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The effects of surface topography on the behaviour of cells attached to percutaneous implants Chehroudi, Babak 1991

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The Effects of Surface Topography on the Behaviour of Cells Attached to Percutaneous Implants By Babak Chehroudi D.M.D., National University of Iran, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Oral Biology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1991 ©Babak Chehroudi, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ii ABSTRACT Epithelial downgrowth on implants can result in deep pockets or sinus tracts which in turn can lead to marsupialization and eventual failure of the implant. One solution to this problem would be to have a surface of an implant that has the ability to impede the apical migration of epithelium. The present studies were aimed to determine whether surface topography could be used to impede epithelial downgrowth on percutaneous implants based on those principles that have been found to control the direction and rate of cell migration in vitro. Studies in culture have indicated that cells can be guided by the grooved surfaces, a phenomenon called contact guidance. In the first series of experiments, the effects of a V-shaped, 10-|im-deep grooved epoxy or titanium-coated epoxy substrata were studied on epithelial (E) cell behaviour. In vitro, grooved surfaces encouraged E cell adhesion and oriented clusters of E cells along their long axis. Seven or 10 days after percutaneous implantation of grooved and control smooth surfaces in rats, grooved surfaces significantly inhibited epithelial downgrowth on the epoxy or titanium-coated epoxy implants. In the second series of experiments, the effects of groove parameters such as depth, spacing and orientation were tested in vivo. Grooves were produced with a 39, 30 and 7 urn pitch and depths of 19, 10 or 3 | im. After 7 days percutaneous implantation of titanium-coated implants epithelial downgrowth was accelerated on the vertically oriented, 3 or 10 urn-deep, grooved surfaces and inhibited on the horizontally oriented grooved surfaces; an observation that could represent the most direct evidence of contact guidance occurring in vivo. In the shallower horizontal grooves [<10 urn-deep] epithelial downgrowth was probably inhibited by contact guidance because there was no evidence of fibroblasts (F) inserting into the implant surface. However, in the 19 u.m-deep grooved surfaces, E cells bridged over the grooves and their migration appeared to be inhibited by the F that inserted into the implant surface. In the third series of experiments, the ultrastructural observations indicated that E cells closely attached to the smooth, and interdigitated with, the 3 um and 10 urn grooved surfaces of titanium-coated implants. This attachment appeared to be through basal lamina and hemidesmosome-like iii structures. The ultrastructural observations on the orientation of E cells and F attached to the implant verified those noted at the light microscopic level. The attachment of F to the titanium surface was mediated by two zones; a thin [=20 nm], amorphous, electron dense zone immediately contacting the titanium surface, and a fine fibrillar zone extending from the amorphous zone to the cell membrane. The objectives of the fourth experiment were [1] to examine cell behaviour on implants in which connective tissue contacted surfaces of various topographies and epithelium encountered only a smooth surface; [2] to compare one-stage and two-stage surgical techniques. Implants delivered micromachined surfaces to the connective tissue and a smooth control surface to the epithelium and implants were removed l,2,and 3 weeks following percutaneous implantation. A complex connective tissue organization that changed with time was noted on the micromachined surfaces whereas a capsule formed on the smooth surfaces. In some cases foci of mineralization were observed on the micromachined surfaces placed using a two-stage surgical technique. Apical migration of the epithelium was significantly (p<.05) inhibited on all surfaces placed by the two-stage technique and by those micromachined surfaces that produced connective tissue ingrowth. In the fifth study, the ultrastructural observations of the mineralized tissue formed on the micromachined surfaces, identified osteocyte-like cells and in some areas revealed close juxtapositioning of collagen and minerals to titanium without an intervening amorphous layer. The findings collectively indicate that contact guidance occurs on artificial surfaces in vivo, and micromachined surfaces could be incorporated advantageously to the design of implant surfaces to optimize their performance. iv TABLE OF CONTENTS P A G E ABSTRACT ii T A B L E OF CONTENTS iv LIST OF FIGURES x L I S T OF T A B L E S xii ACKNOWLEDGEMENT xiii CHAIPTEE 1 INTRODUCTION I. Overview 1 II. Cell Adhesion to Artificial Surfaces 3 A . The cytoskeleton 4 1. Microfilaments or actin filaments. 5 2. Microtubules 5 3. Intermediate filaments 6 B . The adhesion of cells and extracellular matrix to artificial surfaces in vitro 1 1. Interference Reflection Light Microscopy (IRM) 7 a. Focal contacts 7 b. Close contacts 9 c. Extracellular matrix contacts 9 2. Electron microscopy 9 a. Basement membrane 9 b. Hemidesmosomes 10 3. Molecules related to cell adhesion 11 a. Extracellular adhesion molecules 11 b. Cell membrane-bound receptors for adhesion molecules.... 14 c. Cytoplasmic domain of the adhesion molecules 14 C. The adhesion of cells and extracellular matrix to artificial surfaces in vivo 15 D. The role of material properties on cell adhesion 18 1. Mechanical rigidity 18 2. Wettability or status of surface energy 18 3. Surface charge 20 4. Material surface topography 20 a. Smooth surfaces 21 b. Textured surfaces 22 E . Other factors affecting cell/implant adhesion 26 1. Surgical technique. 26 2. Structure of the implant bed 27 F . Summary 27 III. Cell Migration on Artificial Surfaces 28 A . Mechanisms of cell migration 28 B . Cell migration on implants in vivo. 30 C. Summary 31 V IV. Wound Healing 32 A . The inflammatory phase 32 1. Platelets. 32 2. Leukocytes 33 B . Proliferative phase 35 C. Remodelling Phase 36 D. Mechanisms of tissue reorganization around implants 38 1. Haptotaxis. 38 2. Chemotaxis. 40 3. Contact guidance 41 a. Microexudate hypothesis. 42 b. Microfilament bundle hypothesis 43 c. Focal contact hypothesis 43 d. Selective adhesion hypothesis 44 e. Stochastic model hypothesis. 44 4. The role of contact guidance in vivo 45 5. Cell/cell contact inhibition of movement 46 6. Two-centre effect 48 E . Summary 49 V . Complications and failure modes of percutaneous implants 50 A . Marsupialization 51 B . Permigration 53 C. Infection 53 D. Avulsion 54 E . Multiple cause of failure 55 F . Summary 55 CDBAPTEE 2 S T A T E M E N T OF T H E P R O B L E M S 56 C M A P T E E 3 M A T E R I A L S A N D M E T H O D S (General) I. Micromachining 58 A . Cleaning 58 B . Oxidation 59 C . Photolithography 59 E . Oxide patterning 60 F . Final etching. 60 II. Fabrication of the epoxy substrata 61 III. Fabrication of implants for in vivo experiments 63 IV. Preparation and Characterization of Surfaces 64 vi V . Cell Culture 65 VI . Cell attachment 66 VII. Cell Orientation 66 VIII. Implantation Procedure 68 I X . Specimen Collection and Preparation 69 X . Histology and Histomorphometric measurements 70 CUDAIPTEK 4 RESULTS I. Epithelial cell behaviour on grooved or smooth percutaneous implants 72 A . Implants. 73 B . Morphometries 73 C. Observations 74 1. Substratum and implant surface preparation 74 2. In vitro experiments 74 a. Cell attachment 74 b. Cell orientation 76 3. In vivo experiments. 78 a. Clinical observations 78 b. Histology 79 c. Morphometric evaluation 80 i . Tissue attachment and sulcus measurements (epoxy implants) i i . Tissue attachment and recession (titanium-coated implants) D. Summary 87 II. Effects of groove characteristics on percutaneous implant performance 87 A . Implant fabrication 87 B . Preparation and characterization of surfaces. 89 C. Implantation procedure. 89 D. Histology and histomorphometric measurements. 90 E . Statistics 90 F . Observations 92 1. Surface characterization 92 2. Epithelial histology 92 3. Connective tissue histology 94 4. Histomorphometrics 94 a. Depth of attachment 94 b. Recession 97 c. Linear contact-length 97 d. Capsule. 98 G . Summary 99 vii III. The electron microscopy of the soft tissue interface with titanium-coated surfaces 99 A . Implants. 100 B . Observations 100 1. Epithelium 100 2. Connective tissue. 106 C. Summary I l l IV. The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous implants 112 A . Implants. 113 B . Implantation procedures 113 1. One-stage implantation 113 2. Two-stage implantation 115 a. First stage 115 b. Second stage. 115 C. Specimen collection 117 D. Histomorphometrics. 117 E . Statistics 117 F . Observations 119 1. Clinical healing 119 2. Histology 121 a. one-stage Percutaneous implants. 121 b. Two-stage percutaneous implants (First stage). 121 i . One week 125 i i . Two weeks 125 i i i . Three weeks 125 iv. Four-five weeks. 125 v. six-eight weeks. 126 c. Two-stage percutaneous implants (Second stage) 126 3. Histomorphometrics 130 a. Recession 130 i . Effects of stage and time 130 i i . Effects of surface 133 b. Epithelial and connective tissue attachment 133 i . Effects of stage and time 133 i i . Effects of surface 133 c. Connective Tissue Capsule 134 F . Summary 134 V . Electron microscopy of the mineralized tissue found adjacent to micromachined surfaces in vivo 135 A . Sample preparation 136 B . Observations 136 C. Summary 141 viii C J H A P T E E § DISCUSSION I. Epithelial cell behaviour on grooved or smooth percutaneous implants 144 A . Attachment assays 144 B . Orientation assays 145 C. Clinical observations 145 D. Histology of inflammation. 146 E . Tissue attachment and morphometries 146 F . Concluding remark 147 II. Effects of groove characteristics on percutaneous implant performance 148 A . General Results 148 B . Clinical healing. 148 C. Histology 149 1. Epithelial attachment 149 2. Connective tissue attachment 149 3. Cell shape 150 D. Morphometries 151 E . Concluding remark 151 III. The electron microscopy of the soft tissue interface with titanium-coated surfaces 152 A . Tissue preparation 152 B . Histology 153 IV. The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous implants 155 A . General results 155 B . Histology and morphometries. 156 C. Healing. 157 D . Concludin g remark 157 V . Electron microscopy of the mineralized tissue found adjacent to micromachined surfaces in vivo 158 ix C M A P T E E <S I. CONCLUSIONS 160 II. FUTURE DIRECTIONS 161 R E F E R E N C E S 163 APPENDICES 1. Measurements of linear contact length 185 2. Abbreviations used in the thesis 186 3. List of the publications from this thesis 188 X IJST OF FIGURES CHAPTER 1 P A G E Figure 1.1 13 Figure 1.2 17 Figure 1.3 25 Figure 1.4 37 Figure 1.5 39 Figure 1.6 42 Figure 1.7 49 CHAPTER 3 Figure 3.1 60 Figure 3.2 61 Figure 3.3 63 Figure 3.4 67 CHAPTER 4 Figure 4.1 73 Figure 4.2 74 Figure 4.3 75 Figure 4.4 76 Figure 4.5 77 Figure 4.6 79 Figure 4.7 80 Figure 4.8 84 Figure 4.9 85 Figure 4.10 86 Figure 4.11 88 Figure 4.12 93 Figure 4.13.. . . 95 Figure 4.14 98 Figure 4.15 100 Figure 4.16 101 Figure 4.17 102 Figure 4.18 103 xi Figure 4.19 : 1 0 3 Figure 4.20 1 0 4 Figure 4.21 1 0 4 Figure 4.22 1 0 5 Figure 4.23 1 0 5 Figure 4.24 1 0 6 Figure 4.25 1 0 7 Figure 4.26 1 0 7 Figure 4.27 1 0 8 Figure 4.28 1 0 9 Figure 4.29 1 1 0 Figure 4.30 1 1 0 Figure 4.31. 1 1 1 Figure 4.32 1 1 4 Figure 4.33 1 1 6 Figure 4.34 1 1 8 Figure 4.35 1 2 2 Figure 4.36 * 2 3 Figure 4.37 1 2 4 Figure 4.38 •  1 2 6 Figure 4.39 1 2 6 Figure 4.40 1 2 7 Figure 4.41 1 2 8 Figure 4.42 * 2 9 Fgiure 4.43 1 2 9 Figure 4.44 * 3 2 Figure 4.45 * 3 7 Figure 4.46 * 3 7 Figure 4.47 1 3 8 Figure 4.48 1 3 9 Figure 4.49 1 4 1 Figure 4.50 1 4 2 xii L I S T O F T A B L E S P A G E Table 1 83 Table 2 91 Table 3 96 Table 4 120 Table 5 131 Table 6 140 xiii Acknowledgements I wish to thank my supervisors, Drs D.M. Brunette and T.R.L. Gould for their constructive advice, criticism and support throughout the experiments of this thesis. I am also grateful to Dr. Joanne Emerman for her critisicm and helpful suggestions. I would like to express my appreciation to Mrs. L. Weston and Mr. A. Wong for their technical instruction in the histological processing, ultramicrotomy, electron microscopy and photomicrography. Thanks are also due to Drs. L. Young and D. Pulfrey for their generosity in providing access to their laboratories for the preparation of micromachined surfaces, and to Mr. H. Kato for fabricating the silicon micromachined surfaces. I would also like to acknowledge Dr. B. Ratner, director of the National ESCA and Surface Analysis Centre for Biomedical Problems, for characterization of the titanium surfaces. I am grateful to Ms. H. Maledy and Mrs. D. Price for their technical help in the in vitro experiments, and to Miss. P. Kapitan for assissting surgical procedures. In addition, I am indebted to all of the staff and students of the Department of Oral Biology and Faculty of Dentistry for providing a friendly and stimulating work environment. I would like to express my gratitude to my wife Haleh, and to my parents for making the whole thing possible. Finally, I thank the University of British Columbia for providing me with financial support during the course of this investigation. The experiments of this thesis were supported by the Medical Research Council of Canada (MRC #MA7617) and British Columbia Health Research Foundation (BCHRF #5-52880). '1 CMAIPTEE 1 INTRODUCTION PAGE I. Overview 1 II. Cell Adhesion to Artificial Surfaces. 3 III. Cell Migration on Artificial Surfaces. 28 IV. Wound Healing 32 V . Complications and failure modes of percutaneous implants 49 m INTRODUCTION I. Overview An implant is a device or inert substance, biologic or alloplastic, that is surgically inserted into soft or hard tissues, to be used in function or for cosmetic purposes (1). Alloplastic implants are defined as those made of a variety of synthetic materials such as metals, alloys, ceramics, carbons, polymers and composites. A l l implantable materials, no matter what their origin, should be biocompatible, meaning that they do not elicit toxic, carcinogenic or significant local inflammatory reactions (2,3,4). In a broader context, Williams (5) defined biocompatibility as the ability of a material to perform with an appropriate host response in a specific application; moreover, appropriate host response does not imply a zero host response, since every implanted device will initiate a response of some kind. Nor does it necessarily mean a minimal response, since a minimal response does not always lead to optimal performance. Specificity should be included also in the definition of biocompatibility, since an implantable material may be biocompatible in one application for a specific purpose, but not in other applications or at a different site (6). Historically, it is not clear when or who implanted the first artificial device in vivo; however, archaeological evidence indicates that nail-like metallic implants were placed in alveolar bone by ancient Egyptians (7). Some of today's knowledge about biocompatibility and practical applicability of implants results from centuries of trial-and-error attempts using "off-the-shelf materials. However, the field of implantology has become a serious scientific discipline only during the last three decades. Perhaps one reason for this recent progress is that medical advances have prolonged the average life span, which in turn has generated more demand for biocompatible implants that can successfully replace damaged tissues or organs. The growing need for artificial implants can be estimated from an early report released in 1980, which estimated the number of individuals receiving artificial implants in the United States in the millions (8). The following numbers of implants were placed in one year: 400,000 mammary prostheses; 600,000 artificial lenses and corneas; 250,000 total or partial hip implants; 100,000 2 knee prostheses; 400,000 finger joint prostheses; 500,000-1,000,000 cardiovascular shunts, bypasses, catheters, grafts and pace makers, and approximately 300,000 dental implants. In spite of this widespread application of prosthetic devices, there is still insufficient scientific understanding of the specific events happening at the interface between the implanted material and its host tissue. As a general rule, an implant fails most often at this interface (8). In this thesis, I will attempt to describe our current knowledge of the biological phenomena taking place at the interface of tissues with a class of artificial devices termed percutaneous implants. The following topics are covered: the interaction of cells with artificial substrata, including those interactions involved in cell adhesion and cell migration; the events in wound healing that may result in tissue reorganization around an artificial implant. Finally, the relative contributions of these processes in the failure of percutaneous implants will be discussed. Since the terms used in the literature to identify different classes of implants vary, it is necessary to introduce a simple classification that can be used conveniently in this thesis. Implanted devices can be classified by a variety of methods including the type of material, the function, the anatomic location and the surgical method used during implantation. In terms of problems with tissue adaptation, an alternate way of cataloging artificial implants is to divide them into two main categories: fully embedded and partially embedded. Fully embedded implants (FEI) are buried inside the living tissue so that there can be no communication between the inside sterile environment of the body and the outside contaminated environment. Examples of FEI include orthopedic implants such as total or partial hip-replacement implants, and soft tissue subcutaneous implants such as breast implants. It is generally accepted that if the material is biocompatible, FEI enjoy a high success rate. Partially embedded implants (PEI) are placed into the living tissues so that a part of the implant penetrates the covering epithelium and is exposed to the outside contaminated environment. Two important categories of PEI are percutaneous implants (PI) that penetrate through skin epithelium, and dental implants (DI), which penetrate through oral epithelium. PI vary greatly according to their material, location and function. Examples of PI are catheters implanted for peritoneal 3 dialysis and blood access, auditory devices, urinary conduits, attachments for various limb prostheses, and power connectors for artificial organs. Similarly, DI vary in their location, material, function and shape. Currently, subperiosteal, submucosal, endosseous cylindrical, blade and mandibular staple dental implants are used. PEI often face more complications than FEI because of the unique circumstances that arise as a result of the three phase junction, which is the location where the covering epithelium, implant surface and contaminated outside environment meet (9). As a general rule, to be successful an implant should be integrated within the surrounding tissue with minimal inflammation, and have enough stability to serve designated functional tasks. However, most foreign bodies will eventually produce chronic inflammation in the surrounding tissues, and the degree of chronic inflammation wil l depend on the material's degree of biocompatibilty (10,5). The material characters that influence the degree of biocompatibility include the mechanical stability of the implant as well as chemical and physical surface properties (2,5). FEI are usually in contact with soft connective tissue, vascular tissues or bone and are embedded in a sterile environment. In contrast, PEI must penetrate the covering epithelium and naturally are exposed to mechanical forces at the tissue/implant interface, to the hostile contaminated outside environment and to the complex dynamic behaviour of the covering epithelium. Consequently, PEI face a challenging situation that results in a higher risk of failure than do FEI (2-4,9,11-17). To understand specific complications that arise mainly from the interaction of the host cells and implant surface, it is necessary to understand the behaviour of these cells in more detail. II. Cell Adhesion to Artificial Surfaces The ability of cells to attach to the surrounding milieu regulates many important biological functions, such as tissue and organ differentiation, cell migration, cell communication, secretion and extracellular matrix arrangement, as well as cell shape (18-20). The adhesion of cells to implants is a complex affair involving intracellular and extracellular structures, as well as 4 physicochemical forces governing adhesion at the surface of the implant. Much of our knowledge regarding cell/substratum attachment originates from studies of cells in culture particularly fibroblasts and epithelial cells. Using in vitro techniques, substantial insight has been accumulated into how cells interact with their environment at the microscopic and molecular level. The process of cell adhesion in vitro differs in serum-free and serum-containing medium; however, the events happening in the presence of serum are thought likely to resemble the behaviour of cells in vivo. In the presence of serum, the adhesion of cells to a foreign object is a multi-step process. The initial step is the adsorption of the medium and adhesion molecules (to be discussed later) to the surface. The chemical and physical nature of a biomaterial surface can influence the quality and quantity of the adsorbed adhesion molecules (21,22). The second step in cell adhesion is the actual initial contact of the cell with the substratum, a process that may involve electrostatic forces (23). As both the surface of the cell and currently used tissue culture substrata are negatively charged, they would be expected to repel each other and thus inhibit contact or adhesion. However, Weiss (24) suggests that the charges on the surfaces of cells are distributed as patches of greater-and lesser-than-average charge density. Weiss noted that the areas with lower-than-average charge density could approach the substratum closely enough to facilitate adhesion. The third and fourth steps of attachment and spreading are complementary and involve reorganization of the cytoskeleton and direct interaction of the cell membrane with the substratum. Since the cytoskeleton is actively involved in the organization of many attachment structures, it will be discussed briefly. A . The Cytoskeleton The old concept of the cell as a membrane-bound amorphous bag of jelly with wandering organelles no longer stands. New light and electron microscopic techniques have revealed that the cell contains a complex network of protein filaments, called the cytoskeleton, that consists of three types of filaments: microfilaments, microtubules and intermediate filaments (25). 5 1. Microfilaments (MF) or actin filaments M F are approximately 6 or 7 nm in diameter and contain mainly actin and at least 20 actin-binding proteins (25). M F are often found as mesh works in the cortical region of the ruffling membranes of motile cells. They are also seen as a supporting core structure in the microvilli and other cell surface specializations. M F form parallel arrays of stress fibers that appear to attach to the plasma membrane at the region of cell/substratum interface called adhesion plaques or focal contacts, and run obliquely back into the cytoplasm towards a region in front of the nucleus (20,26). Alternatively, stress fibers may extend from one focal contact to another (26). The actin-binding proteins such as alpha-actinin, tropomyosin and myosin are distributed on the stress fibers in a fashion that resembles that of the myofibrils of muscle cells (25,27,28). Such observations led to the hypothesis that stress fibers may have contractile ability and provide the motive force for cell movement. However, Couchman et al. (29), Herman et al. (30) and Burridge et al. (31) questioned this role because of observations that showed stress fibers and focal contacts are more prominent in less mobile cells, and the presence of focal contacts reflected a very tight adhesion to an inflexible substratum. Highly mobile cells contained regions described as close contacts. It appeared later that M F perform other functions, such as giving the cell tensile strength, adhesive capability, as well as elastic and structural support (32,27). 2. Microtubules (MT) M T appear as long, hollow and unbranched cylinders consisting of heterodimers of alpha and beta tubulin (50KD each). The dimers are joined head-to-tail to form protofilaments, and 13 protofilaments form a microtubule with a diameter of 24 to 25 nm (33). The microtubule-organizing center (MTOC) induces the initial organization of the M T network. It appears that the M T O C may signal the direction of cell movement since the M T O C relocates prior to cell movement in a specified direction (34). As is the case with M F , M T are associated with other proteins that regulate their organization (35,36). Dynein and kinesin are proteins that interact 6 with M T and have a function analogous to that of the myosins of M F (36). They bind to the main structural subunit of M T , tubulin, ,and provide power for the beating of cilia and flagella (25,36), or for movements of intracellular organells (37). M T appear to be involved in a variety of cell functions such as ciliary beating, chromosome separation in mitosis, phagocytosis, axonal directional movements, organization of other cytoskeletal filaments, and partitioning of the cytoplasmic organelles (33-40). 3. Intermediate filaments (LF) Intermediate filament is a broad term describing a group of distinct cytoplasmic fibers that average 10 nm in diameter and are thus intermediate in size between M T and M F (41-45). IF are the most enigmatic component of the eucaryotic cytoskeleton and have the reputation of being stable or static structures that lie around and have little impact on cell behaviour. Recent advances in molecular biology have shown that IF are more dynamic structures than was previously thought (42-45). There are at least five distinct types of intermediate filaments expressed in different cell types. They are type I and type II, keratins in epithelial cells; type III LF consisting of vimentin, desmin, glial fibrillary acidic protein and peripherin; type IV, neuronal IF and type V , nuclear lamins. Type III, vimentin and type V , lamins have been shown to undergo a phosphorylated-mediated reversible disassembly during mitosis (44). It appears that LF interact with M T and M F , since disruption of the organization of M T and M F can cause a dramatic reorganization of the IF system (42,44). However, this interaction appears to be unidirectional. Similar to the case of M F and M T , IF are associated with regulatory proteins (IFAP); functions of these proteins include lateral aggregation (filaggrins), cross-linking (synemin,plectin) and capping (spectrin, ankirin) (42,45). There are also other IFAP such as epinemin whose functions are unknown (43). The dynamic organization of IF has been studied by microinjection techniques and genetic engineering. It appears that both vimentin and cytokeratin accumulate primarily near the nuclear surface and extend vectorially from the nucleus outward towards the cell periphery (44). 7 Recently, the possible binding of IF with the cell membrane or nuclear lamina has been investigated in more detail. Cytokeratin and desmin filaments appear to anchor indirectly to the desmoplakin I and II of the cytoplasmic face of cell adherence junctions (42,43,46). Vimentin and desmin filaments also may anchor to the plasma membrane through the interaction of their N -terminal domains with a protein called ankirin (43). It has been shown further that vimentin may bind to the nuclear lamina at its C-terminal tail domain (43). Such a connection may transmit extracellular signals to the cell nucleus. IF are not obligatory for cell migration; however, they appear to participate in a variety of events, including partitioning cytoplasmic organelles, maintaining structural integrity of the cell or tissue, controlling axonal caliber, transducing extracellular signals, and possibly regulating gene expression (41-46). B. The adhesion of cells and extracellular matrix to artificial surfaces in vitro The adhesion of cells to the substratum has been studied at the light microscope, electron microscope and molecular levels. The principle light microscopic technique used to study the cell substratum interface is interference reflection microscopy (IRM). 1. Interference Reflection Microscopy (IRM) IRM enables an observer to determine how closely various areas of a cell adhere to optically clear substrata (47). Using IRM, Izzard and Lochner (48) classified the contact between cells and their substratum into three main types. a. Focal contacts Focal contacts appear as black areas in I R M that are 0.25-10 urn wide and 2-10 urn long. They represent regions where the distance of the cell membrane from the substratum is 10-15 nm. Focal contacts were first observed in the electron microscope by Abercrombie et al. (49), who called them "adhesion plaques". They speculated that stress fibers associated with the focal 8 contacts generate tension that pulls the whole cell forward. In agreement with Abercrombie, Heaysman and Pergum (50) reported that fibroblasts can move only over a substratum to which they form focal contacts, and it has also been reported that epithelial cells, which migrate in the form of a sheet, develop focal contacts primarily at the periphery of the sheet in the leading front (41). These findings led to the hypothesis that focal contacts were also involved in the migration of epithelial cells, since it is believed that the motile power of an epithelial sheet is located at its free edge close to the ruffling membrane (41). It also should be noted that some degree of adhesion is generally required for most cell types to migrate, but if adhesion becomes excessive it may be inhibitory. In this connection, Singer (26) reported a cell adhesion molecule, fibronectin, at some focal contacts of fibroblasts. He suggested that perhaps two kinds of functionally distinct focal contacts exist: [1] fibronectin-negative focal contacts that might be relatively unstable adhesion sites, characteristic of moving cells, [2] fibronectin-associated focal contacts that might be characteristic of stationary fibroblasts. Other studies have demonstrated various proteases associated with focal contacts (51) that apparently participate in the degradation of the extracellular matrix under these contacts (52). These findings indicate that cells may induce detachment of focal contacts via their proteases so that they can "pick up their feet to take another step". Involvement of proteases in focal contact-like structures has been shown in transformed and metastatic fibroblasts. Chen (53) termed these contacts "invadopodia" and hypothesized that they may be involved in invasive movements of transformed cells. Despite growing evidence of the role of focal contacts in cell locomotion, several studies indicate that focal contacts are more prominent in cells displaying little or no motility, and that highly motile cells can migrate without forming apparent focal contacts (54,55). Thus focal contacts may not be an absolute requirement for the locomotion of some cell types. It is possible that the variation in the role of focal contacts may reflect the properties of different types of cells. 9 b. Close contacts Close contacts also mediate cell/substratum attachment and are characterized as broad areas approximately 30 nm from the substratum that appear grey in the IRM. Close contacts are larger and more diffuse than focal contacts, and their biochemical composition has been more difficult to define (27). It has been suggested that they may be involved in generating traction for forward protrusion of the lamellipodium (26,56). Close contacts are the only form of attachment observed in some highly mobile cells (54,55), suggesting that close contacts alone may be adequate for rapid cell migration. c. Extracellular matrix contacts (ECMC) A third class of cell/substratum attachment, the E C M C , appear white in the IRM, indicating approximately 100 nm or more separation from the substratum. When viewed in the electron microscope, the space between the cell and the substratum contains strands of extracellular matrix. In their investigation of E C M C , Singer et al. (57) reported close transmembrane association of fibronectin-containing fibers and actin filaments, as well as an accumulation of heparan sulfate proteoglycans. They have termed the E C M C "fibronexus". They speculated further that these sites could function during cell migration along tracts of fibronectin-containing extracellular matrix in vivo. E C M C have been reported in fibroblasts and epithelial cells attaching to various substrata such as epoxy, titanium and gold (58,59). 2. Electron microscopy Basement membrane and hemidesmosomes are two structures associated with attachment of epithelial cells to various substrata observed in the electron microscope (EM). a. Basement Membrane The basement membrane (BM) is a three layer, complex mixture of mainly non fibrillar collagen, glycoproteins and proteoglycans. B M is always found between epithelium and 10 connective tissue and is frequently reported at the epithelium/inert-substratum interface (60-64). B M is usually organized into three layers: [1] the "lamina lucida" appears as a 10-50 nm-thick pale layer associated with the epithelial cell membrane; [2] the "lamina densa" is a 20-300 nm-thick electron-dense layer closer to the substratum; and [3] the "lamina fibroreticularis" or "sublamina densa" is a poorly defined layer with fine anchoring fibrils and collagen fibers, that probably originates from the connective tissue. The main components of the B M are: laminin, collagen type IV, heparan sulphate proteoglycans, fibronectin, nidogen, entactin and antigenic structures such as bullous pemphigoid antigen (65). In addition to mediating adhesion of epithelial cells, numerous other functions have been attributed to the B M , including a role as a selective molecular barrier, and participation in tissue morphogenesis and cell differentiation or regeneration (66). b. Hemidesmosomes (HD) HD have been reported regularly at the epithelial cell/implant interface (60-68). HD appear as dense plaques, 0.1 u.m in diameter and 20 nm from the substratum (25,41). The hemidesmosome was named for its morphological similarity to one half of the "desmosomes" that attach epithelial cells together (69). HD, like desmosomes, consist of an electron-dense plaque at the cytoplasmic side of the plasma membrane, into which intermediate filaments are attached. On the extracellular side of HD, fine filaments, possibly composed of collagen type VII (70), cross the lamina lucida and attach to the lamina densa and may extend into the sublamina densa (71). In contrast to the extensive studies on the biochemical characterization of the desmosome, little is known about the individual components of H D . Various desmosomal proteins have been isolated; they include desmoplakin I and II, present in the electron dense plaque (72,73); desmoglein I, likely to be transmembranous (74), and desmoglein II and III, located in the intercellular space (74,75). Although it had been suggested that H D and desmosomes are formed from similar components (76), recent reports have shown that HD lack desmoplakins I and II, plakoglobin and desmoglein I (77,78). New biochemical and 11 immunogold electron microscopic techniques have identified three polypeptides in HD which were not found in desmosomes. These are 240 K D , 180 K D and 125 K D , of which the 180 K D polypeptide specifically associated with HD formed by mouse primary epidermal cells cultured on glass substrata (79). HD were identified at the interface of the epithelial cells cultured on several implant surfaces such as epoxy, carbon, ceramics and titanium (60,62,63). The attachment of cells to biomaterials can be approached from a molecular perspective, which will now be briefly described. 