Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effect of implant surface roughness on the NFkB signalling pathway in macrophages Ali, Tarek Adel 2008

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2008_spring_ali_tarek_adel.pdf [ 5MB ]
Metadata
JSON: 24-1.0066657.json
JSON-LD: 24-1.0066657-ld.json
RDF/XML (Pretty): 24-1.0066657-rdf.xml
RDF/JSON: 24-1.0066657-rdf.json
Turtle: 24-1.0066657-turtle.txt
N-Triples: 24-1.0066657-rdf-ntriples.txt
Original Record: 24-1.0066657-source.json
Full Text
24-1.0066657-fulltext.txt
Citation
24-1.0066657.ris

Full Text

EFFECT OF IMPLANT SURFACE ROUGHNESS ON THE NFKB SIGNALLING PATHWAY IN MACROPHAGES by TAREK ADEL ALI BDS, Alexandria University - Egypt, 1995 DDS, University of Alberta - Canada, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF COMBINED MASTER OF SCIENCE / DIPLOMA IN PERIODONTICS in THE FACULTY OF GRADUATE STUDIES THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2008 © Tarek Adel Ali, 2008 ABSTRACT Physical stress such as the surface roughness of the implants may activate the NFKB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which the macrophage interacts with the surface through serum proteins and/or the formation of membrane rafts. This thesis examines the role of surface topography on activation of the NFKB signalling pathway in macrophages. We examined the effect of implant surface topography on activating the NFKB signalling pathway in the RAW 264.7 macrophage cell line. We also examined the effect surface roughness had on the adhesion of the macrophages using the different media. To finish, we observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. Activation of the NFKB pathway was surface topography dependent. The Smooth surface showed the highest level of activation followed by the Etched then the SLA. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. Activation of NFKB occurred in the absence of fetal calf sera, although to a lesser extent. All three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. After 30 minutes, translocation reached comparable levels to those surfaces tested with complete medium. Disruption of the lipid rafts affected the triggering and signalling of Page i the NFKB pathway. This inhibitory effect was concentration and time dependent. Smooth surfaces bound more macrophages in the 30 minutes assay. Fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Removal of cholesterol did not affect adhesion or spreading on their respective surfaces. We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in-vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s) / surface structure the signal pathway is initiated. Page I ii TABLE OF CONTENTS Abstract^  i Table of Contents^  iii List of Tables vii List of Figures^ viii List of Abbreviations  x Acknowledgements^ xii Dedication^ xiv Chapter 1: Introduction^  1 1.1 Overview^  1 1.2 Dental Implants  3 1.2.1 Biofunctionality and Biocompatibility of Implants^  3 1.2.2 Osseointegration^  3 1.2.3 Surface Topography  4 1.2.3.1 Blasted Surface^  4 1.2.3.2 Acid-Etched Surface  5 1.2.3.3 Sand Blasted and Acid-Etched Surface (SLA)^ 5 1.2.3.4 Biological Responses to Surface Topography  7 1.3 Wound Healing^  8 1.3.1 Macrophages Participate in the Integration of Implants^ 9 Page 1 m 1.4 The Macrophage^  10 1.4.1 Origin and Tissue Distribution^  10 1.4.2 Macrophage Adhesion^  13 1.4.3 Macrophage Plasticity  13 1.4.4 Macrophage Activation^  17 1.4.5 NFKB signalling pathway  20 1.4.5.1 Activation of Nfic.13 in macrophages by LPS^ 22 1.5 Lipid Rafts and Signal Transduction^  26 Chapter 2: Hypothesis and Rationale  30 2.1 Hypothesis^  30 2.2 General approach  30 2.3 Rationale^  31 Chapter 3: Materials and Methods^  32 3.1 Preparation of replica surfaces  32 3.2 Cell culture^  32 3.3 Immunoflourescent labelling^  33 3.4 Cholesterol Depletion  34 3.5 Immunofluorescence^  34 3.6 Adhesion^  34 3.7 Scanning Electron Microscopy^  35 3.8 Statistical Analysis^  36 Page 1 iv Chapter 4: Results^  37 4.1 Activation of the NFKB Pathway^ 37 4.1.1 Effect of Surface Roughness on NFKB Signalling with Complete Medium ^ 37 4.1.2 Effect of Surface Roughness on NFKB Signalling using Complete Medium supplemented with a Suboptimal Dose of LPS^ 39 4.1.3 Effect of Surface Roughness on NFKB Signalling using Serum free Medium41 4.1.4 Effect of Mr3CD on NFKB Signalling^ 43 4.1.4.1 Polished Surfaces^ 43 4.1.4.2 Etched Surfaces 45 4.1.4.3 SLA Surfaces ^ 46 4.2 Effect of MI3CD on Cell Adhesion  47 4.3 SEM of Macrophages on the Different Surfaces in Different Media^ 49 4.3.1 Serum free medium^ 49 4.3.2 Complete medium 49 4.3.3 Complete Medium with Mi3CD^ 50 Chapter 5: Discussion^ 54 5.1 General Discussion 54 5.1.1 Activation of the NFKB Pathway^  55 5.1.2 Cholesterol Depletion^ 58 5.1.3 Adhesion and Morphology 60 5.2 Overall Model of the Possible Function of Surface Roughness on NFKB Nuclear Translocation^  61 Page 1 v 5.2.1 Effect of Surface Roughness on Cellular Receptors-What is the activating Signal?^ 61 5.2.2 Consequences of Surface Roughness on Signal Transduction^ 63 5.2.3 Effect of Surface Roughness on Pro-Inflammatory Cytokine Production^ 64 5.2.4 A Hypothetical Model^ 65 5.3 Summary and Conclusions 67 5.3.1 Macrophage Activation^ 67 5.3.2 Adherence and Morphology 67 5.3.3 Conclusions^  68 5.4 Future Directions 68 BIBLIOGRAPHY^ 71 Page I vi LIST OF TABLES Table 1: TLRs Involved in Innate Immunity in Humans^ 19 Page I vii LIST OF FIGURES Figure 1: Macrophage-family cells^  12 Figure 2: Innate and acquired immune activation of macrophages^ 16 Figure 3: Activation of NFid3^ 24 Figure 4: Raft microdomains 27 Figure 5: A model for cytoskeleton-driven assembly of raft macrodomains^ 29 Figures 6a & b: Effect of Surface Roughness on NFKB Signalling with Complete Medium ^38 Figures 7a & b: Effect of Surface Roughness on NFKB Signalling using Complete Medium Supplemented with a Suboptimal Dose of LPS^ 40 Figures 8a & b: Effect of Surface Roughness on NFKB Signalling with Serum free Medium ^ 42 Figures 9a & b: Effect of Mr3CD with various concentrations on NFKB Signalling on Polished Surfaces 44 Figure 10: Effect of Mi3CD with various concentrations on NFKB Signalling on Etch Surface 45 Figure 11: Effect of Mr3CD with various concentrations on NFicB Signalling on SLA Surfaces 46 Figure 12a: Effect of serum free medium on macrophage adhesion^ 47 Figure 12b: Effect of complete medium on macrophage adhesion  48 Figure 12c: Effect of complete medium + Mi3CD on macrophage adhesion^ 48 Figure 13: Cells on Polished, Etched & SLA in descending order using Serum free Medium after 30 minutes^ 51 Page viii Figure 14: Cells on Polished, Etched & SLA in descending order using Complete Medium after 30 minutes^ 52 Figure 15: Cells on Polished, Etched & SLA in descending order using Complete Medium with Mf3CD (13 mcg/ml) after 30 minutes^ 53 Figure 16: Activation of the NFKB Pathway^ 56 Figure 17: Hypothetical Model of Effect of Surface Roughness on NFKB Signalling^ 66 Page 1 ix List of Abbreviations APC^ Antigen-presenting cell BCR B-Cell Receptor CD14^ CD14 molecule CSF Colony-Stimulating Factor DAPI^ 4', 6'- diamidino-2-phenylindole DNA Deoxyribonucleic acid FCS^ Fetal Calf Serum GM-CSF^Granulocyte-Macrophage Colony-Stimulating Factor IFN- clip Interferon-alpha/beta IFNy^ Interferon-gamma IL Interleukin IicB^Nuclear factor of kappa light polypeptide gene enhancer inhibitor LBPs LPS-binding proteins LIF^ Leukemia Inhibitory Factor LPS Lipopolysaccharide MAPK^ Mitogen-Activated Protein Kinase mcg microgram(s) M-CSF^ Macrophage Colony-Stimulating Factor MPS Mononuclear Phagocytes System Page I x MR^ Mannose Receptor MPCD Methyl-beta-Cyclodextrin NF KB^ Nuclear Factor kappa Beta MyD88 ^myeloid differentiation primary response gene NO^ Nitric Oxide PPl.  ^ Pyrazolo-Pyrimidine 1 RES^ Reticulo-Endothelial System RNA Ribonucleic acid ROS^ Reactive Oxygen Species SCF Stem Cell Factor SEM^ Scanning Electron Microscope SLA Sand-blasted, Large grit, Acid-Etched SR-A^ Scavenger receptor-A TCR T-Cell Receptor TGF-p^ Transforming growth factor beta TiO2 Titanium oxide TLR^ Toll-like Receptors TPS Titanium Plasma Spray TRAF^ TNF Receptor Associated Factor Page 1 xi ACKNOWLEDGMENTS First And Foremost I Would Like To Thank God For All His Blessings. I would like to express my deep gratitude and appreciation to Dr. Doug Waterfield. You are an extremely helpful and understanding gentleman. You guided me through this entire process, were always there whenever I needed you and I would imagine that without your incredible support this would have been a very different experience. I admire and respect you greatly, both as a teacher and a person. THANK YOU. I would like to thank my committee members, Dr. Don Brunette and Dr. Ed Putnins for their generously given time and expertise to better my work. I thank them for their contribution and their good-natured support. My particular thanks goes to my colleague and good friend Salem Ghrebi who spent precious long hours helping and aiding me. I would like to thank Dr. Babak Chehroudi for his assistance with the statistics and Mr. Andre Wong for his technical assistance. I would also like to thank Mandana Nematollahi, Doug Hamilton and Bahadoor Baharloo for their contributions throughout this thesis. Special thanks go to Dr. Tassos Irinakis for his support and guidance throughout my program. As for Farzin Ghannad, my friend, classmate and Teri° Brother', you made this journey a bearable one. The long hours we spent together working our way through Page 1 xii 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. Your motherly love and care is beyond what words can describe, thank you. A very special thanks and acknowledgment goes to my in-laws for believing in me and for their immense support. And finally, the closest people to my heart, my family. I cannot express my love or give enough credit to my mother and father. 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 and partner in life, she was the one who had to go through all the ups and downs 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. My gorgeous daughters Layla and Yasmeen, you are my life and the love I have for you in my heart is unparalleled. Big cheers and utmost love goes to Asser and Ahmed; my two wonderful brothers, friends and confidants. God bless you both and guide you through your own individual journeys. Page xiii DEDICATION THIS THESIS IS DEDICATED TO MY FATHER AND MOTHER. Page 1 xiv Chapter 1: Introduction 1.1 Overview Ever since Branemark, in the 1960's, was able to place dental implants into a patients' mouth without rejection and with what he termed `osseoeintegration', the implant has pretty much revolutionized our practice of dentistry. Needless to say it has also been a major area of research in order to uncover the secrets of "so called" osseointegration and make it happen possibly faster and with complete predictability. In order to do that, we need to better understand what happens between the recipient tissues and the surface of the implant. This zone is a composite entity of numerous cells, proteins and molecules in close proximity to a polycrystalline surface of titanium (Sennerby et al. 1991). Commercially pure titanium implants form a metal oxide on the surface as a result of exposure to the atmosphere. The relationship of an implant with the surrounding tissue is highly dependent on the interaction between a titanium oxide (Ti0 2) layer which is formed on the surface of a titanium implant, and biological elements such as collagen, osteoblasts, fibroblasts and blood constituents (Schroeder et al. 1981). The oxide layer is very stable and corrosion resistant. It is thought to play a key role in the successful osseointegration of implants (Albrektsson et al. 1983). Macrophages play a critical role in the body's response to the "foreign body". They respond to virtually all implanted devices and materials, which include dental implants. Therefore, the role of macrophages in the hosts' inflammatory response, and possible implant rejection has attracted wide attention. Since macrophages arrive rapidly to the area of implant placement, it is critical that we better understand the role that it plays in the hosts' response and in turn, osseointegration. Page 1 1 A major function of macrophages is to mediate host immune and inflammatory responses against foreign objects. They also play an important role in angiogenesis and repair which may also include possible bone formation. For this reason, a clear understanding of the complex interaction between macrophages and biomaterials is crucial for the improvement of materials employed in the construction of these biomedical devices (Kao 1999). It remains unclear how macrophages respond to implanted materials and whether they do so, for example, in a similar manner to their response to a foreign body, the Gram negative bacterium. It may be possible that implants activate macrophage responses in a similar way as bacterial pathogens; activation through Toll and pattern recognition receptors. However, it is also possible that implants activate macrophages through an entirely different mechanism. Over the past few years, surface topography has shown to play an important role in how the body reacts to the implant (Brunette 2005, Hamilton 2006, and Hamilton 2007). Rougher surfaces have shown to better induce bone formation through its direct effect on the osteoblasts. As previously noted, since the macrophages play an important and 'early' role in the body's response to the implant, it is critical to understand what role surface topography has in the macrophage activation. This is the aim of our study. Page 1 2 1.2. Dental Implants Dental implants have been widely used for over 3 decades to help restore both function and aesthetics for patients who have lost some or all of their teeth due to various reasons. 1.2.1 Biofunctionality and Biocompatibility of Implants Biofunctionality is defined as the ability of the device to perform the required function while biocompatibility is the ability of the device to perform its intended function, with the desired degree of incorporation in the host, without eliciting any undesirable local or systemic effects in that host (Cook 1992). The insertion of the implant into the human body evokes a series of host responses known collectively as inflammation (Suska 2001). The use of the biocompatible materials in implants generally results in a progression of the inflammatory response from a high intensity, acute phase to a low-activity, quasi-equilibrium state, called the foreign body reaction (Jenney 1998). 1.2.2 Osseointegration This process was first described by Branemark and co-workers in 1977. The term was first defined in a paper by Albrektsson et al. in 1981 as direct contact (at the light microscope level) between living bone and implant. Osseointegration is histologically defined in Dorland's Illustrated Medical Dictionary as the direct anchorage of an implant by the formation of bony tissue around the implant without the growth of fibrous tissue at the bone—implant interface. Since the histological definitions have some shortcomings, mainly that they have a limited clinical application, another more biomechanically oriented Page I 3 definition of osseointegration has been suggested: "A process whereby clinically asymptomatic rigid fixation of alloplastic materials is achieved, and maintained, in bone during functional loading" (Zarb and Albrektsson 1991). A number of important concepts pertaining to dental implants are discussed below. 1.2.3 Surface Topography Commercially, there are many different types of dental implant surfaces available, rough surfaces being among the most popular. These rough surfaces can be divided into two groups: 1. Roughened surfaces produced by mechanical and chemical means such as the acid-Etched, sandblasted or a combination of both (SLA). 2. Surfaces roughened by the addition of a coating such as the hydroxyapatite coated implants. Our studies will concentrate on the first group. 