS mildly enhanced the response by signaling through the Toll receptor. Activation of NFκB occurred in the absence of fetal calf serum, although to a lesser extent. Disruption of the lipid rafts in the membranes of the cells inhibited the triggering and signaling of the NFκB pathway. Similarly, smooth surfaces bound more macrophages in the 30-minute assay. This in vitro study has demonstrated that surface topography modulated activation of the NFκB signaling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s)/surface structure the signal pathway is initiated (Tarek A et al theiss 2008).  150 7.4 Determination of the effect of macrophage cytokines produced on the test surfaces in the induction of osteogenesis  The historic role of macrophages was to induce immumno-inflammatory responses. With regard to joint prostheses, the macrophage response to particulate materials shed from the implant is involved in fibrosis and bone loss, leading to the so-called aseptic loosening of the prosthesis (Mohanty M 1996). However, some of the cytokine macrophages produced are involved in the healing response, and there are indications that they can have a beneficial effect on bone regeneration. Control of bone formation at the endosseous implant interface is essential to the success of dental implants (Cooper LF 1998; Stanford CM and Brand RA 1999). Macrophages may be involved in this process (Thomsen P and Ericson LE 1991; Sennerby L, et al 1993; DiPietro LA 1995). Recent studies have shown that macrophage cell lines are able to produce osteoinductive signals, including bone morphogenetic proteins (BMPs) and the osteogenic cytokine (TGF-β), osteopontin (OPN) (Champagne CM, et al 2002; Takebe J, et al 2003), and their production was topographically dependent (Takebe J, et al 2003). Furthermore, bone formation has also been observed in some cases of biomaterial implantation with extensive FBGC reaction (Vogel M, et al 2001).  In the third component of this thesis, we investigated whether cytokines from the RAW 264.7 macrophage cell line, stimulated by surfaces of varying degrees of roughness, affected osteoblast differentiation and production of mineralized tissue.  151 A transwell model was used in this study. Osteoblasts cultured from the calvaria of newborn rats were separated by a membrane from the murine macrophage cell line (RAW 264.7) that was plated on different surface topographies. It has been reported that cytokines can function across species barriers (Scheerlinck JP 1999). The rat osteoblasts were chosen for this study because they form bone-like nodules, unlike immortalized osteoblastic cell lines, which may express bone markers but do not produce bone-like nodules. Newly isolated rat osteoblasts were also used in this study to avoid osteoblast heterogeneity as a result of cell passage selection of different osteoblast subpopulations. Details of in vitro models testing of biologic responses to implants have been reviewed by Brunette DM (1999).  In our model, diffusible factors produced by the macrophages on surfaces of different roughness could be assessed. Macrophages were plated on several surface topographies, including SLA and PO. In contrast, the osteoblasts were grown on a single surface (titanium glass cover slips), which produces few bone-like nodules in the absence of exogenous stimulation. The objective was to investigate whether macrophage secretions, possibly cytokines, stimulated by surface topography have an inductive effect on the mineralization processes in terms of alkaline phosphatase (ALP) expression and/or bone- like nodule formation.  At the end of both the first and second weeks, ALP activity increased on surfaces in transwells containing macrophages plated on SLA surfaces compared with macrophages plated on PO surfaces and control surfaces (membranes alone and macrophages alone).  152 By the end of 4 weeks, ALP activity increased significantly on surfaces in transwells containing the membrane-alone control when compared with macrophages plated on SLA or PO surfaces or the macrophage–alone control surfaces. Interestingly, the increase of ALP in the control wells was higher than that in the SLA wells during the 4 weeks. There are two possible explanations for the increase of ALP in the control wells. First, osteoblasts secrete factors that act in an autocrine fashion to increase the differentiation of their progenitor cells, which ultimately results in an increase in ALP expression. Second, the continuous presence of macrophages has negative effects on osteoblast differentiation and ALP expression.  In a second series of experiments, we investigated possible time-dependent effects of macrophages on bone-like nodule formation. Macrophages were (a) added early, (b) added at a later time, or (c) present throughout the culture period. It was noted that the formation of bone-like nodules was greater in number on SLA surfaces than on the polished surfaces when macrophages were present either early or continuously, although these differences were not statistically significant. Similar to ALP, the increase in the number of bone nodules in the control wells was as high as that in the SLA wells during the 4 weeks. In summary, the number of bone-like nodules formed was not affected by the duration of the macrophages in the co-culture model for all tested periods (early effect, late effect, or continuous effect). A larger sample and longer time points might be required to elicit significant differences in bone nodule formation.In addition, a large number of culture ( propably conditions media from different macrophages cells lines)  153 and complementary experiements could be done to further investigate the osteoblasts and macrophages interactions.  