3. Molecules related to cell adhesion Cell membrane does not attach to biomaterials direcdy, but rather attachment is mediated by extracellular, membrane-bound and cytoplasmic adhesion molecules. Many extracellular adhesion molecules possess a tripeptide sequenced, arginine-glycine-aspartic acids (RGD) (25,41,66,80-82), which can be recognized by specific receptors on the cell membrane. a. Extracellular adhesion molecules Glycoproteins are probably the most important extracellular adhesion molecules. Fibronectin, a high molecular weight (440,000 D) glycoprotein that was discovered in the early seventies, is perhaps the most comprehensively studied glycoprotein. It was initially assumed to be the adhesion molecule of connective tissue cells; however, subsequent reports indicated fibronectin-mediated adhesion of other cell types, such as epithelial and blood cells (83). Fibronectin has the ability to bind to both the cell membrane and other extracellular components such as collagen and proteoglycans. Adhesion glycoproteins in other tissues and structures include: laminin in the basement membrane, osteonectin in mineralized or soft connective tissue, chondronectin in cartilage and tenascin in embryonic tissue (66,84). In addition other adhesion glycoproteins such as epibolin, vitronectin, epinectin and entactin have been isolated from diverse tissues including basement membrane, epithelial cells, serum and carcinoma cells (85-87). 12 Proteoglycans are another family of extracellular adhesion molecules that can associate with the cell membrane. New molecular, genetic and biochemical isolation procedures allow the recognition of a variety of backbone proteins. Structurally, proteoglycans consist of a protein core in which serine-glycine sequences serve as attachment sites for one or more glycoseaminoglycan (GAG) chains (66,88). The carbohydrates or G A G chains of the proteoglycans are well-known and include chondroitin-4 or 6-sulfate, heparan sulfate, dermatan sulfate and keratan sulfate ( 89). Previously G A G chains were used to categorize different classes of proteoglycans; however, currently these molecules are described according to their function, location or structure (66). These proteoglycans include: decorin (88,90,91), biglycan (92,93), versican (94-96) and perlican (66,97-99). They function by means of their high affininty binding sites to collagen, fibronectin, growth factors and cell membrane. Fibrous components of the extracellular matrix, collagens and elastin(s), are primarily structural proteins, which also participate in the cell adhesion process. Recent studies have identied 15 different collagen types in mammalian tissues (66). A l l collagens are composed of three polypeptide chains called alpha-chains that are coiled around each other to form a triple-helical conformation typical of collagen. Non-helical sequences are also found, mainly at the ends of the molecules, and in some collagens also within the main body interrupting the triple-helix. The triple-helix protein contains variable amounts of tripeptides, sequenced "Gly -X-Y" where X and Y are commonly proline and hydroxyproline. Collagens can be divided into fibril-forming types such as I,II,III,V,XI and nonfibril-forming types such as IV,VI,VII ,XII . Collagen fibers observed under the transmission electron microscope have a characteristic cross banding, resulting from their alignment, that has been described as a modified quarter stagger array with a hole between the ends of molecules (figure 1.1) (25). Collagens can bind to the cell membrane through two mechanisms, either through their RGD sequence or indirectly by means of other attachment molecules such as proteoglycans or glycoproteins (66). Normal or transformed fibroblasts can attach and generate tension when 13 cultured on a collagen gel. This may lead to reorientation of the collagen fibers, which in turn, can influence the spatial orientation and morphology of cells (100). It has been postulated that cells may transmit information concerning the shape of their environment by attaching and applying tension to collagen fibers in their extracellular matrix (100). region with no gap (32 um), appears as light-staining area in electron micrographs i i • i i i I\AAAAAAAXAAA|AAAAAAAAI I IVVVVVVVT sszs&zsaftt- ftAAAAl gap (35 nm) between individual collagen molecules Figure 1.1- The staggered arrangement of collagen bundles. Adjacent molecules are shown as arrows. Elastin(s), another fibrous component of the connective tissue, is present in small quantity in a variety of tissues, most particularly in elastic tissues including ligaments, aorta, pulmonary arteries, epiglottis, lung, bronchi, skin and gingiva (101,102). Fibroblasts, smooth muscle cells and highly metastatic malignant cells are able to attach strongly to elastic fibers. This attachment may be mediated by a 120 KDa glycoprotein designated as elastonectin (103), or a cell membrane receptor mediated mechanism, involving a 67 KDa lectin-like protein (104) on the surface of chondroblasts, or a 59KDa protein on the surface of lung carcinoma cells (105). These receptors appear to be RGD independent, and in carcinoma cells, the elastin-specific receptor appears to bind to an elastin-derived hexapeptide (VGVAPG) (105). 14 b. Cell membrane-bound receptors for adhesion molecules Cell surface receptors are essential for mediating the attachment of the extracellular matrix to intracellular components. The most important group of these receptors is a family of related molecules termed integrins (81,106-108). Integrins are hetero-dimeric cell surface glycoproteins assembled of an alpha subunit (140,000-180,000 MW) and a beta subunit (105000-125000 MW). There are three major types of beta-chain and an ever-growing number of alpha-chains. Morphologically, integrins are composed of a large extracellular domain, a small transmembrane domain, and an intracellular tail (25,31,66,81,107). The extracellular domain of the integrin can recognize the RGD sequence in a variety of extracellular adhesion molecules, and in mammalian cells there are specific integrin families that bind to a particular attachment molecule (109,110). However, there are also multiligand integrins that serve as receptors for many molecules such as laminin, collagen and fibronectin and may have attachment sites that are not RGD dependent (111). The intracellular portion of the integrin molecule appears to link to the cytoskeleton through other cytoplasmic proteins such as talin, alpha-actinin, vinculin and paxillin (25,31,66,81,107). Many functions have been listed for integrins; however, most importantly they are sites by which cells closely interact with the surrounding environment. Other cell surface molecules are proteoglycans syndecan and CD44 that contain heparan and chondroitin sulfate chains (112). The extracellular domains of these molecules bind to fibronectin, laminin, tenascin and collagen. As with integrins, the intracellular domains have high affinity to the actin component; however, the mechanisms of such interactions are not clear (66,113). c. Cytoplasmic domain of the adhesion molecules Much of our knowledge of the cytoplasmic domain of attachment molecules comes from observations of the cytoplasmic components of focal contacts in fibroblasts, and hemidesmosomes in epithelial cells. It appears that at focal contacts the actin filaments attach to 15 integrins indirectly via a series of cytoplasmic proteins including vinculin, talin, paxillin, alpha actinin and an actin specific capping protein (107,114). Three polypeptides of 240, 180 and 125-K D are identified to be the cytoplasmic component of hemidesmosomes, of which the 125-KD appears to be associated with IF (79). Any model of cytoskeleton/extracellular matrix or substratum attachment should be treated with caution since the functional mechanism of various cytoplasmic attachment molecules are insufficiendy characterized at the biochemical or molecular level. Moreover, there may be additional molecules associated with these adhesion sites, as yet uncovered. Broader discussions of these molecules are available in many reviews (114-118) and will not be discussed in this thesis. To sum up, it appears that the adhesion of the cell to the extracellular matrix or to an implant is a complex phenomenon involving the cooperation of at least three groups of adhesion-associated molecules. This scenario is even more complicated in vivo , where cells and extracellular matrix interact with biomaterial surfaces in a complex three dimensional environment. C. The adhesion of cells and extracellular matrix to artificial surfaces in vivo The observation of the interface of cells and artificial surfaces in vivo requires histological procedures including perfusion, dissection, fixation, infiltration, embedding, sectioning and light or electron microscopy. However, these procedures are not without problems, and some of these technical problems will be addressed in more detail later in the discussion. A major problem is the difference between the hardness of the artificial implant relative to the surrounding tissue, which renders histological processing a frustrating experience and prone to artifacts. To overcome these problems several techniques have been developed including freeze fracturing (119,120), microseparation (121,122), electropolishing (123), grinding (124) and metal coating (125). However, some of these techniques require several stages and harsh treatments that could lead to artifacts. In agreement with in vitro studies, epithelial cells attach to many implant materials such as plastics, metals and ceramics (119,120,125,126) by means of a basement lamina and 16 hemidesmosomes. This type of attachment resembles that present in at least one natural epitheUum-penetrating device, the tooth. Therefore, epithelial cell attachment to implants in vivo can imitate a type of attachment that normally exists in the biological system. Connective tissue cells also attach to the implant. However, light microscopic observations and limited electron microscopic data have indicated that such an attachment is mediated by extracellular matrix, and direct cell contact to the implant surface has rarely been noted (121-123). The surface of most biomaterials implanted in connective tissue becomes coated with a glycoprotein-like structure. Collagen bundles and fibroblasts are mostly arranged parallel to the long axis of the implant so as to form a capsule (121,122). Unlike naturally occurring, connective tissue structures such as tendon or periodontal ligament which has a specific orientation, the capsule surrounding implants does not appear to have a functional role in transmitting force (2). However, a few researchers have reported an alignment of collagen and connective tissue perpendicular or oblique to the rough or porous surfaces of metallic dental implants at light microscopic level (127,128). Moreover, Weiss (129) also described a fibrous connective tissue interface with some dental implants in which the collagen fibers originate at a trabecula of cancelous bone on one side, weaving their way around the implant and inserting into a trabecula located at the other side of the implant. Although in histological sections such collagen organizations may appear parallel with the implant surface, they are anchored at right angles into the adjacent bone (figure 2.2). When an implant is in function, forces closest to the implant causes a compression of the fibers, with corresponding tension on the fibers placed or inserting into the trabeculae. Weiss believes that such a structure can have a functional role similar to that of the periodontal ligament of a tooth. James (130) has observed a suspensory ligament surrounding some dental implants (figure 2.2). The principle fibers may insert into the bone medial and lateral to the implant and pass apically forming a hammock-like arrangement that is assumed to be able to transfer force to the bone. However, there are also several contradictory findings reported in the literature (121,122,131,132), which raise some doubts on the function of the peri-implant connective 17 tissue. It appears that there is an obvious need for more systematic experiments on the organization, fate and functions of the peri-implant connective tissue attachment. Functional Loading Functional loading Implant bony socket Weiss' view James' view Figure 2.2- Schematic drawing of the functional peri-implant connective tissue as described by Weiss and James. Although the term osseointegration was introduced initially to describe a direct contact/attachment between mineralized tissue and implant surface (1,133), it was subsequently modified to describe an indirect contact/attachment likely through a nonmineralized layer of glycoproteins (134,135). However, the likelihood of this finding depends on factors such as the choice of material, or surface texture, since implants made from bioglass do form a direct contact and possibly a chemically bond with mineralized tissue (136,137). Another factor which can affect the bone/implant interface is the micromotion of the implant at the initial stages of healing (138). Implants placed in loose surgically-created bedding and stressed by external mechanical forces often develop a fibrous connective tissue interface, instead of an osseous interface (139). Currently, other procedures, such as Branemark's two-stage surgical technique, that require a 18 period of non-function during the initial healing, to some extend limit micromotion at the implant surface, and have been more successful (133). The data on the amount of micromovement that wil l result in failure of the osseointegration are insufficient, and well-controlled studies are required. D. The role of material properties in cell attachment The properties of materials affecting cell attachment encompass mechanical rigidity, surface energy or wettability, surface charge and surface topography. 1. Mechanical rigidity Maroudas (140) has emphasized the requirement of cells for some degree of mechanical rigidity for the substratum to withstand the tensile forces exerted by the cells. The innovative experiments of Harris and coworkers (141,142) demonstrated that both fibroblasts and epithelial cells exert tensile forces on deformable substrata as they attach and migrate. Moreover, the forces exerted by epithelial cells tend to be weaker than those of fibroblasts. However, traction force may not be equated with speed of locomotion since fast moving cells such as leukocytes exert only a weak traction force (41). Percutaneous or subcutaneous implants made of soft materials such as silicone may alter the nature of cell adhesions, and it is possible that the cells that exert only weak forces would likely to be the least affected. 2. Wettability or status of surface energy The wettability of a material can be measured by the critical contact angle formed by liquid droplets spreading on its surface (143-145). The data can be expressed in terms of critical surface tension in dynes/cm2. Materials with surface tension at or below 20-30 dynes/cm2 are considered hydrophobic (144,145). Surface wettability is also closely related to the free surface energy of a material. Based on the wettability data, materials can be divided into two major 19 groups: [1] materials such as metals, ceramics and glass that are highly hydrophilic and wettable, and have high surface energy; [2] materials such as waxes, silicone or teflon which are hydrophobic and have low surface energy. It is generally accepted that materials with high surface energy are biologically active and promote cell attachment; whereas materials with low surface energy are more likely to produce a capsule of amorphous scar-like tissue (143-146). Nevertheless, biomaterials with low surface energy do have various applications for implants contacting the blood circulatory system such as heart valves, in which adhesive interactions such as platelet attachment, coagulation and thrombus formation are not desired. The wettability profile of a biomaterial also indicates the cleanliness of a surface, which can be inadvertently contaminated by a waxy overcoating from polishing, handling and sterilization (145). The wettability of a material can be increased temporarily by a treatment termed radio frequency glow discharge (RFGD) (144,146). RFGD is a technique which uses plasma clouds of argon gas to bombard the surface of biomaterials. This procedure ashes away the organic contaminants and renders implants sterile, highly hydrophilic, and very receptive for adhesion (147,148). Pratt et al (149) have shown that the combination of RFGD treatment and precoating the surface with fibronectin enhanced endothelial cell adhesion to Mylar films. Baier et al.(143-146) have provided in vivo evidence that RFGD treatment can promote rapid cell adhesion of the connective tissue to many biocompatible materials or even nonbiocompatible materials such as copper. However, necrosis, tissue destruction and scar formation will follow the initial cell attachment with nonbiocompatible materials. The mechanisms by which a wettable surface promotes adhesion are not clearly understood. However, a possible suggestion is that the conformation of proteins adsorbed onto the surface regulates the adhesion to the hydrophilic surface. Baier (143) believes that on high surface energy materials macromolecules adsorb, bend out of shape, and develop more points of 20 attachment for cells. In agreement with Baier's concept, Grinnell and Feld (150) found that fibronectin adsorbed onto the hydrophilic surfaces was in a different conformation from that adsorbed onto hydrophobic surfaces. 3. Surface charge Usually the chemical groups and ions at the surface of a biomaterial determine its surface charge and bonding potential that affect cell adhesion, spreading and migration (151,152). As noted earlier the cell surface carries a net negative charge and so do most biomaterials (41,153). To take advantage of the electrostatic forces for immediate cell adhesion, a logical approach would be to alter the negative charges of either the cell or the biomaterials. Data obtained mostly in vitro have demonstrated that changing the net surface charge of substrata to positive affects initial cell adhesion, spreading and matrix production for various cell types such as fibroblasts, neural cells and osteoblasts (152-156). However, the application and interpretation of this approach in vivo could be complicated, considering that the characteristics of the surface would be altered by the constituents of blood and plasma. 4. Material surface topography Another major factor affecting cell adhesion as well as cell migration is the macroscopic and microscopic geometry of the implant surface. Surface topography might influence cells in a variety of ways, including confining diffusion channels in and out of cells, limiting access to nutrients and escape of waste products, and inducing stress and strain on the cell membrane (157). Commonly all cell types, excluding those that grow in suspension, live in a milieu with some type of topography. This topography could be provided by other cells, extracellular matrix, other organisms or by artificial materials. Curtis (157) in a recent review, divides the surface topographies of artificial implants into two categories: those produced inadvertantly during the tooling of a prosthesis and those produced deliberately to achieve certain desired end 21 results. This section discusses some common surface topographies and their implications on various implants such as subcutaneous, percutaneous and bone-contacting implants. a. Smooth surfaces Glass, metal, plastic and silicone rubber implants with smooth surfaces have been used as subcutaneous implants in animal models. Subcutaneous implants are the simplest and probably one of the most successful types of implants because they interact only with connective tissue in a sterile environment. Nevertheless, subcutaneous implants with smooth surfaces have been frequently reported to induce sarcomas in rodents (158-160). The same material roughened or implanted as particles did not induce sarcomas (159,160). Several mechanisms have been proposed to explain the phenomenon. Kordan (160) and Oppenhimer et al. (161) emphasize the role of the connective tissue capsule as a possible mechanism of foreign-body cancer formation. Fibroblasts and collagen bundles respond to smooth subcutaneous implants by aligning parallel to the implant surface, forming a capsule (121-123,162). The thickness of the capsule is often correlated to the degree of biocompatibility (2,3). A thick capsule, which has been frequently reported on smooth surfaces, is densely populated with cells and collagen bundles and has an insufficient blood supply. It has been suggested that abberations of cell behaviour affecting cell growth may occur within the capsule, probably on those cells closer to the implant, simply because of hypoxia or inadequate nourishment (161). A second hypothesis stresses the role of macrophages and giant cells that accumulate on rough but not on smooth surfaces, in inhibiting sarcoma induction. A paucity of macrophages on the smooth surface may contribute to cancer formation; in support of this theory Ferguson (163) provides in vitro evidence that macrophages are able to kil l tumor cells by direct contact. Boone et al. (164) speculate that factors related to the geometry and mechanics of cell attachment to a flat surface are somehow associated with an increased frequency of genome replication errors, which may allow these cells to escape growth control mechanisms. Although a few cases of sarcoma have been reported with implanted 22 devices in humans (10,165), there is little evidence for a strong relationship between sarcoma induction and smooth surfaces in humans. One explanation for this may be the existence of a longer latent period for sarcoma formation by smooth surfaces in higher animals. Smooth surfaces have been used in percutaneous or dental implants on the basis that a smooth surface does not accumulate microorganisms at the zone where the implant penetrates the epithelium. A common problem with all percutaneous implants and some dental implant designs has been the tendency of epithelium to migrate down the implant surface, forming pockets or sinus tracts that may accumulate bacteria and their toxins (2-4,9, 11,12). As with natural teeth, where a similar process is operative in periodontal disease (166), the end result may be that the implant becomes completely walled off by the epithelium and eventually is extruded. It has been shown that percutaneous implants with smooth surfaces probably fail because of epithelial downgrowth (2-4,9). Some dental, orthopedic and percutaneous implants are designed so that smooth surfaces contact bone. Smooth surfaces of titanium, vacuum deposited onto polycarbonate, have been shown to contact bone matrix through a layer of non mineralized material [200-400 A] likely to be of proteoglycan origin (167,168). In contrast, smooth surfaces of Bioglass bind to the bone matrix and collagen directly without nonmineralized materials at the interface (136,137). Biomechanical factors with metallic implants indicate that implants with smooth surfaces have lower attachment strength than those of textured implants (167-172). b. Textured surfaces Textured surfaces differ greatly in their topography, mainly because of markedly different fabrication techniques. The simplest method of producing a rough surface is to rub the surface with different size abrasive particles (175). Another method is to bombard the surface of biomaterials such as teflon with charged ions to produce nanoporous surfaces (176-178). A harsher treatment, usually used on metals, is blasting the surface with silica particles (132). 23 Sintering is another highly sophisticated method of surface texturing that is achieved by sintering spherical metal particles onto the surface of solid implants made of titanium alloy, to make an interconnected porous surface (173,174). The fundamental rationale for texturing the implant is to increase the surface area for cell attachment and to achieve mechanical interlock by tissue ingrowth (173,174,179). Although crude, one way of measuring the strength of the tissue attachment to the implant is the pull/push-out test or its modification, the peel test. Several experiments using these tests have shown that greater force is required to detach textured implants from either soft or hard mineralized tissues (169-172,174,180). It might be expected that epithelial cells would also interlock into the textured implants and thus be slowed in their downgrowth. However, epithelium-penetrating devices with interconnected pores are not without problems. In a review on percutaneous devices, von Recum and Park (2) state that implant materials with porous surfaces and interconnected porosity, including open sponges, felts and velours are especially prone to serious implant infection which will never reheal. Another possible advantage of textured surfaces is that the connective tissue might ingrow into the pores and form a barrier to resist the downgrowth of the epithelial cells on epithelial-penetrating implants. Winter (181) found fibrous tissue ingrowth into various porous implants with pore sizes of 10-40 urn, and this tissue appeared to prevent the migration of the epidermis alongside implants placed percutaneously in pigs. Squier and Collins (182) have implanted Millipore filters with various pore sizes percutaneously in the backs of pigs. It was reported that only filters with pore sizes of 3 um and above effectively induced ingrowth of connective tissue cell and collagen, that in turn inhibited epithelial downgrowth. There is not a clear-cut relationship between the pore size and the nature or organization of the connective tissue within the pore. However, one concern is the ability of the surface to allow cell or capillary ingrowth. From their experiments with subcutaneous implants, Chavpil et al. (183) have suggested that a pore size greater than 100 urn is required to obtain loose vascularized connective tissue ingrowth into a collagen/glycol methylmethacrylate sponge. Taylor and Gibbons (184) report that porous 24 methylmethacrylate implants must have an average pore size of 360 um to induce the ingrowth of capillaries and cellular material. Polyurethane sponge subcutaneous implants with large pores of about 3200 um were filled more slowly by less vascular and cellular tissue than those implants with pore sizes from 280 to 600 urn (41). The relationship of textured implants and bone also has been investigated. Pilliar (173) concluded that bone tends not to form if the pore size is less than approximately 50 um. Brunette (162), in a review of the topic concluded that bone ingrowth will be facilitated on surfaces with pores greater than 100 um in size. However, bone ingrowth is reported to take place into porous polyethylene implants with pore sizes as small as 40 um (185). The differences in the estimate of the minimum size of pores or rugosity that promotes connective tissue or bone ingrowth probably arise from the variations in materials, pore shape, thickness and precise topography of the rugosity used in different experiments. Some textured surfaces such as porous or rough surfaces can induce calcification of the soft tissue at the interface when implanted subcutaneously (181,186-192). Although biomaterial-induced calcification could have fatal consequences in heart, vascular or urinary prosthesis (189), it would be a desirable property for bone-contacting implants such as endosseous dental or orthopedic implants. Because of the lack of controlled systems and the complex nature of in vivo experimental models, the mechanism of biomaterial-induced soft tissue calcification is poorly understood. Schoen (189), in reviewing biomaterial-induced calcification, concluded that several factors might be involved, including the host metabolism, the implants' physical and chemical structures, the implantation site, and the type of function. He further speculated that calcification may take place within the fluid and cells imbibed by biomaterials or the tissue growing into interstices of porous materials; usually calcification is associated with the sites of greatest dynamic activity. For example, Coleman (193) reported that calcified deposits on smooth-surfaced blood pump bladders are frequently associated with surface defects or discontinuties, perhaps originating during bladder fabrication or resulting from environmental stress cracking. 25 Although it appears that surface roughness, pores or other defects correlate with biomaterial-induced calcification, no clear cut data is yet available on the precise dimensions of these surface textures. This area certainly needs more attention. One disadvantage of textured surfaces could be rugophilia, particularly on those rough surfaces produced by particle polishing (175,194). This phenomenon deals with the tendency of monocytic phagocytes to detect and attach to rough surfaces (rugophillia). The studies of Rich and Harris (194) and Salthouse (175) have shown that macrophages prefer rough surfaces to smooth surfaces (figure 1.3). Rugophobia Rugophilia (Rich & Harris) Figure 1.3-Schematic showing rugophillia of macrophages, described by Rich and Harris. Although little effort has been devoted to studying the effects of surface topography on the type of cell populations attached to implants, the results of some studies did not support the concept of preferential attachment of phagocytes to rough surfaces placed in bone (171,173,174). However, it may not be an appealing idea to devise implants with rough surfaces that may attract macrophages and their close relative, giant cells, which are known to participate in foreign body rejection. The presence of osteoclasts at the interface should also be considered with caution because if a bone-contacting implant with rough surfaces attracts osteoclasts, bone resorption 26 probably might occur, which would destabilize the implant. Ferguson (163), however, in his study of implant surfaces speculated that macrophage accumulation and their activity probably inhibited cancer formation around roughened implant surfaces in rodents. Another disadvantage, particularly in implants with porous or rough surfaces, may be their corrosion properties in a physiological environment. Naji and Harmand (195) demonstrated a relationship between in vitro cytotoxicity and the surface roughness of implants made from chromium/cobalt alloy. They have concluded that textured surfaces of chromium/cobalt alloy may be more susceptible to corrosion. However, this concept could be questioned in highly self-passivated metals such as titanium, in which the surface is protected by a stable layer of oxide (123). Implants with machined surfaces have parallel circular grooves of varying depth on their surfaces produced during the fabrication process. Most cylindrical dental implants such as Biotes (Branemark Dental Implants, Nobelpharma, Sweden), Core-vent (Core-vent Corporation, Encino, California), and I M Z (Interpore International, Irvine, California) have machining grooves on all or portions of their surfaces (163). Histological data have indicated that soft and mineralized tissue attach closely to these surfaces (196-200). An interesting feature of machined surfaces is that the small grooves influence cell alignment and migratory behaviour by a phenomenon termed contact guidance (163). E. Other factors affecting cell/implant attachment Several material-independent factors can alter the attachment of cells to implants or cause failure, but probably two of the most relevant factors are surgical technique and the structure of the implant bed. 1. Surgical technique High speed drilling in bone produces heat that can result in reduced bone regeneration and attachment. The heat produced by drilling has been suggested as one cause of the aseptic 27 loosening of orthopedic implants (201-203). Surgical technique also can affect healing. Implants placed by atraumatic surgery limited to a small area are likely to heal by first intention, whereas implants placed by extensive traumatic surgery may heal by secondary intention, which is normally accompanied by scar formation. As scar tissue does not represent normal connective tissue (204,205), the orientation, quantity and quality of cells and collagen fibers attached to the implant surface may differ in implants that heal by secondary intention. 