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Implant surface topography affects connective tissue attachment to subcutaneous implants Kim, Hugh 2004

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Implant surface topography affects connective tissue attachment to subcutaneous implants By Hugh Kim DMD, TJniversite de Montreal, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In T H E F A C U L T Y OF G R A D U A T E STUDIES (Dental Science) THE UNIVERSITY OF BRITISH COLUMBIA December 2004 © Hugh Kim, 2004 A B S T R A C T A major concern with the implant-soft tissue interface is the aggressive proliferation of epithelium, which can be prevented in part by a firm attachment between the underlying connective tissue and the implant. Another problematic aspect is fibrous encapsulation of the implant, which denotes a failure of implant integration. The role of substrate texturing in maximizing fibroblast attachment and minimizing fibrous encapsulation has been documented previously. The aim of the present in vivo study is to evaluate the connective tissue attachment to titanium surfaces with varying degrees of roughness, including those found on commercially available dental implants. The roughness of the implant surfaces was previously quantified by numerical roughness (Ra) values. Titanium-coated epoxy replicas were implanted subcutaneously in rats for periods ranging from 1-11 weeks. The implants were then processed for histomorphometric evaluation of connective tissue attachment, capsule thickness, and where applicable, the degree of separation between the tissue and implant. Statistical analysis revealed that the textured and rough substrata, namely the grooved (GR: V-shaped grooves, 30-um deep), titanium plasma-sprayed (TPS: Ra=5.85 um), acid etched (AE: Ra=0.59 um), coarsely blasted (CB: Ra=5.09 um) and blasted and etched (SLA: Ra=4.39 um) surfaces exhibited significantly (p<0.05) greater connective tissue attachment and thinner fibrous encapsulation when compared to the smooth, polished control surface (PO: Ra=0.06 jam). In cases where separation occurred at the tissue-implant interface, this was of significantly (p<0.05) lower magnitude with the rough surfaces than the polished surface. The results indicate that rough implant surfaces are the most amenable to stable connective tissue attachment, which has implications for their use in percutaneous and permucosal applications, such as dental implant abutments. T A B L E OF C O N T E N T S Abstract ii Table of contents iv List of tables vi List of figures vii Acknowledgements viii Chapter One - Review of the Literature 1 I. Overview 2 1. Types of implants 2 2. Commercially used biomaterials 4 3. Methods used to alter surface topography 5 A. Rough and porous surfaces 6 B. Machined surfaces 9 C. Micromachined surfaces 9 II. Implants in soft tissue 10 1. The interface between implants and soft tissues 11 2. Failure at the implant-soft tissue interface 14 III. Cell behavior on titanium surfaces 16 1. Cell attachment 16 2. Cell shape 17 3. Haptotaxis 18 4. Contact guidance 19 5. Fibroblast organization into connective tissue 20 IV. Wound healing: general principles 21 1. Overview 21 2. The phases of wound healing 22 V. Soft tissue healing around titanium implants 24 VI. The fibrous capsule 25 1. Significance of capsule width 25 2. Capsule composition and biocompatibility 27 VII. Surface topography and soft tissue response 28 1. In vitro studies of surface topography and fibroblast behavior 28 2. Surface topography and fibrous capsule formation 30 3. In vivo studies of surface topography and fibrous capsule formation 31 Aims of this project 34 Bibliography 35 Chapter Two - Manuscript 52 I. Introduction 53 II. Materials and methods 56 1. Preparation of the titanium surfaces 56 2. Micropatterning of the silicon surface 57 3. Surface replication and titanium coating 57 4. Implantation procedure 58 i v 5. Histology and histomorphometric analysis 59 6. Statistical analysis 60 III. Results 65 1. Histological observations 65 2. Morphometric results 66 a. Attachment 67 b. Complete attachment and detachment 67 c. Capsule thickness 68 d. Tissue-implant separation distance 69 e. Relationship between measured parameters 69 IV. Discussion 77 Bibliography 86 Chapter Three - Conclusions and future directions 94 I. Conclusions 95 II. Future directions 96 Bibliography 100 v LIST O F T A B L E S Table 1. R a values of the surfaces used in this project. (Page 61) Table 2. Number and fate of each implant surface type tested. (Page 70) Table 3. Effect of surface type and time on attachment. (Page 73) Table 4. Effect of surface type on the incidence of complete attachment and detachment. (Page 75) Table 5. Effect of surface type and time on capsule thickness. (Page 76) Table 6. Effect of surface type and time on tissue-implant separation. (Page 76) LIST OF F I G U R E S Figure 1. Schematic representation of groove dimensions on the micromachined grooved surface. (Page 62) Figure 2. Schematic representation of the experimental implants used. (Page 63) Figure 3. Schematic representation of morphometric parameters used: (a) assessment of attached tissue as a percentage of total implant length, (b) measurement of capsule thickness and tissue-implant separation. (Page 64) Figure 4. Photomicrographs demonstrating fibroblast orientation on the (a) micromachined grooved surface and the (b) machined surface. (Page 71) Figure 5. Photomicrographs showing residual tissue along the coarsely-blasted implant surface in an area of tissue detachment. (Page 72) Figure 6. Photomicrographs demonstrating complete tissue attachment along the lengths of the (a) titanium plasma-sprayed, (b) blasted and etched (SLA) and (c) coarsely blasted surfaces. Complete tissue detachment