1.2.3.1 Blasted Surface This type of surface is blasted with particles such as TiO2 or Al203, a commercial example is TiOblastTM produced by Astratech. The resulting surface from the blasting is an isotropic one. Titanium oxide particles with an average size of 25 pm produce a moderately rough surface in the 1-2 pim range on dental implants. Studies have reported high clinical success rates for titanium grit-blasted implants, up to 10 years after implantation (Rasmusson 2005). In 1996, Wennerberg et al. demonstrated that grit- blasting with TiO2 particles gave similar values of bone to implant contact as a smooth surface but significantly increased the biomechanical fixation of the implants. Although Page I 4 the torque force increased with the surface roughness of the implants, comparable values in bone apposition were observed (Abron 2001). This corroborates that roughening titanium dental implants increases their mechanical fixation to bone but not their biological integration. 1.2.3.2 Acid-Etched Surface Acid-Etching of titanium in a solution of hydrochloric acid and sulphuric acid results in a micro-rough surface. This technique is used with the Osseotite Implant System (Implant Innovations, Inc. (3i), Palm Beach Gardens, Florida). It is worth mentioning that the texture is not uniform over the entire screw surface as the surface area on the top is 1.8 to 2 gm' and decreases to 0.5 to 0.7 1.1m 2 in the valleys and on the flanks (Al-Nawas 2003). These surfaces showed greater bone-implant contact than in machined surfaces even when the quality of bone was described as being poor (Weng 2003). Clinically there was a cumulative success rate of approximately 97% at 6 years (Sullivan 2001). Another study showed that at the 12-month follow-up appointments, cumulative survival rates of 98.0% were recorded for the implants (Drago 2006). 1.2.3.3 Sand Blasted and Acid-Etched Surface (SLA) The SLA (Sand-blasted, Large grit, Acid-Etched) surface was first tested in cell cultures and animals in 1990. The results of these tests inspired the launch of a full scale SLA research project. The SLA surface has proved to be able to be functionally loaded 6 weeks after placement of a 4.1 diameter ITI implant. More than ten years of intensive testing and successfully ongoing clinical and field trials have shown that the SLA implant surface Page I 5 clearly has the potential to replace the rough titanium plasma spray (TPS) implant surface. It is postulated that the macro/micro double roughness of the SLA surface has led to improved osseointegration of ITI implants as well as reduced time to loading of about a maximum of 50%. This allows full prosthetic restoration six weeks after implant placement in healthy patients with adequate bone quality (Scacchi 2000). The SLA surface is produced by a large grit sand-blasting process with corundum particles that leads to a macro-roughness on the titanium surface. This is followed by a strong acid-Etching bath with a mixture of HCl/H2SO4 at elevated temperature for 5 minutes (Wong 1995). The result was macro-roughness achieved by large grit sand-blasting followed by acid-Etching creating the micro roughness superimposed on the macro structure. The chemical composition of the SLA structure was found to be titanium oxide (TiO2) using X-ray photoelectron spectroscopy. This method analyses the first few atomic layers of the surface, and thus the chemical composition of the material which is in direct contact and interacts with tissue fluids and cells. The SLA surface was launched in mid-June 1998 at the ITI World Symposium in Boston. In a direct biomechanical comparison between the SLA and the machined and acid-Etched surface, an animal model for implant removal torque testing was carried out using a split-mouth experimental design (Li 2002). The results of the study demonstrated that the SLA implant produced torque values which were higher by approximately 30%. Recent clinical studies have underlined the effectiveness of SLA implants, which allow restoration after a reduced healing period of only 6 weeks. Success rates of approximately 99% have been reported for clinical follow-up periods of up to 5 years (Bornstein 2005). Page 16 1.2.3.4 Biological Responses to Surface Topography The success of dental implants is dependent on the material properties of the implant material including mechanical properties, surface chemistry, and surface topography. The main goal of implanting any material is to obtain an appropriate host tissue response for the particular application. Surface topography is known as one of the major determinants of implant performance in-vivo and influences cell behaviour in a myriad of ways. This includes cell adhesion, cell selection, mechanical interlocking, cell orientation and topographic (contact) guidance, tissue organization, cell shape, production of local microenvironments (Albrektsson T et al 1981 and Brunette DM 1996) and production of growth factors and cytokines (Kieswetter K et al 1996). The response of surface topography (roughness) and micro machined surfaces to cells including fibroblasts, epithelium 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). Unfortunately, the same may not be said about the relationship between macrophages and the dental implant surface although it has been demonstrated that a clear understanding of the complex interaction between macrophages and biomaterials is crucial for the improvement of materials employed in the construction of biomedical devices (Kao 1999). The processes leading to macrophage adhesion and activation on biomaterials are complex and not yet fully understood. In summary, surface roughness plays a major role in both the quality and rate of osseointegration of titanium dental implants. Highly roughened implants such as TPS or grit-blasted have been shown to favour mechanical anchorage and primary fixation to Page I 7 bone. Topographies in the nanometre range have been used to promote protein adsorption, osteoblastic cell adhesion and the rate of bone tissue healing in the peri-implant region. 1.3 Wound Healing As soon as the implant recipient site is prepared, a cascade of events take place collectively known as wound healing. This may be divided into coagulation/fibrinolysis phase were a blood clot is formed, an inflammatory phase and finally repair. The inflammatory phase seems to be the very critical part of this process. This may be explained by the fact that during this phase not only are the cells involved in the defence mechanisms, but also are critically involved in the next phase which is the repair process. The immune system can be considered as being divided into humoral and cellular defences. The humoral includes antibodies and complement. The cellular part of the immune system consists of neutrophils, macrophages, and lymphocytes. These cell populations appear and peak in the wound in a specific time frame defining and controlling the different yet overlapping stages of wound healing (Witte 1997). Needless to say, the macrophage is an active participant in this process. They reach their maximum number at the site within 24 — 36 hours. During their presence at the scene, they are heavily involved in the phagocytosis process, removal of debris and wound cleaning. As will be detailed in a following section macrophages exhibit "plasticity" and can play a major role both in the induction of the mechanisms of the adaptive immune system as well as in the production of substances that are crucial in the process of wound healing (Tsirogianni 2006). Experiments on animals which were macrophage-depleted, showed that wound repair was defective (Danon 1989). On the other hand, intradermal injections Page 8 of macrophages into cutaneous wounds in rats lead to an increase in collagen synthesis and tissue strength (Casey 1976). Macrophages participate in the repair process through the secretion of cytokines and growth factors. These in turn activate and recruit other cells involved in wound healing, regulate fibroblast chemotaxis, proliferation, and collagen synthesis (Wahl 1985). Through these numerous and various functions, macrophages influence angiogenesis, fibroplasia, and matrix synthesis (Park 2004). 1.3.1 Macrophages Participate in the Integration of Implants Macrophages are detected on different types of material surfaces and in the peri-implant tissue after implant insertion in soft tissue (Ericson 1991). In our Lab (Brunette's Lab, Faculty of Dentistry, UBC), implant surfaces of varying roughness were implanted subcutaneously in rats. The results revealed that macrophage- like cells, as assessed by morphology, were observed on rough SLA surfaces in greater numbers than on Smooth surfaces or grooved micro fabricated surfaces in which each facet of the groove was Smooth. More interestingly, the SLA surfaces (rough) were associated with more bone formation than the Smooth or grooved surfaces (Brunette et al 2003). Therefore, the authors suggested that the presence of macrophages on an implant surface is not necessarily detrimental and it is possible that the expression of proteins by macrophages recruited to the rough surfaces may exert positive effects on bone formation. Page I 9 1.4 The Macrophage Our interest in macrophage interaction with material surface topography is derived from two perspectives. First, we would like to understand what aspects of the cell—surface interaction lead to macrophage activation and signalling. Second, we would like to determine what aspects of surface topography lead to biologically favourable macrophage—surface interactions. In our lab, it was demonstrated that a suboptimal dose of LPS (6.4 ng/ml) and surface topography act in a synergistic manner to activate the macrophage that lead to an increase in the secretion of pro-inflammatory cytokines and chemokines (Refai et al. 2004). Since it has been well established in the literature that one of the ways LPS activates the macrophage is through Toll-like receptors and the NFKB pathway, we set our study to look at this pathway to see if the surface topography could activate macrophages through the same pathway. 1.4.1 Origin and Tissue Distribution Macrophages are part of the reticuloendothelial system, later renamed the mononuclear phagocytes system (MPS) when it was recognized that a subset of these RES cells derive from bone marrow precursors (van Furth 1972). The MPS family of cells comprises bone marrow progenitor cells, pro-monocytes, peripheral blood monocytes, and tissue macrophages. The cells of the mononuclear phagocyte system are derived from pluripotent hematopoietic stem cells in bone marrow that further differentiate into monoblasts, promonocytes and then monocytes which mature to become tissue macrophages (Figure 1). Approximately 24 hours after entering the systemic circulation, monocytes migrate into tissues where they differentiate into macrophages. During this Page 10 maturation process, the cells become larger and the numbers of mitochondria and lysosomes within them increase. The relative amounts of lysosomal enzymes are also notably higher in macrophages than in their precursors. The process of macrophage differentiation is regulated by their microenvironment and by the degree of hematopoietic stimulation within the tissue (Billack 2006). Optimal proliferation and differentiation of mononuclear phagocytes from pluripotent progenitors requires the presence of a combination of polypeptide growth factors (Kaufmann 2004). These include macrophage colony-stimulating factor (M-CSF or CSF- 1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-3 (IL-3), stem cell factor (SCF), interleulcin-1 (IL-1), leukemia inhibitory factor (LIF) and interferon-y (IFN7). Amongst the above factors, CSF-1 is the only one that is clearly absolutely required for macrophage differentiation and proliferation in vivo, and as the sole added factor, CSF-1 can also direct macrophage differentiation from bone marrow progenitors in vitro (Metcalf 1989, Lee 1992, Wiktor-Jedrzejczak 1996, Metcalf 1997). Macrophages are generally a population of ubiquitously distributed mononuclear phagocytes responsible for numerous homeostatic, immunological, and inflammatory processes. Their wide tissue distribution makes these cells well suited to provide an immediate defence against foreign elements prior to leukocyte immigration. Because macrophages participate in both specific immunity and nonspecific immunity against bacterial, viral, fungal, and neoplastic pathogens, it is not surprising that macrophages display a range of functional and morphological phenotypes. Page 1 11 111 TissuesBone marrow^Blood I Pasident mac-optiago^huh alcd dociritic colts yinc3h) rxxias CA111111{3f1 11100d PrWielit7 GM.CSF TL3 KI TNF larnily rrx rttets PU.1 fit - and k - Ille'lrin$ Immurv.31Qwlir -!arr Meri1Der5 SeleCtilS EGF-TM7 tKeptors Endothelial tra:ropt.ape Recrui rnac•cphage ArNen-ior-sper,: , lic e'hnited Oasscally antigen activated I argarbans cell ?,5kin) Kup4fer cell Pet) \ OstEpclasls (Ilene) Microg' a :CMS) Normal macrophages for example include macrophages in connective tissue (histiocytes), lung (alveolar macrophages), spleen (free and fixed macrophages), bone marrow (fixed macrophages), skin (histiocytes, Langerhans's cell) etc. The macrophage population in a particular tissue may be maintained by three mechanisms - influx of monocytes from the circulating blood, local proliferation and biological turnover. Under normal steady-state conditions, the renewal of tissue macrophages occurs through local proliferation of progenitor cells. The viability of macrophages ranges between 6 and 16 days. Nature Reviews I Immunology Figure 1: (Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology: Alternative activation of macrophages 3, 23-35 (January 2003) doi:10.1038/nri978) Macrophage-family cells (cells of the mononuclear phagocyte system) have marked phenotypic heterogeneity. A simplified life history of the macrophage and closely related cells is shown above. Page I 12 1.4.2 Macrophage Adhesion Macrophages express many receptors that mediate their diverse functions. The receptors are located on the surface as well as in vacuolar compartments and the cytosol, thereby mediating recognition of both extracellular and intracellular pathogens. Receptors used for phagocytosis of microorganisms are called opsonic receptors. This group of receptors include complement receptors, Fc receptors (Ig superfamily), and the non-Toll-like receptors, the latter of which include the family of scavenger receptors as well as C-type lectins (Gordon 2007). These receptors function in phagocytosis and endocytosis of complement- or antibody-opsonised particles/microorganisms, respectively (Hawlisch 2006). Some opsonic receptors have a role in cell signalling. For instance, Fc receptors have a modulatory effect on NFKB induction (nuclear transcription factors that regulate production of pro-inflammatory mediators- Hawlisch 2006). Also scavenger receptors have been shown to collaborate with TLR (see below) to induce NFKB. They may also directly mediate NFKB induction upon interaction with their appropriate ligand. Non-opsonic surface receptors that do not mediate phagocytosis/endocytosis but are important sensors of bacteria, fungi and viruses are the Toll-like receptors (TLR) (O'Neill 2006). 