2. Structure of the implant bed Recipient sites for a biomaterial differ in their ability to support implants. For example, an orthopedic implant in a patient who lost a joint due to inflammatory joint disease (rheumatoid arthritis), may have to deal with tissues that are abnormally activated by arrays of immune mediators. Similarly, placement of a dental implant in the extraction socket of a periodontally infected tooth could be questioned. Unfortunately, details of cell/implant interactions in such situations are not clear and more controlled studies are required. F. Summary The field of implant and biomaterial research has become a fast growing and demanding discipline, mainly because of man's longer life expectancy that has generated an increased need for artificial tissues and organs. Cell adhesion to implants as a fundamental criterion of biocompatibilitiy or implant stability in living tissue is discussed. Cell adhesion has been mainly studied at the light, electron and molecular level. These studies suggest that cells attach to a biomaterial by means of a variety of adhesion molecules, which can be categorized as extracellular molecules, cell membrane receptors and their intracellular domains. The mechanical, physical and chemical properties of materials are considered as important factors affecting cell adhesion. Some of these properties are recognized as surface rigidity, surface wettability, surface charge and surface topography. Material-independent factors that may affect cell attachment are the choice of surgical techniques and the structure of the implant bed. 28 III. Cell Migrat ion on Artif icial Surfaces During development, cell migration and constant rearrangement of the extracellular environment are required to form functioning tissues or organs. Implantation of a biomaterial may create a similar situation that requires cell migration and subsequent tissue remodelling to form a stable tissue/implant relationship. As with cell adhesion, work on cell migration has been mainly accomplished in vitro. The mechanisms of cell migration have been studied most extensively in fibroblasts, phagocytes and epithelial cells. Although there is no agreement on the mechanism of cell locomotion, it appears that cell locomotion involves various stages including adhesive interactions, force generation, protrusion of cellular processes and relocation. A. Mechanisms of cell migration Stossel believes that the most serious speculation on the mechanism of cell movement was made in the 18th century (206). It was suggested then that a transition from solid to liquid status could provide the motile force for cells, driving them forward. Although the major cytoplasmic protein actin has the ability to form sol-to-gel transitions, this theory was overshadowed by the discovery of myosin. Myosin, the major protein of muscle cells interacts with actin to produce the sliding movements of the myofibrils and muscle contraction. Since the intermittent distribution of myosin on the actin-containing microfilament shows similarities to that in muscle cells (25), it was proposed that a mechanism similar to that of force generation in muscle cells may be operative in non-muscle cells. Abercrombie at al. (207,208) proposed a model in which continuous contraction of the stress fibers associated with focal contacts of the leading lamellae exerts a force on the substratum and pulls the rest of the cell forward. Although stress fibers have been shown to shorten as the cell moves (209), this theory does not account for cells that do not possess focal contacts and yet are capable of locomotion (210). Huxley (211) and Small (212) elaborated on a theory that proposed that the forward movement of a cell results from a forward flow of the cytoplasmic constituents, that in turn are propelled by the sliding movement 29 of the membrane-bound actin filaments and myosin molecules. A broader theory proposed by Dunn (209) suggests that cells move by the coordination of force generation by the cytoplasmic actin/myosin meshworks, which are distributed at the leading lamellae and throughout a cell. The actin/myosin model of cell movement soon became criticized as new findings revealed that non-muscle cells possess a variety of myosin-like molecules and that many of these molecules differ in structure and function from muscle-cells' myosin (212-214). Moreover, genetic engineering studies showed that mutant amoebae devoid of muscle myosin (myosin-JJ) were still able to migrate. These findings drew attention to the non-muscle myosin that was identified as myosin-I or minimyosin. In contrast to myosin-II, myosin-I exists only as a monomer and is unable to form filaments. However, it has two binding sites to actin filaments and at least one of them is A T P dependent (214,215). Recent studies have shown that myosin-I is associated with the cell membrane and can generate force when interacting with actin (215). New evidence using the amoeba model and immunolocalization techniques has led to the hypothesis that myosin-I interacts with actin at the leading front to produce the force required for cell migration while myosin-II is required at the rear, possibly to stop membrane ruffling or to minimize cell contact in order to support the locomotive activity of the cell front (27). The relatively small proportion of myosin to actin and lack of experimental data on the presence of myosin-I in non-amoeba cells (206,216,217) have raised doubts, leading to other hypotheses. Proponents of these hypotheses boldly ignore the role of cytoskeletal proteins and emphasize the role of direct incorporation of molecules into the cell membrane and subsequent recycling during endo/exocytosis, as a potential mechanism of locomotion (216). Recently the old notion of sol-to-gel transformation of actin has been revived, with a new interpretation that this mechanism generates osmotic forces to drive the cell forward (217). Epithelial cells, however, migrate in a sheet or unit in which cells within the sheet form long-lasting desmosomal attachments. Depasquale (218) and Heath (20) demonstrated that in vitro cells located at the margin of the sheet display marked surface activity including ruffling, and suggested that the marginal cells may provide the motive power for the migration of an epithelial 30 sheet. In their view epithelial cells' locomotion resembles a train in which the marginal cells act like the locomotive and the non marginal cells are dragged passively behind like boxcars. This theory may have in vivo validity in the case of epithelial cells migrating to cover a wound in the cornea (41). The leap-frog or tracked-vehicle model, proposed by Krawczyk (219) and Gibbins (220), suggests that cells several microns behind the leading edge of a migrating epithelium proliferate. These cells then move up, forward and over the basal cells and then move down to attach to the substratum. In this way new cells are layered down in an advancing front. Evidence for this theory is more indirect than the train theory as it rests on observations of histological sections taken at long intervals that had to be interpreted to infer the dynamic events of cell locomotion (219,221). Obviously, we still have to learn a lot about the mechanisms underlying cell migration and factors regulating such mechanisms. It seems probable that not one single mechanism but rather an array of mechanisms may be involved in cell migration, and equally possible that different mechanisms may dominate in a particular population of cells. B. Cell migration on implants in vivo It should be noted that the relationship of cell to implant is not necessarily a static affair but that the process of cell migration and renewal at the interface are constantly taking place. A good example of a dynamic cell system at the interface is the relationship of epithelial cells to dental implants or percutaneous devices. Epithelial cells attach to percutaneous and dental implants at their most superficial zone adjacent to the outside contaminated environment. An important function of epithelial cells in this area is to form a seal protecting the implant from contamination. However, epithelial cells do not become stationary at the interface but continuously migrate and proliferate, and may compromise percutaneous devices and some designs of dental implant. 31 One reason for implant failure is downgrowth of the epithelial cells (2-4,9,11-17). Downward migration of the epithelium results in the formation of sinus tracts or deep pockets on such devices, encouraging bacterial or plaque accumulation that in turn can lead to inflammation and infection and the eventual loss of the implant. This type of migration results from the innate tendency of epithelium to cover a denuded area. Because of the physical presence of the implant, E cells migrate downward on the surface of the implant and this migration continues until E cells are attached to each other from all sides (2). To achieve this, E-cells around the implant must marsupialize the implant, which results in its failure. Epidermal or oral epitheial cells also migrate during cell maturation. The junctional epithelium (JE) of a tooth is an excellent model of renewing and migrating E cells. During normal maturation, cells of the JE migrate upward towards the surface where they shed into the periodontal sulcus (222). Hall and Ghidoni (223), have hypothesized that during maturation of an epithelial attachment to a percutaneous implant, a tight mechano-chemical bond holds between epithelium and the implant surface and causes extrusion and pulling of the implant outward by the migration of cells towards the surface. This process may result in extrusion and failure of the soft-tissue-anchoring, percutaneous implant. A better understanding of the nature of cell migration at the implant interface might be achieved from studies of the events taking place during wound healing. C. Summary Cell migration has been reviewed as an essential requirement that would assist distribution of the tissue at the implant interface. Generally there is not a common agreement as to the mechanisms of cell migration; however, it appears that different cell types may favor a particular mechanism or combination of mechanisms. Much of the knowledge on cell migration at the implant interface in vivo has been obtained from the observations of epithelial cells. Epithelial cells on the implant surface may migrate downward during healing or upward during the maturation process. A better understanding of the migration of mesenchymal cells in vivo may be attained from the observation of processes involved in wound healing. 32 IV. Wound Healing If an implant is to be used internally, some tissue injury wil l be caused by the surgical intervention. Wound healing and tissue repair are composed of sets of interdependent processes that are automatically triggered by the tissue injury. The first defensive mechanism that operates after injury is blood clot formation, which controls bleeding and includes as many as seventeen factors involved in the extrinsic and intrinsic mechanisms of blood coagulation (224). The end result is that to alter soluble fibrin, substrates are converted to insoluble fibrin that, along with platelets, forms a meshwork to seal off the injured area. During these initial events of wound healing, the surface of the implant will be conditioned by components of plasma and the extracellular matrix that may affect its future tissue integration (144,145). To understand the dynamics of the cell/implant interface during wound healing, three interrelated phases of healing should be considered: the inflammatory phase, the proliferative phase (granulation tissue formation), and the remodeling phase. A. The inflammatory phase Clinically, this phase is associated with redness, heat, swelling and pain that occur as a result of cellular and tissue injury. Vessels dilate in inflamed tissues and blood and plasma components leak into the injured tissue. As a result inflammatory cells such as platelets, neutrophils, macrophages, lymphocytes and mast cells accumulate in the area. It is likely that mediators such as cytokines secreted by these cells will subsequently affect the resident cell population. 1. Platelets Platelets are enucleated discoid blood cells, approximately 2 um in diameter that contain at least three types of secretory granules: alpha-granules, dense bodies and lysosomes (25,224). These granules may contain adhesive glycoproteins such as fibrinogen, von Willebrand factor, biogenic amines such as serotonin and prostaglandins, and various growth factors and enzymes. 33 Platelets are specialized for three functions: adhesion, aggregation and secretion (225). The initial role of platelets is achieved through their adhesion glycoproteins, which promote the adhesion of platelets to each other and to the extracellular matrix. The bioactive amines cause blood vessels to dilate and to become permeable, which in turn facilitates the migration of leukocytes to the injured areas. Platelet constituents are also believed to modulate provisional matrix formation in the injured tissue by means of providing a substrate for cell movement, and promoting cell growth with fibronectin or platelet-derived growth factor (PDGF) (226,227). Platelets interact with other inflammatory cells. They can stimulate mast cells to secrete histamine (228); they facilitate macrophage influx to the injured site by chemotactic factors, and promote their adhesion (229). 2. Leukocytes Neutrophils (polymorphonuclear leukocytes, PMN) are cells with multilobulated nuclei, whose primary function is to phagocytose and digest pathogenic organisms and other debris. They are the first type of leukocyte to arrive at an injured site, to which they are attracted by chemotactic factors. Macrophages are differentiated monocytes that arrive at the injured site shortly after neutrophils. Macrophages, like neutrophils, are phagocytic cells and also are known to release a plethora of biologically active substances. These substances can recruit additional inflammatory cells, aid the macrophage to decontaminate the tissue, and initiate the formation of granulation tissues (229). Thus macrophages play an essential role in the transition between the inflammatory phase and the proliferative or granulation tissue formation phase. Mast cells are easily identified by their distinctive metachromatic granules when stained with cationic dyes. These granules contain a variety of biologically active substances including histamine, proteoglycans, serotonin, cytokines (interleukins), prostaglandins, platelet activating factor and various proteolytic enzymes (230,231). Early studies of mast cells emphasized the dramatic pharmacological properties of heparin, histamine and serotonin. Heparin is a potent natural anticoagulant; histamine and serotonin are substances that promote dilation of capillaries 34 and increase their permeability. The release of these substances during the inflammatory phase regulates blood clotting and mobilizes other leukocytes from the blood vessels. Mast cells play a central role in IgE mediated allergic reactions. In the most severe form of the allergic reaction, anaphylactic shock, a sudden release of granules by mast cells takes place that can be fatal (232). For some time mast cells were known only for their pivotal role in allergic reactions; it is now clear that mast cells actively regulate the matrix production of soft and mineralized connective tissue (190,230,233-238). Recent works using in vitro studies have shown that mast cells are heterogeneous, and form two distinct populations, a tissue residing population and a blood residing population (230). Lymphocytes are a special class of leukocytes, which exist in two main types; B-lymphocytes that produce antibodies, and the family of T-lymphocytes that are helper T-cells, suppressor T-cells and killer T-cells. T-cells do not produce antibody, but they regulate the function of other leukocytes or fibroblasts, and participate in the killing of virus-infected cells (25,237,238). T-lymphocytes participate in connective tissue regulation by an array of growth and regulatory peptides, known as lymphokines. Recent studies of the role of lymphokines have shown that transforming growth factor beta (TGF-B) may react as a potent chemoattractant for fibroblasts, perhaps an important recruiting mechanism for fibroblasts to the wound site (237). T-lymphocytes also produce other factors including fibroblast inhibiting factor (FIF) and fibroblast activating factor (FAF); the first would favor localization and accumulation of fibroblasts in the inflammatory site, and the second regulates fibroblast growth (238,239). The role of lymphokines in fibroplasia has been demonstrated in thymic free animals, or in experimentally T-lymphocyte depleted animals. Generally, these experiments indicate that the healing process was slowed, and fibroplasia decreased or disappeared (239). Although T-lymphocytes obviously have some role in the regulation of fibroblasts, it is not yet clear which family of T-lymphocytes contributes the most to this process. Persistance of an antigen source 35 stimulates T-lymphocytes to produce continuously varied lymphokines, including those which stimulate fibroblast proliferation and matrix synthesis, that may lead to excessive fibroplasia and scarring (238,239). The inflammatory phase is thus an orchestrated interaction of many cell types, mainly blood-borne, and their secretory products. In this phase, the injured site wil l be inspected by various leukocytes. Hostile organisms, foreign proteins or particles will be killed or phagocytosed, and a hospitable environment will be provided so that matrix-producing cells can migrate into the wound site. Arrival of fibroblasts or other matrix-producing cells signals the beginning of the proliferative phase. B. Proliferative phase This phase is associated with the migration of undifferentiated mesenchymal cells into the wound and subsequent proliferation of these cells, as well as the accumulation of more macrophages. Along with the movement of these cells, blood vessels begin to sprout branches which migrate into the wound bed to provide an adequate blood supply for the fast growing cell populations. Undifferentiated mesenchymal cells soon adopt the appearance of fibroblasts or myofibroblasts. Fibroblasts begin to synthesize extracellular matrix including collagen (mainly type III), fibronectin and glycoseaminoglycans, in particular hyaluronic acid (240). This type of connective tissue resembles embryonic connective tissue, and may facilitate cell migration into the wounded area (240). As in the inflammatory phase, the proliferative phase relies on harmonized interactions of macrophages, platelets, mast cells, T-lymphocytes, fibroblasts, and endothelium of the blood vessels. As pointed out earlier, fibroplasia and angiogenesis are probably caused by mediators secreted by leukocytes and platelets. Some examples of these mediators are growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha and beta (TGF-a,p) and fibroblast growth factor (FGF). The role of these 36 mediators in wound healing and cell behaviour has been the focus of many research groups, and an excellent review on the role of growth factors in wound healing has been published in Clark and Hanson's (241) recent book on wound healing. However, these factors wil l not be discussed in detail in this thesis, because little information on their function at the tissue/implant interface is available. C. Remodelling phase It is difficult to draw an exact boundary between the proliferative and remodelling phases. In fact, matrix remodeling can start as soon as matrix is formed. However, it should be emphasized that ultimately matrix remodelling should result in a tissue which resembles that which existed prior to injury. The remodelling of connective tissue begins with a decrease in the quantity of fibronectin, hyaluronic acid and type III collagen, and a concomitant increase in the quantity of type I collagen (224,240,242). The collagen bundles grow in size and establish cross-links to provide a three dimensional matrix in association with proteoglycans that are primarily rich in chondroitin sulfate, dermatan sulfate and heparan sulfate (95,242). Full maturation of the connective tissue matrix may take as long as 6-10 months (2). It is now clear that a successful wound repair is heavily dependent on the synchronized influx of specific cell populations into the lesion, and subsequent matrix production. Although the sequence of morphological events occurring in the healing process is well understood, our knowledge of the specific factors that might regulate cell migration and reorganization within the wounded tissue is insufficient. The interpretation of these factors becomes even more complicated when they are interacting with a foreign object. The schematic drawing of figure 1.4 is an attempt to show the complex interaction of cells participating in the healing process in the vicinity of an implant. The next section suggests some phenomena and factors that possibly participate in this complex picture to regulate cell migration and reorganization in an implant-induced wound. Clearly, it is beyond the scope of this thesis to discuss all the possible biochemical/biophysical phenomena that participate in tissue reorganization. (f Microrganisms Epithelial CELLS Microcirculation Fibrin Clot 1 £<§4 Platelets I Polymorphonucleocyti Macrophages Mast cells T Lymphocytes Fibroblasts ^^J^xtjacellular Matrix Figure 1.4- Schematic illustration of complex network of cell/cell interaction adjacent an implant One-way interaction Two-way interaction (feed-back) 38 D. Mechanisms of tissue reorganization around implants Because most tissues are opaque, direct observation of individual cells in situ is difficult. Most of what we know about cell movement and reorganization during wound healing around implants is based on indirect observations, generally of fixed tissues. The arrangement of cells and extracellular matrix around an implant appears to be the result of the cooperation of a number of cell/implant surface-dependent phenomena, including haptotaxis, chemotaxis, contact guidance, cell/cell contact inhibition of movement, and the two-centre effect. Although several of these phenomena have been reviewed recently (162), some of them are central to this thesis and will be considered explicitly. 1. Haptotaxis Carter (243) produced an adhesive gradient by evaporating palladium across a cellulose acetate substratum that was nonadhesive. Cells plated on such a substratum migrated towards the area of greater adhesivity (figure 1.5). Carter termed this directed migration "haptotaxis". The mechanism appeared to be that the stronger attachment of those lamellipodia which extended towards the area of the substratum with greater adhesivity exerted a dominant tension on the remainder of the cell, and inhibited migration away from the adhesive gradient (244). Another study of haptotaxis was performed by Harris (245), who demonstrated that haptotaxis was directly related to the hydrophilicity of the substratum. In his experiments, fibroblasts did not show haptotaxis when cultured on substrata that had an adhesive gradient produced on hydrophilic polystyrene. However, he observed a strong haptotaxis when adhesive gradient was produced on nontreated hydrophobic polystyrene. Rich and Harris (194) on the other hand, demonstrated that not all cells move towards the more hydrophilic substrata. Macrophages prefer hydrophobic substrata. Thus haptotaxis is a phenomenon that relates to substratum characteristics as well as to cell type, perhaps to a cell-specific membrane property. 39 Palladium Figure 1.5-Schematic drawing showing Carter's view of haptotaxis. Cellulose acetate Haptotatic gradient In vivo, adhesive gradients may be provided by adhesion molecules such as fibronectin and laminin. Embryonic evidence for such an assumption has been provided by Linask and Lash (246,247). They observed that precursor cells of the heart in the chick embryo migrate anteriorly over endoderm to form paired endocardial tubes. Fibronectin was found associated with endoderm in a gradient which increased in the anterior direction correlating with the direction of heart-cell migration; antibody blockage of the R G D sequence and fibronectin stopped directed heart cell migration. Another example has been provided by Shi et al. (248) in urodele embryo. They noted that gastrulation proceeds by involuting mesoderm cells moving individually along the roof of the blastocoel, which is decorated with fibronectin and laminin fibrils. In addition, Hammarback et al. (249) demonstrated that neurites preferentially extend along the adhesive gradients provided by the biopassages of laminin. These observations may indicate that a similar mechanism could be operative in a wound. For example, cells migrating into the wound initially may leave various footprints that dictate the directional migration or the type of the cells that should subsequently migrate into the wound. 40 As described by Brunette (162), the insertion of an implant may produce an analogous situation to an adhesive gradient. It would be expected that cells would preferentially migrate and attach to an implant surface i f the surface were more adhesive for cells than other cells or extracellular matrix. Conversely, a material with low adhesivity would be expected to encourage cells to attach and migrate towards other cells or extracellular matrix. This situation may be responsible for the formation of capsules observed with poorly adhesive materials in vivo. To take practical advantage of the principle of haptotaxis, it seems logical to coat the surface of biomaterials with adhesive biomolecules. This approach has been reported in the periodontics literature where fibronectin or laminin were coated on the root surface to promote fast connective-tissue reattachment and bone refill. Although several reports favored such an approach on partially demineralized roots (250-252), other publications did not find significant improvement in fibroblasts' attachment or bone-refill when a coating technique was used (253,254). Wasefi et al. (177) have shown that autologous fibroblasts cultured on the implant surface might have produced factors or conditions that promoted fast connective tissue ingrowth in vivo. In contrast, von Recum et al. (2-4) camouflaged the implant surface with the main component of connective tissue, collagen, and reported a delay in connective tissue ingrowth. The reasons for these differences may be lack of a standard and efficient technique for stable surface coating, immediate deterioration by the proteolytic enzymes that exist in the wound, and finally lack of knowledge of the detailed conformation of these molecules at the implant/tissue interface. 2. Chemotaxis Chemotaxis is defined as the directed migration of cells in response to a concentration gradient of a soluble chemoattractant. Chemotaxis has been mainly studied in neutrophils and monocytes; however, it is now clear that other cells such as fibroblasts respond to chemotactic signals as well (255). The mechanism of chemotaxis is poorly understood, but it appears that specific 41 membrane receptors may be involved that can recognize a gradient of chemoattractants. The signal is perhaps transmitted to the cell by a change in the membrane potential (256) or ion influx (257). In a wound, bacteria and members of the clotting cascades may send chemotactic signals to neutrophils and monocytes. Many of these signal, not only enhance directed cell movements, but may specifically affect other functions such as oxidative metabolism and exocytoses of lysosomal enzymes (258,259). Thus chemotaxis achieves two goals; it facilitates the invasion of inflammatory cells to the wound and enhances their bactericidal properties. If the surface structure of an implant is unstable it is possible that materials released into the tissues could act as chemoattractants. The release of calcium and phosphate from implant materials such as bioactive glass and hydroxy appatite may act as chemotactic factors for cells that have the potential to differentiate into osteoblasts. 3. Contact guidance Although first observed by Harisson (260), contact guidance was named and substantially studied by Weiss (261,262), who defined contact guidance as the tendency of cells to be guided by the shape of the substratum to which they are attached (figure 1.6). He observed that cells preferentially orient and migrate along parallel lines engraved in glass or the oriented bundles of collagen naturally present in fish scales. Later studies indicated that not only grooved surfaces oriented and directed cell migration but substrata made from uniform glass cylinders also demonstrated the same ability (263-267). Contact guidance has been observed in diverse cell populations including fibroblasts, neutrophils and epithelium (263-266). Recent studies using more controlled microfabrication techniques have shown that fibroblasts respond hieratically to grooves of different dimensions, with large grooves dominating the effects of smaller grooves (263). Epithelial cells on grooves 3-60 um-deep, with 30-220 um-spacing, appear to be affected by the repeat spacing of the grooves more than by their depth, and more pronounced alignment has been noticed on densely spaced grooved surfaces than on surfaces with widely spaced 42 grooves (264). Recently Clark et al. (264) reported that on shallow grooves [0.2-1.9 urn] , repeat spacings between [4-24 urn] have little effect on fibroblasts' and epithelial orientation, and groove depth determines the extent of cell alignment. A number of mechanisms has been hypothesized to explain contact guidance. a. Microexudate hypothesis This theory, proposed by Weiss (262), suggests that colloidal exudates secreted from the cells first become oriented along the long axis of the grooves. Subsequently, the leading edge of a cell would track on these molecules and thus orient the locomotion of the cell. Weiss (270,271) also proposed that there may be orientation of micellae of the colloidal ground substance in vivo that could provide ultrastructural guides for cell locomotion within the organism. Trinkaus (272) believes strongly that this theory is the best hypothesis yet available for giving orientation to the movements of most cells in vivo. In Trinkaus' view, Weiss's hypothesis could be the readiest explanation in the directed migration of pigment cells along blood vessels, the movement of nerve axons along blood vessels, and the movement of neural crest cells along the side of the neural tube. Figure 1.6- Schematic drawing of contact guidance on grooved surfaces (Weiss). 43 This theory, however, has not been endorsed by in vitro experimental tests. Curtis and Varde (273) showed orientation on uniform glass cylinders of cells that would not expect to produce any microexudate orientation. Another flaw is that cells orient with the grooves in serum-free medium faster than the time normally required for protein synthesis and secretion (262). b. Microfilament bundle hypothesis This theory, originally suggested by Curtis and Vade (273), was supported by Dunn and Heath (274) who observed the behaviour of fibroblasts on simple patterned substrata with sharp changes in inclination. Their theory was based on the relative rigidity as well as contractile ability of stress fibers inserting into the focal contacts. Dunn and Heath proposed that stress fibers of fibroblasts sense the geometry of a substratum, and are unable to bend over sharp changes of inclination in a substratum. Thus fibroblasts could not locomote efficiently in a direction that involves crossing such a sharp inclination. A criticism of this theory arises from observations that indicate not all cells that move develop focal contacts and stress fibers, and yet such cells exhibit contact guidance (275,276). Another problem is the observation of Brunette (264) that showed cells can be oriented on grooves of small dimension, which are less likely to interfere with stress fibers. c. Focal contact hypothesis Ohara and Buck (277) suggested that focal contacts are linear and rigid. There is a tendency for focal contacts to form and align in the direction of the grooves and ridges, simply because the substrate available for focal contact formation would be unrestricted. This would result in alignment and polarization of the cytoskeleton, and ultimately the entire cell itself. This theory, however, does not explain the contact guidance of some cell types, such as leukocytes or fungal hyphae, which are contact guided and yet do not seem to have focal contacts (157). This theory was later modified by Curtis (157), who described contact guidance as the ability of cells to react to discontinuities by forming attachment sites to them. In contrast to Ohara and Buck, Curtis 44 made no presumption about the structure of these adhesion sites; however, he showed that the attachment sites frequently associated with actin condensation may be disturbed as the size of the discontinuities becomes comparable to or greater than a cell's dimensions. This interference with the adhesion sites would be enough to exert an effect on cell orientation. d. Selective adhesion It has also been suggested that contact guidance occurs because cells may show preferential adhesion to a specific area or to a particular shape on the substratum, a similar theory to that of haptotaxis. For example, with grooved substrata cells may adhere selectively to the walls of the groove, the ridges or the edges where the ridges and groove walls meet. Selective adhesion as a mean of orienting cells has been shown in several experiments in which channels were cut or scratched in a nonadhesive surface coating to expose the more adhesive underlaying surface. These experiments used glass substrata coated with chrome (278), gold (279), or brain phospholipids (280). Polystyrene substrata were also used, in which case substrata were coated with fine lines of silicon monoxide (281) or selectively sulphonated (245). These experiments indicate that adhesive heterogeniety of the substratum can alone induce orientation in spreading cells and subsequently influence their directions of translocation. Nevertheless, this theory appears unsatisfactory in the case of contact guidance of cells cultured on uniform glass cylinders, where differential adhesion could not play a role (268). e. Stochastic model This model, which has been suggested recently (162), proposes that cells react probablistically to the topographical features of a surface. The probability of a cell making a successful protrusion and adhesion in a given direction may be reduced by specific features of a surface topography, and similarly protrusions and adhesion made in other directions might be favored. Thus cell shape and direction of locomotion would be determined probablistically by the surface. A slight deviation in any steps involved in cell locomotion has the chance of being sufficiently 45 magnified to induce cell orientation. The probablistic nature of cell response to a topographical feature was also addressed by Clark et al. (269), who suggested that specific topographical features could reduce the probability of a cell making a successful protrusion and/or contact in a given direction. This reduction could be due to a number of factors; however, Clark et al. favored Dunn and Heath's (274) proposal on relative inflexibility of microfilament bundles as the most economical explanation. Thus, it appears that no single hypothesis can sufficiently explain contact guidance, and it may be plausible that more than one mechanism is operative. 4. The role of contact guidance in vivo There is some evidence indicating that contact guidance may be operative in vivo. One example is the directed migration of neural crest cells along the notochord in the embryonic stage of development (157). Lofberg (282) showed that neural crest cells are aligned by the matrix in the region of the notochord. Bard and Higginson (283) also have shown that fibroblasts in chick cornea become aligned by the orthogonal array of collagen fibrils during the development of the cornea. There are also cases where the cell movement is guided and oriented by the shape of a cellular substratum. Haan (284) has reported that the oriented endodermal cells' arc provides a guidance signal for the migration of precardiac mesodermal cells in the early chick embryo. Another important example of contact guidance in vivo may be in wound healing, in that fibroblasts or other cells orient and migrate along parallel tracks on the fibrin clot and extracellular matrix produced by the retraction of platelets and contraction of myofibroblasts (285). This mechanism may in fact account for the highly oriented appearance of the infiltrating fibroblasts as these cells enter the granulation tissue (286,287). It has also been proposed that the oriented fibroblasts and myofibroblasts in the granulation tissue facilitate wound contraction by inducing tension along the lines on which they were migrating. Indeed, Repesh et al. (288) have shown that as a wound heals, lines of stress and patterns of orientation form along axes parallel to the wound surface. These lines of stress may also direct the movement of the epithelial cells at the edge of the wound. However, in all these in vivo instances, there is the possibility of other 46 effectors of cell behaviour such as chemical gradients being present as well, so these observations may not provide definitive evidence for contact guidance. Contact guidance may happen on the surface of implants because many commercially available dental, orthopedic and percutaneous implants possess grooves and geometries that might be expected to influence the behaviour and organization of cells attached to the implant. Consistent with this notion is the observation that fibroblasts exhibit contact guidance when cultured on these implants in vitro (162). 