It is clear from our study that macrophage mediators have an effect on in vitro bone formation by osteoblasts. Although the results cannot necessarily be applied to the in vivo situation, the results nevertheless suggest that the early macrophage responses after implantation may have an impact on the process of osseointegration. However, it is extremely difficult to model the complex in vivo state, where different cell types and their associated factors are involved in bone formation. Moreover, the complexity of  the coculture system leads to another consideration, namely the possibility that not only did macrophage secretions influence the osteoblast, but the osteoblast secretions might affect the macrophages. This possibility could explain why the influence of the macrophages tended to decrease with time.  Previous studies have suggested that macrophages may be involved in bone formation. Champagne and Cooper (2002 and 2003) reported that the addition of conditioned media from macrophage cell lines increased ALP activity when measured at days 1 to 4.They showed that induction of ALP activity was due to presence of the osteoinductive cytokine BMP-2. Takebe and Cooper (2003) also showed that the production of BMP-2 from macrophage cell lines was topography dependent. It should be noted that the previous studies used a different macrophage cell line, J774A.1, and a different target cell, hMSCs, than those in the present study. Therefore, the differences between the two cell populations may explain some of the differences between our findings. Moreover, the  154 previous authors did not employ the co-culture transwell technique used in our study; they treated the hMSCs directly with conditioned media from J774A.1. Our study used the target osteoblast cells on a smooth surface because a recent study showed that osteoblasts plated directly on SLA surfaces developed more and larger nodules than osteoblasts cultured on PO surfaces (Schwarz F, et al 2007). By keeping the target cells on a smooth surface, we avoided the complication of the direct effects of the topography. Recently, several studies have also shown a link between macrophages and macrophage products and vascular calcification (Shioi A 2002; Tintut Y et al 2002). Vascular calcification often includes fully formed bone tissue, and its formation mimics embryonic bone development (Yu SY 1974; Mohler ER  2001). Tintut Y, et al (2007) postulated that macrophages augment in vitro vascular calcification via two independent mechanisms: cell-cell interaction and production of pro-inflammatory cytokines such as TNF-α secreted from LPS-activated macrophages. Similarly, Shioi A (2002) suggested that macrophages participate in the vascular calcification through production of inflammatory mediators such TNF-α and oncostatin M. ALP expression was used as a marker to assess the mineralization process in both studies. TNF-α has been shown to be involved in osteoblast proliferation (Frost A 1997) and blocking signaling through TNF-α receptors attenuated bone repair (Gerstenfeld LC 2001). Our study showed that unstimulated macrophages and LPS-stimulated macrophages on the SLA surface modulated secretion of TNF-α. Thus, normal bone formation and vascular calcification share some processes, but there is too little data to prove that they are identical.   155 In conclusion, macrophages comprise a heterogeneous population of cells that undergo environmentally induced differentiation into functionally distinct populations. my research has shown that at early times the macrophages respond to surface topography by altering their morphology and their secretory profile inflammatory cytokines and chemokines. At later times, the cytokines produced by such cells may be involved in the endosseous wound healing. A better understanding of the effect of surface topography in macrophages behaviour may enable improved surface design to specifically direct cell function and improve implant performance too .                 156 7.5 Future directions  In my collaboration with different institutes in my countery such as King Faisl Specialist Hospital and Research Centre, Schools of Pharmacy, Medicine, and Science I hope to be able to establish a small lab to continue my research and obtain guidance, suggestions from my supervisors Drs Waterfield and  Brunette with exchange visit. I feel a number of studies should be carried out to expand on the findings of my thesis. The cell-surface topography interaction should be investigated in greater detail. More specifically we need: 1. to determine  whether surface topography has an effect on functional activation of the M2 macrophage phenotype; the phenotype responsible for immunosuppression and healing.  This can be carried by two experimental designs. 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