1.4.3 Macrophage Plasticity As is apparent from the previous section on the origin and tissue distribution of macrophages lineage-defined populations of macrophages have not been identified. The heterogeneity of the macrophage function instead reflects the plasticity of the macrophage. This plasticity in function is determined by both the tissue and the immunological Page 1 13 microenvironment in which the macrophage exists. The diversity seen in macrophage differentiation to these signals is ever increasing. A number of models have been put in place to simplify this diversity of function. I will be discussing one of these models (Figure 2). In response to cytokines and microbial products in vitro macrophages can demonstrate specialized (polarized) functional properties. Five types of macrophage activation have been elucidated. The first pathway is called Innate Activation. In this case microbial stimuli are recognized by pattern-recognition receptors, such as Toll-like receptors (TLRs), CD14/lipopolysaccharide-binding protein and a range of non-opsonic receptors. These stimuli induce the production of pro-and/or anti-inflammatory cytokines, such as interferon-cc/13 (IFN-«/a), and reactive oxygen species (ROS) and nitric oxide (NO), followed by a regulated inflammatory response. Enhanced expression of co-stimulatory surface molecules favours antigen presentation and activation of specific immunity. Scavenger receptor-A (SR-A) and mannose receptor (MR) promote the phagocytosis and endocytosis of host, as well as microbial ligands. The second form of activation is Humoral Activation. Humoral activation and phagocytosis is mediated by some Fc and complement receptors, whereas other receptors down-regulate responses. This activation results in cytotoxic function of macrophages such as those seen in response to tumours. Again, as above, the immune response can be modulated by the production of pro-and/or anti-inflammatory cytokines. Classical activation is the third form of activation. The macrophages are activated by the priming stimulus IFN- v, followed by a microbial trigger (lipopolysaccharide, LPS). This form of activation produces effecter cells with a greatly enhanced capability for Page 1 14 killing intracellular microbes such as Mycobacterium tuberculosis. These macrophages are also highly pro-inflammatory as a result of the pro-inflammatory cytokines they secrete. The fourth form of activation is Alternative Activation. Alternative activation is mediated by interleukin-4 (IL-4) and IL-13, acting through a common receptor chain (IL-41Zok). This response is involved in activation of humoral immunity as well as anti- parasitic responses. It has been also implicated in the repair process. Due to the induction of arginase these cells are implicated in tissue remodelling and angiogenesis. The final form of activation is referred to as the Innate/acquired Deactivation state. The uptake of apoptotic cells or lysosomal storage of host molecules generates anti- inflammatory responses using through production of the anti-inflammatory cytokines TGF-I3 and IL-10. Cellular activity is modulated by the interactions of macrophages with T cells, fibroblasts and matrix, through a range of receptors. Cytokines and glucocorticosteroids are potent modulators of activation. Page 1 15 C014 il'glucan 0 rPoentor TREM^""N__ b Hiurrioral activation Fc ^oceptcrs ENC:i , _ zym CrOrripi:rtiVil receptor • Ciasa►cal activation IN y recep'01( d P I1omativ activation IL-4 colL•13 M17 CD2ONI. - Storage al and geop- oarebrosides Cytokine ^voelotor for IL-10. TGF IFN-40 oeho,CSF Gkumccattoostered recootoir e Innateiacquirod deactivation 1;.pat...03m MI-IC class it Gowivity-ialiOn Art rillianvnatcev cytoknes TGF p „0 IL-'0 Lb ▪ PGE7 Irrevurio Supp•aSSO1 Nature Review2 I Immunology Co stimulatory tnoiecilieN • Mcrobai Ingoer by u-,s ••■■•••• Pro- ellarivrarlory C$1''ICkflet3 0 I -6 Trif 0 II NO and r5prratory tx1r-st Microb•colai Tissue darrage GNU& arwreurkty DTH cass 4,  0-14rnoral ir-murkty Alier9c and anti-pareWe responses Repar (argresef 111 Innate activation M■CrMal :Taws Figure 2: Innate and acquired immune activation of macrophages. ((Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology: Alternative activation of macrophages 3, 23-35 (January 2003) doi:10.1038/nri978). Page I 16 At the present time there have been no studies on the plasticity of macrophage responses to dental implant surfaces. 1.4.4 Macrophage Activation Macrophages need a stimulus to be activated. Such stimuli may come from diverse sources. For example a "foreign body", microorganisms and/or bacterial products can trigger a macrophage response. Similarly, macrophages can be triggered by stresses such as found by encountering pro-inflammatory cytokines. As mentioned above when stimulated they respond by either phagocytosis, binding to an invading pathogen releasing a wide array of mediators including reactive oxygen, enzymes, bioactive lipids, and cytokines and chemokines. Macrophages are also found to actively respond to almost all biomaterial implants in vivo, including metal (Takebe et al. 2003). The macrophage response to modern synthetic biomaterials has much in common with its response to a much older foreign body, the Gram-negative bacterium. This response includes recruitment and activation of other leukocytes, increased matrix turnover, angiogenesis, promotion of cell survival, and stimulation of bone resorption (Garrigues et al 2005). As such a similarity exists, it warrants a detailed discussion of how macrophages respond to Gram-negative microorganisms. Lipopolysaccharide (LPS) is a part of the outer membrane of gram-negative bacteria which contributes to the structural integrity of the organism. After extracellular lyses of the Gram-negative bacteria by the complement system, LPS is released into the circulation where it becomes bound to plasma proteins designated as LPS-binding proteins (LBPs). In Page 1 17 this conjugated form, LPS is presented to specific receptors on monocytes and macrophages. Since the early 1990s, the effects of LPS on macrophages were known to be mediated by the activation of the CD14 receptor. The CD14 receptor is an outer membrane protein that neither traverses the membrane nor has an intracellular effector domain. Therefore, it was believed that a second molecular component of the receptor was present to enable the LPS-mediated signalling to be transduced across the membrane, and ultimately, to effect gene activation. The existence of this additional LPS-receptor component, designated as the "Toll-like" receptor 4 (TLR4), has recently been confirmed and found to be itself a transmembrane receptor with an extracellular LPS-binding domain and a cytoplasmic domain that serves as a platform for the recruitment of protein kinases involved in cell signalling. Once LPS binds to CD14, LBP dissociates and the LPS-CD14 complex physically associates with Toll 4. An additional accessory protein MD2 is required for the final signal induction. In humans, TLRs participate in the innate immune response (Blach-Olszewska 2005). They are type I membrane-associated receptors characterized by an extracellular leucine- rich repeat signature, a transmembrane cysteine-rich flanking region, and a cytoplasmic domain referred to as the Toll-IL-1 receptor (TIR). Eleven TLRs have been identified in humans (Table 1). Among them, TLR1, TLR2, TLR4, TLR5, TLR6, TLR10 AND TLR11 are cell surface receptors, whereas the others are associated with the membrane of intracellular organelles such as endosomal vesicles (Iwasaki 2004, Billack 2006, Carmody 2007, Uematsu 2008). This family of receptors is capable of recognizing conserved microbial patterns including components of the bacterial cell wall, microbial nucleic acids, Page ! 18 and bacterial motility (Krutzik 2004, Billack 2006). Hirschfeld et al. (2001) demonstrated that signalling by the mammalian TLR2 and TLR4 lead to the activation of overlapping but non-identical genes. For instance, TLR2 activation causes the expression of IL-113, but not IFNY, whereas TLR4 activation leads to the expression of both IL-1I3 and IFNY. This demonstrates that activation of different TLRs by different types of pathogens promotes the generation of non-identical signalling pathways. These signals will then tune the immune system to activate the appropriate effector responses. Known ligands of TLRs include LPS, peptidoglycan, lipoteichoic acid, and stress proteins (Blach-Olszewska 2005). 111111111111 Cell- Surface^Mycobacterial products Cell Surface Lipopol■ saecharide Cell t surfacc & IrstrarceW4 Cell Surface Cell Steaec Cell Surface Intracel Intracellular Cell Surface Cell Surface era ai Lipopolvsaccharide Bacterial flagellin Bacterial lipopeptides e$ (antiviral agents), Single-stranded RNA Bacterial Unknown Profilin-like molecule Table 1: TLRs Involved in Innate Immunity in Humans (Billack 2006, Carmody 2007, and Uematsu 2008) TLRs are now known to have dynamic functions going beyond pattern recognition that trigger host defence mechanisms against microbial invasion. Following TLR activation, largely defined signal transduction cascades through the recruitment of differential adapter proteins to the cytosolic TIR domain is induced. Subsequently, this leads to the activation Page I 19 of NFKB signal transduction pathway, mitogen-activated protein kinase (MAPK), and interferon (IFN)-regulatory factor 3 (IRF3) in a specific manner determined by the nature of the ligand and the engaged TLR (Hoffmann 2006, Kaisho 2006 and Beutler 2006). The intent of this thesis is to look at the effect of surface topography on activation of the NFKB signal pathway. 1.4.5 NFKB signalling pathway Ever since it was first described in 1986 by the Noble prize recipient David Baltimore, the NFKB signalling pathway has attracted many researchers to further explore and understand the nature and significance of this pathway. The activation of this pathway has been shown to be significant in regulating various target genes leading to numerous biological functions. It has often been referred to as the central mediator of the immune response since its activation controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. NFKB is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA- binding proteins that recognize a common sequence motif. Activators of NFKB signalling include bacteria, bacterial products, viruses, viral products, chemotherapeutic agents as well as mitogens and growth factors. Also, it has been demonstrated that NFKB activity may be induced by physical stress (Pahl 1999). Therefore, it has been concluded that NFKB functions generally as a central regulator of stress responses. There are two signalling pathways leading to the activation of NFKB known as the classical and the alternative pathway (Karin 1999; Tergaonkar 2006). The alternate Page I 20 pathway involves ligation of the cell surface receptors LTI3R, BAFFR, and CD4OR. Intracellular signalling by phosphorylation and processing of transcription factors is significantly different than that used by the classical pathway and culminates with the binding of ReIB and P52 to promoters in the nucleus. The classical pathway is activated by a number of different mechanisms. The most common classical signalling pathways are those that originate through ligation of TNFR To11R/IL-1R and T/B cell receptor. NFKB is normally sequestered in the cytoplasm of non-stimulated cells and consequently must be translocated into the nucleus to function. Translocation capability of NFic13 is halted by its association with IkB family of inhibitory proteins (Verma et al. 1995). In both pathways, when a stimulus leads to the activation of NFKB, the IkB proteins are degraded and NFKB is released to translocate into the nucleus where it regulates gene transcription (Karin 2000, Gilmore 2006, and Tergaonkar 2006). More than 700 blocking agents have been described in the literature that have the ability to inhibit the NFKB pathway. These compounds include antioxidants, peptides, small RNA/DNA, microbial and viral proteins, small molecules, and engineered dominant- negative or constitutively active polypeptides. These inhibitors (in the form of compounds and agents) seem to block the pathway either generally or partially by blocking only a part of the pathway (Gilmore 2006). However, there has not been any description in the literature of the effect dental implants and more specifically the surface topography, on the activation and/or inhibition of the NFKB pathway. From the second a dental implant is placed into the prepared bony socket, millions of cells quickly migrate to the surface of the implant and start responding to it in various ways. The traditional inflammatory response is initiated through the activation of Page 21 these cells followed by a phase of repair which may occur concomitantly with the inflammatory cycle. Therefore, the role of the dental implant in this process of inflammation/repair appears to be very intriguing. 1.4.5.1Activation of NFKB in macrophages by LPS In mammals, TLR4 is required for LPS-mediated signalling and is believed to directly recognize LPS. LPS is a potent activator of innate immunity, and excessive exposure to LPS, or endotoxin, causes serious pathological effects (Beutler 2000). During a microbial infection, LPS-binding protein (LBP) binds to circulating LPS and presents it to the CD14/MD2 which then binds TLR4 receptors. The TLR4 is then activated, initiating a phosphorylation cascade that results in the phosphorylation of and its dissociation from the complex. In mammals, the first intracellular event following LPS treatment is the recruitment of the adaptor protein MyD88 to TLR4 (Medzhitov et al. 1998). MyD88 is one of four adaptor proteins that can be used for Toll signalling. MyD88 is also required for IL-1 and IL-18 signalling and activation of NFKB (Adachi et al. 1998). MyD88 negative mice and macrophages were also found to be unresponsive to LPS. However, MyD88 negative macrophages still activate NFicB DNA-binding activity in response to LPS, albeit with delayed kinetics (Kawai et al. 1999). This unexpected finding suggests that LPS signalling, presumably through TLR4, requires MyD88 for its full biological response, but that macrophages do not entirely require MyD88 to degrade IkB and activate NFKB. The mechanism by which LPS activates NFKB in a MyD88-independent manner remains unknown. One possibility is that the LPS induced NFKB response observed in MyD88- deficient cells reflects NOD signalling rather than TLR4 signalling. Another possibility is Page 1 22 that TLR4 uses another adapter molecule in addition to MyD88. It is also not clear why macrophages from MyD88 knockout mice are unresponsive to LPS but can still activate NFKB (Silverman 2001). MyD88 is crucial for the assembly of downstream signalling complexes, which included protein kinases (IRAK family members) and TRAF. The major downstream event of Toll 4 signalling is activation of the NFKB transcription factor. Under normal circumstances, the NFKB transcription factor, a dimer made up of two subunits called p50 and p65, is observed in the cytoplasm joined to an inhibitor protein called IKB (Figure 3). The activation of NFKB is initiated by the signal-induced degradation of IKB. The p50/p65 activated dimer translocates into the nucleus where it binds to specific DNA sequences called kappa B sites (KB) and promotes gene transcription. Page I 23 Endosomal TLR778/9 Lbiqumnabon P65 Pro4nnammatory cytolcines 0 = Phosphoryanon TRW Bacterial^Plasma memIxang,-- lipopeptides TLR4 dsRNA Endosoma TLR3 IKK complex NI- se. 5.,e2Tt^v Set 38 lay /4-^Ac//' Jr' LPS Figure 3: (Reprinted from Doyle S.L., O'Neill L.A.J. Toll-like receptors: From the discovery of NFKB to new insights into transcriptional regulations in innate immunity(2006) Biochemical Pharmacology, 72 (9 SPEC. ISS.), pp. 1102-1113. with permission from Elsevier): Activation of NFKB in macrophages by LPS. Under normal circumstances, the NFKB transcription factor, a dimer made up of two subunits called p50 and p65, is observed in the cytoplasm complexed to an inhibitor protein called IKB. During a microbial infection, LPS-binding protein (LBP) binds to circulating LPS and presents it to the CD14 and TLR4 receptors (Step 1). The TLR4 is then activated, initiating a phosphorylation cascade that culminates in the phosphorylation of IKB (Step 2). Phosphorylation of IKB results in the dissociation of IKB from the complex (Step 3). Interestingly, the phosphorylated IKB peptide is degraded by the 26S proteasome. The p50/p65 dimer translocates into the nucleus where it binds to specific DNA sequences called kappa B sites (KB) and promotes gene transcription (Step 4). Proinflammatory proteins such as NOS2 and TNFa are coded for by genes that are transcriptionally regulated by NFKB. (Janeway 2002, Beutler 2005, Takeuchi 2002 and Karin 2000). Page I 24 Surface topography of dental implants may activate proinflammatory cytokine production in macrophages in a similar way that gram-negative bacteria or lipopolysaccharide (LPS) activate macrophages -via the Toll-like receptor 4 (TLR4) (Refai 2004). 