5. Cell/cell contact inhibition of movement This phenomenon was first described by Abercrombie and Heaysman (289,290) in a series of experiments that are now regarded as classic. In these experiments fibroblasts populations were arranged so that the cells collided. After collision, cell movements halted, cells became stationary and remained more or less as a monolayer on the substratum. Because the inhibition of movement resulted from cell contact, this phenomenon was termed "contact inhibition". The initial response of a fibroblast colliding with another fibroblast is an immediate halt in the activity of the ruffled membrane of the leading edge of the cell, and further movement in that direction ceases. In a few minutes, ruffling starts to gain dominance at other edges of the cells, away from the point of contact, so that the cells move apart from each other (291,292). Contact inhibition of movement is often confused with a totally different phenomenon named contact inhibition of mitosis, which has subsequently been more aptly named by Stoker and Rubin (293) as density dependent mitosis. This phenomenon refers to the reduction of the frequency of cell division that occurs in dense cultures of many cell types. In contrast, contact inhibition of movement deals with behavioural changes in the motility of colliding cells. Contact inhibition of movement is not specific for fibroblasts but occurs in epithelial cells as well. Collision of epithelial cells leads to the formation of an adhesion and local paralysis of the locomotory machinery, but there is no retraction phase (294). Moreover, contact inhibition of movements is not restricted to collisions of homogeneous cells, it also occurs in heterogenous 47 cell collisions, although slightly modified. For example, when epithelial cells collide with fibroblasts, only the fibroblasts are affected, leading to an inhibition and retraction of the fibroblasts' lamellipodia (295). The non responsiveness of epithelial cells to colliding fibroblasts, however, may be because of the angle at which the collision occurred. Experimental data indicate that fibroblasts may present a different degree of contact inhibition of movement if collided with head-on or at a different angle (296). Elsdale (297,298) reported that if two arrays of fetal lung fibroblasts collide at an angle greater than 20°, they cease forward movement, whereas if the angle of collision is less than 20°, they line up and merge into one. There have been several hypotheses put forward to explain contact inhibition of movement. One explanation put forward by Abercrombie (299) was that the inhibition of locomotive activity results from the action of molecules diffusing through gap junctions formed between colliding cells. However, the signal hypothesis has been ruled out by Heaysman & Turin (300), who observed that fibroblasts exhibited contact inhibition when they collided with dead fibroblasts which had been fixed by membrane stabilizing agents and presumably could not transmit signals. A second proposal emphasized the adhesivity of the membrane (301). In this view, cells are less likely to establish an efficient attachment to the surface of other cells than to the substratum. This mechanism, which is based on differential adhesion, is similar to haptotaxis. In support of this hypothesis, fibroblasts cultured on nonadhesive substrata have been shown to clump together instead of following the principle of contact inhibition of movement according to which cells would disperse on the substratum (292,302). Another similar hypothesis (292), interprets contact inhibition of movement as a consequence of mechanical failure of the attachment site between two cells. In an attachment site two focal contacts often form with microfilament bundles inserting from both cells. Two bundles of microfilaments may exert more stress on the adhesion plaque than one. This possibly results in failure of the attachment and the diversion of cells towards the substratum. Whatever the mechanism might be, contact inhibition of cell movement plays a significant role in many biological systems in vivo. For example, when the population density of cells is not 48 uniform, contact inhibition causes cells to move away from a densely populated area towards a sparsely populated area. A n example may be wound repair, in that cells will migrate into the denuded area to repopulate an injured site. Similarly, in vitro cells grow centrifugally from the free edges of an explant (302). Cell movement during embryonic morphogenesis, leukocyte migration and malignant cell invasion (292,302) are other examples where contact inhibition of movement may be involved. Contact inhibition can be operative in two ways with respect to tissue reorganization on an implant. Initially it mobilizes cells at the edge of the wound to reach the implant surface; then it may regulate cell migration on the surface of the implant. One important example is the heterogeneous contact inhibition that occurs in collisions between epithelial cells and underlying fibroblasts on the surface of percutaneous or dental implants. As discussed earlier, the downgrowth of the epithelial cells on epithelium-penetrating implants contributes to their failure. Migration of epithelial cell may be inhibited by the fibroblasts colliding with them on the surface of the implant. 6. Two-centre effect Another phenomenon first investigated systematically by Weiss (18) is the two-centre effect. In his original experiments, Weiss observed that when two explants of chick embryonic tissue were placed a small distance apart on a thin layer of clotted plasma, the outgrowth of cells from each explant was oriented toward the other. His explanation was that the cells within the explant produced stress on the fibrin fibers, producing oriented lines between the two explants. Fibroblasts migrated from one explant, aligned parallel to the stress lines and migrated preferentially towards the other explant. Such a phenomenon may take place within a wound. In this case, adjacent edges of the wound act as centres, a fibrin clot being already present on which fibroblasts exert stress and then migrate along the lines of stress. A similar mechanism might have been operative when porous surfaced titanium disks were placed on a lawn of fibroblasts (303). Fibroblasts aligned 49 perpendicular to the implant surface while attached to the floor of the culture dish (first centre) and to the implant surface (second centre). It is possible that similar mechanism may align fibroblasts attached to implants in vivo. Cellular bridge Figure 1.7- Schematic drawing of the two-centre effect. E. Summary Every wound triggers three phases of tissue response which leads to healing. The inflammatory phase mainly involves blood-bom cells which control bleeding and decontaminate the wound; in addition, cytokines produced by platelets, macrophages and lymphocytes attract parenchymal cells. The proliferative stage concerns activities of the matrix-producing cells that are probably controlled by a variety of growth and regulatory peptides. The remodelling phase overlaps with the proliferative phase and is characterized by an increase in cross-banded collagen type I and a reduction in fibronectin, hyaluronate and type III collagen. Reorganization of the cells and extracellular matrix with an implant surface may depend on a variety of mechanisms acting in harmony to establish a biological interface with the implant surface. 50 V. Complications and failure modes of percutaneous implants Teeth, deer antlers and shark scales are the only naturally occurring structures which permanently penetrate through stratified squamous epithelium such as gingiva or skin (2,9). A variety of artificial devices also must penetrate through stratified epithelium, including dental implants, maxillofacial reconstructive implants, auditory prostheses, catheters for perfusion or dialysis, power connectors for artificial organs, electrical connectors for signals, probes and detectors as well as some apparatus used in drug delivery systems. However, the junction between such percutaneous devices and stratified epithelium has proved problematical, and consistent long-term viability of the PD/soft tissue interface has not been achieved (2-4,9,11,12,304). Skin-penetrating devices anchored in the bone have proved to have greater longevity compared to those anchored in soft tissues (305,306). Dental implants integrated in the alveolar bone (osseointegrated) also appeared to have greater long term success compared to implants integrated in soft tissue (133). Nevertheless, unfortunately not all implants can be osseointegrated. For example, the anatomical location of the skin-penetrating device may not allow it to be anchored in the bone. Another problem to the osseointegration in that any technique that involves implanting a device in bone requires considerable removal of healthy bone, which induces excessive trauma, and thus is not considered a conservative procedure. It should also be noted that the success of osseointegrated systems is not necessarily the result of their design, but also results from the rigorous criteria employed for patient selection, a technically demanding surgical technique, and a redundant-design philosophy in which 4 or 5 implants are placed so that the loss of one or two can be tolerated. A caveat with bone integrated dental implants is that their long-term application has so far been documented convincingly in fully edentulous patients. The complex distribution of the occlusal forces in the oral cavity may favor a soft tissue interface similar to that of teeth for those implants opposing to, or used as abutments with the natural dentition (129,130,307-310). Ankylosed dental implants could impose abnormal forces on the soft tissue interface of opposing teeth, or if used as a bridge abutment with natural teeth could result in pathological conditions leading to the 51 loss of the periodontal supports of the involved teeth (310). This thesis, however, specifically deals with skin-penetrating devices which are anchored in soft tissue, and only the phenomena that may lead to the failure of these devices will be discussed in detail. Typical skin-penetrating devices generally fail to establish a stable interface with the integument and the device soon needs to be removed or replaced. After 10 years of experimental, surgical and histological studies, von Recum et al. (3,4) stated that "all classical percutaneous devices have a common end point: they are extruded within days or weeks". This report also pointed out that only a few investigators who exercised extreme surgical, medical and sanitary precautions had been successful in prolonging the life of these devices up to a year. The failure modes that lead to extrusion of a device include marsupialization, permigration, infection, avulsion, and a combination of these. A . Marsupialization Marsupialization was described earlier in the section (III-B.) on cell migration (pp. 30-31); however, in brief, it relates to the tendency of the epithelial cells to migrate and proliferate horizontally to cover the denuded area. This migration and proliferation stop only when cells meet each other and achieve mutual contact. Skin-penetrating implants hamper the epithelial cells' efforts to cover the denuded area. As a result of this, epidermis surrounds a smooth surfaced implant and continues to grow towards the only alternate direction, downward into the area of least resistance that is offered by the underlying inflamed connective tissue at the implant interface (2). Consequently, epithelium creates a sinus tract also called a trough or a pocket around the implant. This process may continue, until the entire implant is walled off and failure occurs. Marsupialization has been reported to occur in some designs of dental implants (13-17,311). For example Mack (312) in his master's thesis introduced the term "exteriorization", which is similar to the definition of marsupialization, and recognized the process as a possible failure mode of subperiosteal dental implants. 52 One possible solution to this problem has been based mainly on an analogy to naturally occurring percutaneous devices such as teeth and antlers, von Recum and Park (2) suggested that perhaps tight attachment between a healthy periodontal membrane and the tooth and similar connections between the skin and the bony antlers do not allow the gingival or epidermal basal cells to penetrate deeper along the surface of the percutaneous organ or to form a sinus tract. This notion is supported by the observations of several authors (313-315,166) who have shown that persistent inflammation in the periodontal connective tissue results in a loss of tissue integrity and epithelial downgrowth leading to deep pocket formation around the tooth. Winter (181) observed that fibrous connective tissue grew into pores of polyetrafluorethylene or hydron sponge coincidental with a stable ring of epidermal attachment. In contrast, epidermis grew downward into the dermis on smooth surfaced implants that did not show connective tissue ingrowth. In addition, the work of Kantrowitz et al. (178) with nanoporous implants, as well as that of Squier and Collins (182) with Millipore filters have provided evidence for the pivotal role of connective tissue attachment in inhibition of epithelial downgrowth on percutaneous implants. A potential problem with this strategy is posed by the histochemical observations of von Recum (3), Feldman and von Recum (316,317), and Schreuders et al. (318), who have shown that abnormal or immature collagen and other connective tissue components, as seen in a wound of less than 36 hours, are permanently present within the pores of the implant. This immature connective tissue may not stop epithelial proliferation and migration efficiently. However, it is possible that the degree of connective tissue maturity within the pores depends on the choice of the implant materials, on the shape and location of the implant, on the size and geometry of the pores, and possibly on the differences between species. Another approach to the problem of marsupialization is that in which a porous or textured surface is used to achieve tight attachment of the epithelium and tissue interlock (2,3). However, this approach may cause PD failure by permigration. 53 B. Perrnigration von Recum et al. (2,3,4) described perrnigration as the tendency of epithelial cells to migrate throughout the entire porosity of a porous implant surface to encapsulate and surround it. Perrnigration is a result of normal maturation of epidermal basal cells within the pores or surface discontinuities of textured surfaces, von Recum (3) hypothesized that maturing and proliferating epithelial cells within the pores of Dacron velour implants are pushed in the direction of the least resistance, which is towards the underlying immature connective tissue to displace it. Hall and Ghidoni (223), however, hypothesized that epithelial cells attach strongly to porous surfaces such as Dacron velour, and in normal function epithelial cells mature, migrate, die and finally desquamate as the cells move towards the surface. When attached to a porous surface, the cells pull the implant upwards, resulting in the implant's eventual extrusion. Hall and Gidoni point out the following dilemma. If good skin interfacing is accomplished, PDs wil l eventually become extruded. In contrast, if good skin interfacing is not accomplished as with smooth surfaced implants, a sinus tract will form that also can lead to failure. Thus either good or poor attachment leads to failure. However, the accuracy of Hall and Gidoni's theory needs to be proven, since no studies have shown systematically the pattern of epithelial cell migration during the maturation process at the implant interface. C. Infection A third mode of failure with percutaneous device is infection, which is believed to be more common in implants with interconnected pores (2,3,4) because the pores possibly provide an easy pathway for microbial invasion. Feldman and von Recum (316,317) suggested that PDs with interconnected pores never completely fill with tissue and, instead, fill with intercellular fluids. Thus such PDs may serve as a wick and allow bacteria free access to subepidermal tissue through the interconnecting porosity. Another view was suggested recently by Nowicki at al. (319) who experimented with porous vitreous carbon PDs and reported that i f the tissue integrated into the porous surface, infection was never noticed even following inoculation of 54 known pathogens such as Staphylococcus aureus or Escherichia coli. There could be many factors affecting the probability of infection, including the choice of the materials, the geometry and size of the pores, the diameter of the conduit traversing the skin, the time during which the device must remain implanted, the presence of clotting around the implant site, the surgical technique and the post surgical maintenance (320). Gristina et al. (321,322) have compared the attachment phenomena at the biomaterial interface to a race between tissue cells and bacteria. Bacteria, which can use similar adhesion molecules to those of host tissue cells to bind to the surface, may win the race for the surface and thus cause infection, instead of tissue integration. Once bacterial adhesion has occurred, it is unlikely that tissue cells will be able to displace these primary colonizers to occupy and integrate the surface. As a result biomaterials-centered infection occurs, which is notoriously resistant to antibiotics and host defenses and usually tends to persist until the biomaterial or foreign body is removed (321,322). It should be noted that each time the attachment of the tissue/biomaterial is disturbed, the cycle of the race for reattachment between bacteria and host cells is also initiated. D. Avulsion Percutaneous devices are subjected to a variety of forces that can lead to mechanical disruption of the interface, and device failure (2,304). Hall et al. (304) described these forces as lateral, axial and torsional, and pointed out that they often act simultaneously. Lateral loading consists of a force parallel to the skin and perpendicular to the long axis of the PD. Axial loading consists of loads perpendicular to the plane of the skin and parallel to the long axis of a PD. Torsion occurs if the PD is rotated about its long axis. Such forces can result in tearing or shearing at the skin interface. For the most part, failure by avulsion may be the result of gross difference between the modulus of elasticity of the skin and that of most implant materials. The few elastomers, foams or porous materials that do approach such an elastic match, however, may fail because of other nonmechanical reasons (304). 55 E . Multiple cause of failure Because, in vivo, PDs face a complicated environment, it is likely that they often fail for multiple reasons. For example, mechanical forces may create an unstable interface encouraging epithelial downgrowth, which results in sinus tract formation. Bacteria and their toxins may accumulate in the sinus tract and lead to a biomaterial-centered infection that wil l further complicate this scenario and result in implant failure. F. Summary Percutaneous implants, in particular soft tissue, skin-penetrating implants, eventually fail because of marsupialization, permigration, infection, avulsion or a combination of these processes. Although several solutions to these problems have been suggested, there is insufficient information on what constitutes the optimal surface of a percutaneous implant to ensure a successful and long-term functioning device. ^ 6 CHAFFEE 2 S T A T E M E N T O F T H E P R O B L E M S 56 (\ STATEMENT OF PROBLEMS Percutaneous devices and dental implants are widely used for a variety of applications, however, there is still insufficient scientific understanding of the specific events happening at the interface between the implanted material and its host tissue. Dental implants and percutaneous devices encounter a complex situation in which they interact with soft and sometimes hard connective tissue, as well as the epithelium. One reason for the failure of some dental implants and percutaneous devices is epithelial downgrowth (2-4,9,11-17). Downward migration of the epithelium on such devices results in the formation of sinus tracts or deep pockets, encouraging bacterial or plaque accumulation, which in turn can lead to inflammation and infection, with the eventual loss of the implant. One method to control epithelial downgrowth on such devices is to alter the surface topography of a device so that cell migration could be guided or redirected by a phenomenon called contact guidance. Contact guidance is defined as the ability of the surface topography to orient and direct cell migration. Although contact guidance has been mainly studied in vitro, there is some evidence indicating contact guidance could operate in vivo. However, in all these in vivo instances, there is the possibility of other effectors of cell behaviour such as chemical gradients being present as well, and this important aspect of cell behaviour has never been tested directly in vivo. Thus, one objective of the experiments described in this thesis was to test whether contact guidance occurs on grooved surfaces produced by micromachining in vivo, and to incorporate the findings into the design of percutaneous implants in order to inhibit downward migration of the epithelial cells. In vitro, parameters of grooved surfaces such as depth , spacing and orientation are known to effect contact guided cell migration and orientation (263,264,269). Because nothing is known about the role of such parameters in vivo, a second objective was to investigate the effects of groove parameters on epithelial and connective tissue cells. Detailed morphological observations of epithelial and connective tissue interfaces with the implant surface require electron microscopy. However, a difficult task in electron microscopy is to obtain thin or ultrathin sections of the 57 interface of typical implant materials with tissues. The method of Gould et al. (60,125) was modified to allow detailed electron microscopic observations of the interface of tissue attachment to titanium with minimal artifacts. The role of connective-tissue attachment to the percutaneous implant also is not well understood. It has long been assumed in the periodontics literature that an intact and uninflamed connective tissue inhibits the downgrowth of the junctional epithelium (14-17). By analogy, it has been postulated that long term normal function for dental implants could be obtained through a similar functional peri-implant connective tissue attachment (129,130). Although the Millipore filter model of Squier and Collins (182) indicated that the connective tissue attachment inhibited epithelial downgrowth, detailed investigation of the role of connective tissue has not been investigated on solid implant surfaces which represent a more realistic model of percutaneous device. Thus, experiments were designed to study the role of the connective tissue attachment to the implant in inhibiting epithelial downgrowth. A possible weakness in surgical procedures currently used to insert percutaneous devices is that they are frequentiy placed in one stage so that the connective-tissue and epithelial attachments are established concurrently. A procedure that vastly improves the success rate of endosseous dental implants is the method, introduced by Branemark and co-workers (133), in which implants are placed in two stages. In the first stage, the root part of the implant is buried in the jaw bone for a period of 2-3 months, during which time healing occurs at the bone/implant interface. When healing is complete and the implant is immobilized, a second surgery establishes the pergingival portion of the implant. Although Grosse-Siestrup and Affeld (9) have hypothesized that two-stage surgery may increase the success rate of PDs, this notion has never been adequately tested. An experiment described in this thesis compared the effects of one and two stage surgical technique on the extent of tissue attachment and implant failure. C M A J P T E E 3 M A T E R I A L S A N D M E T H O D S (Cxeneral) PAGE I. Micromachining 58 II. Fabrication of the epoxy substrata 61 III. Fabrication of implants for in vivo experiments 63 IV. Preparation and Characterization of Surfaces 64 V . C e l l Culture 65 VI. Cell attachment 66 VII. Cell Orientation 66 VIII. Implantation Procedure 68 I X . Specimen Collection and Preparation 69 X . Histology and Histomorphometric measurements 70 58 MATERIALS AND METHODS (General) I. Micromachining Micromachining is a technique that produces grooves, slots or pits with precise dimensions in silicon or gallium arsenide wafers. This technology has been utilized in a number of applications including: solar cells, high value capacitors, solid state inductors, charging plates of computer printers, miniature gas chromatography systems, integrated circuit isolation, infrared polarizers, and vertical channel thrystors. The particular micromachining technique employed in these studies adopted from that developed in the Department of Electronical Engineering of the University of British Columbia by Camporese et al. (323) for the fabrication of high quality photomasks for solar cells. Micromachining allows excellent control over the production of grooved or pitted surfaces because different aspects of the grooves and pits are controlled by different steps involved in the micromachining process. The n-type (100) silicon wafers* were used in these studies. These wafers were 5 centimetres in diameter, 200-350 [im in thickness, which had polished front surfaces and bright-etched rear surfaces. The micromachining process involves several stages: A. Cleaning A vigorous cleaning criterion was used to obtain highly clean silicon wafers. The method involved the following steps: [1] ten minutes in a solution of (300 ml H2O, 60 ml H2O2, 60 ml NH4OH) at 75°-85° C, [2] thirty seconds in 10% HF, and [3] ten minutes in a solution of (300 ml H2O, 60 ml H C L , 60 ml H2O2). Wafers were rinsed for at least ten minutes in distilled water, after each step of cleaning. Finally, wafers were immersed in isopropyl alcohol for four minutes and blow dried in filtered nitrogen. 'Virginia Semiconductor Inc., Fredericksburg, Va, USA. 59 B. Oxidation The objective of this stage was to grow a layer of silicon dioxide on the front and rear surfaces of the silicon wafer. This was achieved by using wet oxygen at 1150° C for two hours in a furnace*. This procedure produced oxides of 0.6 urn thick on each side of the silicon wafer. C . Photolithography This stage begins with production of a computer generated master pattern to produce photomasks. In these studies, two master patterns were utilized. The first, which produced grooved surfaces, contained arrays of parallel stripes cut in Ruby-Lith™t and reduced by step and repeat photography. The second, which produced the tapered pitted surfaces, contained arrays of equally spaced clear squares. Currently, photomasks are produced by computerized optical pattern generator and the pattern are then etched in chromium-gold. This technique produces photomasks with higher resolution and are less prone to fabrication errors than those produced using Ruby-Li th™. The next step of this phase involved coating the front surface (polished side) of the silicon wafer with the negative photoresist*. Wafers were then exposed through the photomask with a 320 nm wave length UV-light, developed and baked at 160°C. It should be noted that the alignment of the photomask on the silicon wafer plays a significant role in obtaining precise groove dimensions dictated by anisotropic etching, slight misalignment of the photomask may cause a less predictable etching. T w o inch tube furnaces (No. 7), Fairchild Semiconductor Corporation, USA. t Ulano Corporation, 255 Butler Street, Brooklyn, N Y , USA. ¥Microposit S-1400 Series (Shipley), Newton, Massachusetts, USA. 60 D. Oxide patterning Buffered H F was used to remove the unprotected oxide layers resulted from the U V light exposure. Then the remaining developed photoresist was removed in microstrip (NMP) solvent*. This procedure produced a silicon wafer whose front surface was patterned with oxide layer. For example wafers used for grooved surfaces had strips of silicon oxide. E. Final etching The wafers were preferentially etched in 19% potassium hydroxide solution at 80°C. The potassium hydroxide etches away the silicon, leaving the oxide layer intact. The speed of etching varies according to the crystalline arrangement of the silicon wafers. The etch rate is typically 1.4 ujn/minute which will decrease 300 times, if silicon wafer "111" is used. The shape of the grooves or pits is dictated by the crystal orientation of the silicon wafer, for example silicon wafer 110 produces vertical walled grooved surfaces and silicon wafer 100 produces v-shaped grooves. The depth of grooves or pits can be controlled by the time of etching; and the desired repeat spacing, the "pitch" (comprising one groove and one ridge between the grooves) can be incorporated in the design of the master pattern. Figure 3.1 shows an example of a micromachined surface with V-shaped grooves. Under ideal conditions, anisotropic etching produces grooves or pits whose walls form a 55° angle with the surface. In these experiments, possibly because of a slight misalignment of the photomask on the silicon wafer and/or long etching time adopted to obtain 19 and 30 urn-deep grooves with 39 urn-pitch, slight deviation of the angle of etching (58° ± 3° SD) was noted when cross sections of grooved silicon wafers were observed under the scanning electron microscope (SEM). The dimensions of the grooves and tapered pits used in these studies are shown in their cross sections in figure 3.2. The dimensions of all surfaces measured in the S E M were within ±1 jim (SD), except for the grooved surfaces referred to as 19 and 30 urn-deep. The former surface had grooves which were 19 ± 3 | im (SD) deep, 20 ± 3 (SD) urn wide at the top and were 'Microstrip 2001, Olin Hunt Specialty Inc., West Paterson, NJ, USA. 61 separated by 19 ± 2 u.m flat ridges. The latter surface had grooves which were 30 + 8 (SD) u.m deep, 35 + 7 (SD) | im wide at the top and separated by 4 ± 2 u.m flat ridges. Figure 3.1- Example of a micromachined surface with V-shaped grooves. II. Fabrication of the epoxv substrata Although micromachining uses silicon in the production of topographies with desired properties, the topography can be transferred to other materials that are more suitable for biological experimentation. Vinyl silicone base impression materials* were used to take the impression of the micromachined silicon wafers and impressions were used to cast replicas of the "Exaflex (G-C) Dental Industrial Corporation, Tokyo, Japan. 62 © © © © © 4 | i m 3 | i m E a . E a . o VAA/VVVVVVVVVVVVVVVVVVVVVVVV" 19 n m 11 Lim E 22 LUII 17 LUII \_y—w 17 LLm 13 n m A - ^ ^ W — V / W V / 20 Lim 35 (im 19 n m 4 n m ® 270 | l m 115 n m Figure 3.2- Schematic diagram of cross sections of micromachined grooved and pitted surfaces with dimensions. 63 original micromachined surfaces in epotek*, a biocompatible epoxy resin. The epoxy replicas of micromachined surfaces baked at 60°C for 3 days, and then used for in vitro and in vivo experiments. III. Fabrication of imnlants for in vivo experiments The principles used to design the implants were relatively similar in all phases of experiment. In general, implants were U-shaped and in most cases had two protruding percutaneous components connected to each other by a flat pedestal (figure 3.3). On each protruding component there was an outer test surface, which faced laterally and an inner test surface, which faced medially. The fabrication of the implant initially started with replication of the original silicon substrata with the desired surface geometry in epoxy resin. The area with the chosen surface topography was then cut in slices of =1 cm2. The non-patterned sides (the back side) of the cut slices were thinned to the thickness of =0.5-1 mm by a soft-grain sand papert. Because it was desired to have test surfaces on both surfaces of the cut slices, two slices made in this way were then glued together under the dissecting microscope using a polyether impression material so that grooves or rows of tapered pits of one side were aligned parallel to that of the other side. The two-sided slices were then shaped roughly with a high-speed dental drill and mounted on the stage of an electric saw to create a longitudinal cut along the long axis of a groove or along a row of pits. This cut created a straight edge that would be joined to the implant pedestal. This junction was later used as a reference to measure the extend of the connective tissue attachment. The replicas of two-sided, trimmed, slices with designated size and test surfaces were placed parallel to each other on a glass cover slip from the trimmed edges. Slices were then glued to the *Epotek 302-3 (Epoxy Technology), Billerca, Mass, USA. tBuehler ltd., Evanston, 111., USA. *Permadyne- ESPE, Praparate G M B H . , D-8031 Seefeld, Oberbay, W. Germany. 64 glass using Exaflex*. The spacing of one slice from another varied depending on the experimental criteria. Finally, light body Exaflex was poured on the slices to make the first impression of an implant. The impression was poured with Epotek to cast the master pattern of an epoxy implant. The implant was then shaped to the desired size, and the roughnesses were removed with ultrafine sand paper. The trimmed implant was replicated to produce the number of implants required for a given phase of the experiment Figure 3.3- Schematic diagram of an implant model. Two protruding parts of this particular design are about 7 mm apart and have equal areas of grooved and smooth surfaces. IY. Preparation and Characterization of Surfaces Epoxy substrata and epoxy implants were cleaned using a detergentt (specifically formulated for tissue culture) under ultrasonication, rinsed ten times with sterile distilled water, and then incubated for two hours with serum free tissue culture media in which the antibiotics were raised "Exaflex (G-C) Dental industrial Corporation, Tokyo, Japan. t7X Cleaning Solution, Flow Laboratories, Mclean, V A 22102, USA. 65 to ten times the concentration used in normal growth media. After a further five rinses with sterile distilled water, the epoxy substrata or implants were stored in individual sterile petri dishes. Epoxy substrata and implants were coated with 50 nm titanium using a sputter coater*. The titanium-coated surfaces were cleaned by ultrasonication for twenty minutes in a detergent*. After being rinsed twenty times with deionized sterile distilled water, they were dried overnight in a tissue culture laminar flow hoodt and sterilized by gamma radiation* or by an argon-gas glow-discharge chamberl At least one randomly chosen sample from each batch of titanium-coated implants was inspected by scanning electron microscopy (SEM) for the fidelity of the replica to the original micromachined surfaces as well as the smoothness and continuity of the titanium coating. Titanium surfaces resulting from the coating procedure as well as surfaces treated by glow discharge were analyzed by X-ray Photoelectron Spectroscopy (XPS) at the National ESCA and Surface Analysis Center for Biomedical Problems, Seattle, Washington. V. Cell Culture Epithelial (E) cells derived from porcine epithelial rests of Malassez (a group of cells found in the periodontal ligament) were cultured as described by Brunette et al. (324). This technique results in the growth of both epithelial cells and fibroblasts. The two cell types could be separated by the tendency of fibroblasts to be less resistant to detachment by trypsin (325). In brief, the cells were cultured in alpha Minimal Essential Medium supplemented with 15% fetal *Randex 3140 Sputtering system, 815 San Antonio Rd, Palo Alto, Calif., USA. tCanadian Cabinet Co Ltd., Ottawa, Canada. vGamma Cell 220, Atomic Energy of Canada (=650000 rads). URandex 3140 Sputtering system, 815 San Antonio Rd, Palo Alto, Calif., USA. 66 bovine serum and antibiotics [penicillin G* 100 p-g/ml, gentamicin 50 |ig/ml, amphotericin Bt 3 mg/ml] at 37°C in a humidified atmosphere of 95% air 5% Co2. E cells were removed from the growth surface and suspended using a trypsin solution* [0.25% trypsin, 0.1% glucose, citrate-saline buffer, p H 7.8] and plated onto the substrata at a cell population density of 2.5 x 10* cells/cm2. vi. Cell attachment As in most assays of cell attachment, an attached cell was arbitrarily and operationally defined according to the shear force it must resist to avoid being dislodged (19). The cells were allowed to attach on the smooth or grooved titanium-coated, or uncoated, epoxy substrata for various periods of time as noted, and the dishes placed on a linear shakerU so that the grooved substrata were aligned with or against the direction of motion of the linear shaker. The linear shaker was set at 150 or 36 strokes per minute and an amplitude of 1 cm for 20 seconds. The shaker produces shear forces that encourage cell detachment from the surface. The cells remaining on the substrata were stained with Richardson's stain, and counted in twenty microscopic fields of 6.25 X 10-2 mm2 at 200 times magnification. The number of cells attached at specific time periods to grooved or smooth surfaces were compared statistically using student t-test. VII. Cell Orientation Even when seeded at low population densities, E cells normally form clusters or groups because of the tendency of the cells to form strong lateral adhesions. Unlike fibroblasts, where *Sigma, St. Louis, M O , U S A tGibco, Grand Island, N Y , U S A ¥Worthington TPCK ^Precision Scientific Co., Chicago, USA. 67 the orientation of individual cells can be examined, the orientation of clusters of E cells on epoxy or titanium-coated substrata was measured using an orientation index (01) similar to that employed by Dunn and Heath (274). The 01 for a cluster of the epithelial cells is defined as the value of the longest segment of the cluster in the direction of the grooves divided by the width of a cluster, at its widest point, at right angles to the grooves (figure 3.4). A value of 1.0 indicates no preferred orientation of a cluster; in other words, the width of the cluster is the same as its length. In contrast, an 01 value greater than 1 indicates that the cluster is preferentially oriented in the direction of the grooves. For statistical analysis, log 01 is the most suitable variate; the log 01 values of cells cultured on grooved epoxy and titanium-coated substrata and those on adjacent smooth epoxy or titanium-coated surfaces were compared statistically using student t-test. Figure 3.4- Photomicrograph of epithelial cells cultured on a titanium-coated micromachined grooved surface. The orientation index (01) is calculated as the length (L) in the direction of the grooves of the longest segment of the epithelial cells cluster divided by the width (W) at right angles to the grooves of the cluster at its widest point. 68 VIII. Implantation Procedure Implantation procedures varied slightly in different experiments and notable variations will be discussed individually in each experiment. In general, inbred male Sprague Dawley rats, weighing from 450-600 g were intubated and anesthetized using halothane*. The fur above the parietal area was shaved and treated with a depilatory t, and the underlying skin scrubbed with Betadine* for one minute followed by 70% ethanol for one minute. An access incision was made distally from one ear to the other. In front of the access incision, two parallel test incisions, just sufficient in size so that the two protruding components of the implants could be accommodated, were made. The epidermal and the dermal layers over the periosteum were reflected by blunt dissection from the access incision towards the test incisions. The implant was then placed through the access incision into the test incisions so that the test surfaces penetrated through the skin. Finally the access incision was sutured using 5-0 silkH and the animals received a prophylactic intramuscular injection of antibiotics §. A l l surgeries were done under full sterile conditions, and animals kept in 60 cm tall cages to reduce the chances of rubbing the implant against the ceiling of the cage. Some animals were fitted with Elizabethan collars (11) for 48 hours after surgery to reduce their ability to disturb the implant. 'Ayerst Laboratories, Montreal, Canada. tNeet, Whitehall Laboratories ltd.,Mississauga, Ontario, Canada. ¥Purdue Feredrich Inc., Toronto, Canada. fEthicon, Peterborough, Ontario, Canada. §Pen-Di-Strep, Roger/STP, London, Ontario, Canada. 69 IX. Specimen Collection and Preparation Animals were killed by an overdose of Sodium Pentobarbital*. Prior to complete collapse of the heart, perfusion was carried out through the left ventricle with a solution of 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, preceded by a 5 minute flush of warm heparinized normal saline into the blood circulation. The implant and the tissue around it were gently removed using a No. 15 Scalpel bladet and placed in Karnowsky's fixative for 24 hrs at 4° C followed by 2% buffered O S O 4 for 2 hrs. At this stage the two arms of the U-shaped implants were separated with a diamond disc, and the O S O 4 fixation was allowed to continue for a further 2 hrs. Because the standard dehydrating agent, alcohol, has been found to damage the groove pattern and the titanium coating, Aquembed ¥, a water miscible resin, was used. Specimens were dehydrated in graded Aquembed (50%-100%, lhr in each change), then infiltrated with graded Aquembed/Eponll (50%-100%, lhr in each change) on a rotator. The specimens stayed on the rotator overnight in the fresh Epon, after which, infiltration was continued with 3 changes of fresh Epon without accelerator (2 hr in each change), followed by 3 changes of Epon with accelerator (2 hr in each change). Specimens were then stored in the vacuum at 4° C overnight and finally embedded in Epon. *M.T.C. Pharmaceutical, Mississauga, Ontario, Canada. tSkidmore Instruments, Sheffield, England. ''Aquembed, Ladd res. Ind., Burlington, V T , USA. liJ.B.EM Inc., B.P./P.0.693 Pointe-Claire/ Dorval, Quebec, Canada. 70 X . Histology and Histomorphometric measurements Sections were taken every 100 urn throughout the length of the implant using a Sorvall MT2* microtome. Two-u.m-thick sections were cut in an orientation such that the horizontal grooves and tapered pits were cross sectioned and the vertical grooves were longitudinally sectioned. Sections were stained with Toluidine Blue and examined under the light microscope t. For transmission electron microscopy (TEM), fifty-to sixty-nanometer-thick sections of the implants were cut with a diamond knife from various regions of the epithelial and connective tissue attachment. The sections were then stained with alcoholic uranyl acetate and aqueous lead citrate, and viewed under an electron microscope*. For histomorphometric measurements, the distance occupied by the epithelial and connective tissue attachment, as well as the length of recession were measured on two-u.m-thick sections using a graphics tablet, computed, and appropriate software! The means of the epithelial and connective tissue attachments, recession and capsule thickness were calculated on each test surface. The unit for statistical analysis was the test surface. That is, the measurements made in the sections for an implant surface were averaged to give the mean that was then combined with the values of other implants having the same surface in the statistical calculations. Data were entered into the University of British Columbia main frame computer ( A M D A H L ) for statistical analysis using the SPSS** statistical package. 'Sorval MT-2, Porter-Blum Ultra-Microtome. tZeiss Photomicroscope, Oberkoken, W. Germany. vPhillips 300 HApple Computer, Cupertino, Ca., USA. §Rocky Mountain Software. "SPSS Inc., Chicago, 111, USA. CHAIPTEIE 4 RESULTS P A G E I. Epithelial cell behaviour on grooved or smooth percutaneous implants 72 II. Effects of groove characteristics on percutaneous implant performance 87 III. The electron microscopy of the soft tissue interface with titanium-coated surfaces 99 IV. The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous implants 112 V . Electron microscopy of the mineralized tissue found adjacent to micromachined surfaces in vivo 135 71Ps R E S U L T S This chapter is divided into several sections based on the individual experiments performed. It should be noted that specific modifications or additions to the general methodology are discussed for each experiment. In the first experiment (section I), epithelial (E) cells were shown to be contact guided by a 10 u.m-deep grooved epoxy or titanium-coated epoxy implant surface in vitro, and it was noted that the downward migration of the epithelium was inhibited by the grooved surfaces placed horizontally on the surface of a percutaneous implant. The second experiment (section II) investigated the effects of several parameters of grooved surfaces such as orientation, depth and spacing on epithelium and connective tissue. Contact guidance was directly demonstrated in vivo from the observations that the migration of E cells were affected by the groove orientation. Horizontally placed grooved surfaces inhibited E cells downgrowth whereas vertically oriented grooved surfaces accelerated downgrowth of the epithelium. In addition, different mechanisms of inhibition of epithelial downgrowth were noted in deep grooves (19 (im-deep) where connective tissue ingrowth indirectly inhibited E cells migration, and shallow grooves (< 10 u.m-deep) where contact guidance was the cause of the inhibition. As detailed observations of the tissue/implant interface require electron microscopy, the third experiment (section HI) investigated this aspect of the interface. Besides verifying what had been observed at the light microscopic level, it was further found that the epithelium of the skin attaches to titanium surfaces through hemidesmosomes and a basal lamina. In the area of the connective tissue, F attachment to the titanium surfaces was mediated through two zones, an amorphous and a fine fibrillar zone. In above experiments it was impossible to determine unequivocally the effects of micromachined surfaces on E cell and connective-tissue-cell behaviour separately, because connective tissue and epithelium interacted with the same surface topography. In the fourth experiment (section IV), therefore, an implant system was designed which permitted the connective tissue to contact surfaces of various topographies, whereas the epithelial cells contacted only a smooth flat surface. Thus, the effect of surface topography could be evaluated 72 directly on connective tissue attachment and its further role on the epithelial downgrowth. It became clear that 30-u.m and 19-um-deep grooved surfaces and surfaces with 120-u.m-deep tapered pits formed an interface with connective tissue that inhibited epithelial downgrowth. The second purpose of this study was to compare one and two-stage surgery for percutaneous implant placement It was noted that implants placed by means of a two-stage surgical method in which the connective-tissue/implant interface was allowed to heal prior to connection of the percutaneous component had more connective tissue attachment and less recession than the implants placed by a one-stage surgical method. Because foci of bone-like tissue were observed in some micromachined surfaces in the study of section IV, the final section of this chapter (section V) focuses on the detailed electron microscopic observations of the interface of this tissue with the titanium surfaces. I. Epithelial cell behaviour on grooved or smooth percutaneous implants These experiments were designed to investigate the effects of a 10 urn-deep, 39 urn- pitch grooved surface (figure 3.2c) on the behaviour of epithelial cells in vitro and in vivo. The groove size was selected based on previous experiments in this laboratory, which suggested that contact guidance occurs on grooves of that size on titanium surfaces in vitro. For these experiments, it was hypothesized that grooves of 10 Jim-deep can influence E cells migration in vivo, resulting in the inhibition of the epithelial downgrowth on a percutaneous implant. A biocompatible epoxy was initially used to test the validity of the hypothesis. Titanium is the material of choice in many implant systems, however, its presence complicates histological preparation. Subsequently techniques were developed to enable histological processing of titanium-coated epoxy implants. Then, the effects of 10 um-deep grooves on epithelial downgrowth was studied using titanium-coated epoxy implants. 73 A. Implants Figure 3.3, shows a schematic diagram of the implant used in these experiments. Each test surface of the implant consisted of equal smooth and grooved areas. The grooves were 10 |im-deep truncated V-shaped and placed horizontally on the implant so that they would be parallel to the surface of the skin when placed in situ. Some implants were later coated with 50 nm of titanium. Figure 4.1- Diagram illustrating the histomorphometric measurements used in this study; epithelial attachment, connective tissue attachment (measured from the end of the epithelial attachment to the bottom of the implant), recession and sulcus depth. B. Morphometries Two |im-thick sections were taken every 100 urn throughout the length and the sections were then stained with Toluidine blue and examined under the light microscope. Figure 4.1 illustrates 74 the histomorphometric measurements used in these experiments. The distance occupied by the epithelial attachment, connective tissue attachment and sulcus depth were measured on the test surfaces of epoxy implants. An additional measurement, recession (tissue downgrowth) was made on titanium-coated implants. Recession was measured from a reference line placed at the level of the tissue on the implant during the surgery. C. Observations 1. Substratum and implant surface preparation S E M observations of the test surfaces indicated that grooved surfaces were replicated with high fidelity to the master pattern on the silicon wafers. The titanium coating was smooth and continuous with no evidence of cracks. Figure 4.2 shows the titanium-coated epoxy grooved surfaces of implants prior to surgical placement. Figure 4.2- Uniform and continuous titanium coating of the epoxy grooved surfaces of an implant before surgery. 2. In vitro experiments-a. Cell attachment Generally, it can be concluded from figure 4.3 that more E cells remained attached to the grooved part of the epoxy substrata than to the smooth portion. The difference was statistically significant (p<.()5) when tested by student t-test. Similar results were obtained when a more vigorous force was used to wash the cells off the substratum (i.e., 150 strokes/min. as opposed 75 to 36 strokes/min.). Moreover, more cells attached to grooved surfaces regardless of whether the grooved substrata were aligned with or against the direction of motion of the linear shaker. Time. min. Time, min. Figure 4.3- Curves indicating the number of cells (mean ± SD) attached per microscopic field as a function of time after plating. Cells were operationally defined as being attached if they resisted removal after incubation in a linear shaker at either 150 strokes/min (designated as the fast condition) or 36 strokes/min (designated as the slow condition). In addition the grooved surfaces were aligned so that the washing media flowed either with or across the grooves: (a) slow with grooves; (b) fast with grooves; (c) slow across grooves; (d) fast across grooves. (Dotted line, grooved substratum; solid line, smooth substratum). It should be pointed out that cell numbers were counted per microscopic field. However a microscopic field of the grooved surface is a flat projection of the three dimensional surface and thus underestimates the true surface area. For microscopic fields of equal area, the 10 urn-deep 76 grooved surface used in these experiments had ==1.2 times the surface area of a smooth flat surface (appendix 1). Even allowing for this correction, however, more cells were found attached per unit area on the grooved surface. Thus grooved substrata used in these studies did not decrease and probably increased cell attachment. Figure 4.4 shows the number of cells remained attached to the grooved titanium and smooth titanium surfaces after shaking at 36 strokes/minute against the grooves. When titanium-coated epoxy and uncoated epoxy substrata were compared, more cells were attached to titanium surfaces than to epoxy surfaces (p<0.05), regardless of whether the surface was smooth or grooved. 60-i "3 40 -1. J3 3 20-x Grooved 50 100 Time, m i n . 150 200 Figure 4.4. Curves indicating the average numbers of cells attached per microscopic fields of titanium-coated grooved or smooth surfaces as a function of time for attachment. Attached cells were defined as cells that resisted removal after incubation in a linear shaker at 36 stroke/min. b. Cell orientation Figure 4.5 shows light micrographs of the oriented clusters of E cells on grooved and smooth portions of titanium-coated surfaces. On the smooth portions of the epoxy substratum the OI of 85 E cells clusters was 1.07 ± .04 (SE) and on the smooth titanium-coated surface was 1.03 + 77 0.03 (SE) indicating that the clusters of E cells had no preferred orientation on a smooth, flat surface. On the grooved portions of the epoxy substratum the OI of 98 E cell clusters was 1.75 ± .07 (SE) and on the titanium-coated substratum the OI value of 104 clusters of E cells on the grooved area was found to be 2.14 + 0.1 (SE). When the log OI value of the grooved and smooth surfaces of epoxy and titanium-coated surfaces were compared using the student t-test, a statistically significant difference (P< .05) was found. Thus the E cell clusters were oriented with the long axis of grooves. The difference between the OI on grooved and smooth surfaces was significant even when the foreshortening effect, noted above, was considered in the microscopic measurement of length at right angles to the long axis of the grooves. Figure 4.5- (a) Clusters of epithelial cells cultured on grooved titanium substrata show orientation along axis of grooves, (b) Randomly oriented epithelial cell clusters on smooth titanium substrata (X350, Richardson's stain) 78 3. In vivo experiments In these experiments, it was hypothesized that if grooved surfaces are placed horizontally on the implant surfaces, which is parallel to the skin, grooves are able to inhibit downgrowth of the epithelial cells. The mechanism of such inhibition is probably based on the phenomenon of contact guidance. The implant design was that shown in figure 3.3. In this design, equal areas of horizontally oriented grooves were compared directly to the equal areas of smooth control surfaces, which were placed adjacent to the grooves. This design allowed direct comparisons of grooved and smooth surfaces. a. Clinical observations There are many advantages in placing the implant in the parietal area. For example, bone support under the implant may reduce the possibility of implant dislocation and the bone of the skull provides a good reference surface so that the implants could be placed at the same depth. Moreover, as reported by Jansen and de Groot (305), percutaneous implants placed in the cranial area in the vicinity of bone heal better than those in other areas such as the dorsum. Commonly, for the first two days following surgery most of the animals appeared to be irritated by the implants and rubbed the implant against the cage wall. Some animals disturbed the implants with their paws during grooming. Although implants were placed in proximity to the bone at the parietal area to reduce the possibility of dislocation, the soft tissue became separated from the implant in some instances. However, implants that were not disturbed during the period of the experiment exhibited excellent healing and tissue adaptation (figure 4.6). As a general rule, healing was excellent and uneventful in these animals as judged by tissue texture, contour and colour, as well as the absence of overt bleeding or exudates. My subjective impression was that the titanium-coated implants healed faster than the epoxy implants. Of the 6 epoxy implants in place for 7 days, 5 showed evidence of attachment on at least one of their test surfaces, but only a total of four proved suitable for morphometric evaluation. Of the 79 5 epoxy implants left in place for 10 days, 3 showed evidence of attachment but only 2 were measurable. A total of 18 titanium-coated implants were placed for 7 days, 12 showed evidence of attachment, but only 7 had at least one test surface suitable for morphometric measurements. The connective tissue attachment could not be measured on one titanium-coated implant because the base of the implant was lost during block preparation. Among 12 titanium-coated implants placed for 10 days, 8 showed evidence of attachment but only 6 had at least one surface suitable for histomorphometric measurements. Figure 4.6- Titanium-coated implant in situ after 7 days. The healed access incision is shown by arrows and two sutures around the access incision are still present. b. Histology The tissue-processing methods used in this study produced sections in which a clear outline of the cell nucleus and often the entire cell boundary could be traced under light microscope. The zones of the epithelial and connective tissue attachments were well-defined and preserved intact. Both epithelium and connective tissue were found closely attached to the grooved and smooth surfaces. Nevertheless, differences were found in cell arrangement, orientation and location on grooved and smooth epoxy or titanium-coated implants. 80 I M P L A N T I M P L A N T After 7-days percutaneous implantation, E cells were closely attached to both the grooved and the smooth portions of epoxy or titanium-coated implants. As a general rule, the E cells attached to the smooth portion of the implant were arranged in a multilayer of elongated cells. In contrast, 81 on the grooved portion of the implant the integrity of the layers of elongated cells with elliptical nuclei was broken by groups of cells with circular nuclei in or above the grooves (figure 4.7). As the shape of the nucleus often indicates the shape of the whole cell (268), this observation suggests that the epithelial cells within the grooves were more spherical than the cells on the ridges, or that the cells were elongated perpendicular to the plane of section. After 10-days in situ, although epithelium was frequently found attached to the grooved portion of the epoxy implants, the total extent of the tissue attachment decreased in the areas nearest the smooth surfaces. Contrary to the titanium-coated implants in which epithelium remained attached to both grooved and smooth portions of the implant 10 days after implantation, epithelial attachment was not found on the smooth portion of the epoxy implants; instead, epithelial cells were found attached to the underlying pedestal. Thus the decrease in epithelial attachment level in the grooved portion of the implant immediately adjacent to the smooth may be related to the tight adhesion between epithelial cells, so that rapid migration in one area affects the location of cells in an adjacent area. Some sections contained inflammatory cells, comprising mainly macrophages, a few neutrophils, and rarely giant cells. The number of the inflammatory cells in the smooth or grooved portions of the implant did not noticeably differ, neither were differences noted between the titanium or epoxy implants. c. Morphometric evaluation Not all implants used in these experiments were suitable for morphometric evaluation. In some instances, thrombus or bacterial accumulations were found between the implant and the tissue so that a suitable attachment for morphometric measurements did not form. In other instances, although the epithelial and connective tissue was attached to the implant in situ , they split during processing so that only parts of tissue remained attached to the implant. These implants were not suitable for morphometric measurements because the extent of the epithelial and connective tissue attachment could not be measured accurately. Thus the specimens chosen 82 for morphometric evaluation were free of thrombus or bacterial accumulations, and showed no overt signs of artifacts introduced by histological processing. Moreover, measurements were made on those implants, which demonstrated a continuous epithelial attachment between the grooved and smooth portions of the implant. It should be noted that the term "depth" refers to the distance occupied by the epithelium and connective tissue on the long axis of the implant. Because the grooves have a well defined geometry, the linear contact length of the attachment on the horizontally placed grooved surfaces can be calculated. For example, in these experiments, the linear contact length of grooves would be 1.2 times the depth if cells were to adhere tightly to all surfaces (i.e. groove walls and ridges). The calculations for the linear contact length for all surfaces used in these experiments are shown in appendix 1. Statistical comparisons were performed in two ways. Student t-test was utilized initially to compare sulcus depth, epithelial and connective tissue attachment as well as recession on successive sections of grooved and adjacent smooth surfaces of each implant placed. Then all the means from the grooved or smooth surfaces of all implants were pooled and compared with a paired t-test. Table 1 shows the morphometric measurements and statistical comparisons for epoxy and titanium-coated epoxy implants. i. Tissue attachment and sulcus measurements (epoxy implants) The morphometric measurements could be only performed on epoxy implants in place for 7 days since after 10 days, epoxy implants were devoid of attachment on their smooth test portions (figure 4.8). Figure 4.9a presents the depth of epithelial attachment measured in sections, separated by 100 (im, of an epoxy percutaneous implant that had been in place for seven days. It can be seen that the depth of epithelial attachment is greater on the smooth than on the grooved portions of the implant, indicating that epithelial cells had migrated deeper into the connective tissue on the smooth surface. This impression was confirmed when the amount of implant connective tissue attachment was measured (figure 4.9b). A greater depth of connective tissue attachment was found in the grooved portions of the epoxy implants. The mean depth and linear TABLE 1. Histomorphometric Comparison of Grooved VS. Smooth Surfaces. Measurements Mean of Grooved*±(SE) Mean of Smooth*±(SE) Mean of (G-S)t 1 (SE) number of implants p value Epoxy Ti-coated Epoxy Ti-coated Epoxy Ti-coaled Epoxy Ti-coated Epoxy Ti-coated Sulcus Depth 268 + 44 ~ 269 1 75 - -1 ±43 - 4 - NS Epithelial Attachment 7 davs: Depth 3061 38 199 ± 30 457 ±45 295 ± 30 -151 ±31 -96 ± 24 4 7 <.025 <.005 Length 367146 238 1 36 457145 295 1 30 -901 33 -57 127 4 7 <.05 <.05 1Q days; Depth - 198122 - 2831 38 - -85122 - 6 - <.01 Length - 237 1 32 - 283 1 38 - -46121 - 6 - <.05 , Connective Tissue Attachment LP i 7 days: Depth 855170 934 1210 580155 829 1 208 2751 58 105 1 30 4 6 <.01 <.01 Length 1027 + 84 1070 1 250 580 + 55 829 1 208 447 1 68 241 144 4 6 <.005 <.002 10 days: Depth - 711 1 125 - 571 1 101 - 1401 57 6 - <.05 Length - 853 1 150 - 571 1 101 - 2821 74 - 6 - <.0l Recession 7 davs: - 10601 168 - 1221 1 158 - -161 148 - 7 - <.01 10 davs: - 1809159 - 2061 1 160 - -2521 115 - 6 - <.05 * Mean of attachment and recession of the grooved and smooth surfaces of all implants, t Mean of the differences between smooth and grooved portions from each implant. 84 contact length of epithelial and connective tissue attachment were calculated by averaging the values in the sections obtained for the grooved and smooth portions of each implant. Combining the results of the four successful implants using the paired t-test, a statistically significant (p<.05) difference was found between the smooth and grooved surfaces for both the depth and contact length of epithelial attachment and connective tissue attachment. These data indicate that the 10 |im-deep grooved epoxy surfaces used in this study inhibited epithelial cell downward migration, because the depth and contact length of epithelial attachment was less, and the depth and contact length of connective tissue attachment was greater, on the grooved surfaces than on the smooth surfaces. Sulcus depth, however, did not differ significantly on the smooth or grooved portions of the implant. Figure 4.8- Since the attachment level of the smooth surface was located on the pedestal, an accurate morphometric comparison to the grooved surface could not be carried out. (solid bars, epithelial attachment; striped bars, connective tissue attachment). 85 600 -| I I I ! I 0 10 20 30 40 50 Interval, 100 i i m Figure 4.9- Histograms illustrating the depth of the epithelial attachment (a) and connective tissue attachment (b) to the smooth and grooved areas of an epoxy implant in place for seven days. 86 ii. Tissue attachment and recession (titanium-coated implants) The depth of the epithelial attachment, measured on seven individual titanium-coated implants after seven days, showed a shorter depth of attachment on the 10 urn-deep grooved used in these experiments compared to the smooth surface (p<.05) on each implant. Conversely, connective tissue attachment, was found to be longer on the grooved than on the smooth surfaces (p<.01). In addition tissue recession from a reference line that was placed at the level of tissue attachment at the time of surgery, was greater on the smooth than on the grooved surfaces (p<.05). Taken together these results indicate that epithelial cells had migrated deeper on the smooth than on the grooved surface in each epoxy or titanium-coated implant. Similar to epoxy implants, when the data from individual implants were pooled, by averaging the values of sections obtained for the grooved and smooth portions of each implant, a paired t-test showed a statistically significant (p<.05) difference between the smooth and grooved surfaces for depth of epithelial tissue attachment, depth of connective tissue attachment, and recession. The average difference in depth of epithelial attachment between the grooved and smooth portions of implants are shown in table 1. Similar comparisons were performed on titanium-coated implants in place for 10 days (figure 4.10a,b,c) where epithelial and connective tissue attachment was evident on the smooth surface. There was a statistically significant (p<.05) difference between the smooth and grooved surfaces for all variables (table 1). These data indicate that the epithelial downgrowth is inhibited on the grooved epoxy or titanium-coated surfaces relative to that observed on smooth epoxy or titanium-coated surfaces. As in the case of depth of attachment, the contact length of the connective tissue attachment was significantly (p<.01) longer on the grooved than on the smooth surface, and the contact length of the epithelial attachment was significantly (p<.05) shorter on the grooved than the smooth surface at both seven and 10 days. — o -500 n Grooved Smooth a. c Qi s u a. ST 400 H 300 — 200 ioo A IS Interval, 20 100 fj.m 2000-1 3500-3000 2500 s a. C- 2000 o en $ 1500 1000 500 0 0 5 10 15 20 25 30 35 Interval, 100 L i m Figure 4.10 - Histograms illustrating the depth of the epithelial attachment, connective tissue attachment and the length of recession on the smooth and grooved portions of a titanium-coated implant in place for 10 days. 87 A D. Summary The effects of a 10 urn deep, micromachined, grooved epoxy and titanium-coated epoxy substratum were studied on E cell behaviour in vitro and in vivo. More E cells were found attached to the grooved substrata than adjacent smooth surfaces. In comparison to the smooth surfaces where clusters of E cells were randomly oriented, on the grooved surfaces, clusters of E cells were markedly oriented along the long axis of grooves. Grooved and smooth surfaces were implanted percutaneously in the parietal area of rats. Light microscopy indicated that E cells were closely attached to the implant surfaces and interdigitated into the grooves. Histomorphometric measurements after 7 and 10 days indicated that epithelial downgrowth was inhibited on the grooved portions of both epoxy and titanium-coated epoxy implants. II. Effects of groove characteristics on percutaneous implant performance Although the experiments of section I demonstrated that horizontally placed, V-shaped, micromachined grooves 10 um deep, 39 (im spacing inhibited apical migration of the epithelium, nothing is known of the effect of varying groove parameters such as depth and spacing on the behaviour of either E cells or fibroblasts (F) on implants in vivo. In these experiments the effects of grooves of different depths, spacings and orientations were studied on the behaviour of cells abutting titanium-coated percutaneous devices. A. Implant fabrication The epoxy implants had two protruding components connected to each other by a flat pedestal. Each protruding component had an outer test surface that faced laterally and an inner test surface that faced medially, and overall the implant was smaller in size than the implants used in the previous section. Moreover, only one surface topography was placed on each test surface of an implant. Thus an implant had a total of four surface topographies distributed equally on the 88 laterally or medially facing test surfaces. The topographies used on the test surfaces are shown in figure 3.2a-f and include 19 um or 10 urn deep grooves with a 39 urn pitch, 10 urn or 3 urn deep grooves with a 30 urn pitch as well as 3 urn deep grooves with a 7 um pitch. These grooved surfaces were placed horizontally on the four test surfaces of implants so that they would be parallel to the surface of the skin when placed in situ (figure 4.11a). On another group of implants, 10 | im or 3 um grooved surfaces with 30 um pitch size were placed vertically on the implant so that the grooves were oriented parallel to the long axis of the protruding component. This arrangement would be perpendicular to the surface of the skin when placed in situ (figure 4.1 lb). Smooth surfaces were also placed on some implants to serve as controls. Implants were baked at 60° C for 3 days, and then coated with = 50 nm of titanium using a sputter coater*. Figure 4.11- Schematic diagram of the implant model (a) implant with horizontally oriented grooves, (b) implants with vertically oriented grooves. 