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 and T lymphocytes, while implantation of most synthetic biomaterials causes inflammation and foreign body responses without the involvement of antibody-producing cells such as B cells and T cells. There are at least three possible mechanisms whereby surface topography may affect macrophage activation and secretion: 1. The surface roughness itself has been found to affect the spreading, proliferation, and differentiation of cells in vitro (Martin 1995). 2. The increase of surface area characteristic of a rough surface has been found to affect cytokine production (Shanbhag 1994). 3. Quorum sensing, a phenomenon whereby gene expression responds to population interaction (Greenberg 1994) on the different surfaces. Just what receptor(s) and intracellular pathways are associated with the above possibilities are matters of current investigation. Page i 25 1.5 Lipid Rafts and Signal Transduction Cell membranes are composed of separate domains that have unique physical and biological properties. One class of membrane domain is the sphingo-lipid and cholesterol enriched membrane rafts. Lipid rafts are dynamic micro-domains in the membranes of living cells, composed of cholesterol and sphingo-lipids. Cholesterol and sphingolipids carrying saturated hydrocarbon chains assemble to form tightly packed sub-domains corresponding to liquid-ordered phases biophysically characterized in model membranes. In immune cells and other cell types, many of the proteins that reside in rafts function in cell signalling. Accordingly, it has been proposed that rafts act as specialized signalling compartments in cell membranes (Simons 2000). This hypothesis has been supported by studies showing that lymphocyte signalling is inhibited by removing cholesterol from rafts thereby inhibiting raft macro-domain formation (Xavier 1998). A significant feature of raft macro-domains is that they are reflective of the signals initiated by surface receptors. An example of this principal is the immune synapse of T cells stimulated with antagonist ligands to the TCR. Such ligands cause altered signalling events that fail to activate the cell (Sloan-Lancaster 1996). The failure of a ligand to generate a mature immune synapse in T cells might be due to the signals not being sufficient to cause the actin reorganization that is necessary for raft assembly beyond a certain threshold (Figure 4). In any event, failure to form an immune synapse inhibits the prolonged signalling needed for the induction of T-cell proliferation (Lee 2002). Accordingly, actin-dependent assembly of rafts for T-cell activation could serve as a gatekeeper to ensure that signals arising from the TCR are suitable for a full response by the cell (Rodgers 2005). Page 1 26 Actin cytoskeleton • fir In ce Paft macrodoman ^• Raft micmdDrnain Raft—cytosk;10on linking nachi i;r ye TREIZSjii :nironotiogy Figure 4: (Reprinted from Rodgers W, Farris D, Mishra S. Merging complexes: properties of membrane raft assembly during lymphocyte signalling. Trends Immunol. 2005 Feb;26(2):97-103. With permission from Elsevier): Raft micro-domains (coloured circles) are 50-100 nm in size, with separate micro-domains having distinct protein compositions. Still larger are raft macro-domains, which are micrometers in size. The macrostructures form through association of rafts with the underlying actin cytoskeleton and these interactions are mediated by raft—actin-binding machinery, such as the ERM family of proteins. Molecular mobility experiments show raft-associated proteins exchange with non-raft regions of the plasma membrane and, based on the rate of diffusion, this probably occurs by diffusion of protein monomers or small raft structures that contain no more than several raft proteins (black circles). Single particle tracking experiments suggest that proteins and lipids reside in raft micro-domains for 5 to 10 s. However, the lifetime of the micro-domains and raft micro-clusters is largely unknown. Macro-domains, by contrast, can remain assembled for hours. Page I 27 Although biochemical and genetic studies have provided compelling evidence for raft— actin interactions, the mechanism governing assembly of rafts into macro-domains has been less clear. One hypothesis is that raft macrostructures are assembled through migration of smaller rafts in the plasma membrane to discrete regions on the cell surface (Rodgers 2005). The recruitment of rafts to the antigen receptors during cell activation, as evidenced by imaging experiments underscores an important synergy between rafts, the cytoskeleton and lymphocyte signalling (Figure 5). In this model, engagement of surface receptors that initiate signals for actin polymerization and raft migration results in delivery of raft-associated signalling proteins to the site of cell signalling. The merging of rafts and accumulation of signalling proteins amplifies the initial signals from the surface receptors, resulting in a cascade of continued raft assembly and signal amplification. The cascade continues until inhibitory signals are delivered that attenuate cytoskeleton dynamics, and this could include protein tyrosine phosphatases that function to down regulate signalling proximal to the antigen receptors (Mustelin 2003). The notion that cell signalling and activation is coordinated with assembly of raft macro-domains is supported by genetic data showing that efficient capping of rafts is necessary for robust activation of T cells (Holsinger 1998, Fischer 1998). Similarly, immune synapses mature through discrete stages of recruitment, rearrangement and activation and inactivation of signalling proteins at the site of T cell—APC interaction (Freiberg 2002), thus further underscoring the dynamic interplay between raft assembly and cell signalling. Page I 28 4q, (,) Ado bona! sgna's 77,?'1.7.70.1::, 2)....ow...too..v.. InTacelltfar slna'ng a nd actin pioymenzation Receptor—ligand interactions ^ Rat migration ^ Signal amps fication Figure 5: (Reprinted from Rodgers W, Farris D, Mishra S. Merging complexes: properties of membrane raft assembly during lymphocyte signalling. Trends Immunol. 2005 Feb;26(2):97-103. with permission from Elsevier): A model for cytoskeleton-driven assembly of raft macro-domains. Assembly of raft macro-domains occurs in three stages, beginning with engagement of receptors by ligands expressed on secondary cells. Interaction between multiple species of receptor—ligand pairs might occur, and the receptors might be distributed within (blue arrow) and outside (purple arrow) of raft micro-domains (coloured cylinders and circle). Once the receptors have bound their respective ligands, intracellular signalling cascades are initiated that lead to actin polymerization and an actin- and myosin-dependent flow of micro-domains to the site of cell signalling. As the raft micro-domains merge during the migration phase, signalling proteins that were formally segregated between discrete domains are now proximal within the same microenvironment (beige cylinder). This results in enhanced protein interactions and amplification of the initial signals that result in further recruitment of rafts and associated signalling proteins to the merged signalling complex. This continues until arrested by stop signals. Cessation of raft accumulation probably coincides with formation of a mature immune synapse in T and B lymphocytes. Page I 29 Chapter 2: Hypothesis and Rationale This thesis examines the role of surface topography on activation of the NFKB signalling pathway in macrophages. 2.1 Hypothesis Physical stress such as the surface roughness of the implants may activate the NFKB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which macrophages interact with the surface through serum proteins and/or the formation of membrane rafts. 2.2 General Approach Three epoxy surfaces of different roughness were used in our experiments: Polished, Etched and SLA. The first aim of this study was to examine the effect of implant surface topography on activating the NFic13 signalling pathway in the RAW 264.7 macrophage cell line. This study addressed several concepts: 1. The ability of the surfaces to activate NFKB in the presence or absence of suboptimal stimulatory concentrations of LPS. 2. The ability of the surfaces to activate NFKB in the presence or absence of FCS. 3. The ability of the surfaces to activate NFKB in the presence of Methyl-f3- Cyclodextrin (M(3CD) a compound that removes cholesterol from raft components of the cell membrane. Page 30 The second part of this study examined the effect surface roughness had on the adhesion of the macrophages using the different media described above. The third and final study observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. 2.3 Rationale The studies presented in this thesis will markedly increase our knowledge on the mechanism(s) by which surface roughness affects signalling pathways in macrophages; the pathways that in turn initiate either inflammatory or anti-inflammatory sequellae post implant placement. Such knowledge is important for development or surface topography that will selectively down-regulate the inflammatory response while up-regulating the healing/osseointegration response. Page 1 31 Chapter 3: Materials and Methods 3.1 Preparation of replica surfaces Ti disks (15 mm in diameter and 1-mm thickness) of three surface topographies: mechanically Polished (PO), acid Etched (AE), and SLA were provided by Institute Straumann (Waldenburg, Switzerland). Impressions of the four surfaces were made with vinyl polysiloxane impression material (PROVIL Light; Dormagen, HK, Germany). Vinyl polysiloxane negative replicas were used to cast epoxy-resin (EPO-TEK 302-3; Epoxy Technology, Billerica, MA) positive replicas of these surfaces, and then left in a fume hood for 24 h for initial polymerization. For polymerization completion, positive replicas were baked at 58°C for 3 days. The epoxy replicas were then cleaned by ultrasonication in a detergent (7X) (ICN Biomedicals, Inc., Costa Mesa, CA). Finally, replicas were treated for 3 min in an argon-gas glow-discharge chamber and placed into 24-well culture plates (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) for use in cell experiments. 3.2 Cell culture Murine macrophage-like cells RAW 264.7 (ATCC, Manassas, VA) were cultured in 75- cm3 tissue culture flasks (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with antibiotics [penicillin G 100 mg/ml, gentamycin 50 mg/ml (Sigma-Aldrich, St. Louis, MO), amphoteracin B (Gibco, Grand Island, NY) 3 mg/m1] and 10% heat-inactivated fetal bovine serum (Cansera, Rexdale, Ontario, Canada). The cultured cells were incubated at 37 °C in 95% air and 5% CO2. Cells were routinely passaged by harvesting Page 1 32 using a cell scraper and re-plated in tissue culture flasks at a ratio of 1:5. For use in experiments confluent cultures were harvested as above. The cells were then plated onto the epoxy-resin replica surfaces at a population density of 7 x 10 5 cells/ml/well for 5, 15 and 30 minutes. They were then processed for indirect immunoflourescent labelling. In one experiment, a suboptimal stimulatory concentration of LPS (6.4 ng/ml) was added to the test surfaces. This concentration was defined by a lack of cytokine production over 48h. 3.3 Immunoflourescent labelling Surfaces with cells were washed in 0.1 M Phosphate Buffer Saline (PBS), pH 7.3, twice for 2 minutes. Afterwards, the cells on the surfaces were immediately fixed in 4% formaldehyde (Fisher Scientific, Nepean, Ontario, Canada) for 30 minutes and washed 3 times for 10 min each in PBS. The surfaces were then incubated for 2 min in 0.2% Triton X-100 (Fisher Scientific). Next, all samples were incubated for 20 min in 3% bovine serum albumin (Sigma-Aldrich), 0.1% glycine (Electron Microscopy Sciences, Hornby, Ontario, Canada), 0.2% Tween 20 (Fisher Scientific), and 5% normal goat serum (Sigma-Aldrich) for blocking of nonspecific immunoglobulin binding. All samples were incubated with of NFKB p65 (H-286) (Santa Cruz Biotechnology) rabbit polyclonal antibody raised against amino acids H-286 of activated NFKB p65 (1:50 dilution in PBS) for 1 hour in 37 °Celsius. Samples were washed three times with PBS for 5 min each, and incubated with Alexa Fluor 546 F(ab')2 fragment of goat anti-rabbit IgG secondary antibodies (1:600 dilution in PBS) (Molecular Probes, Burlington, Ontario, Canada) for 60 minutes at room temperature. Nuclei were counterstained with Page 1 33 DAPI (4', 6'- diamidino-2-phenylindole) chromosomal staining (Molecular Probes Inc., Eugene, OR). Finally samples were washed 5 times for 2 min each in PBS. 3.4 Cholesterol Depletion Prior to placing the cells on the surfaces, samples that were to have the cholesterol content removed from the rafts were treated with M7564 Methyl-(3-cyclodextrin (M(3CD) (Sigma-Aldrich Canada Ltd. Oakville, Ontario). Cells were incubated at 37 °C for 15 minutes with different concentrations of M(3CD mixed with complete medium. The cells were then placed onto the surfaces and then fixed and stained as described above. 3.5 Immunofluorescence All samples were observed with the AXIO Skop 2 epifluorescence microscope (Zeiss, Oberkochen, Germany), using a 63x oil objective using 540 nm wavelength excitation filters and a barrier filter for the desired emitted wavelength (red light). Digital images were captured with a low light-intensity CCD camera (PentaMax 12 bit CCD; Princeton Instrument, Trenton, NJ) using Northern Eclipse 6.0 software (Empix Imaging, Mississauga, Ontario, Canada). Immunofluorescence data analysis involved counting the number of cells displaying nuclear translocation of NFKB. Nuclear translocation was considered present if any red staining was observed within the nucleus. An average of 150 cells counted for each group. 3.6 Adhesion Cells were seeded onto the three different surfaces in three different media: with complete medium, with serum free medium and complete medium with Mr3CD (13 Page 1 34 mcg/ml). The cells were incubated for 30 minutes on the surfaces and the number adherent cells assessed. Ten random fields on each surface in each of three experiments were observed with the epifluorescence microscope and nuclei counterstained with DAPI were counted. 3.7 Scanning Electron Microscopy In order to observe possible morphological differences at high resolution, cells were seeded for 30 minutes onto the three different surfaces in the three different media as described above. After thirty minutes incubation, the cells were fixed with 4% formaldehyde (Fisher Scientific, Nepean, Ontario, Canada) for 10 minutes. This was followed by 2.5% glutaraldehyde in distilled water at 0°C. The samples were then washed with distilled water 3 times at room temperature for 5 minutes each. Next was secondary fixation using 1 % osmium tetroxide in 0.1 M PBS at room temperaturefor 1 hour. The surfaces were washed 3 times in PBS for 5 minutes then rinsed in 50%, 70%, 90% and 95% ethanol once 10 minutes each. The surfaces were then soaked in 100% ethanol from a newly opened bottle twice for 10 minutes each. Air drying of specimens can cause deformation and collapse of structures, the primary cause of such damage being the effects of surface tension. Therefore, the next step was critical point drying in order to prevent damage of the specimens by placing the specimen in an environment where the fluid within the specimen can pass from the liquid to gas phase with zero surface tension. The Supercritical Autosamdri0-815, Series B (Tousimis Research Corporation, Rockville, MD) was used according to the manufacturer's directions. Newly dried specimens are highly hygroscopic and must be coated with a thin layer of Page 1 35 metal or carbon as soon as possible. Therefore, the final step was to sputter coat our specimens with gold alloy. Surfaces were then observed using a Cambridge 260 Stereo scanning electron microscope at 10 kV accelerating voltage. 3.8 Statistical Analysis Data are presented as the mean and standard error of the mean of 2 independent studies. For statistical comparison, data were analyzed using the software program SPSS 16 for Mac (SPSS Inc., Chicago, IL). Statistical comparison was determined by using the two- way analysis of variance (ANOVA) including interaction effect (two tailed and p < or = 0.05) and post hoc Tukey multiple comparison testing. Page 1 36 Chapter 4: Results 4.1 Activation of the NFiB Pathway In order to look at the effect of surface roughness under a variety of conditions cells were plated on the various test surfaces as follows. 4.1.1.Effect of Surface Roughness on NFKB Signalling with Complete Medium NFKB signalling in RAW 264.7 cells was quantified on the three surfaces at three time points (Figure 6a). Three random fields were selected on each surface and 50 cells were counted to quantify nuclear translocation. The mean of the three fields was calculated and SD was obtained. Two independent experiments were carried out. It is clear that the over the 30 minute interval the Polished surface had a statistically higher percentage of cells exhibiting nuclear translocation of NFKB (p <0.01) followed by the Etched and the SLA (although the difference between the latter two was not statistically significant). A gradual increase in the number of cells showing nuclear translocation was observed with time with all three surfaces. At 30 minutes NFKB nuclear translocation was observed in 100% of the cells. On the Etched and SLA surfaces nuclear translocation was observed in 70 % and 80% respectively of the cells. Representative pictures for the 30 minute incubation time are shown in Figure 6b. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2-phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti-p65 NFicl3 antibody (red) only. Page 1 37 Effect of Surface Roughness on N11:11 signalling with Complete Medium Etched^SLA 50 45 40 35 311 se- /11 ou 15 10 :et^5 0 5 minutes^15 minutes^30 minutes Complete Medium — 30 minutes Polished Etched SLA Figure 6: a) NFKB signalling in RAW 264.7 cells on the three surfaces at three time points using complete medium; b) Representative pictures for the 30 minute incubation time point. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2-phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti-p65 NFicB antibody (red) only. Page 38 4.1.2 Effect of Surface Roughness on NFKB Signalling using Complete Medium Supplemented with 6.4 ng/mL of LPS As endotoxin contamination of all wound sites is a reality after implant placement NFKB signalling in RAW 264.7 cells was quantified in the presence of the sub-stimulatory (6.4 ng/mL) of LPS on the three surfaces at the three time points (Figure 7a). Three different random fields were selected and 50 cells were counted to quantify nuclear translocation. The mean of the three fields was calculated and SD was obtained. Two independent experiments were carried out. It is clear that over the 30 minute interval the Polished surface had a statistically higher percentage of cells exhibiting nuclear translocation of NFKB (p <0.01) followed by the Etched and the SLA (again the difference between the latter two was not statistically significant except at the 5 minute interval with Etch showing more nuclear translocation than SLA (p<0.05)). Indeed 100 % of the cells demonstrated nuclear translocation at all time points on the Polished surface. Again it was noted that a gradual increase in the number of cells showing nuclear translocation was observed with time on the Etched and SLA surfaces. It appears that addition of the sub-stimulatory (6.4 ng/mL) of LPS only mildly enhanced the signalling. Representative pictures for the 30 minute incubation time are shown in Figure 7b. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2- phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti- p65 NFKB antibody (red) only. Page I 39 Effect of Surface Roughness on NFkB signalling with Complete Medium + LPS —4,-- Polished^—Etched^SLA 50 45 411 35 30 25 211 15 10 5 (1 •  • 5 minutes^15 minutes^30 minutes Complete Medium with LPS — 30 minutes Polished Etched SLA Figure 7: a) NFKB signalling in RAW 264.7 cells on the three surfaces at three time points using Complete Medium supplemented with 6.4 ng/mL of LPS; b) Representative pictures for the 30 minute incubation time point. The top row pictures show the cells stained for anti-p65 Nficl3 antibody (red) and DAPI (4', 6'- diamidino-2- phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti- p65 NFxB antibody (red) only. Page I 40 4.1.3 Effect of Surface Roughness on NFiB Signalling using Serum Free Medium As Fetal Calf Serum (FCS) may contain endotoxin as well as other molecules used by the macrophage for adhesion to substrates, NFKB signalling in RAW 264.7 cells was quantified in the absence of FCS on the three surfaces at the three time points (Figure 8a). Three different random fields were selected and 50 cells were counted to observe whether nuclear translocation was seen or not. The mean of the three fields was calculated and SD was obtained. Two independent experiments were carried out. It can be seen that all three surfaces had very few cells with nuclear translocation at the 5 minute time point with no significant statistical differences between the surfaces. However, the signalling started to pick up at 15 minutes and then reached comparable numbers to the experiment with complete medium after 30 minutes. Again it appears that the Polished surface had significantly more NFicl3 translocation at 30 minute than the etched or SLA surface (p< 0.01). Interestingly, it was observed that the number of cells adhering to the surface without FCS was much less than the number seen in the other two experiments. Representative pictures for the 30 minute incubation time are shown in Figure 8b. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2-phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti-p65 NFKB antibody (red) only. Page 1 41 Effect of Surface Roughness on NFkB signalling with Serum Free Medium Poh.I^—*— Etched^SLA 511 45  - 4I1 - 35 - 341 25  - 20  - 15  - 111 5 11  5 minutes^15 minutes^30 minutes Etched SLA • • • Polished O•• • • • •• Serum Free Medium — 30 minutes Figure 8: a) NFKB signalling in RAW 264.7 cells on the three surfaces at three time points using Serum Free Medium; b) Representative pictures for the 30 minute incubation time point. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2-phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti-p65 NFKB antibody (red) only. Page I 42 4.1.4 Effect of M/JCD on NFKB Signalling It has been proposed that many molecules involved in cell signalling reside in sphingo- lipid and cholesterol enriched cell membrane rafts implying that such rafts act as specialized signalling compartments involved in cellular activation. In order to test if these rafts are involved in signalling in our experimental system, NFKB translocation in RAW 264.7 cells was quantified in the presence of varying concentrations of Mr3CD (a cholesterol depleting raft disrupting chemical) on the three surfaces at the three time points. 4.1.4.1 Polished Surfaces It was observed that nuclear translocation on Polished surfaces remained absent using the 13 meg and 6.5 mcg/ml even after 30 minutes (p<0.05 - Figure 9a). This was also true for all concentrations of IVII3CD at the 5 minute time point. Very few cells showed nuclear translocation when treated with lower concentrations of the chemical at the 15 minute time point. The number of cells showing nuclear translocation increased with time and with decreasing concentrations of MI3CD. It was noticed that the intensity of the signalling in the cytoplasm was very high compared to the intensity seen within the nucleus. Unlike figure 8b, the number of cells adhering here appears to be comparable to the numbers noted using the complete medium. Representative pictures for the 30 minute incubation time for the three surfaces are shown in Figure 9b. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2-phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti-p65 NFKB antibody (red) only. It is noticed that the intensity of the red staining is very low. Page 1 43 Effect of AI PCI) With decreasing concenItal•  s on NFkB signalling on Polished surfaces 13 mcg --s— 6 5 mcg —41.— 3 25 mcg ^ 1 625 mcg^0 8125 mcg 50 45 411 35 30 4.1 ?II bk 15 10 5  5 minutes^15 minutes^311minutes b • MfiCD— 30 Minutes *4. • • • •  •• •• • •^I • •• ^••• • • • • TO • • 46 • IL 41.4^•• 5^•• • eD• Jr. 4. Polished ^ Etched ^ SLA Figure 9: a) NFKB signalling in RAW 264.7 cells on the Polished surface at three time points using Complete Medium and decreasing concentrations of M(3CD; b) Representative pictures for the 30 minute incubation time point using Complete Medium with a 13 mcg/ml concentration of Mf3CD on all 3 surfaces. The top row pictures show the cells stained for anti-p65 NFKB antibody (red) and DAPI (4', 6'- diamidino-2- phenylindole) staining for nuclei (blue). The bottom row show the cells stained for anti- p65 NFKB antibody (red) only. It is noticed that the intensity of the red staining is very low indicating that signalling is absent. Page I 44 Effect of AIDCD with decrea sing concentrations on NFkB signalling on Etched surfaces r. • 50o 45 • 40Tr, ▪ 35 30 Z • /11 15 ..1 10 1"; 0  —40— 13 mcg —1•— 6 5 meg --A-- 3 25 mcg^I 625 mcg^0 8125 mcg 5 minutes^15 minutes ^30 minutes 4.1.4.2 Etched Surfaces It was observed that nuclear translocation remained absent using the 13 mcg and 6.5 mcg/ml concentrations even after 30 minutes (p<0.05 - Figure 10). This was also true for all the concentration at the 5 minute time point. Very few cells showed nuclear translocation at lower concentrations at the 15 minute time point. The number of cells showing nuclear translocation kept increasing with time and with decreasing concentrations of MftCD. It was noticed that the intensity of the signalling in the cytoplasm was very high compared to the intensity seen within the nucleus. Overall, the results here are similar to what was observed in Figure 9a but with an overall fewer number of cells showing nuclear translocation. Figure 10: NFic13 signalling in RAW 264.7 cells on the Etched surface at three time points using Complete Medium and decreasing concentrations of MI3CD. Page I 45 Effect of NIDCD with decreasing concentrations on NFU signalling on SLA surfaces 13 mcg t 65 mcg —*— 3.25 mcg - 1 625 mcg^0 8125 mcg 50 0 45 40 35 30 257.7 4 /0 15 E 10 -077 5 minutes 15 minutes 30 minutes 4.1.4.3 SLA Surfaces Unlike on the Polished or Etched surfaces, nuclear translocation was not observed on the SLA surface at any concentration throughout the 5 and 15 minute time points (p<0.05 - Figure 11). Nuclear translocation remained absent from all cells using all concentrations except for the lowest one (0.8125 mcg/m1) at the 30 minute time point. Even with the lowest concentration, the number of cells showing nuclear translocation remained lower than what was observed on the other surfaces using the same concentration at the same time point. It must be noted however, that there was a very weak signalling intensity observed in the cytoplasm of these cells starting at the 5 minute time point using a concentration of 1.625 mcg /ml but with no nuclear translocation. Figure 11: Nfic13 signalling in RAW 264.7 cells on the SLA surface at three time points using Complete Medium and decreasing concentrations of Mf3CD. Page I 46 Adhesion of Nlacrophages on Polished surfaces after 30 minutes 50 45 - 40 35 7,;" 30 /0 15 10 5 Serum Free Medium Alediu►^NI(WI) 4.2 Effect of M/JCD on Cell Adhesion As MI3CD had a pronounced effect on NFic13 translocation and hence cell signalling it was important to determine whether the compound had an effect on adherence to the various surface topographies. In order to test this possibility, the number of RAW 264.7 cells was quantified in the presence of varying concentrations of Mf3CD. The number of cells was counted after culturing them for 30 minutes on the 3 different surfaces. Figures 12a, 12b & 12c indicates that when complete medium and complete medium + MI3CD (13 mcg/m1) were used, adherent cell numbers were comparable to each other (difference was not statistically significant). The interesting finding was that the cells cultured in serum free medium did not adhere very well. It was observed as well that the cells adhered most on the Polished surfaces followed by the Etched and least on the SLA. Figure 12a: Number of adherent RAW 264.7 cells on the Polished surface after 30 minutes using Serum Free Medium, Complete Medium, and Complete Medium with a 13 mcg/ml concentration of MI3CD. Page I 47 Adhesion of Macrophages on Etched surfaces after 30 minutes 50 45 40 35 30 25 20 15 10 5 0  Serum Free Medium^Medium^ 'D Adhesion of Macrophages on SEA surfaces after 3(1minutes 7", 30 25 ,f; 20 15 10 5 0 Serum Free Medium Medium 50 45 - 40 - 35 - Alp( 'I) Figure 12b: Number of adherent RAW 264.7 cells on the Etched surface after 30 minutes using Serum Free Medium, Complete Medium, and Complete Medium with a 13 mcg/ml concentration of MI:3CD. Figure 12c: Number of adherent RAW 264.7 cells on the SLA surface after 30 minutes using Serum Free Medium, Complete Medium, and Complete Medium with a 13 mcg/ml concentration of Mi3CD. Page I 48 4.3 Scanning Electron Microscopy of Macrophages Plated on the Different Surfaces in Different Media As Mf3CD did not have a pronounced effect on cell adhesion but still affected cell signalling we decided to look at the effect of the compound on cellular morphology on the test surfaces. In order to determine the effect on morphology RAW 264.7 cells were plated on the three surfaces for 30 minutes using three sets of media; media without FCS (Serum free Media), Media with FCS (Complete Media), and Complete Media with mpap (13 mcg/ml). The cells were then processed for scanning electron microscopy. 4.3.1 Serum free Medium Cells in Serum free Medium plated on the 3 different surfaces were round and spaced distant from each other especially on the Etched and SLA surfaces (Figure 13). The cells hardly showed any spreading except for the Smooth surface; although this was very minimal compared to what was observed using the other media. 