'Randex 3140 Sputtering System, 815 San Antonio Rd., Palo Alto, Calif. 94303. 89 B. Preparation and characterization of surfaces The titanium-coated implants were cleaned by ultrasonication for twenty minutes in a detergentt specifically formulated for tissue culture. After twenty rinses with deionized, sterile dis t i l led water, they were dried overnight in a tissue culture laminar flow hood¥, sterilized for 3 minutes in an argon-gas glow-discharge chamber and immediately stored in deionized, sterile distilled water in exhaustively-cleaned, water-tight glass vials. At all stages, implants were manipulated by sterile, clean, titanium forceps. Randomly chosen samples from each batch of the titanium-coated implants was inspected by scanning electron microscopy and analyzed by X-ray Photoelectron Spectroscopy (XPS). C . Implantation procedure A l l surgeries were done under full sterile conditions, and the implantation procedure used was similar to that described in the chapter 3. Animals kept in 60 cm tall cages to reduce the chances of rubbing the implant against the ceiling of the cage, and were fitted with Elizabethan collars (2) for 48 hours after surgery to reduce their ability to disturb the implant. Thirty seven implants were placed; each implant contained four test surfaces. After histological processing, ten implants demonstrated epithelial and connective tissue attachment that was suitable for histology and morphometric measurements on all four test surfaces. Another five implants had three test surfaces, fourteen implants had two test surfaces and five implants had one test surface suitable for such observations and measurements. Three implants were outright clinical failures. One implant failed as a result of overt infection at the tissue/implant interface. Two other failing implants demonstrated microhematomas between the tissue and the implant that likely prevented the establishment of a proper tissue/implant attachment. Such implants were probably disturbed by the rats who, in the first day following surgery, often 17X Cleaning Solution, Flow Laboratories, Mclean, Virginia, 22102 ¥Canadian Cabinets Co. Ltd., Ottawa, Canada 90 attempted to rub the implants with their paws. The surfaces that were excluded from histomorphometric analysis frequently demonstrated remnants of tissue attached, but the location of the epithelial and connective tissue interface could not be determined accurately in these specimens. In these specimens, I suspect that the excision of the implant and tissue or tissue processing caused separation of the attached tissue from the implant surface. Table 2 outlines the number and fate of each type of surface tested. Overall, 92% of the implants yielded at least one usable test surface for histomorphometric measurements. This represents a substantial increase in the success rate over that observed in the section I. D. Histology and histomorphometric measurements Two-um-thick sections were taken every 50 um throughout the length of the implant. Sections were cut in an orientation such that the horizontal grooves were cross sectioned and the vertical grooves were longitudinally sectioned. Sections were then stained with Toluidine Blue and examined under the light microscope. In addition to the histomorphometric parameters described earlier that are epithelial, connective tissue attachment and recession, the thickness of the connective tissue capsule was also measured adjacent to the grooved or smooth surfaces. In surfaces having horizontal grooves, the thickness of the capsule was measured over the flat ridges between the grooves. However, comparable measurements could not be made in the implants having vertical grooves because the position of the cells relative to grooves or ridges is not evident in this plane of section. E . Statistics Between 15-30 sections were collected from each surface of each implant. The means of the epithelial and connective tissue attachments, recession and capsule thickness were calculated on each test surface. Multi-factorial analysis of variance was used for the main effects of the surface and position (i.e. lateral or medial) on the attachment level and capsule thickness. The data were tested for statistical significance using one way A N O V A and the Table 2. The total number and fate of each type of surface tested. Type of Surface Placed Clinical Failures * Damaged in Processing Histomorphometrics Smooth 21 2 3 16 Vertical Grooves 10d30pt 16 2 7 7 3d30p 16 2 7 7 Horizontal Grooves 19d39p 21 2 1 18 10d39p 21 2 7 12 10d30p 18 0 7 9 3d30p 16 0 9 7 3d7p 21 2 7 12 * Total of 12 surfaces were outright clinical failures. Since each implant had four test surfaces, total of 3 implants clinically failed, t The letter "d" stands for deep and "p" stands for pitch. For example 10d30p stands for a 10 um-deep, 30 um-pitch grooved surface. 92 Student-Newman-Keuls multiple-range test. The unit for statistical analysis was the test surface. That is, the measurements made in the sections for each test surface were averaged to give the mean, which was used as the value for that surface in that implant in the A N O V A calculations. F. Observations 1. Surface characterization S E M observation of surfaces supported the findings of the previous study and indicated that grooves were not distorted and titanium coating was smooth and continuous. However, slight rounding of the groove edges was noticed in the 3-um-deep, 7-um-pitch grooves. For all surfaces, X-ray Photoelectron Spectroscopy (XPS) analyses of the outer 50-100 A of the surface indicated that the major titanium species detected on the implants was Ti+4 (Binding Energy=459 eV). The large oxygen signal also present in these samples suggests that the Ti+ 4 species probably exists as T1O2. At least three or four different carbon species were present on the implants. The predominant carbon signals were representative of C-C (Binding Energy=285 eV) and C-0 (Binding Energy=286.5 eV) bonds. Other elements found on the implant surface included silicon and traces of nitrogen. 2. Epithelial histology E cells were closely attached to all the horizontal and vertical 10 um and 3 um deep grooves as well as to the smooth surfaces. On the smooth surface, E cells were arranged in a multilayered fashion with elongated nuclei. Cells with elongated nuclei were also lined up along the surface of the vertical grooves. In contrast, in the sections prepared from the horizontal grooved surfaces, cells with elongated nuclei were found only over the flat ridges between the grooves. The nuclei located within the 10 um-deep, or 3 um-deep, grooves tended to be rounded. A different situation pertained in 19 um deep horizontal grooves where E-cells often contacted only the flat ridges between the grooves, and cell nuclei were rarely found inside the grooves (Fig 4.12). 93 Figure 4.12- Photomicrographs of 2 urn-thick sections obtained from epithelial attachment to the smooth control surface (a) of the implant (Im), as well as to the horizontally oriented 3 um-deep, 7 um-pitch (b); 3 um-deep, 30 um-pitch (c); 10 um-deep, 30 um-pitch (d); 10 um-deep, 39 um-pitch (e) and 19 um-deep, 39 um-pitch (f) grooved surfaces. 94 3. Connective tissue histology Two distinct regions were observed on the smooth, 3 um-deep grooved and 10 um-deep grooved surfaces; a dense region close to the implant surface and a loose connective tissue region farther from the implant surface. The width of the dense region, which we have termed the connective tissue capsule, varied with the smooth surface having the thickest and the 10 um-deep the thinnest capsule. Although F were more numerous close to the implant surface, no distinct zones could be identified accurately on the 19 um-deep grooved surface. Commonly, on smooth surfaces or surfaces with horizontal or vertical, 10 um or 3 um-deep grooves, fibroblasts were arranged parallel to the long axis of the implants. In contrast, fibroblasts were inserted obliquely into the implants with 19 um-deep horizontal grooves. The cell nuclei found within the horizontal 10 um and 3 um-deep grooves with 30 um-pitch appeared either rounded or elongated, with half of the nucleus in the groove and half over the adjacent, flat ridge. Occasionally cell nuclei were found inside the horizontal, 3 um-deep, 7 um-pitch grooves (figure 4.13). Inflammatory cells, mainly macrophages, a few neutrophils, and rarely giant cells were noticed in some sections, but there was no noticeable difference in the number of inflammatory cells between smooth and grooved surfaces. Numerous small capillaries and blood vessels were present close to the implant surfaces. 4. Histomorphometrics-a. Depth of attachment Table 3 and the diagrams in figure 4.14 summarize the morphometric measurements and statistical analyses. Multi-factorial A N O V A indicated no difference in depth of attachment or recession between the laterally and medially facing surfaces. However, there were significant differences among grooved and smooth surfaces (p<.05). The shortest E-cell attachment was found on the horizontal 19 um-deep grooves, whereas the longest E-cell attachment was 95 Figure 4.13- Photomicrographs of 2 urn-thick sections obtained from connective tissue attachment to the smooth control surface (a) of the implant (Im), as well as to the horizontally oriented 3 um-deep, 7 um-pitch (b); 3 um-deep, 30 um-pitch (c); 10 um-deep, 30 um-pitch (d); 10 um-deep, 39 um-pitch (e) and 19 um-deep, 39 um-pitch (0 grooved surfaces. Table 3. Histomorphometric Measurements Epithelial Attachment Connective Tissue Attachment Recession Capsule Surfaces MeanlSD (urn) Mean ± SD (urn) Mean±SD(um) Mean ± SD (urn) Smooth 248 ±30 609 ± 94 1146±150 25 ± 7 V10d30p* 304 ±37 473 ± 80 1381 ±119 -V3d30p 319 ±26 453±119 1299 ± 9 3 -Pepth Length Pepth Length H19d39pt 121 ±31 - 812±81 1310 ±131 774±116 8 ± 2 H10d39p 159 ±24 200 ±29 720 ±128 908 ±160 944 ± 123 17±4 H10d30p 152 ±30 212 ±41 731 ±89 1024±125 915 ±86 13 ± 6 H3d30p 207 ±49 249 ±59 622 ±71 747 ± 85 1011 ±83 17 ± 5 H3d7p 195 ±48 289 ±73 576 ±81 853 ±120 1075 ±79 2 2 ± 5 The letter "V" represents surfaces with vertically oriented grooves, the lower case "d" stands for deep and "p" stands for pitch. For example, V10d30p stands for a 10 um-deep,30 urn-pitch, vertically-oriented grooved surface. t The letter "H" represents surfaces with horizontally oriented grooves. For example, H19d39p stands for a 19 pm-deep, 39 urn-pitch, horizontally-oriented grooved surface. 97 observed on vertical grooved surfaces. Epithelial attachment on horizontal, 10 um-deep grooves was shorter than on horizontal, 3 um-deep grooves, but both grooved surfaces had less depth of attachment than the smooth surface, which in turn was less than the vertical grooves. The pitch of the grooves was not found to exhibit a statistically significant effect on the depth of the attachment (p>0.05). The longest connective-tissue attachment was on horizontal 19 um-deep grooves and the shortest connective-tissue attachment was on vertical grooves. Taken together these results indicate that the extent to which the epithelium migrated downward varied inversely with the depth of the grooves, and that the range of pitches used in this experiment was not a significant factor. b. Recession The reference line that was placed during the surgery could be traced in each section as a semilunar dent at the top of the protruding parts of the implant. The recession measurements were made from the bottom of the dent to the beginning of the epithelial attachment. Statistical analysis indicated that surfaces could be divided into four categories, which differed significandy (p<0.05) in recession. The most recession was found in surfaces with vertically-oriented grooves. The second group comprised horizontally-oriented, 3 um-deep grooved and smooth surfaces. Horizontally-oriented, 10 um-deep grooved surfaces constituted the third group. The fourth group comprised of horizontally-oriented, 19 um-deep, 39 um-pitch grooved surfaces that produced the least recession. c. Linear contact-length Because the grooves have a well defined geometry, the linear contact length of the attachment on the horizontal grooved surfaces can be calculated (see appendix 1). The linear contact length was measured for epithelial and connective tissue attachments on all the horizontal grooved surfaces. However, as the E-cells did not enter the 19 um-deep grooves, the linear contact length for the epithelial attachment of this surface was not calculated. Statistical analyses revealed the 98 same pattern as that observed for depth of attachment with the exception that the length of the epithelial attachment on 3 um-deep, 30 um-pitch grooves was not statistically different from that on the smooth control surfaces, and the linear-contact length on 3 um-deep, 7 um-pitch grooved surfaces was significandy larger than those observed on the smooth and 3 um-deep, 30 um-pitch grooved surfaces. d. Capsule The thickness of the capsule varied between 8 to 24 um among surfaces. The horizontal 19 um-deep grooves had the thinnest connective tissue capsule (8±2 um) whereas the smooth and the horizontal, 3 um-deep, 7 um-pitch grooved surfaces had thicker capsules (24±7 um). Epithelial Attachment 400 |V3d30p |V10430p Smooth I H10d39p |H10d30p 300 W 200 -1 H19d39p ioo 0 J Scales \aa CennectlTC Tiosae Attachment 900 H19d39p H10d30o |H10d39p H3d30p Smooth H3d7p 800 600 V10d30p I V3d30p 500 . 400 300 . Scate= tun Receeiien |V3d30p , „ - „ , . 1300 -V10d30p Smooth H3d7p H3d30p 1200 -1100 -1000 _ |H10d39p lH10d30p H19d39p 900_ 800 -700 . Scale* \un Capsule Thlckne 30. I Smooth 2 3 . H3d7p 20 |H10d39p |H3d30pK , 5 H10d30p 10. H19d39p 0 Saks pm Figure 4.14- Diagrams showing values of the epithelial attachment, connective tissue attachment, recession and capsule thickness. The scaled vertical lines indicate the values for the various measurements. A small vertical line to the left connecting two or three surfaces indicates that there is no significant difference between the values. Surfaces not connected by a vertical line have values that differ significantly (p<.05). "V" represents surfaces with vertically oriented grooves, "H" represents surfaces with horizontally oriented grooves, "d" stands for depth and "p" stands for pitch. 99 G. Summary Experiments of this sections were designed to investigate the effects of varying groove parameters such as depth, spacing and orientation on epithelial downgrowth and attachment of E cells and F to percutaneous implants in vivo. Grooves were produced with a 39-um pitch and depths of 19 um or 10 um. In addition, 10-um- and 3-um-deep grooves were made with pitches of 30 um and 7 um. Epoxy implants with grooves oriented either horizontally or vertically to the long axis of the implant, as well as smooth control surfaces were coated with 50 nm of titanium and placed in the parietal area of rats for a period of 7 days. Histomorphometric measurements indicated that epithelial downgrowth was accelerated on the vertically oriented grooved surfaces and inhibited on the horizontally oriented grooved surfaces; an observation that could represent the most direct evidence of contact guidance occurring in vivo. E cells bridged over the 19-um-deep grooves and their migration appeared to be inhibited by the F that inserted into the implant surface. In the shallower horizontal grooves, however, epithelial downgrowth appeared to be inhibited by contact guidance because there was no evidence of F inserting obliquely into the implant surface and E cells were interdigitated into the grooves. III. The electron microscopy of the soft tissue interface with titanium-coated surfaces The observations of the interface of epithelium and connective tissue with micromachined surfaces were made by light microscopy. As little is known about the ultrastructure of the cell/implant interface on textured surfaces, a separate experiment was designed to study the detailed organization of the epithelium and connective tissue adjacent to micromachined-grooved, titanium-coated, percutaneous implants at the ultrastructural level. Although obtaining ultrathin sections for transmission electron microscopy proved to be technically demanding because of differences in the hardness' of titanium, tissue and embedding plastic, the methods used in this study modified from those of Gould et al. (60,125), resulted in satisfactory ultrathin sections from the cell/titanium-coated implant interface. 100 A. Implants Implants were made from epoxy resin and coated with =50 nm titanium. The implant design, surface treatments and implantation procedures were identical with the experiment described earlier in section II. The following surfaces were examined: surfaces with grooves, 3 um-deep, 7 um-pitch; 10 or 19 um-deep, 39 um-pitch, and a smooth control surface. B. Observations 1. Epithelium Epithelial cells could be easily identified by an abundance of cytoplasmic tonofilaments and presence of desmosomes. The presence of tonofilaments, which consist of cytokeratin, is characteristic of E cells. E cells were found tighdy attached to the titanium coating of the smooth or the grooved surfaces via hemidesmosome-like structures (figure 4.15). Moreover, the titanium surface was covered by a basal lamina-like structure (approximately 30 nm thick) (figure 4.16). 0.5um imp — Figure 4.15- Electron micrograph of the interface between titanium-coated (T) implant and epithelial cells in vivo showing hemidesmosome-like (HD) structures. 101 0.2 pm Figure 4.16- Electron micrographs showing a basement membrane-like structure at the area of epithelial attachment to the titanium surface. Note tonofilaments (T) are parallel with the implant surface. Elongated E cells were found closely adapted to the smooth surface and flat ridges between the grooves (figure 4.17). E cells found inside the 3 urn-deep and 10 um-deep grooves were usually round (figure 4.18,4.19), however, as noted by light microscopy, cells or cell remnants were not found inside the 19 um-deep grooves (figure 4.20) and E cells bridged from one ridge to the next. The cytoplasm contained numerous ribosomes, some rough endoplasmic reticulum, mitochondria and abundant tonofilaments. It appeared that those E cells attached closely to the titanium surface had their tonofilaments oriented parallel to the implant surface (figure 4.16). Inside the 10 um-deep grooves, where cells were round, tonofilaments appeared to follow the round outline of the cell membrane (figure 4.21). Occasionally macrophage-like cells were noted penetrating epithelial layers and contacting the titanium surface. An example is shown in figure 4.22 where an E cell and a monocyte in close proximity were observed attached to the titanium surface inside a 10 um-deep groove. 102 In some specimens much of the epithelium was pulled away from the implant, possibly during tissue preparation and sectioning. However, in such specimens, a monolayer of E cells remained attached to the titanium surface (figure 4.23,4.24). Figure 4.17- Electron micrographs of epithelial (E) cells attached to the smooth titanium (Ti)-coated surface of the implant (imp). Desmosomes (D) are shown between E cells. 103 Figure 4. 18- (right) Electron micrograph of epithelial (E) cells attached to the 3-um-deep grooved surface. Note E cell rounded within the groove. Tonofilaments (T) and desmosomes (D) are marked with arrows. Figure 4.19- Electron micrograph of epithelial (E) cells attached to the 10-um-deep grooved surface. Note E cells appear round within the groove and elongated outside the grooves. 104 Figure 4.21- Electron micrograph of a rounded E cell within a 10-um-deep groove. Tonofilaments (T), titanium (Ti), implant (imp). 105 Figure 4.22- Electron micrograph showing an epithelial cell and a monocytic leucocyte attached inside a 10-um-deep groove. Arrow in the tissue marks a monocyte. imp m Figure 4.23- Electron micrograph from a 10-um-deep grooved implant showing an E cell left attached on the titanium, while the rest of the epithelium was pulled away from the implant during processing. 106 Figure 4.24- Higher-power electron micrograph showing pieces of the E cells left attached to the titanium surface. Desmosome (D), Titanium (T) 2. Connective tissue As was observed for E cells, F attached to the smooth surfaces were elongated and in some areas came in close contact with the titanium surface (figure 4.25). In contrast to E cell behaviour, F penetrated deep inside the 19 urn-deep grooves and the long axis of the cell had an oblique or a nearly perpendicular orientation to the implant surface (figure 4.26). Figure 4.27a illustrates the oblique orientation of F adjacent to the leading front of the E cells on a 19 um-deep grooved surface. A basement membrane separated F and epithelium in the vicinity of the groove (figure 4.27b). The oudine of F located within the 3 um-deep and the 10 um-deep grooves were more round (figure 4.28). 107 Figure 4.26- Electron micrograph showing a fibroblasts (F) penetrated deeply into the 19-um-deep grooved titanium (Ti)-coated implant (imp) so that it is aligned almost perpendicular to the implant surface. 108 Figure 4.27- Electron micrographs showing the oblique alignment of fibroblasts (F) with 19-um-deep grooved titanium (Ti)-coated implant (imp) under the leading front of the E cells (4.27a). (White arrows mark the extent of the flat ridges between two adjacent grooves). Higher power shows E cells with well-defined desmosomes (D) and basement membrane (BM) contacting a fibroblast (F) obliquely aligned to the 19-um-deep titanium (Ti)-coated grooved implant (4.27b). 109 Figure 4.28- Electron micrograph showing round nucleus of a fibroblast (F) inside, and an elongated nucleus of a F outside the 10-um-deep titanium (Ti)-coated grooved implant (imp). Note the presence of numerous microvilli and filopodia extending inside the groove and over the ridge. Fibers with the cross-banding typical of collagen could be identified in the vicinity of the implant surface but were never observed in direct contact with the implant surface. These dense collagen bundles ran mainly parallel to, and approximately 4 um from, the smooth-surfaced implants (figure 4.29). Collagen bundles were more scarce on grooved surfaces and had no apparent preferred orientation relative to the grooves. A striking characteristic of F within the grooved surfaces was the presence of numerous microvilli and filopodia extending inside the grooves and over the ridges (figure 4.28,4.30). An abundance of rough endoplasmic reticulum, golgi apparatus, ribosomes and mitochondria were evident in F at the vicinity of implants implying highly active, synthesizing cells. 110 Figure 4.29- Electron l m p micrograph of elongated fibroblasts (F) on smooth titanium (Ti)-coated implant (imp). Note parallel alignment of collagen bundles (C) with the implant surface. 4 r Figure 4. 30- Electron micrograph of a fibroblast (F) on the 3-um-deep titanium (Ti)-coated implant (imp). Note the cell process bending over the acute angle between the groove and the ridge. I l l The membrane of F was rarely found in direct contact to the titanium surface. In most cases such contacts were mediated by two distinct zones (figure 4.31); one zone comprised a thin (= 20 nm), amorphous, electron-dense layer that covered the titanium surface and the second zone consisted of a fine fibrillar structure extending from the amorphous zone to the cell membrane. The thickness of the amorphous zone was constant on the grooved or smooth surfaces; however, the thickness of the fine fibrillar zone varied extensively in different areas of the implant. Figure 4.31- Electron micrograph of the titanium (Ti) surface at the area of connective-tissue attachment. Note the amorphous zone (AZ) immediately contacting the surface and the fine fibrillar zone (FFZ) extending from the A Z to the cell membrane (CM). C. Summary The experiments of section III investigate the ultrastructure of the epithelial and connective -tissue attachment to titanium-coated micromachined grooved, as well as smooth control, implant surfaces. V-shaped micromachined grooves,3,10, or 19 um deep, were replicated in epoxy resin, and coated with 50-nm titanium. These grooves, as well as smooth surfaces were implanted percutaneously in the parietal area of rats for 7 days. The tissue preparation technique used in these experiments allowed ultrathin sectioning for electron microscopic observations. 112 These observations indicated that E cells closely attached to, and interdigitated with, the 3-um and 10-um grooves and the attachment appeared to be through basal lamina and hemidesmosome-like structures. In contrast, E cells were not found inside the 19-um-deep grooves and made contact only with the flat ridges between the grooves. As a general rule, F were oriented parallel to the long axis of the implants produced a connective tissue capsule with 3-|im and 10-um-deep grooved surfaces. On the 19-um-deep grooved surfaces, however, F inserted obliquely into the implant. The attachment of F to the titanium surface was mediated by two zones; a thin (== 20 nm), amorphous, electron dense zone immediately contacting the titanium surface, and a fine fibrillar zone extending from the amorphous zone to the cell membrane. IV. The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous imnlants The experiments of sections I and II provided evidence that micromachined surfaces with horizontally-oriented grooves, 3 or 10 um deep, inhibited epithelial downgrowth, through the mechanism of contact guidance. Furthermore, it was demonstrated in section II that 19-um-deep micromachined grooved surfaces produced obliquely oriented connective-tissue ingrowth, that had the potential to inhibit epithelial downgrowth. However, in all these studies it was not possible to determine unequivocally the effects of these surfaces on epithelial-cell and connective-tissue-cell behaviour separately, because connective tissue and epithelium interacted with the same surface topography. It was decided, therefore to design an implant system which permitted the connective tissue to contact surfaces of various topographies, whereas the epithelial cells contacted only a smooth flat surface. Thus, the effect of surface topography on connective tissue organization and on epithelial downgrowth could be evaluated direcdy. The second purpose of the experiments in this study was to compare one and two-stage surgery for percutaneous implant placement. The idea for two-stage surgery was drawn from Branemark's two-stage surgical technique for endosseous dental implants, which has enjoyed considerable success (133). 113 A. Implants Implants were made of three separate components; a base component (BC), which would contact connective tissues, and two skin penetrating components (SPC) (figure 4.32). The BC was U-shaped and had two protrusions connected to each other by a flat pedestal. SPCs were fabricated so that each SPC could be matched precisely to a protruding part of the BC. The SPC and BC were joined by means of a titanium screw pin*. V-shaped grooves 19 |im-or 30 um-deep, and 120 um-deep tapered pits (figure 3.2e-g) as well as smooth control surfaces were oriented on the BC so that the long axis of grooves and the rows of pits would be parallel to the surface of the skin when placed in situ. The SPCs had smooth surfaces. All BCs and SPCs were coated with =50 nm titanium. B. Implantation procedures Implantation was accomplished using either a one-stage or a two-stage technique that will be described separately: 1. One-stage implantation The two SPCs and the corresponding BCs were pre-screwed with titanium screw pins in the laboratory and used as a single implant. The access incision was made distaUy from one ear to the other and two parallel test incisions, just sufficient in size so that the two protruding components of the implants could be accommodated, were created in front of the access incision. Following the reflection of the epidermis and dermis, the periosteum was removed and the bony bed of the implant was smoothed by a bone file so that the implant rested firmly on the surface. The implant was placed through the access incision into the test incisions so that the SPCs penetrated through the skin. The tissues in the front and the end of SPCs were sutured by 6-0 'Filpin 0^ -Series), Vivadent, Tonawanda, NY, 14151-0304, USA. 115 silk* and the end of the thread was tied to the titanium pin. This type of sling suturing maintained the tissue at the very top of the SPCs just under the flat pedestal where the reference line for recession measurements was located. The access incision was sutured with 5-0 silk* and the animals received a prophylactic intramuscular injection of antibioticst. Animals were kept in 60-cm-tall cages to reduce the chance of rubbing the implant against the ceiling of the cage. In addition, custom made Elizabethan collars were fitted to the animals, to reduce their ability to disturb the implant. 2. Two-stage implantation- a. First stage BCs were implanted in the parietal area through an access incision, which was similar to that for the one-stage implants. The epidermal and dermal layers were reflected and the periosteum was removed by a combination of blunt and sharp dissection to form a pouch of sufficient size to accommodate the B C . As in the one-stage surgery, the bone under the B C was smoothed with a bone file so that the implant rested firmly on the surface. The B C was then placed on the parietal bone and the access incision was sutured using 5-0 silk. The animal received a prophylactic intramuscular injection of antibiotics. b. Second stage Eight weeks was chosen as the time to insert the SPC, on the basis of data from a pilot experiment, which indicated that by eight weeks the acute inflammation from surgical trauma from the first procedure had resolved and the peri-implant connective tissue had healed. Animals with an implanted B C were anaesthetized and the corresponding SPCs were screwed to the BC in situ by a titanium pin through a small cutaneous incision. SPCs and B C were secured by spot *Ethicon,Peterborough, Ontario, Canada. tPen-Di-Strep, Roger/STP, London, Ontario, Canada. 116 application of the tip of a heated forceps to the SPCs pin hole adjacent to the titanium pin. Further stabilization of the SPC was achieved by a thick mix of glass ionomer cement* placed over the rubber stopper of an endodontic file which had been threaded through the pin and fixed over the pin hole of the SPC. Sling suturing was used to place the tissue at the reference line. The same postoperative precautions as those used in the second stage of the two-stage percutaneous implantation were taken. Figure 4.33 illustrates the implantation procedures. A total of 53 complete implants (BC & SPCs) and 22 BCs were placed. Figure 4.33- Clinical Pictures showing the second stage of the two-stage implantation, (a) the fit of the SPC to a protruding part of the BC is tried with the titanium pin, (b) one SPC is secured and the pin-hole of the other protruding part of the BC is examined with the titanium pin, (c) both SPCs are secured. Note that the titanium pins were leveled off later and sutures were placed as described in the text. 'Miracle Mix, GC International Corp., Scotsdale, A Z , USA, 85260 117 C . Specimen collection Two subcutaneously placed BCs were removed every week up to 11-weeks postimplantation. Complete implants (BC+SPC attached) were removed one, two and three weeks after implantation. D. Histomorphometric measurements Sections were taken every 100 um throughout the length of the implant using a Sorvall MT2 microtome. Two-um-thick sections were cut in an orientation such that the grooves and tapered pits were cross sectioned. Sections were stained with Toluidine Blue and examined under a light microscope*. The distance occupied by the epithelial attachment and connective tissue attachment, as well as the length of recession from a reference line located at the top of the SPCs, were measured using a computer with a graphics tablett, and appropriate software*. In addition, the thickness of the connective tissue capsule adjacent to the grooves, tapered pits or smooth surfaces was also measured at three different locations. In grooved or pitted surfaces, the thickness of the capsule was measured over the flat ridges between the grooves or pits. Figure 4.34 shows the morphometric measurements on the implants. E . Statistics Between 10 and 15 sections were collected from each surface of each implant. The means are calculated for the epithelial and connective tissue attachment, recession, and capsule thickness for each test surface. The unit for subsequent statistical analysis was the test surface. That is, the measurements made in the sections for one implant surface were averaged to give the mean, which was then combined in the A N O V A calculations with the values of other implants having "Zeiss Photomicroscope (Zeiss, Oberkoken, W. Germany). tApple Computer, Cupertino, Ca, USA. *Rocky Mountain Software. Figure 4.34- Schemaric diagram shows the cross section of the implant (BC+SPC), and the morphometric measurements. 119 the same surface. Multi-factorial analysis of variance was used for each dependent variable (recession, epithelial attachment, connective tissue attachment and capsule thickness) to examine the main effects of surface, stage and time. In addition, for each factor the data were tested for statistical significance, using one-way A N O V A and the Student-Newman-Keuls (SNK) multiple-range test. A two-by-two contingency table was used to analyze the failure rate of implants with smooth surfaces and surfaces with grooves or pits. F. Observations 1. Clinical healing One-stage percutaneous implants demonstrated some evidence of inflammation such as swelling, redness and bleeding, which were resolved within 3-days of surgery. In implants placed by the two-stage surgical technique, soft tissue edema and redness were prominent features of the first surgery. The edema gradually disappeared over two weeks. The least inflammation and tissue reaction was noted in the second stage of the two-stage technique when the SPCs were connected to the subcutaneously implanted B C . At this time (8 weeks post subcutaneous implantation), BCs were found firmly attached to the underlying connective tissue and were relatively immobile. As indicated in Table 4, clinical failures were mainly associated with smooth-surfaced BCs, three weeks after either one or two-stage implantation. This was statistically significant (p<.05) when compared to the number of failures for other surfaces. T a b l e 4. The total number and fate of each type of surface tested. O n e - s t a g e s u r g e r y T w o - s t a g e s u r g e r y Total Failed Successful Histological Sections Total Failed Successful Histological Sections S u r f a c e s Acceptable Unacceptable Acceptable Unacceptable S M O O T H 1-week 8 0 8 7 1 10 0 10 8 2 2-weeks 8 2 6 5 1 10 0 10 6 4 3-weeks 10 5 5 5 0 8 4 4 4 0 G R O O V E S 19 u m - d e e p 1-week 8 0 8 7 1 10 0 10 7 3 2-weeks 8 0 8 6 2 10 0 10 7 3 3-weeks 10 1 9 6 3 8 2 6 6 0 30 u m - d e e p 1-week 6 0 6 6 0 10 0 10 9 0 2-weeks 10 0 10 8 2 8 0 8 5 3 3-weeks 10 1 9 7 2 8 0 8 5 3 TAPEREP PITS 1-week 6 0 6 6 0 10 0 10 7 3 2-weeks 10 0 10 6 1 8 0 8 5 3 3-weeks 10 1 9 6 3 8 0 8 5 3 o T o t a l s 104 10 94 75 16 108 6 102 74 27 121 2. Histology-a. one-stage Percutaneous Implants After one week implantation, a firm epithelial attachment was noted on all SPC and the connective tissue on either SPC or B C could be categorized as a loose, immature, granulation tissue. Fibroblasts with large nuclei and some inflammatory cells such as neutrophils and macrophages contacted the implant surface at 1 week (figure 4.35a,b,c,d). By two weeks some fibroblasts had adopted a perpendicular orientation to the 19 and 30 um deep grooved surfaces, whereas a capsule had formed on the smooth surfaces (figure 4.36a,b,c). After three weeks, implants with grooved surfaces had a thin capsule (4-5 layer of cells), and the cells varied in orientation: fibroblasts with a perpendicular and oblique orientation to the implant, as well as cells with rounded nuclei inside the grooves were observed (figure 4.37a,b,c). After two weeks, fibroblasts on the surfaces with tapered pits were parallel to each other and bridged from one edge of the pit to the other edge in a hammock-like arrangement (figure 4.36d). By three weeks more cells formed the hammock-like structure, which penetrated deeper into the pit (figure 4.37d). As a general rule, there was a gradual downward migration of the epithelium, which replaced the connective tissue during the first two weeks after implantation. By three weeks, many implants that had smooth surfaces contacting the connective tissue had either failed because of epithelial marsupialization, or had a very short connective tissue attachment that encapsulated the implant. b. Two-stage percutaneous implants (First stage) In the pilot study that was performed to determine the healing process of the connective tissue attachment to the subcutaneously placed B C , the tissue/implant interfaces were removed and processed for weekly histological observations. 