4.3.2 Complete Medium The cells in Complete Medium were more elongated on the 3 different surfaces compared to the serum free medium especially on the Smooth surface on which the cells showed the most spreading (Figure 14). The cells had projections extending onto the surfaces; the spreading being more evident on the Etched and SLA surfaces. The cells appeared to be more in number and in close proximity to each other. It was apparent that serum components increased the binding of macrophages to the various test surfaces. Page I 49 4.3.3 Complete Medium with M/JCD The cells seen on the surfaces appeared to be very similar to those on complete medium (Figure 15). The cells on the smooth surface were well spread and branched out. The cells on the Etched and SLA surfaces were rounder than on the smooth, however, the cells had numerous projections extending to the surface and spreading out similarly to what was observed with the complete medium only. Compared to the serum free medium, the cells were in closer proximity to each other and more spread. A common feature in all 3 experiments was that Smooth surfaces bound more macrophages in the 30 minute assay. Generally, it did not seem that removal of cholesterol by MI3CD treatment affected either the morphology of the macrophages nor their ability to bind to their respective surfaces. Page I 50 I OFIJ^lekt) WO Snm^S.00000 P , 00017 a  • 4 Figure 13: Cells on Polished, Etched & SLA in descending order using Serum free Medium after 30 minutes. Page 51 Figure 14: Cells on Polished, Etched & SLA in descending order using Complete Medium after 30 minutes. Page I 52 Figure 15: Cells on Polished, Etched & SLA in descending order using Complete Medium with MliCD (13 mcg/m1) after 30 minutes. Page I 53 Chapter 5: Discussion 5.1 General Discussion Macrophages play a central role in host defence. This population of leukocytes was first reported to act as non-specific phagocytic cells capable of engulfing and digesting particles. Later work indicated the macrophage also has more sophisticated functions. Bacterial recognition triggers the production of many pro-inflammatory chemokines and cytokines. Moreover, the macrophage has a role as an antigen-presenting cell for the initiation of the adaptive immune response. An additional complexity has recently been reported in that the macrophage exhibits a significant degree of "plasticity". In this instance the cytokine milieu in which they exist will determine the form of activation and hence function (inflammatory or healing) in its specific location. In the case of non-biologic foreign bodies such as dental implants macrophages are implicated in either the rejection or acceptance of that material. Historically, the macrophage was looked upon as an undesired and unwelcomed visitor when implants were placed as they can activate local inflammatory responses. Having said that, we must remind ourselves that numerous studies have investigated the role of the macrophage in wound healing and concluded that wounds heal poorly in its absence. This led to the discovery the macrophage is a key orchestrator of tissue repair (Leibovich 1975, Korn 1980, Diegelmann 1981) through the release of several growth factors and cytokines. Page I 54 It is critical that we try to understand the role the macrophages play when implants are placed in the body. Such knowledge is important for development or surface topography that will selectively down-regulate the inflammatory response while, at the same time, up-regulate the healing/osseointegration response. In this study, we examined the ability of the surfaces to activate NFKB in the presence or absence of suboptimal stimulatory concentrations of LPS and in the presence or absence of FCS (fetal calf serum). We also examined the ability of the surfaces to activate NFKB in the presence of Methyl-b-Cyclodextrin (M13CD), a compound that removes cholesterol from raft components of the cell membrane. The second part of this study examined the effect surface roughness had on the adhesion of the macrophages using the different media described above. The third and final study observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. 5.1.1 Activation of the NFKB Pathway The NFKB pathway can be activated through either of two pathways; the classical pathway and the alternate pathway (Figure 16). The alternate pathway involves ligation of the cell surface receptors LTPR, BAFFR, and CD4OR. Intracellular signalling by phosphorylation and processing of transcription factors is significantly different than that used by the classical pathway and culminates with the binding of RelB and P52 to promoters in the nucleus. The classical pathway is activated by a number of different mechanisms. As can be seen from Figure 17 the most common signalling pathways are those that originate through ligation of TNFR To11R/IL-1R and TB cell receptor. Page I 55 TNF-R TLR/ IL-1R TCR/BCR Classical Pathway St' ,s receptors for UV and ROI's ROSS-TALK LTR (Lymphotoxin) Alternate Pathway BAF FR (B-cell activating factor) C 114OR CROSS-TALK However, a number of other stimuli can activate the NFKB pathway; cell stress (UV irradiation, reactive oxygen species), phagocytosis of foreign particles, and cross-talk from activation of the MAP kinase pathway. The pathway is mediated by 11(1(13 and leads to phosphorylation of IKB as detailed in the introduction. Signalling Pathways to 1L_...■IfJ(B Figure 16: Activation of the NFKB Pathway This thesis investigates the effect of surface roughness on its ability to activate the NFKB pathway. I found that surface roughness selectively decreases NFKB translocation to the nucleus. This is the first demonstration of this unique finding. The smooth surface had a significantly higher degree of translocation over the 30 minutes test period. The two Page I 56 rough surfaces (Acid etched and SLA) demonstrated similar translocation in spite of there being significant differences in the integral roughness parameters. Endotoxin has been shown to be present in the placement of implants as bacteria contaminate the wound site during surgery. Moreover, endotoxin has been shown to be a powerful activator of the NFKB pathway. Endotoxin or LPS activates the macrophage through complex binding with LPS-binding protein and the specific glycosol phosphatidylinositol (GPI)-anchored cell membrane receptor, CD14 (Hailman 1994). Binding of endotoxin to LPS-binding protein and CD14, in turn, results in mobilization of TLR4 to lipid rafts and subsequent GPI-receptor complex assembly. In my study, I found that the NFKB pathway was activated when a suboptimal dose of LPS was added to the cells prior to seeding them onto the surfaces. Although this was expected, as LPS is an NFKB activator, our study demonstrated that even with the addition of the LPS to the cells, only the Polished surface had a 100 % nuclear translocation at all time points. A gradual increase in the number of cells showing nuclear translocation was observed with time on the Etched and SLA surfaces; however, the addition of the sub-stimulatory concentrations of LPS only mildly enhanced the signalling on those rough surfaces. In order to explore this further, I examined the extent of activation using serum free medium (media without FCS). In these experiments the number of cells showing nuclear translocation was very low at the 5 minutes mark compared to the other two forms of media. However, the signalling started to pick up at 15 minutes and then reached comparable numbers to the experiment with complete medium and medium with LPS after 30 minutes. Again it appears that the Polished surface had significantly more NFKB translocation^at^30^minute^than^the^etched^or^SLA^surface. Page I 57 Thus, the presence of serum optimized cell adherence and spreading on the different surfaces. This is probably due to the presence of fibronectin and other components within the serum necessary for cellular adhesion. 5.1.2 Cholesterol Depletion The use of Mf3CD has been a subject of wide controversy, since it has the ability to remove cholesterol not only from the cholesterol-rich membrane rafts but also from non- raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. Also, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. However, exposing cells to low concentrations for a short period of time may have preferential effect on membrane fractions proposed to contain membrane rafts (Zidovetzki 2007). The use of Mf3CD to remove cholesterol from the cell membrane was done using the method previously described by Pierini 2003. She reported that this method removed approximately 20.9 ± 3.6% of the cholesterol content in neutrophils. However it is unclear whether the same amount of cholesterol was removed in our macrophage line. It is important to note that the degree of cholesterol depletion may differ significantly between cell types even when comparable cyclodextrin concentrations and exposure times are applied. The results of this study turned out to be very interesting. I found that the treatment of the macrophages with Mi3CD prior to seeding onto the different surfaces resulted in a significant alteration in the rate of signalling activation. I also observed that higher concentrations completely abrogated NFKB signalling until 30 minutes. Lower concentrations led to the gradual return of translocation over the 30 minutes period. The Page I 58 SLA surface showed the least amount of NFKB translocation even with the lowest concentration used when compared to the etched and Polished surfaces. One explanation of this finding is that efficient assembly of rafts requires a necessary threshold that must be surpassed to initiate a signalling activation (Rodgers 2005). Therefore, when a higher concentration was used, rafts available for assembly were non existent due to cholesterol depletion and we did not obtain any signalling. When the dose of cyclodextrin was reduced, more rafts were made available. However, they may not have reached a size necessary for initiation of a full activation signal resulting in downstream nuclear translocation of NFKB. This was evident in the form of various signalling intensities observed within the cytoplasm despite the absence of nuclear translocation. When the cells were treated with a low enough dose, nuclear translocation was observed and interestingly it varied from surface to surface. The fact that the SLA surface had the least amount of nuclear translocation even though the signalling almost returned to normal after 30 minutes on the Polished surface again suggests that the rough surface played a role in how the NFKB pathway was being activated. However, it remains unclear in what form or capacity. Another study was done to examine whether different cyclodextrins inhibited the production of nitric oxide and proinflammatory cytokines in murine macrophages stimulated with LPS (Arima 2005). They concluded that DMA7-13- cyclodextrin lowered nitric oxide and various proinflammatory cytokines' production in murine macrophages stimulated with LPS and lipid A, probably by suppressing the binding of LPS to its LPS receptors on the cells. They also reported that it was the least cytotoxic of the cyclodextrins tested. My study differs in that the concentration of MPCD we used was much lower and despite that we did see a major reduction in the Page 1 59 NFKB activation without having a cytotoxic effect on the cells (Kilsdonk 1995, Christian 1997, Keller 1998, Sheets 1999, Levitan 2000, Fulop 2001, Grimmer 2002, Niu 2002, Dreja 2002, Matthews 2003, Romanenko 2004). In conclusion, I have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. However, at present it is unclear through which receptor(s)/ surface structure the signal pathway is initiated and whether it involves the Classical and/or Alternative pathways of NFKB activation. 5.1.3 Adhesion and Morphology As Mr3CD had a pronounced effect on NFKB translocation and hence cellular signalling it was important to determine whether the compound had an effect on cellular morphology and adherence to the various surface topographies. The interesting finding was that the cells adhered in the presence of complete medium with and without MI3CD. In serum free medium the cells did not adhere or spread very well. When the three surfaces were compared it was observed that the cells adhered most on the Polished surfaces then the Etched and least on the SLA. These findings were somewhat a surprise to us. We assumed that after removal of the cholesterol from the rafts the cytoskeleton of the cell would be affected resulting in a change in 'normal' adhesion and spreading onto the surfaces. The findings showed otherwise. Even though the rafts were disrupted enough to prevent NFKB signalling activation, the scanning electron microscopy of the cells did not show signs associated with loss of morphology or viability. One explanation is that the dose, duration and type of cell used did not affect adhesion unlike what was reported by Yanagisawa 2004. His Page I 60 group demonstrated that the use of Mf3CD on neuro-epithelial cells (NEC) adhesion is dependent on lipid rafts. As was previously demonstrated using confocal microscopy scanning electron microscopy confirmed that the presence of serum appeared to be necessary for cells to adhere and spread on the different surfaces. 5.2 Overall Model of the Possible Function of Surface Roughness on NFKB Nuclear Translocation The experiments reported in this thesis beg the question as to just how surface roughness can deliver the signal at the level of the cell membrane. They also have to explain the effect of the surfaces on production of pro-inflammatory cytokines reported by a number of different laboratories (Brunette 1996, 1999, 1999 and Kieswetter 1996). 5.2.1 Effect of Surface Roughness on Cellular Receptors-What is the Activating Signal? Figure 17 demonstrates the myriad of ways in which the NFKB signalling pathway can be activated. At this point we can't exclude any of the aforementioned pathways. However, one can logically argue against a number of them. Very little is known about activation by the alternative pathway and at present the effect of the surfaces on this pathway can't be discounted. Classical activation by ubiquitous stressors (ROI's) is a possibility. Khadaroo et al 2003 demonstrated that antecedent oxidative stress reprograms the LPS signalling pathway leading to NFKB translocation such that it involves activation of Src family kinase members. Activation of PI 3-kinase appears to be a consequence of Src activation and is clearly involved in the downstream signalling events leading to NFKB translocation. In our system this would seem an unlikely possibility given that membrane raft integrity is required to initiate the signalling Page 1 61 cascade. Likewise, phagocytosis is arguably not in the running as there are no particles from the surfaces present to engulf This leads us to the known receptors such as TNFR To11R/IL-1R and TB cell receptor. These receptors normally require ligation by their respective ligands followed by an allosteric change in the molecules; after which there is a selective recruitment of cytosolic adaptor molecules that will initiate the signalling cascade. It is hard to envisage that surface roughness can mimic these allosteric changes. The only explanation that I can thing of is the "freezing" of the portions of the membrane that come in contact with the surface and a concomitant accumulation of the above receptors at the contact point. This could hypothetically lead to the pre-requisite allosteric changes. The most likely explanation for the signalling we report is the "cross-talk" model from activation of the MAP kinase pathway. It has been reported that integrins could directly activate the MAP kinase family of transcription factors. Integrins are a family of heterodimeric transmembrane proteins consisting of a and 0 subunits that are involved in cellular adherence to substrates. Engagement (clustering) of integrins in focal adhesions couples the extracellular matrix with the actin cytoskeleton of the cell. Integrins do not have a kinase domain for signalling as such. Signalling through integrins relies on other signalling molecules. The intracellular molecule Talin forms a direct interaction with the integrin cytoplasmic domain and the actin stress fibres inside the cell. Paxillin and other molecules may interact with conventional signalling molecules such as FAK, CAS and Src using actin as a scaffold for signal molecule assembly. Thus, integrin clustering at the focal adhesions upon engagement of the surfaces would interact with Focal Adhesion Kinase (FAK) causing FAK phosphorylation (Schaller 1992). Activation of Src by FAK links integrin binding to the Page 62 MAP kinases; in particular activation of the Ras-ERK12 signalling pathway (Schlaepfer 1994, Schlaepfer 1996). Moreover activation of NFicB, in turn, has been linked to activation of Src family kinase members as mentioned above. In our activation scenario integrin clustering could consequently lead to NFKB translocation. 5.2.2 Consequences of Surface Roughness on Signal Transduction Why do the different surfaces behave differently in terms of signal transduction. One explanation for the differences seen is that rough surfaces have early inhibitory effects on the activation of the NFKB pathway. Alternatively, the smooth surface could provide more stimulatory signals. This cannot be resolved at the present time. Regardless it is imperative to ask just what the consequences are to the cells displaying different level of signal transduction on the test surfaces. To discuss this we must consider just how the NFKB is regulated. NFKB is controlled by two negative feedback loops involving two molecules; IkBa and A20. Both these molecules function by limiting the nucleus to cytoplasm oscillations in NFKB. As mentioned in the introduction the IkBa suppresses the NFKB transcriptional activity. Once phosphorylated by IKK it undergoes a process of ubiquitination and is degraded by the proteosome. The NFKB is then translocated to the nucleus effecting activation of "early" genes. These "early" genes encode IkBa and A20 as well as inflammation controlling cytokines (Lipniacki 2007). IkBa recycles to the cytoplasm together with the NFKB and as a consequence inactivates the transcriptional factor. A20 is a zinc finger that inactivates the IKK signal. Together these molecules provide a dual negative feedback loop controlling NFKB signalling. In our experimental system the smooth surface gave the strongest NFKB signal. The Page 1 63 transcription of responsive genes from cells plated on the smooth surface would thus activate this feedback loop providing a greater down-regulation of the response down the line. Conversely, the rough surface generated a weaker NFKB response and would be expected to have a lesser degree of NFKB signalling down regulation. 5.2.3 Effect of Surface Roughness on Pro-Inflammatory Cytokine Production A number of investigators have demonstrated that surfaces of different roughness have the ability to stimulated different amounts of pro-inflammatory cytokines in macrophage cell lines (Champagne 2002, Takebe 2003, Refai 2003, and Tan 2006). In our laboratory Refai et al. demonstrated that the roughest surface (SLA) had a higher cytokine and chemokine production than the Polished and acid etched surface at 24 and 48 h. Although, on the surface it seems that our test results at 30 minutes are not in agreement with his work we feel that this is explainable. Given that the smooth surface generated a major NFKB response within 30 minutes this would lead to an early activation of the negative feedback loop. This in turn would lead to a down-regulation of signalling and concomitantly a down-regulation cytokine/chemokine production at 24 hours. It should be noted that this simplistic explanation can be impacted by two confounding factors. The first is that co-activation of the MAP kinase pathway would also have an impact on inflammatory cytokine production. The second is that early production of cytokines will have an amplifying effect; triggering more cytokine production through autocrine interaction with cytokine receptors on the surface of the macrophage. Page 1 64 5.2.3 A Hypothetical Model Our model for what is happening can be summed up in figure 17. The macrophage cell line adheres to the three surfaces by their focal adhesions. The Polished surface, the acid/etched surface and the SLA surface have significantly different roughness parameters; with the acid/etched and SLA being significantly rougher than the Polished. Given the nature of the different surfaces the cellular adhesions will interact with these surfaces in distinct manners. As a consequence we propose that integrin clustering will be different on the different surfaces. Within 10 minutes integrins clustering on the smooth surface provides for a more effective signal than the other two surfaces. This clustering will lead to activation of FAK. The linkage of FAK to phosporylation of IKK in the NFKB complex NFKB is as yet undetermined (Src-pathway 1 or PI3K-pathway 2). Translocation of NFKB into the nucleus will activate a series of early genes that allow for transcription of NFKB regulatory proteins as well as pro-inflammatory cytokines. The regulatory proteins will, in the case of the smooth surface, provide tighter regulatory control of transcription resulting in a decrease of pro-inflammatory cytokines down the line. Pathway 3 is an amplification pathway triggered by autocrine recognition of pro- inflammatory cytokines. Page I 65 C y10 kinesNUCLEUS • Figure 17: Hypothetical Model of Effect of Surface Roughness on NFKB Signalling. Pathways 1 and 2 depict integrin signalling to NFkB. Pathway 3 depicts the cytokine amplification pathway. Page 66 5.3 Summary and Conclusions 5.3.1 Macrophage Activation 1. Different surface topography activates NFKB pathway differently. The Smooth surface showed the highest level of activation followed by the Etch then the SLA. 2. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. 3. Triggering of the macrophages occurred in the absence of fetal calf sera, although to a lesser extent. This indicates that components of sera were partially responsible for activation of the NFKB. It can be seen that all three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. However, the signalling started to pick up at 15 minutes. After 30 minutes it reached comparable levels to those surfaces tested with complete medium. 4. Disruption of the lipid rafts in the membrane through removal of the cholesterol content affected the triggering and signalling of the NFKB pathway. This inhibitory effect was concentration and time dependent. 5.3.2 Adherence and Morphology 1. Smooth surfaces bound more macrophages in the 30 minutes assay. 2. The presence of fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Serum components were also found to increase binding of macrophages to the various test surfaces. Page 1 67 3. Removal of cholesterol did not affect the ability of macrophage to bind and spread on their respective surfaces. 5.3.3 Conclusions We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present it is unclear through which receptor(s) / surface structure the signal pathway is initiated and whether it involves the classical and/or alternative pathways of NFicB activation. Further research will be required to determine mechanisms regulating specificity and selectivity of NFKB function, as well as its role in different cell types. 5.4 Future Directions The exact mechanism by which the surface of the implant activates NFKB remains unknown. Future experiments need to be directed towards finding out the receptor(s), pathways and crosstalk involved in this process of activation. We suggest that through the use of blockers, some of these mysteries may be solved. In the last few years great progress has been made in elucidating the mechanisms of signal transduction by integrins. Integrin function is especially important in macrophages since integrin signalling is critical for cellular functions such as adhesion, spreading, chemotaxis and the release various cytokines (Berton 1999). Ming and Page 1 68 Lowell in 1998 demonstrated in the absence of the predominant Src-family kinases, integrin signalling is blocked and macrophages manifest impaired migration. They also reported that these mutations did not affect integrin-mediated MAP kinase activation or NF-KB translocation, suggesting that the integrin signalling pathways which regulate cytoskeletal changes and cell migration are different from the pathways which activate MAP kinases and NF-KB. Therefore, further studies on integrins and their role in adhesion and NFKB activation in the presence of the implant surface is very important. The signalling pathways elicited by integrins are extremely complicated. Certainly many of these pathways contribute independently to aspects of the biologic response to cell adhesion (for example regulating gene transcription without affecting cytoskeletal rearrangements) while other physiologic responses will require the coordinated action of several different integrin signalling pathways (Berton and Lowell 1999). A better understanding of these signalling pathways is critical because of the central role that macrophages play in response to the insertion of implants. Specific inhibition of integrin-mediated signalling responses will optimistically shed some light on this rather complex response. Future experiments could include the selective blocking of the Src family of signalling molecules (using the specific inhibitor PP1) on the test surfaces. The inhibitors effect on NFKB would confirm the linkage. In order to dissect this pathway further specific inhibitors for ERIC1/2, JNK and p38 could be tested individually to determine where full activation of these MAP kinases is required for NFKB activation. It would also be interesting to test the effects of NFKB inhibitors on these surfaces to determine the effect of NFKB on subsequent transcription of early cytokine genes. Page I 69 It would be interesting to evaluate NFkB translocation at later time points. This could allow us to correlate translocation with cytokine secretion (either inflammatory or reparative). The involvement of stress receptors such as the ones involved with UV and ROI's need to be investigated keeping in mind that the process by which ROI's are regulated is highly complex and consequently the mechanism by which NFKB is activated via ROI's may be equally multifaceted and should be investigated. A final avenue to explore could be the ligands involved with activation of the alternative pathway (LTbR, BAFFR, and CD4OR). It is important to note that the alternative pathway stimuli also activate the classical pathway (crosstalk) which only makes things that much more complicated. Also, activation kinetics of NFKB via these two different pathways is remarkably different since activation via the classical pathway occurs within minutes and is highly dynamic. Activation via the alternative pathway, however, occurs within hours and is generally persistent (Hoffman 2006). Notably, the alternative pathway requires new protein synthesis, unlike the classical pathway that typically utilizes pre-existing signalling components (Wu 2007). Multiple blockers used simultaneously may need to be incorporated to narrow down the receptors and pathways involved in this process. Finally, as the macrophage functional phenotype changes with time from an inflammatory phenotype to a reparative one the patterns of cytokine production and in turn feedback to the activation/inhibition of the NFKB pathway needs to be investigated over short and long time durations. Page I 70 BIBLIOGRAPHY • Abron A., M. Hopfensperger, J. Thompson and L. Cooper, Evaluation of a predictive model for implant surface topography effects on early osseointegration in the rat tibia model, J Prosth Dent 85 (2001), pp. 40-46. • Akira S. TLR signalling. Curr Top Microbiol Immunol 2006;311:1-16. • Albrektsson T, Wennerberg A. The impact of oral implants - past and future, 1966- 2042. J Can Dent Assoc. 2005 May;71(5):327. • Albrektsson, T., Hansson, H., Kasemo, B., Larsson, K., Lundstrom, I., McQueen, D. & Skalak, R. (1983) The interface of inorganic implants in vivo; titanium implants in bone. Annals of biomedical engineering 11,1-27. • Al-Nawas B, Gotz H. Three-dimensional topographic and metrologic evaluation of dental implants by confocal laser scanning microscopy. Clin Implant Dent Relat Res 2003;5(3): 176-83. • Arima H, Motoyama K, Matsukawa A, Nishimoto Y, Hirayama F, Uekama K. Inhibitory effects of dimethylacetyl-beta-cyclodextrin on lipopolysaccharide-induced macrophage activation and endotoxin shock in mice. Biochem Pharmacol. 2005 Nov 15;70(10):1506-17. Epub 2005 Oct 7. • Aschoff, L. (1924). Lectures in Pathology, New York: P. B. Hoeber. • Berton, G. & Lowell, C.A. Integrin signalling in neutrophils and macrophages. Cell. Signal. 11,621-635 (1999). Page 1 71 • Beutler B, et al. Genetic analysis of host resistance: Toll-like receptor signalling and immunity at large. Annu Rev Immuno12006;24:353-389. • Beutler B. The Toll-like receptors: analysis by forward genetic methods. Immunogenetics. 2005;57:385-92. • Beutler, B. Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity. Curr. Opin. Microbiol 2000. 3: 23-28 • Billack B. Macrophage Activation: Role of Toll-like Receptors, Nitric Oxide, and Nuclear Factor kappa B Am J Pharm Educ. 2006 October 15; 70(5): 102. • Blach-Olszewska Z. Innate immunity: cells, receptors, and signalling pathways. Arch Immunol Ther Exp. 2005;53:245-53. • Brunette D.M., Hamilton D.W., Chehroudi B. and Waterfield J.D., Update on improving the bio-implant interface by controlling cell behaviour using surface topography, Int Congr Ser 1284 (2005), pp. 229-238. • Brunette DM, Chehroudi B. The effects of the surface topography of micromachined titanium substrata on cell behaviour in vitro and in vivo. J Biomech Eng 1999; 121(1): 49-57. • Brunette DM. Effects of surface topography of implant materials on cell behaviour in vitro and in vivo. In: Hoch HC , Jelinski LW , Craighead HG , editors. Nanofabrication and biosystems. New York: Cambridge University Press; 1996. p 335-355. Page I 72 • Brunette DM. In vitro models of biological responses to implants. Adv Dent Res 1999; 13: 35-37. • Carmody RJ, Chen YH. Nuclear factor-kappaB: activation and regulation during toll- like receptor signaling. Cell Mol Immunol. 2007 Feb;4(1):31-41. Review. • Casey W.J., Peacock Jr E.E., and Chvapil M., Induction of collagen synthesis in rats by transplantation of allogenic macrophages. Surg Forum 27 (1976), pp. 53-55. • Champagne CM, Takebe J, Offenbacher S, Cooper LF. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone 2002; 30: 26-31. • Christian A.E., Haynes M.P., Phillips M.C. and Rothblat G.H., Use of cyclodextrins for manipulating cellular cholesterol content, J. Lipid Res. 38 (1997), pp. 2264-2272. • Cook SD, Dalton JE. Biocompatibility and biofunctionality of implanted materials. Alpha Omegan. 1992;85(4):41-7. • Creagh, E. M. and O'Neill, L. A., TLRs, NLRs and RLRs: A trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol. 2006. 27: 352-357. • Danon D., Kowatch M.A and Roth G.S., Promotion of wound repair in old mice by local injection of macrophages, Proc Natl Acad Sci USA 86 (1989) (6), pp. 2018— 2020. • Diegelmann R.F., Cohen I.K. and Kaplan A.M., The role of macrophages in wound repair. A review. Plast. Reconstr. Surg. 63 (1981), pp. 107-113. Page I 73 • Dietrich C. et al., Relationship of lipid rafts to transient confinement zones detected by single particle tracking, Biophys. J. 82 (2002), pp. 274-284. • Doyle S.L., O'Neill L.A.J. Toll-like receptors: From the discovery of NFKB to new insights into transcriptional regulations in innate immunity (2006) Biochemical Pharmacology, 72 (9 SPEC. ISS.), pp. 1102-1113. • Drago CJ, Lazzara RJ. Immediate occlusal loading of Osseotite implants in mandibular edentulous patients: a prospective observational report with 18-month data. J Prosthodont. 2006 May-Jun;15(3):187-94. • Dreja K., Voldstedlund M., Vinten J., Tranum-Jensen J., Hellstrand P.and Sward K., Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction, Arterioscler. Thromb. Vasc. Biol. 22 (2002), pp. 1267-1272. • Eriksson AS, Thomsen P. Leukotriene B4, interleukin 1 and leucocyte accumulation in titanium and PTFE chambers after implantation in the rat abdominal wall. Biomaterials. 1991 Nov;12(9): 827-30. • Fischer K.D. et al., Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor, Curr. Biol. 8 (1998), pp. 554-562. • Fulop Jr. T., Douziech N., Goulet A.C., Desgeorges S., Linteau A., Lacombe G.and Dupuis G., Cyclodextrin modulation of T lymphocyte signal transduction with aging, Mech. Ageing Dev. 122 (2001), pp. 1413-1430. Page I 74 • Fuqua WC, Winans SC, Greenberg EP. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 1994;176(2):269 —275. • Garrigues GE, Cho DR, Rubash HE, Goldring SR, Herndon JH, Shanbhag AS. Gene expression clustering using self-organizing maps: analysis of the macrophage response to particulate biomaterials. Biomaterials. 2005 Jun;26(16):2933-45. • Ghosh, S., & Karin, M. (2002). Missing pieces in the NF-kappaB puzzle. Cell, 109(Suppl), S81—S96. • Gilmore TD, Herscovitch M. (2006) Inhibitors of NF-kappaB signalling: 785 and counting. Oncogene. 2006 Oct 30;25(51):6887-99. • Gordon S. The macrophage. Bioessays. 1995 Nov;17(11):977-86 • Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003 Jan;3(1):23-35. Review. • Gordon S. The macrophage: past, present and future. Eur J Immunol. 2007 Nov;37 Suppl 1:S9-17. Review. • Gordy C. et al., Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs, J. Immunol. 172 (2004), pp. 2030-2038. • Grimmer S., van Deurs B.and Sandvig K., Membrane ruffling and macropinocytosis in A431 cells require cholesterol, J. Cell Sci. 115 (2002), pp. 2953-2962. Page 1 75 • Hailman E., Lichenstein H.S., Wurfel M.M., Miller D.S., Johnson D.A., Kelley M. et al., Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD 14. J Exp Med 179 (1994), pp. 269-277. • Hamilton D.W., Wong K.S. and Brunette D.M., Microfabricated discontinuous-edge surface topographies influence osteoblast adhesion, migration, cytoskeletal organization, and proliferation and enhance matrix and mineral deposition in vitro, Calcif Tissue Int 78 (2006) (5), pp. 314-325. • Hamilton DW, Chehroudi B, Brunette DM., Comparative response of epithelial cells and osteoblasts to microfabricated tapered pit topographies in vitro and in vivo. Biomaterials. 2007 May;28(14):2281-93. • Hawlisch, H. and Kohl, J., Complement and Toll-like receptors: Key regulators of adaptive immune responses. Mol. Immunol. 2006.43: 13-21. • Hayden MS, Ghosh S. Signalling to NF-kappaB. Genes Dev. 2004 Sep 15;18(18):2195-224. Review. • Hirschfeld, M., Weis, J.J., Toshchakov, V., Salkowski, C.A., Cody, M.J., Ward, D.C., Qureshi, N., Michalek, S.M., and Vogel, S.N. 2001. Signalling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69: 1477-1482 • Hoffmann A, Baltimore D. Circuitry of nuclear factor kappaB signalling. Immunol Rev 2006;210:171-186. Page 176 • Hoffmann, A., Natoli, G., and Ghosh, G. (2006) Transcriptional regulation via the NF-kappaB signalling module. Oncogene 25: 6706-6716. • Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987-995. • Janeway CA, Jr., Medzhitov R. Innate immune recognition. Ann Rev Immunol. 2002;20:197-216. • Jenney CR, DeFife KM, Colton E, Anderson JM. Human monocyte/macrophage adhesion, macrophage motility, and IL-4-induced foreign body giant cell formation on silane-modified surfaces in vitro. Student Research Award in the Master's Degree Candidate Category, 24th Annual Meeting of the Society for Biomaterials, San Diego, CA, April 22-26,1998. J Biomed Mater Res. 1998 Aug;41(2):171-84. • Jordan S. and Rodgers W., T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses, J. Immunol. 171 (2003), pp. 78-87. • Kaisho T, Akira S. Toll-like receptor function and signalling. J Allergy Clin Immunol. 2006 May;117(5):979-87; quiz 988. Epub 2006 Apr 3. Review. • Kao WJ. Evaluation of protein-modulated macrophage behaviour on biomaterials: designing biomimetic materials for cellular engineering. Biomaterials 1999; 20(23- 24): 2213-2221. • Karin M, Ben Neriah Y. Phosphorylation meets ubiquitination: the control of NF- [kappa]B activity. Ann Rev Immunol. 2000;18:621-63. Page I 77 • Karin M. (1999) How NFkB is activated: the role of the IkB kinase (IKK) complex. Oncogene. 18, 6867-6874. • Kaufmann S., S.Gordon & R.Medzhitov. The Innate Immune Response to Infection. Eds. Am. Society for Microbiology Press 2004. pp71-94. • Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11: 115-122 • Keller P.and Simons K., Cholesterol is required for surface transport of influenza virus hemagglutinin, J. Cell Biol. 140 (1998), pp. 1357-1367. • Khadaroo Rachel G., Kapus Andras, Powers Kinga A., Cybulsky Myron I., Marshall John C., and Rotstein Ori D., Oxidative Stress Reprograms Lipopolysaccharide Signalling via Src Kinase-dependent Pathway in RAW 264.7 Macrophage Cell Line J. Biol. Chem., Nov 2003; 278: 47834 - 47841. • Kieswetter K, Schwartz Z, Hummert TW, Cochran DL, Simpson J, Dean DD, Boyan BD. Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells. J Biomed Mater Res 1996; 32(1): 55-63. • Kilsdonk E.P., Yancey P.G., Stoudt G.W., Bangerter F.W., Johnson W.J., Phillips M.C. and Rothblat G.H., Cellular cholesterol efflux mediated by cyclodextrins, J. Biol. Chem. 270 (1995), pp. 17250-17256. • Korn J.H., Halushka P.V. and LeRoy E.C, Mononuclear cell modulation of connective tissue function. J. Clin. Invest. 65 (1980), pp. 543-554. Page I 78 • Krutzik S.R. and Modlin R.L., The role of Toll-like receptors in combating mycobacteria, Semin Immunol 16 (1) (2004), pp. 35-41. • Lee FD. The role of interleukin-6 in development. Dev Biol. 1992;151:331-338 • Lee K.H et al., T cell receptor signalling precedes immunological synapse formation, Science 295 (2002), pp. 1539-1542. • Leibovich SJ, Ross R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am J Pathol. 1975 Jan;78(1):71-100. • Levitan I., Christian A.E, Tulenko T.N. and Rothblat G.H., Membrane cholesterol content modulates activation of volume-regulated anion current (VRAC) in bovine endothelial cells, J. Gen. Physiol. 115 (2000), pp. 405-416. • Li D, Ferguson SJ, Beutler T, Cochran DL, Sittig C, Hirt HP, Buser D. Biomechanical comparison of the sandblasted and acid-Etched and the machined and acid-Etched titanium surface for dental implants. J Biomed Mater Res. 2002 May;60(2):325-32. • Lipniacki T, Kimmel M. Deterministic and stochastic models of NFkappaB pathway. Cardiovasc Toxicol. 2007;7(4):215-34. Epub 2007 Oct 18. • Lipniacki T, Paszek P, Brasier AR, Luxon BA, Kimmel M. Stochastic regulation in early immune response. Biophys J. 2006 Feb 1;90(3):725-42. Epub 2005 Nov 11. • Martin JY, Schwartz Z, Hummert TW, Schraub DM, Simpson J, Lankford J Jr, Dean DD, Cochran DL, Boyan BD. Effect of titanium surface roughness on proliferation, Page 1 79 differentiation, and protein synthesis of human osteoblast-like cells (MG63). J Biomed Mater Res 1995;29(3):389-401. • Martini, FA. Fundamentals of Anatomy and Physiology. 6th ed. New York: Benjamin Cummings; 2004. pp. 791-812. • Matthews V., Schuster B., Schutze S., Bussmeyer I., Ludwig A., Hundhausen C., Sadowski T., Saftig P., Hartmann D., Kallen K.J. and Rose-John S., Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE), J. Biol. Chem. 278 (2003), pp. 38829-38839. • Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., and Janeway, C.A., Jr. 1998. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signalling pathways. Mol. Cell 2: 253-258 • Meng, F. & Lowell, C.A. A betal integrin signalling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J. 17,4391-4403 (1998). • Metcalf D. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature. 1989;339:27-30. • Metcalf D. The molecular control of granulocytes and macrophages. Ciba Found Symp. 1997;204:40-50; discussion 50-46. • Mustelin T. and Tasken K., Positive and negative regulation of T-cell activation through kinases and phosphatases, Biochem. J. 371 (2003), pp. 15-27. Page i 80 • Niu S.L., Mitchell D.C. and Litman B.J., Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation, J. Biol. Chem. 277 (2002), pp. 20139-20145. • O'Neill, L. A., How Toll-like receptors signal: What we know and what we don't know. Curr. Opin. Immunol. 2006. 18: 3-9. • Pahl HL (1999) Activators and target genes of Rel/NF-kB transcription factors. Oncogene 18:6853. • Park J.E. and Barbul A., Understanding the role of immune regulation in wound healing, Am J Surg 187 (2004), pp. S11—S16. • Pierini LM, Eddy RJ, Fuortes M, Seveau S, Casulo C, Maxfield FR. Membrane lipid organization is critical for human neutrophil polarization. J Biol Chem. 2003 Mar 21;278(12):10831-41. • Rasmusson L., Roos J. and Bystedt H., A 10-year follow-up study of titanium dioxide-blasted implants, Clin Implant Dent Relat Res 7 (2005), pp. 36 1̂2. • Refai AK, Textor M, Brunette DM, Water -field JD. Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines. J Biomed Mater Res A. 2004 Aug 1;70(2):194-205. • Rodgers W, Farris D, Mishra S. Merging complexes: properties of membrane raft assembly during lymphocyte signalling. Trends Immunol. 2005 Feb;26(2):97-103. Page 1 81 • Rodgers W. and Zavzavadjian J., Glycolipid-enriched membrane domains are assembled into membrane patches by associating with the actin cytoskeleton, Exp. Cell Res. 267 (2001), pp. 173-183. • Romanenko V.G., Fang Y., Byfield F., Travis A.J., Vandenberg C.A., Rothblat G.H. and Levitan I., Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels, Biophys. J. 87 (2004), pp. 3850-3861. • Scacchi M., (2000) The development of the ITI ® DENTAL IMPLANT SYSTEM . Part 1: A review of the literature Clinical Oral Implants Research 11 (s 1) , 8-21 • Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192-6. • Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994 Dec 22-29;372(6508):786-91. • Schlaepfer DD, Hunter T. Signal transduction from the extracellular matrix--a role for the focal adhesion protein-tyrosine kinase FAK. Cell Struct Funct. 1996 Oct;21(5):445-50. Review. • Schroeder, A., Van der Zupen, E., Stich, H. & Sutter, F. (1981) The Reactions of Bone, Connective Tissue and Epithelium to Endosteal Implants with Titanium Sprayed Surfaces. Journal of Oral and Maxillofacial Surgery 9,15-25. Page i 82 • Schuster JM, Nelson PS. Toll receptors: an expanding role in our understanding of human disease. J Leukoc Biol. 2000;67:767-73. • Scotchford CA, Ball M, Winkelmann M, Weis J, Csucs C, Brunette DM, Danuser G, Textor M. Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions. Biomaterials. 2003 Mar;24(7):1147-58. • Sennerby, L., Ericson, L., Thomsen, P., Lekholm, U. & Astrand, P. (1991) Structure of the bone-titanium interface in retrieved clinical oral implants. Clinical Oral Implant Research 2,103-111. • Shanbhag AS, Jacobs JJ, Black J, Galante JO, Giant TT. Macrophage/particle interactions: effect of size, composition and surface area. J Biomed Mater Res 1994;28(1):81-90. • Sharma P.et al., Nanoscale organization of multiple GPI-anchored proteins in living cell membranes, Cell 116 (2004), pp. 577-589. • Sheets E.D, Holowka D.and Baird B., Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes, J. Cell Biol. 145 (1999), pp. 877-887. • Sheets E.D. et al., Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane, Biochemistry 36 (1997), pp. 12449-12458. Page I 83 • Silverman N, Maniatis T. NF-kappaB signalling pathways in mammalian and insect innate immunity. Genes Dev. 2001 Sep 15;15(18):2321-42. • Simons K. and Toomre D., (2000) Lipid rafts and signal transduction. Nature Mol. Cell Biol. Rev. 1,31-39. • • Simons K. and Toomre D., Lipid rafts and signal transduction, Nature Mol. Cell Biol. Rev. 1 (2000), pp. 31-39. • Sloan-Lancaster J. and Allen P.M., Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology, Annu. Rev. Immunol. 14 (1996), pp. 1-27. • Sullivan DY, Sherwood RL, Porter SS. Long-term performance of Osseotite implants: a 6-year clinical follow-up. Compend Contin Educ Dent 2001;22(4):326— 328,330,332-324. • Suska F, Kalltorp M, Esposito M, Gretzer C, Tengvall P, Thomsen P. In vivo/ex vivo cellular interactions with titanium and copper. J Mater Sci Mater Med. 2001 Oct- Dec;12(10-12):939-44. • Takebe J, Champagne CM, Offenbacher S, Ishibashi K, Cooper LF. Titanium surface topography alters cell shape and modulates bone morphogenetic protein 2 expression in the J774A.1 macrophage cell line. J Biomed Mater Res 2003; 64: 207-216. • Takeuchi 0, Akira S. Genetic approaches to the study of Toll-like receptor function. Microbes Infect. 2002;4:887-95. Page I 84 • Tan KS, Qian L, Rosado R, Flood PM, Cooper LF. The role of titanium surface topography on J774A.1 macrophage inflammatory cytokines and nitric oxide production. Biomaterials. 2006 Oct;27(30):5170-7. • Tergaonkar V. (2006) NFkB pathway: A good signalling paradigm and therapeutic target. Int J. Biochem. Cell Biol. 38,1647-1653. • Testori T, Meltzer A, Del Fabbro M, et al. Immediate occlusal loading of Osseotite implants in the lower edentulous jaw. A multicenter prospective study. Clin Oral Implants Res 2004; 15(3):278-84. • Tsirogianni AK, Moutsopoulos NM, Moutsopoulos HM. Wound healing: immunological aspects. Injury. 2006 Apr;37 Suppl 1:S5-12. • Uematsu S, Akira S., Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol. 2008;(183):1-20. Review. • van Furth R, Cohn Z, Hirsh J, Humprey J, Spector W, Langevoort H. The mononuclear phagocyte system: a new classification of macrophages, monocytes and their precursors. Bull. WHO. 1972;46:845-852 • van Furth R, Cohn ZA, Hirsch JG, Humphrey JH, Spector WG, Langevoort HL. Mononuclear phagocytic system: new classification of macrophages, monocytes and of their cell line. Bull World Health Organ. 1972;47(5):651-8. • Varma R. and Mayor S., GPI-anchored proteins are organized in submicron domains at the cell surface, Nature 394 (1998), pp. 798-801. Page I 85 • Verma I. M., Stevenson J. K., Schwarz E. M., Van Antwerp D., & Miyamoto S. (1995). Rel/NF-kappa B/I kappa B family: Intimate tales of association and dissociation. Genes Dev., 9,2723-2735. • Wahl SM. Host immune factors regulating fibrosis. In: Evered D, Whelan J, eds. Fibrosis. Ciba Foundation Symposium Series, No. 114. London: Pitman, 1985:175— 195. • Weng D, Hoffmeyer M, Hurzeler MB, et al. Osseotite vs. machined surface in poor bone quality. A study in dogs. Clin Oral Implants Res 2003;14(6):703-8. • Wennerberg A.,. Albrektsson T, Albrektsson B.and Krol J.J, Histomorphometric and removal torque study of screw-shaped titanium implants with three different surface topographies, Clin Oral Implant Res 6 (1996), pp. 24-30 • Wiktor-Jedrzejczak W, Gordon S. Cytokine regulation of the macrophage (M phi) system studied using the colony stimulating factor- 1 -deficient op/op mouse. Physiol Rev. 1996;76:927-947 • Wilson B.S. et al., Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes, Mol. Biol. Cell 15 (2004), pp. 2580-2592. • Winkelmann M, Gold J, Hauert R, Kasemo B, Spencer ND, Brunette DM, Textor M. Chemically patterned, metal oxide based surfaces produced by photolithographic techniques for studying protein- and cell-surface interactions I: Microfabrication and surface characterization.Biomaterials. 2003 Mar;24(7):1133-45. Page I 86 • Witte M.B and Barbul A., General principles of wound healing. Surg Clin North Am 77 (1997), pp. 509-528. • Wong M, Eulenberger J, Schenk R, Hunziker E. Effect of surface topology on the osseointegration of implant materials in trabecular bone. J Biomed Mater Res. 1995 Dec;29(12):1567-75. • Wu ZH, Miyamoto S. Many faces of NF-kappaB signalling induced by genotoxic stress. J Mol Med. 2007 Nov;85(11):1187-202. Epub 2007 Jul 3. • Wulfing C. and Davis M.M., A receptor/cytoskeletal movement triggered by costimulation during T cell activation, Science 282 (1998), pp. 2266-2269. • Xavier, R. et al. (1998) Membrane compartmentation is required for efficient T cell activation. Immunity 8,723-732. • Yanagisawa M, Nakamura K, Taga T, (2004) Roles of lipid rafts in integrin- dependent adhesion and gp130 signalling pathway in mouse embryonic neural precursor cells. Genes to Cells 9 (9) , 801-809 • Zarb, G. & Albrektsson, T. (1991) Guest Editorial: Osseointegration: A Requiem for the Periodontal Ligament? The International Journal of Periodontics & Restorative Dentistry 11,88-91. • Zidovetzki R, Levitan I.Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim Biophys Acta. 2007 Jun;1768(6):1311-24. Epub 2007 Apr 6. Review. Page I 87

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0066657/manifest

Comment

Related Items