122 Figure 4.35- Photomicrographs of 2-um-thick sections obtained from the area of connective-tissue attachment to the smooth control surface (a), to the 19-um-deep (b) and 30-um-deep grooved (c), as well as to the 120-um-deep (d) pitted surfaces of the BC (imp) after one week, one-stage, percutaneous implantation (Original magnification for a,b,c X500, and ford X400). Note the distance from one groove to the next groove (pitch) is 30 um. 123 Figure 4.36- Photomicrographs of 2-um-thick sections obtained from the area of connective-tissue attachment to the smooth control surface (a), to the 19-um-deep (b) and 30-um-deep grooved (c), as well as to the 120-um-deep (d) pitted surfaces of the BC (imp) after two weeks, one-stage, percutaneous implantation. (Original magnification for a,b,c X500, and for d X400) 124 Figure 4.37- Photomicrographs of 2-|im-thick sections obtained from the area of connective-tissue attachment to the smooth control surface (a), to the 19-um-deep (b) and 30-um-deep grooved (c), as well as to the 120-um-deep (d) pitted surfaces of the B C (imp) after three weeks, one-stage, percutaneous implantation. (Original magnification for a,b,c X500, and for d X400) 125 i. One week A n immature granulation tissue was formed that conformed remnants of red blood cells, fibrin meshwork, neutrophils, macrophages and occasional large fibroblasts. There was no apparent difference in the population or density of cells identified at the interface of grooved, pitted or smooth surfaces. ii. Two weeks The dominant cells were large fibroblasts, macrophages and few neutrophils. Evidence of a capsule was observed on the smooth surface. On the grooved and pitted surfaces, fibroblasts were found in contact to the implant and a few cells were located within the grooves and pits. iii. Three weeks On the smooth surface the capsule became thicker and on the grooved surfaces a thin capsule comprising ~4 layers of fibroblasts was formed. Fibroblasts within the grooves were either rounded or oriented obliquely or perpendicularly to the long axis of the implant. Pitted surfaces produced a hammock-like structure similar to that observed in the one-stage percutaneous implants. iv. Four - five weeks Although the thickness of the capsule continued to increase on the smooth surfaces, no apparent change was noted on the thickness of the capsule on the grooved or pitted surfaces. However, the cytoplasm of cells located inside the grooves became condensed and the cell boundary became indistinct. These cells stained distinctively darker than other cells located farther from the implant surface (figure 4.38, 4.39). On the pitted surfaces, the hammock like structure became populated with more cells and penetrated deeper into the pit. At five weeks, half of a pit was filled with cells. An interesting feature of this period was an increase in the number of mast cells with distinct cytoplasmic granules on all surfaces. 126 Figure 4.38 Figure 4.39 Light micrographs of 2-um-thick sections obtained from 19-um-deep (4.38) and 30-um-deep (4.39) grooved BC (imp) placed subcutaneously during the first stage of the two-stage implantation. Note the cytoplasm of fibroblasts located inside the grooves appears condensed, and the cell boundary is indistinct (original magnification X650). v. Six - eight weeks The thickest connective tissue capsule was noted at this time period on the smooth surfaces of BC. However, similar to observations of the 3-5 weeks postimplantation period, no noticeable change was noted on the thickness of the capsule on the grooved or pitted surfaces. The novel observation of this time period was the appearance of densely stained foci of mineralized tissue contacting the 19-um and 30-um-deep grooved as well as 120-um-deep tapered pitted surfaces. This tissue will be discussed in detail in the next section (section V). c. Two-stage percutaneous implants (Second stage) Epithelial cells were observed tightly attached to the smooth test surfaces of the SPC after one week. The connective tissue attachment comprised two parts; a loose connective tissue capsule in contact with the smooth surface of the SPC under the epithelial attachment, and a healed 127 connective tissue in contact with the micromachined or control smooth surfaces of the B C of which the dominant cells were fibroblasts; macrophages and some mast cells were also seen on the micromachined and smooth control surfaces of BC. Osteoblast-like and osteocyte-like cells were also noted on those micromachined surfaces of BCs in which mineralization took place (figure 4.40). A thick capsule (= 10-20 layer of fibroblasts) was found on smooth surfaced BCs, (figure 4.41). On B C s with grooved surfaces, fibroblasts contacted the implant forming a thin capsule comprising =3-5 layers of cells, and frequently fibroblasts penetrated within the grooves. The nuclei of fibroblasts within the grooves were either rounded or elongated obliquely or nearly perpendicular to the long axis of the implant (figure 4.42). The cytoplasm of fibroblasts located inside the grooves appeared condensed, and the cell boundary was indistinct. These cells stained distinctively darker than other cells located farther from the implant surface. On surfaces with tapered pits, fibroblasts formed a hammock-like fashion similar to that observed in the one-stage percutaneous and subcutaneous implants. By two and three weeks, although the capsule on BCs with smooth surfaces had become thicker, no apparent changes were noted in the thickness of the capsule on the grooved or pitted surfaces. However, on the pitted surfaces the hammock-like structure penetrated deeply into the pits so that by three weeks most pits were filled with connective tissue (figure 4.43). Figure 4.40- Light micrograph obtained from 30-um-deep grooved BC (imp), one week post SPC implantation. Note the osteocyte-like cells adjacent to the implant surface. imp 128 Figure 4.41-Light micrograph of smooth-surfaced B C (imp), two weeks post SPC implantation. Note the thick capsule adjacent to the implant surface, (original magnification X500). At two weeks, the downgrowing epithelial attachment on implants with smooth BCs had replaced the connective tissue attachment on the SPC and/or half of the BC. In contrast, the epithelial attachment had just reached either the junction of the BC and SPCs on implants with 30-um-deep grooves or 120-um-deep pits. At three weeks, generally, epithelial attachment was found on the BC except for implants that had a 30 um deep grooved surface on the BC, in which the epithelial attachment was located at the SPC/BC junction. 129 Figure 4.42- Light micrographs of 19-um-deep grooved BC (imp), one week (a) and three weeks (b) post SPC-implantation. Note perpendicular arrangement of some fibroblasts within the capsule to the grooves (a), (original magnification X650). Figure 4.43- Light micrograph of 120-um-deep tapered pitted B C (imp), three weeks post SPC implantation. Note the fibrous connective tissue ingrowth inside a tapered pit. (original magnification X200). 130 3. ffistomorphometrics Table 5 summarizes the morphometric measurements of this study. Multifactorial A N O V A indicated that although stage (one-stage/two-stage) and time significantly affected connective-tissue attachment and recession (p<.05), they had no significant effect on the epithelial attachment. Surface topography significantly affected epithelial attachment, connective-tissue attachment and recession (p<.05). There was a significant interaction between stage and time only. A more detailed analysis of the data, using one-way A N O V A and S N K multiple-range comparisons, provided more information on the effect of each factor on the outcome. Figure 4.44 provides the means and the results of statistical analyses for capsule thickness, recession, epithelial and connective-tissue attachment, for surfaces placed by one or two-stage surgical technique. a. Recession The reference line for recession appeared in each histological section as a distinct point located on the SPC. The measurements were made from this point to the epithelial attachment (figure 4.34). i. Effects of stage and time At either two or three weeks post implantation, there was significantly less recession (p<.05) on two-stage percutaneous implants than on one-stage implants, when comparing implants with the same topography. As a general rule and as expected, recession increased with time and the greatest rate of recession occurred between the first and second weeks of implantation. Table 5. Histomorphometric measurements of epithelial and connective tissue attachment, recession, and capsule thickness for all implants. Surfaces Epithelial Attachment Connective-Tisue Attachment Recession Capsule (umlSD) (umlSD) (umlSD) QimlSD) 1 stage 2 stage 1 stage 2 stage 1 stage 2 stage 1 stage 2 stage Smooth 1 week 469±76 4231160 13581400 16511248 16231501 14501127 25111 41110 2 weeks 421±73 346±100 511167 12421466 25501105 18601459 2615 3417 3 weeks 475±100 583±152 182186 6481294 29841201 22671400 4612 31112 Grooves 19 um-deep 1 week 336±94 288±74 17221510 19461360 14311418 13221348 1813 1914 2 weeks 321±104 253±104 6361178 13241297 25201304 19691251 1613 1913 3 weeks 251±90 3271121 3551264 8351210 28711325 23171341 27114 1416 30 um-deep 1 week 270±100 189163 22471344 21061399 7501302 9321313 1615 2014 2 weeks 311±194 178154 9971331 16411392 19761401 14221423 1616 1917 3 weeks 197+94 199179 6001310 15801105 23881377 16701112 1714 2315 TaDered Pits 1 week 356±164 192163 25021282 20531455 6891227 12521473 2213 33111 2 weeks 266±62 2521145 8761202 15481452 23481222 16011243 2317 2016 3 weeks 265±111 2841115 6911207 12071178 26191229 21271121 2513 2112 H M 1- Week 2- Weeks U J 600 500 — a J 400 o co *. < _ 300 CD = 200 100 S- 1St S- 2SI 120P- 1St 19G- 1 SI 19G- 2SI 30G- 1 St 120P- 2St SOG- 2St S- 1St S- 2St 19G- 1St 30G- 1St 120P- 1St 19G- 2St 120P- 2St 30G- 2St 3- Weeks S- 2St S- 1St 19G- 2St 120P- 1 St 120P- 2St 19G- 1St 30G- 2St 30G- 1St c <D E .c o CO < a> 3 CO CD > 2500 2000 1500 ^ m 1- Week 120P- 1 St 30G- 1St 30G- 2St 120P- 2St 19G- 2St 19G- 1 St S- 2St S- 1St 2- Weeks 3- Weeks 1000 -o CD C e O 500 120P- 2S1 30G- 2St 19G- 2St S- 2St SOG- 1St 120P- 1 St 19G-1St S-1St SOG- 2St 120P- 2St 19G- 2St 120P- 1S1 S- 2St 30G- 1St 19G- 1St S- 1St 3000 -2500 -1- Week g 2000 © o CB CC 1500 1000 _ S- 1St S-2S1 19G- 1St 19G- 2St 120P- 2St 30G- 2St I 30G-1SI I 120P- 1St 2- Weeks s- 1St 19G- 1 St 120P- 1 St 30G- 1St 19G- 2 St S- 2St 120P- 2St 30G- 2St 3- Weeks s- 1St 19G- 1 St 120P- 1 St SOG- 1 St 19G- 2St S- 2St 120P- 2 St 30G- 2 St 0) « fl> c o 3 M a. CD O |j.m 50 — 40 30 — 20 — 10 1- Week S- 2St 120P- 2St S- 1St 120P- 1 St 30G- 2St 19G- 2St 19G- 1St 30G- tSt 2- Weeks S- 2St S- 1St 120P- ISt 120P- 2St 30G- 2St 19G- 2St 19G- 1 St 30G- ISt 3- Weeks s- 1St S- 2St 19G- 1St 120P- 1 St 30G- 2St 120P- 2St 30G- ISt 19G- 2St Figure 4.44- Diagrams showing values of the epithelial attachment, connective tissue attachment, recession and capsule thickness for one-stage and two-stage implants placed at 1,2 and 3 weeks. Small vertical lines to the left connecting two or more surfaces indicate that there is no significant difference among the values. Surfaces not connected by a vertical line have values which differ significantly (p<.05). "p" represents tapered pits, "G" for grooves, and "St" stands for stage. For example "30G-2St" stands for implants with 30-um-deep grooved surfaces on tne BC, placed with two-stage implantation procedure. 133 ii. Effects of surface In two-stage implants the 30-um-deep grooved and 120-um-deep pitted surfaces had significandy less recession (p<0.05) than other surfaces at two and three weeks. In one-stage implants, less recession occurred on grooved and pitted surfaces than the smooth surfaced implants, but these differences were not statistically significant at 3 weeks post implantation. b. Epithelial and connective tissue attachment As described in previous sections, the term "depth of attachment' refers to the distance occupied by either the epithelium or connective tissue on the long axis of the implant. When epithelium migrates downwards it displaces connective tissue. Thus, a greater depth of connective tissue attachment implies less epithelial downgrowth. Conversely, a greater depth of epithelial attachment is often associated with rapid epithelial downgrowth. i. Effects of stage and time In the two and three-week periods, the two-stage implants had significantly greater connective-tissue depth (p<.05) than the one-stage implants. In contrast, statistical analysis did not demonstrate significant differences in depths of the epithelial attachment between one-stage and two-stage implants. As was observed for recession, the depth of the connective tissue attachment in most cases decreased gradually with time. ii. Effects of surface The two-stage, 30-um-deep grooved implants demonstrated a stable connective tissue attachment after 2 weeks; there was no significant decrease in the connective tissue attachment between 2 and 3 weeks for these implants. Although the two-stage, pitted implants lost some connective-tissue attachment during the third week of implantation, this surface ranked as the second best surface in terms of amount of connective-tissue attachment. In terms of the depth of epithelial attachment, no significant differences were noted among surfaces implanted for 1 week. 134 However, the depths of epithelial attachments were shorter on grooved and pitted surfaces than smooth surfaces at 2 and 3 weeks. The shortest depth of epithelial attachment was found on the 30-um-deep grooved surfaces at two and three weeks post implantation. c. Connective Tissue Capsule There was no significant difference in the thickness of the capsule in the three«zones that were measured on all surfaces. Therefore, the values of the three zones were averaged to obtain one value for the capsule thickness. As a general rule, One-stage percutaneous implants had thinner capsules than two-stage implants. The smooth-surfaced implants had thicker capsules than other surfaces. For example the smooth-surfaced one-stage implants at 3 weeks and the smooth-surfaced two-stage implants at 1 week had capsules that were significantly thicker than those on other surfaces (p<0.05). F. Summary In studies of sections I, II and III connective tissue and epithelium interacted with the same surface, so that the effects of the surfaces on each population could not be determined separately. The objective of the experiments of the section IV were [1] to examine cell behaviour on implants in which connective tissue contacted surfaces of various topographies and epithelium encountered only a smooth surface; [2] to compare one-stage and two-stage surgical techniques. Implants had a base component (BC) which was either smooth or had a surface with 18-um or 30-um-deep grooves or 120-um-deep tapered pits, and a skin-penetrating component (SPC) which was smooth. In the two-stage technique, the B C was implanted subcutaneously for 8 weeks, which permitted the healing of the peri-implant connective tissue. In the second stage the SPC was connected to the BC. For one-stage implants, B C & SPC were connected and implanted percutaneously. Implants (BC & SPC) were removed 1,2 or 3 weeks after percutaneous implantation; and histological sections were measured for recession, connective tissue and epithelial attachment as well as capsule thickness. Light microscopy indicated that both grooved 135 and tapered pitted surfaces encouraged connective tissue ingrowth. On the grooved surfaces, the orientation of fibroblasts changed from an oblique to a more complex pattern which included cells having round nuclei within the grooves, as well as cells oriented obliquely or perpendicular to the grooves. In the tapered pits a hammock-like arrangement of fibroblasts was observed. In some cases foci of mineralization and formation of bone-like tissue were noted on the grooved and pitted surfaces. The apical migration of the epithelium was significantly (p<.05) inhibited by those micromachined surfaces which produced connective tissue ingrowth to the BC. This study found that placing the implants in two stages improved the performance of percutaneous devices, and that a further improvement was achieved if the implant had a surface promoting connective tissue ingrowth. V . Electron microscopy of the hone-like tissue found adjacent to micromachined surfaces in vivo. The last two decades have witnessed the development of biomaterials that have surface chemical or biomechanical properties that would be expected to promote bone formation. An example of a material with specialized surface chemistry is the bioactive coating devised by Hench and coworkers (326). A n example of a surface that has a specific biomechanical advantage is the porous coated system developed at the University of Toronto (174), which integrates tissues closely in a three dimensional interlocking system. The experiments described in sections II, III and IV of this chapter demonstrated that the connective tissue attachment grew into micromachined surfaces and likely inhibited epithelial downgrowth. Moreover, light microscopy indicated that interestingly, a bone-like tissue was formed within or adjacent to some micromachined surfaces. This observation may indicate that micromachined surfaces have the potential to induce bone formation in rats. To verify such possibility, the first step would be to characterize the structural details of this mineralized tissue. This goal was achieved using T E M . 136 In this study the subcutaneous implants used in the experiments of section IV were removed after 10 weeks and prepared for electron microscopy using similar methods to those explained in section III. The implant surfaces prepared had 19 and 30 um-deep grooves and 120 um-deep tapered pits. A . Sample preparation As discussed earlier obtaining ultrathin sections for T E M was technically demanding because of differences in the hardness' of epoxy, titanium, epon, soft tissue and mineralized tissue, nevertheless, with extreme care, satisfactory ultrathin sections were obtained from the soft-and-mineralized-tissue/implant interface. The surface of some epon embedded subcutaneous implants were demineralized by sodium ethoxide (10 minutes), and E D T A (1-week). This procedure resulted in acceptable ultrathin sections from the bone-like tissue adjacent to the implant surface. B. Observations After six weeks of subcutaneous implantation densely stained foci appeared on some of the 30-um-deep grooves. These foci were located half way up the surface of the implant, and cells around them had the appearance of osteoblasts. By seven weeks these foci became mineralized, however, no osteocytes could be found within the mineralized matrix (figure 4.45a). By 9 weeks foci were extended on the implant surface and osteocyte-like cells could be found within the lacunae inside the 30 um-grooves (figure 4.45b). Mineralization also was noticed on some pitted surfaces after 8 weeks of implantation and on few 19 um deep grooved surfaces after 10 weeks of implantation (figure 4.46,4.47). No instances of mineralized tissue forming adjacent to smooth surfaces were observed during the entire experiment. 137 Figure 4.45-Photomicrographs obtained from 30-um-deep grooved B C (imp) placed subcutaneously during the first stage of the two-stage implantation. Note two calcification foci (a) and osteocyte-like cells inside the foci (b). (original magnification X500). Figure 4. 46- Photomicrograph obtained from 120 um-deep tapered pitted surfaces of BC (imp) placed subcutaneously during the first stage of the two-stage implantation. Note calcified tissue and osteocyte-like cells (arrows) adjacent to the implant. 138 Figure 4.47- Photomicrograph obtained from 19 um-deep grooved surfaces of BC (imp) placed subcutaneously during the first stage of the two-stage implantation. Osteocyte-like cells are marked by arrows. Table 6 gives the distribution of number of implants exhibiting bone-like tissue for each type of surface. When the data on the micromachined surfaces were grouped together, bone formation differed significantly between smooth and textured surfaces ( X 2 test, p<.05). It should be noted that the bone that formed in response to the surfaces was located approximately 2000 um from the parietal bone and was not likely to be an outgrowth from the periosteal tissue. These data suggest that implant-surface topography can affect bone formation, but sufficient data are not yet available to determine whether the micromachined surfaces differ significantly in this property. The sections obtained from demineralized and undemineralized tissues indicated that the cells surrounding the mineralized foci had the characteristics of osteoblasts (327) including cuboidal shape, abundant rough endoplasmic reticulum, golgi apparatus, secretory vesicles and cellular processes. Cells within the mineralized foci resembled osteocytes, as they resided in distinct lacunae and had several extended cell processes (figure 4.48). Thus, because these foci contained osteoblast-like and osteocyte-like cells and mineralized extracellular matrix, they are referred to as bone-like tissue in this thesis. Osteoblast-like cells were located approximately 50-100 um away from the implant surface whereas bone-like tissue and osteocyte-like cells were found within the grooves and pits or in close proximity to the flat ridges between grooves and pits. 139 140 Table 6. Effects of surface topography on bone-like tissue formation in vivo Surface Bone Present Bone Absent Total implants Smooth 0 12 12 Micromachined 19 um-deep Grooves 2 10 12 30 um-deep Grooves 4 10 14 120 um-deep Tapered pits 3 9 12 The appearance of the extracellular substance varied with location. In the areas of tissue not in intimate contact with the implant the major component was collagen fibres, which could easily be identified from their size and typical banding pattern. In some areas of undemineralized sections this periodicity was indistinct, probably as a result of the plane of section or incipient mineralization. Areas of tissue undergoing mineralization exhibited roughly circular aggregates of electron-dense crystalline needle-like material scattered in the collagen matrix. In the body of the mineralized foci, the matrix resembled woven bone. The appearance of the extracellular matrix adjacent to the surface also varied in a manner that suggested a progressive process of mineralization. Matrix vesicles were observed subjacent to some connective tissue cells on some micromachined surfaces as early as seven days after implantation, and some of the vesicles were found in direct contact with the titanium surface (figure 4.49). At later times contact was established in some areas by collagen bundles with an oblique or perpendicular orientation abutting directly to the surface. At other mineralized sites the mineralization front approached the titanium-coated micromachined surfaces, and projections of the mineralized tissue directly contacted the titanium surface. Most commonly, however, 141 mineralized tissue was in direct contact with the titanium-coated, micromachined surface, without any detectable zone of nonmineralized material (figure 4.50). C. Summary In this section the ultrastructure of the bone-like tissue formed in response to 19-um and-30-um-deep grooved and 120-um-deep pitted surface was examined. The bone-like tissue contained characteristic osteoblast/osteocyte-like cells adjacent to the implant surface. The interface in some areas revealed close juxtaposition of collagen to titanium without an intervening amorphous layer. 142 Figure 4.49- Electron micrographs obtained from undemineralized specimens. Matrix vesicles (MV) found as early as seven days post implantation contacting the titanium (Ti) coating of a grooved implant (imp). 143 Figure 4.50- Composite figure of electronmicrographs obtained from undemineralized specimens, (a) Close contact of collagen [C] bundles with the surface of titanium [Ti] coating within a 30-um-deep groove, note mineralized [M] tissue in close proximity, (b) Typical cross banded collagen bundles with the surface of titanium coating of a 30-um-deep grooved implant, (c) Mineralized tissue approaching a grooved implant, note direct contact with the titanium coating has been established in a few areas, (d) Mineralized tissue contacting titanium coating directly, arrows mark the borders of the titanium coating. CIHIAPTEE § DTSCIISSTON PAGE I. Epithelial cell behaviour on grooved or smooth percutaneous implants 144 II. Effects of groove characteristics on percutaneous implant performance 148 III. The electron microscopy of the soft tissue interface with titanium-coated surfaces 152 IV. The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous implants 155 V . Electron microscopy of the mineralized tissue found adjacent to micromachined surfaces in vivo 158 144A DISCUSSION Similar to the approach used in chapter 4, this discussion is divided into five main sections based on the individual experiments performed during the course of this thesis. It should be noted at the outset that the objective of these studies was to determine the effects of micromachined surfaces on the interaction of percutaneous implants with epithelial and connective tissue cells. However, certain design features that are used in percutaneous implants could not be accommodated within these experiments. For example, the skin-penetrating component of percutaneous devices usually has a circular cross section, but such a configuration would not have been optimal for in vitro studies of cell attachment or orientation, which are most readily accomplished on a flat surface. Also a circular design could complicate the in vivo histomorphometric measurements and subsequent statistical analyses. Thus for these experiments a U-shaped implant was developed; this proved to be the most suitable design to deliver the test surfaces to tissues with maximum stability in the parietal area of rats. Although a basic U-shape design was employed consistendy in all experiments, some changes were made in the implant design to accommodate specific questions asked in the different experiments. For example, in the experiments of section I, U-shaped implants had equal area of micromachined grooved and smooth areas on each face to allow a direct comparison. I. Epithelial cell behaviour on grooved or smooth percutaneous implant A. Attachment assays Titanium substrata promoted more E cell attachment than epoxy substrata. E cells attached to the 10 um-deep grooved epoxy surfaces in greater numbers than to adjacent smooth surfaces. Similarly more E cells attached to the grooved than smooth titanium-coated surfaces. Part of the increase may be attributable to the greater surface area of the grooved substrata. Another explanation is that the grooves provide mechanical protection of the cells from removal by the washing media in the cell attachment assay. An observation, which argues against the latter 145 possibility is that similar results were found when the substrata were washed so that the wash medias flowed in the same direction as the grooves. Thus, overall it appears that grooves of the dimensions and shape used in these experiments do not impair, and may enhance, epithelial cell attachment in vitro. B. Orientation assays Both titanium and epoxy 10 um-deep grooved substrata caused a significant orientation of E cells in the direction of the long axis of the grooves. The numerical value of the orientation index for titanium grooved substrata was greater than that of the epoxy grooved substrata of identical dimensions. Some of the effects of the grooved substrata observed in vivo could be predicted from in vitro studies. For example , in cell culture, fibroblasts always, and epithelial cells often, move in the direction of their long axis. Because cell orientation is linked to direction of cell locomotion, the orientation data could be used to predict the epithelial migration across the grooves, i.e., down the long axis of the implant, would be inhibited. The morphometric measurements, in vivo , showed inhibition of the epithelial migration down the long axis of grooved epoxy and titanium-coated implants that are in accord with such a hypothesis. C. Clinical observations For the undisturbed implants, there was excellent clinical healing and a firm implant/tissue attachment during the short period of the experiment. However, some implants were lost, mainly as a result of infection, microhematomas, thrombosis and fractures. Because implant failure did not occur in those animals restricted by the Elizabethan collar , it is likely that mechanical disturbance of the implant by the animal was responsible for most of failures. As noted by Powers et al. (328) for the titanium tympanic membrane device, lack of mechanical trauma is clearly correlated to a high rate of success and absence of acute and chronic inflammation. Although the subjective impression was that the titanium implants healed faster than the epoxy 146 implants, there was not a demonstrable difference between the number or distribution of inflammatory cells on epoxy or titanium implants, or on smooth and grooved surfaces. D. Histology of inflammation There was no evidence of toxicity caused by the epoxy or titanium-coated implants, but some inflammation of the tissues in contact with implants was noted. Inflammatory cells, which mainly consisted of macrophages and neutrophils were not preferentially located in the grooved portion of the implants neither there was any apparent difference on the degree of the inflammation between epoxy or titanium implants. Salthouse (175) has described the presence of macrophages and giant cells at rough implant surfaces and suggested that superior tissue compatibility might be obtained with smooth well-contoured implants with no acute angles. For the micromachined grooved substrata used in this study, it should be noted that although there are edges where the walls of the groove meet the ridges and floor of the groove, the walls, ridges, and floor themselves are all smooth. This geometry results from the micromachining process because the shape of an anisotropically etched hole is determined by the orientation of the silicon wafer relative to its crystallographic axis. Grooved substrata replicated from micromachined surfaces may not attract macrophages in the same manner as irregularly shaped surfaces produced by abrasion used in the study of Salthouse (175). E . Tissue attachment and morphometries The morphometric measurements of epithelial attachment, connective tissue attachment, and recession all showed less epithelial downgrowth on the grooved surfaces of epoxy or titanium-coated implants. However, implants with titanium-coating had more tissue attachment compared to that of epoxy implants and in contrast to epoxy implants that had no tissue remained attached to their smooth surfaces after 10 days. Titanium-coated implants had both connective and epithelial attachment to smooth and grooved surfaces after 10 days. The biocompatibility of the titanium 147 may be better than epoxy in the long term and indeed the clinical observations of this study indicated that the healing takes place much faster with titanium implants compared to epoxy implants. The behaviour of E cells on the smooth titanium surfaces influenced the E cells on the adjacent grooved surfaces after 10 days. The epithelial recession and length of attachment was greatest for the grooved surface in those areas that were close to the smooth surface (figures 4.9,4.10). It thus appeared that the portion of the epithelial sheet on the smooth surface was dragging downward the more slowly migrating epithelium on the grooved surface. F. Concluding remark In several implant systems surface texture has been specifically designed to aid performance (2,173,196,329). In some instances, the methods of production probably affect cell behaviour advantageously. For example the highly successful Biotes titanium dental implants have fine circular grooves located in the region where the implant contacts gingival tissue (162). It is possible that these fine grooves inhibit epithelial downgrowth by the mechanism of contact guidance. As the titanium-coated grooved substrata used in this study had larger grooves than those present in the Biotes implants, it appears likely that a range of groove dimensions can affect epithelial cell behaviour (264). However, the optimal dimensions of the grooves, i.e. those that are most effective in inhibiting downgrowth, are not known, and more studies are required to resolve this issue. Because micromachining produces surfaces with well defined and specifiable properties, it is a technique of choice for investigating systematically the topographical properties of surfaces that influence cell behaviour at the implant-tissue interface, as was done in the experiments of section U. 148 II. Effects of groove characteristics on percutaneous implant performance A . General results A unique feature of the studies reported in section II was that it tested the effect of the orientation of the grooves on cell behaviour on the implant. The results of the experiments of section I demonstrated that horizontal grooves inhibited epithelial downgrowth; a finding that would be expected i f contact guidance were operative. However, it could be also speculated that it was the grooved surface per se that inhibited migration, perhaps by altering other aspects of cell behaviour such as cellular attachment. If grooved surfaces inhibited cell migration nonspecifically then one would expect no difference in epithelial downgrowth between horizontally and vertically oriented grooves. This study, however, clearly demonstrated that the orientation of the grooves was important. The vertically oriented grooves promoted epithelial downgrowth relative to the smooth surface whereas the horizontal grooves inhibited downgrowth. Thus, epithelial tissue appeared to exhibit contact guidance on implants in vivo. B. Clinical heating The techniques used in this study produced a higher success rate with fewer clinical complications and faster healing than that achieved in the experiments of section I. One possible reason for the higher success rate may be that implants were glow-discharge treated. It has been presented that glow discharge treatment increases the surface-energy of materials and encourages connective-tissue/implant attachment (145). The results of this study supported this concept. Connective tissue appeared to adapt more closely to the glow discharge treated surfaces than to the surfaces used in the previous study, in which implants were sterilized by ( 6 0 CO) gamma irradiation. However, the smaller size of the implants, as well as the use of Elizabethan collars to prevent the animals from interfering with the implants, might have also contributed to the higher success rate. 149 C. Histology-1. Epithelial attachment A distinct feature of horizontal 19-urn-deep grooves in the epithelial-attachment region was that epithelial cells were rarely found inside the grooves. One explanation may be simply that the cells degenerated inside the grooves, perhaps on account of a lack of nutrients, or a metabolic alteration that resulted in cell death. However, this explanation is unlikely, since no noticeable cell debris was found within the grooves and the grooves were mostly filled with amorphous material. Another possible explanation may be related to a lack of flexibility in the E-cell sheet that prevented the E-cell sheet from accommodating to the grooves. This explanation conflicts with the in vitro findings on these surfaces, where E-cells exhibit considerable flexibility and easily migrate in grooves of similar size (264). Nevertheless, it should be considered that in vivo E-cells form a three-dimensional tissue and their flexibility may be modified by cell-cell interactions. The three dimensional structure of the epithelial tissue in vivo might have limited the flexibility of individual E cells. The epithelial tissue behaved like a sheet under tension so that the movement of individual E cells into the groove was restricted. 2. Connective tissue attachment Unlike the other surfaces that produced parallel alignment of cells and capsule formation, 19 um-deep grooves induced fibroblasts to orient obliquely with the implant surface. The significance of the connective tissue organization with 19 um-deep grooves becomes clear when one considers that there was not a close attachment and interdigitation of the epithelial cells to these grooved surfaces, and yet epithelial downgrowth was most effectively inhibited. Thus, the underlying organization of the connective tissue per se was most likely responsible for stopping the down growing epithelium. Squier and Collins (182) have reported the inhibition of epithelial downgrowth by connective tissue on Millipore filters with pores >3 um, which had been implanted percutaneously in the skin of pigs. Although Squier and Collins' study and the 150 study described in this section indicated that appropriately oriented connective tissue can control epithelial downgrowth, the differences in optimal size probably arise from the different characteristics of the surfaces. In particular, cells and fibers can penetrate completely through the pores of Millipore filters, but are restricted to surface attachment on micromachined surfaces. In any case, it seems likely that the surface geometries that promote oblique or perpendicular orientation of F and fibers with respect to the implant surface have the potential to improve the long-term performance of percutaneous devices. A situation contrary to that of the 19 um-deep horizontal grooves prevailed on the 10 um and 3 um-deep, horizontal grooves where E-cells attached closely to the surface, and the connective tissue capsule was not oriented in a manner to prevent epithelial downgrowth. In these instances, contact guidance of the epithelium by the substratum was the probable cause of the inhibition of epithelial downgrowth. However, it should be noted that these experiments did not distinguish unequivocally the effects of surfaces on E cells and F behaviour separately, because epithelium and connective tissue interacted with the same surface topography. This problem is studied in the experiments of section IV. 3. Cell shape The shapes of the nuclei of E-cells and F in 10 and 3 um-deep grooves were similar and consisted of either round nuclei inside the groove or a nucleus that bent over the groove edge. The latter shape was found more frequently in F, which may indicate more flexibility of F in vivo. Since the shape of the entire cell can often be predicted by the shape of the nucleus (268), the rounded appearance of cells within the grooves may indicate either spherical cells or elongated cells aligned with the long axis of the grooves. Spherical E-cells and elongated, aligned F have been found on micromachined grooved surfaces in vitro (263,264). However,the oblique alignment of F found in the 19 um-deep grooves in vivo would not be predicted by the in vitro observations. One possible explanation is the two-centre effect (162) which might produce cells 151 with this orientation. The two-centre effect refers to the orientation of cells from one anchoring centre to another. In vitro the two-centre effect was first shown when oriented cells grew from two explants towards each other on a plasma clot (18). As pointed out in introduction, the two-centre effect also appears to be operative when a porous titanium implant surface is placed on a lawn of fibroblasts (303). It has been hypothesized that the two centre effect can occur in vivo with one centre being the implant surface and the second centre being a particular component of the extracellular matrix or the periphery of the blood vessels (162). Another possibility for the oblique alignment of the fibroblasts may be simply that the cells did not have enough time to become aligned with the long axis of the grooves; F attached for a longer period of time might demonstrate a different the alignment predicted by contact guidance. D. Morphometries In this study an important factor in controlling epithelial migration on horizontally-oriented grooved surfaces in vivo was groove depth. Shallow grooves were the least effective in inhibiting epithelial downgrowth. No difference in epithelial downgrowth was discerned between the 39 um and 30 p:m pitches in 10 pm-deep horizontal grooves nor between the 30 um and 7 um pitches in 3 um-deep horizontal grooves. E . Concluding remark The surface topographies used on dental implants and percutaneous devices include rough, porous, microporous, nanoporous, sintered, plasma sprayed and etched surfaces. The intent of all these designs is to optimize tissue adaptation to the implant . Although some designs of implant offer different surfaces to bone and the covering soft tissues, it is rare for any distinction to be made between the major components of soft tissue, epithelium and connective tissue. This study implies that the optimal surface that inhibits epithelial downgrowth direcdy may not be 152 optimal for connective tissue, and vice versa. Thus, the design of an ideal implant might incorporate specific surface topographies for each cell population that the implant encounters. III. The electron microscopy of the soft tissue interface with titanium-coated surfaces A . Tissue preparation The histological preparation of the interface of typical implant materials, such as hard plastics, ceramics and metals, with tissues for thin or ultrathin sectioning is a potentially difficult task in implant research. Mckiney and Steflik (119,120) have introduced a modified cryofracturing technique to obtain ultrathin sections following the separation of the tissue from the implant surface. Another method, introduced by Kasemo et al (123) is electropolishing. In this method, which was applied to titanium implants, the bulk of the titanium can be removed, leaving a thin layer of titanium oxide suitable for ultrathin sectioning. However, the several stages involved in the sample preparation and the paucity of electron micrographs published in the literature indicate the complexity of this technique. An alternate approach is to use epoxy substrata (60,125,126) on which test materials such as titanium can be deposited. In this manner the interface of the metal and the tissue can be processed directly for ultrathin sectioning. The histological methods used in these studies were modified from those of Gould et al. (60,125) to prepare with minimal artifacts sections of tissue attached to implants that had a precise surface geometry. An important consideration in histological preparation of implants is to identify and eliminate artifacts, which can be easily introduced during many stages of tissue preparation such as tissue/implant excision, dehydration and embedding, as well as sectioning (122,330). In this study it was noted that extreme caution had to be exercised during the excision of the implant and the soft tissue, since soft-tissue organization was easily disrupted by the force of the scalpel. To prevent this disruption, the tissues were perfused with glutaraldehyde to provide rigidity to the soft tissue around the implant and make it less vulnerable to the shearing force of a scalpel. A second preparative problem was that the conventional dehydrating agent, alcohol, distorted the 153 implant, so that the grooves were irregular and the titanium-coating lifted from the surface of the epoxy implants. Aquembed, a completely water miscible epoxy resin was used as a replacement for alcohol, as it did not distort the grooves or the titanium coating and dehydrated the tissue in a satisfactory manner. A third challenging technical problem was to match the hardness of the implant and the embedding medium, because differences in hardness often resulted in folds and chatter on the histological sections. In this study I attempted to adjust the hardness of the embedding medium by using Epon 812* 58%, Nadic Methyl Anhydride* 28%, and Dodecenyl Succinic Anhydride* 14% (by weight), to match that of the epoxy implants. However, I was unable to compensate totally for the greater hardness of the titanium-coating, and occasional artifacts, such as folds and scratch lines were observed even on carefully sectioned tissues. These folds and scratches detracted from the appearance of some micrographs but did not render the micrographs uninterpretable. B. Histology That the epithelial attachment to titanium is a strong one is indicated by the electron microscopic observations. In a few specimens, although the majority of the epithelium had pulled away from the implant, a monolayer of E cells remained closely attached to the titanium surface. Thus the attachment of E cells to the titanium was probably stronger than that found between the E cells themselves. The strong attachment of the epithelial cells to titanium is not surprising since it is known from previous studies (60,125) and was confirmed in this work, that E cells attach to titanium via hemidesmosomes and basal lamina. However, it should be noted that, hemidesmosomes were not observed in all sections. One possibility for this result is that offered by Gibson et al. (61) who noted that even small changes in the plane of section away from 90 degrees to the cell membrane can result in the ultrastructure of the hemidesmosomes being indistinct *J.B. E M Inc. Pointe-Claire-Dorval, Quebec, Canada, H9R 4S8. 154 The importance of the influence of connective tissue organization on the oral epithelium has long been noted in the periodontics literature, where it is presumed that intact and uninflamed periodontal ligaments and connective tissue inhibit the downgrowth of the junctional epithelium (166,313-315). Similar to the previous light microscopic studies, the electron microscopic observations indicated that the 19 um-deep but not the 3 um-deep and 10 p.m-deep grooved surfaces induced oblique or nearly perpendicular orientation of F. Morphometric measurements demonstrated that epithelial downgrowth was inhibited on 19 um-deep micromachined grooved surfaces, it seems likely that the surface geometries that promote oblique or perpendicular orientation of F with respect to the implant surface have the potential to improve the long-term performance of percutaneous devices. XPS analysis demonstrated that the surface of these implants consisted largely of titanium oxide and in this respect was similar to some commercially available titanium implants (123). It has been repeatedly reported that in vivo the surface of biomaterials including titanium is covered by a conditioning film, which regulates cell/implant attachment (123,145,197,198). Moreover, it has been postulated that the thickness of the coating correlates with the degree of biocompatibility of a material (123). In the present study, the grooved and smooth titanium surfaces interfaced with connective tissue by means of two distinct zones; an amorphous zone in immediate contact with the titanium and a fine fibrillar zone in contact with the cell membrane. It is clear that in vivo as in vitro (41,162), cells do not attach directly to the surface of a biomaterial but instead to a conditioning film. More sophisticated methods such as immunogold ultramicroscopy are needed to provide specific information on the molecular composition of these conditioning films. Immunogold ultramicroscopy requires thin sectioning and minimal tissue/implant disruption. The technique described in this study has the potential to provide satisfactory specimens with few artifacts, and with appropriate modifications might prove useful for identifying the molecules mediating cell/implant attachment. 155 I V . The role of the connective tissue in inhibiting epithelial downgrowth on percutaneous implants A. General results Natural teeth, dental implants and percutaneous devices encounter a complex situation in which they interact with soft and sometimes hard connective tissue, as well as the epithelium. The downgrowth of epithelium that occurs during periodontal disease jeopardizes the survival of teeth as well as implants and it has long been assumed in the periodontics literature that an intact and uninflamed connective tissue inhibits such downgrowth (166,313-315). By analogy, it has been postulated that long term normal function for dental implants could be obtained through a similar functional peri-implant connective-tissue attachment (129,130,331). Similarly, in a study of percutaneously implanted Millipore filters, Squier and Collins (182) concluded that the connective-tissue attachment inhibited epithelial downgrowth. The experiments described in sections II and IU provided indirect evidence that the connective-tissue attachment to 19 um-deep grooved titanium-coated percutaneous implants impeded epithelial downgrowth, but in those studies the effects of these surfaces on the connective tissue were confounded with their effects on the epithelium, because both tissues contacted the same surface topography. In the present study the effect of surface topography on connective tissue could be studied direcdy, because the B C , which had various precisely-controlled surface topographies, contacted only connective tissue, and the epithelium initially at least contacted only a smooth surface (the SPC). Thus, differences in epithelial downgrowth on the implants could be attributed direcdy to the effects of the surface topography of the B C on connective tissue organization. A second feature of this study was that it compared a conventional one-stage surgery with a two-stage surgical technique that permitted the connective tissue to heal and organize before the epithelium established attachment to the SPC. The results indicated that less epithelial downgrowth occurred on micromachined surfaces placed with such the two-stage surgical technique. 156 B. Histology and morphometries Fibroblasts adopted an oblique orientation to the 19 um and 30-um- deep grooved surfaces two weeks after implantation. This arrangement was later replaced by a complex organization of fibroblasts comprising groups of cells with rounded nuclei inside the grooves, others perpendicularly oriented to the grooves and others half bent between the grooves and ridges. However, unlike the experiments of sections II and HI, an oblique orientation of the connective tissue was not prominent on the 19-um-deep grooves after one week of implantation. The differences in the surgical procedures and implant sizes could be responsible for this variability. These observations indicated that the organization of the connective tissue attached to an implant with grooved surfaces is complex and variable in time course. Nevertheless, it was found that connective-tissue attachment in various forms inhibited epithelial downgrowth. There are several possible, and not mutually exclusive, mechanisms for the inhibition of the epithelial downgrowth. Direct inhibition of epithelial locomotion might have occurred through contact inhibition of cell movement, a well documented aspect of cell behaviour in vitro (281,292,299). Another possibility is that the young granulation tissue adjacent to the one-stage implants produced more stimulatory factors, such as interleukins and other inflammatory mediators, that can stimulate epithelial proliferation and migration, than did the more mature connective tissue found adjacent to the two-stage implants. A third possibility is that the difference in downgrowth resulted from the greater stability of the two-stage implants, which were markedly less mobile in the first two weeks of the study. However, the mobility of these implants increased with time, so that there was no discernible difference between the mobility of two and one-stage implants at 3 weeks. Taken together these observations indicate that although the relatively small mobility of the two-stage implants during the first and second week of 157 implantation might have contributed to their better performance initially, other mechanisms such as cell-contact inhibition of movement or the organization of the connective-tissue matrix contributed to their success at later times. The data for implants in place for three weeks indicated that one or two-stage implants with 19-um-deep grooves and 120-um-deep tapered pits maintained their attachment level by means of a long connective-tissue attachment and a short epithelial attachment. In contrast, implants with smooth surfaces maintained their attachment level by means of a long implant-junctional epithelium and a short connective-tissue attachment. The higher failure rate associated with smooth surfaces suggests that such an attachment is not as effective a means of attachment as the oriented connective-tissue ingrowth. C. Heating A striking difference between fibroblast interaction with SCI and percutaneous implants was that the cells took much longer to form a tight attachment to the SCI. This delay in colonization was probably related to the surgical technique employed as the SCI was located in a pouch formed by blunt dissection and healing in this area could perhaps be considered as healing by secondary intention. In contrast tissues were tightly adapted to the percutaneous implants and fibroblasts attached to the surfaces much sooner. D. Concluding remark A procedure that vastly improves the success rate of endosseous dental implants is the method, introduced by Branemark and co-workers (133), in which implants are placed in two stages. In the first stage, the root part of the implant is buried in the jaw bone for a period of 2-3 months during which time healing occurs at the bone/implant interface. When healing is complete and the implant is immobilized, a second-stage surgery establishes the pergingival portion of the implant. Percutaneous devices, in contrast, are typically placed in one stage so that the connective-tissue and epithelial attachments are established concurrently. The acute 158 inflarnmation resulting from surgery as well as the probable mobility of the device in the soft tissue might jeopardize the possible benefits that could be obtained from proper connective-tissue attachment. Although Grosse-Siestrup and Affeld (9) have hypothesized that two-stage surgery may increase the PD's success rate, the experiments of this thesis have provided direct evidence that a two-stage implantation technique has the potential to improve the performance of the percutaneous devices, and that further improvement might be achieved if optimal surface topographies were provided for the peri-implant connective tissue attachment. V. Electron microscopy of the hone-like tissue in contact with micromachined surfaces Pathologic calcification, defined as the deposition of crystalline calcium-phosphate material consisting primarily of hydroxyapatite in an ectopic site (i.e., a site other than in normally mineralized tissue such as bone), has been observed to occur adjacent to a wide variety of artificial implants, including percutaneous devices (181), subcutaneous implants (186), and mammary prostheses (187). A number of animal models have been studied but Schoen (188,189) has advocated the use of rat subcutaneous implantation as a rapid, convenient and economical means of investigating host and implant determinants of mineralization. In this study, unlike many instances of ectopic mineralization, the tissue response to the grooved implants was not limited to the development of crystalline material but rather resulted in the production of bone-like tissue. Moreover it should be emphasized that bone-like tissue was not produced on all the subcutaneously implanted grooved substrata; a situation that indicates that full experimental control of the phenomenon has not yet been achieved. Nevertheless, data on the 19 um-deep, 30 um-deep grooved and 120 um-deep pitted surfaces indicate that mineralization occurred on a statistically significandy greater proportion of micromachined surfaces than on smooth surfaces. In fact mineralization of a smooth surfaced SCI was not observed in these experiments. The observation that surface topography can affect mineralization is not novel and has been observed in a number of systems. Perhaps the earliest example of the possible effects of an implant forming a bone-inductive microenvironment in vivo is the work of Selye et al. (190), 159 who implanted glass cylinders of various shapes into rats so as to direct tissue ingrowth in desired directions. A striking observation was that bone and cartilage formation occurred but only in some sizes and shapes of cylinders. Bone formation has been observed in conjunction with a variety of porous and textured surfaces, as well as machined surfaces, having microscopic grooves, on a number of dental implant systems. Such textured substrata could act through several not mutually exclusive mechanisms such as by allowing a bone-inductive microenvironment to be established, or by inducing cell polarity that facilitates extracellular matrix secretion, or by selecting for cells with osteogenic properties by their locomotory or attachment properties. It may be possible to distinguish between those possibilities by implanting surfaces with systematically varying surface topographies. Because micromachining can produce surfaces with precisely controlled topography that can be varied in shape depth and spacing, the implantation of micromachined surfaces could be a valuable method of investigating the topographical control of bone formation on artificial implants. It was observed that mineralized tissue and collagen bundles contacted the titanium surface in a manner similar to the close collagen-biomaterial interaction demonstrated by Matsuda and Davies (136) on bioactive glass substrata. Previous reports on the tissue-titanium interface have noted a layer of amorphous biomolecules, perhaps glycoproteins, in the immediate vicinity of the titanium (134). Reasons for the difference between the observations of this study, and those of others could be attributed to the many differences between the experimental systems employed, including the animal model, surgical technique, and surface properties of the implant However, equally it should be recognized that the examination of the titanium-tissue interface is a challenging technical task and the number of reports, and presumably specimens examined, is small. It is possible that close contact between collagen and titanium surfaces in other systems has not been reported because of the small number of samples studied. Finally it should be noted that the electron microscopic techniques used in this study do not have the resolution to rule out the presence of substrate adhesion molecules, such as fibronectin, that might mediate the attachment of collagen to the titanium. \(oO CMAIPTEE <£ P A G E I. C O N C L U S I O N S 160 II, FUTURE DIRECTIONS 161 160 A I. CONCLUSIONS The experiments of this thesis at the outset were designed to investigate whether the phenomenon of contact guidance occurs in vivo and i f so, would it be possible to take advantage of the principles of contact guidance to control cell orientation and cell migration on the surfaces of percutaneous implants. Grooved surfaces which were produced with micromachining were used to study contact guidance. Grooves of different dimensions on which epithelial (E) cells attached and demonstrated contact guidance in vitro, were found to affect cell migration and orientation on the surface of percutaneous implants in vivo. Specific conclusions of the experiments can be summarized as following statements: 1. Horizontal grooves, 10 um deep, inhibit epithelial downgrowth on percutaneous devices, probably by the mechanism of contact guidance. 2. Vertical grooves, in contrast, encourage epithelial downgrowth, an observation that could be considered the most direct evidence of contact guidance occurring in vivo on artificial substrata. 3. It appears that E cells interdigitated into shallow grooves (<10 um deep), whereas, deeper grooves (>19 um deep) resulted in E cells bridging over the edges of the grooves. 4. Fibroblasts, in contrast, appear to penetrate and orient obliquely with deep (>19 um) grooves. 5. Epithelial downgrowth appears to be inhibited directly by shallow grooves through the mechanism of contact guidance. 6. Epithelial downgrowth appears to be inhibited indirectly by connective-tissue ingrowth into the deep (>19 um) horizontal grooves. 7. A two-stage percutaneous implantation technique appeared to be more successful than a one-stage implantation technique. 8. A greater success rate of percutaneous implants was achieved if implants had a surface promoting connective tissue ingrowth. 161 9. Some micromachined surfaces appeared to have the ability to induce bone-like tissue formation. Ultrastructural studies indicate that this bone-like tissue is formed from mineral crystals, osteoblast and osteocyte-like cells. 10. Although at seven days, collagen bundles were not found contacting titanium surfaces, after eight or nine weeks, collagen bundles appeared to contact the micromachined titanium surfaces direcdy that is without an intermediate layer. 11. The mineralized tissue in some areas appeared to contact the micromachined titanium surfaces direcdy without an apparent non-mineralized interface. II. FUTURE DIRECTIONS The studies in this thesis mainly concerned the morphological aspects of the tissue/implant interface and the experimental implant model used proved to be a simple means of delivering different surface topographies to the tissues. There are many possible applications of these methods, but I will briefly discuss only two categories of experiments: 1. Research should be directed towards placing micromachined surface on currently used implants to optimize their performance. Different surface topographies could be placed at different parts of an implant depending on which tissue was present. For example, percutaneous devices anchored in soft tissue might be made of two components. The first component would provide a surface suitable for connective tissue, and the second component would convey a surface topography desirable for epithelium. Because dental implants contact bone, connective tissue and epithelium, they might be fabricated with three different surfaces, each optimal for the tissue the surface contacts. However, this approach may introduce mechanical, surgical and biological complications, which would have to be investigated in future experiments. 2. Research should be directed towards understanding the requirements for a physiologically acceptable interface between tissues and biomaterials at the molecular level. A promising 162 technique that could provide detailed information is in situ hybridization which could be used to detect messenger R N A sequences in cells adjacent to an implant Using this technique, it may be possible to identify precisely the location and types of cells which are making R N A sequences that code for specific molecules. These experiments might be able to distinguish specific cell activities that occur as a result of the presence of a biomaterial and to modify the surfaces to obtain acceptable cell behaviour. Another equally useful technology is immunogold electron and light microscopy which allows precise localization of biological molecules. This technique can be used to identify those molecules that condition the surface of a biomaterial. A third useful technique is the optical sectioning technology used in confocal microscopy. The confocal microscope allows optical sectioning of a three-dimensional transparent or semi-transparent object. 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List of the publications from this thesis 188 185 A Appendix 1 Calculation of linear contact length, (Schematic example for 10 um-deep, 39 um pitch groove L2 L4 L2 22 um LI Sin 54.7 = 10 + LI LI = 10+ 0.82 LI = 12.2 L2 Sin 35.3= L2 + 12.2 L2 = 0.57X12.2 L2 = 7.04 L4 = 17-(L2X2) L4 = 17-14.08 L4 = 2.92 Conversion factor for linear contact length (CFCL) CFCL = [(LI X2) + L4 + 22] +39 CFCL = 49+ 39 CFCL = 1.25 Linear contact length measurements for 19 um-deep. 39 um-pitch grooves LI = 19 + 0.82 Ll=23 CFCL = [(LI X 2)+ 15]+39 CFCL = 1.6 Linear contact length measurements for 10 um-deep. 30 um-pitch grooves LI = 10+ 0.82 = 12.2 CFCL = [(LI X 2) +L4 + 17] + 30 CFCL = 1.4 Linear contact length measurements for 3 um-deep. 30 um-pitch grooves LI = 3+0.82 = 3.6 CFCL = [(LI X2) + L4 + 19] + 30 CFCL = 1.12 Linear contact length measurements for 3 um-deep. 7 um-pitch grooves LI =3 + 0.82 = 3.65 CFCL =[(LlX2)+3]+7 CFCL = 1.48 186 Appendix 2 Abbreviations used in the thesis AZ: Amorphous zone BC: Base component B M : Basement membrane C: Collagen C M : Cell membrane D: Desmosomes DI: Dental implants E cells: Epithelial cells ECMC: Extracellular matrix contacts EGF: epithelial growth factor E M : Electron microscope F: Fibroblasts FAF: Fibroblasts activating factor FEI: Fully embedded implants FFZ: Fine fibrillar zone FGF: Fibroblast growth factor G A G : Glycoseaminoglycan HD: Hemidesmosomes IF: Intermediate filaments IFAP: Intermediate filaments associated proteins IRM: Interference reflection microscopy MF: Microfilaments MT: Microtubules MTOC: Microtubule-organizing centre 187 01: Orientation index PDGF: Platelets-derived growth factor PEI: Partially embedded implants PI: Percutaneous implants P M N : Polymorphonuclear leukocytes RFGD: Radio frequency glow discharge RGD: Arginine-glycine-aspartic SEM: Scanning electron microscopy SPC: Skin penetrating component TEM: Transmission electron microscopy TGF: Transforming growth factor Ti: Titanium X P S : X-ray photoelectron spectroscopy 188 Appendix 3 List of the publications from this thesis Papers 1. B . Chehroudi, T.R.L. Gould and D . M . Brunette, "Effects of grooved epoxy substratum on epithelial cell behaviour in vitro and in vivo"", J. Biomed. Mater. Res., V o l . 22, 459-473 (1988). 2. B . Chehroudi, T .R.L. Gould and D . M . Brunette, "Effects of grooved titanium-coated implant surface on epithelial cell behaviour in vitro and in vivo" ", J. Biomed. Mater. Res. , Vol . 23, 1067-1085 (1989). 3. B . Chehroudi, T.R.L. Gould and D . M . Brunette, "Titanium-coated micromachined grooves of different dimensions affect epithelial and connective-tissue cells differently in vivo", J. Biomed. Mater. Res. Vol . 24:9, 1202-1219, 1990. 4. B. Chehroudi, T.R.L. Gould and D . M . Brunette, "A light and electron microscope study of the effects of surface topography on the behaviour of cells attached to titanium-coated percutaneous implants", / . Biomed. Mater. Res., 25, 387-405, 1991. 5. B. Chehroudi, T.R.L. Gould and D . M . Brunette, "The role of connective tissue in inhibiting epithelial downgrowth on titanium-coated percutaneous implants" / . Biomed. Mater. Res., accepted for publication. Chaper of Book 1. D . M . Brunette, B . Chehroudi and T.R.L. Gould, "Electron microscopic observations on the effects of surface topography on the behavior of cells attached to percutaneous and subcutaneous implants", Proceedings 2nd Int. Cong. Tissue Integration in Oral, Orthop. and Maxillofac. Reconstruction, In press. 2. D . M . Brunette, J. Ratkay and B . Chehroudi, "The behaviour of osteoblasts on micromachined surfaces", Proceedings l*t Bone-Biomaterial Interface Workshop, In Press. Abstracts 1. B. Chehroudi, T.R.L. Gould and D . M . Brunette "Effects of grooved titanium substratum on cell behaviour in vivo and in vitro, J. Dent. Res., Vol . 66, 114, (1987). 2. B . Chehroudi, T.R.L. Gould and D . M . Brunette "Grooves inhibit epithelial downgrowth on implants", J. Dent. Res., Vo l . 67, 348, (1988). 3. D . M . Brunette, N . Schindelhauer, B . Chehroudi and T.R.L. Gould "Effects of grooved titanium substrata on cell shape in vivo and in vitro, J. Dent. Res., Vol . 67, 347, (1988). 4. D . M . Brunette, K.S . Wong, N . Schindelhauer, B . Chehroudi and T.R.L. Gould, "Effects of grooved titanium substrata on cell shape in vivo and in vitro " 4th Inter. Cong. Cell Biol., p. 156 (1988). 5. B. Chehroudi, T.R.L. Gould and D . M . Brunette "Effects of surface topography on epithelial and connective tissue attachment", J. Dent. Res., Vol . 68, 306, (1989). 189 6. B . Chehroudi, T.R.L. Gould and D . M . Brunette "Surface topography affects orientation of cells attached to titanium-coated implants", J. Dent. Res., Vol . 69,291, (1990). 7. D . M . Brunette, B . Chehroudi and T.R.L. Gould, "Evidence for the Occurrence of contact guidance and two-centre-effect on artificial substrata in Vivo ", Trans. Soc. Biomat., p. 297, 16th annual meeting (1990). 8. D . M . Brunette, B . Chehroudi and T.R.L. Gould, "Electron microscopic observations on the effects of surface topography on the behavior of cells attached to percutaneous and subcutaneous implants", 2nd Int. Cong. Tissue Integration in Oral, Orthop. and Maxillofac. Reconstruction, Rochester, Minnesota, September 23-27, (1990). 9. D . M . Brunette, J. Ratkay and B . Chehroudi, "The behaviour of osteoblasts on micromachined surfaces", 1st Bone-Biomaterial Interface Workshop, Toronto, Cannada, December, (1990). 10. B . Chehroudi, T .R.L. Gould and D . M . Brunette "Connective tissue ingrowth inhibits epithelial downgrowth on titanium-coated implants", / . Dent. Res., vol 70, 366, (1991). 11. B. Chehroudi, J. Ratkay and D . M . Brunette,"Electron microscopic observation of cells attached to titanium surfaces", Proceedings Scanning Microscopy 1991, In Press. 

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