is noted along the (d) polished surface. (Page 74) ACKNOWLEDGEMENTS I would first like to thank my supervisors, Dr. Babak Chehroudi and Dr. Donald M . Brunette for their guidance, support and humor throughout this project. It has been a privilege to work in their laboratory. I am indebted to Dr. Hiroshi Murakami for performing the animal surgeries in these experiments. I would also like to thank Dr. Douglas Waterfield for serving on my committee and Dr. Doug Hamilton for his advice during the writing of the thesis. Thanks are due to Mr. Andre Wong for his assistance in microtomy, to Mr. Colin Ng for his computer expertise, and to Ms. Mabel Cho for providing a figure used in this thesis. Thanks go to all of my friends, colleagues, and professors from our lab, the UBC graduate periodontics program and from my dental school days at the Universite de Montreal. I am particularly grateful to Dr. Rene Voyer for encouraging my entry into periodontics and to Dr. Ira Sy for his continued mentorship and support. Thanks to Christine Lee for her support and friendship as well as to her parents, Drs. Graham and Loretta Lee for opening their home to me when my computer went down during the critical stages of the thesis preparation! Finally, I would like to thank my parents, my siblings and their families. Their importance need not be explained. Vll l Chapter One - Review of the Literature Chapter One - Review of the Literature I. O V E R V I E W Biomaterials are widely used in medicine and dentistry today, where they have a multitude of applications. Devices are implanted in the various hard and soft tissues of the human body for the purpose of restoring function or esthetics. The clinical success of these devices ultimately depends on the biocompatibility of their materials, defined by Williams (1987) as the ability of a material to perform with an appropriate host response in a specific application. The host response should allow for an optimal wound healing process leading to integration of the biomaterial into the body tissues. Although biomaterials may fail because they elicit an overexuberant inflammatory reaction from the host, biocompatibility is not to be confused with inertness. Despite the fact that the majority of first generation biomaterials were chosen for their inertness, this property actually precludes their full integration into the host body tissues (Williams, 2001). An implanted material represents a foreign body to the host, which eventually responds by encapsulating the implant with fibrous tissue. The most biocompatible materials should thus evoke a controlled inflammatory reaction with minimal fibrosis, or even lead to regeneration of the original host tissues surrounding the implant (Ratner, 2001). These are the properties sought for tissue integration with the myriad of implant materials used in medicine and dentistry today. 1. Types of implants As our population continues to age, injury and disease to human organs and tissues are inevitable. The use of implanted devices to replace organs or to restore their 2 function has gained popularity over the years. According to a 2001 review by Ratner, over 2.7 million intraocular lenses are placed in the US annually, along with 1.7 million cardiovascular stents, 300 000 orthopedic prostheses and 300 000 dental implants. Implants may be classified according to their chemical composition, e.g. plastic, metal or ceramic. Secondly, they may be classified according to the type(s) of tissue they interface with, e.g. bone or soft tissue. Thirdly, implants can be classified based on their physical location. Partially embedded implants, which include dental implants and percutaneous catheters, cross the epithelial barrier and are simultaneously exposed to the outside environment. Fully embedded implants, such as orthopedic or mammary prostheses, are completely enclosed within the body. IMPLANTS Fully embedded implants Partially embedded implants - Orthopedic implants - Dental implants: penetrate oral mucosa - Breast implants - Percutaneous implants: penetrate the skin • Catheters for dialysis/vascular access • Auditory devices 3 2. Commercially used biomaterials The spectrum of biomaterials used for implantation includes silicone, cellulose, zirconia, aluminum oxide, cobalt-chromium alloys, stainless steel, titanium and various polymers (Ratner, 1993). Among metals, titanium and its alloys are believed to be among the most biocompatible materials (Morehead et al, 1994). They are used extensively in orthopedic surgery for knee and hip arthroplasty (Windier et al, 2001), as well as in implant dentistry (Branemark et al 1969, Esposito et al 2001). Titanium's attractive qualities include superior corrosion resistance, mechanical strength and biological safety. The latter refers to titanium's relative lack of cytotoxic, carcinogenic or immunogenic properties (Williams, 2001). Since titanium satisfies the primary criteria as a biologically safe material capable of withstanding mechanical stress, the question then turns to titanium's role at a higher echelon of biomaterials research: tissue engineering. Williams defines this term as the persuasion of the body to heal itself through the delivery to the appropriate site of cells, molecular signals and supporting structures. In other words, researchers are determining whether implanted titanium surfaces can be modified to play a role in functional integration of devices and regeneration of the native tissues. With that goal in mind, alterations to titanium surfaces have been extensively researched (Brunette 2001, Lausmaa 2001). Modifications to the titanium surface are grouped according to two major themes. The first refers to changing the chemistry of the Ti surface by coating it with ceramics, proteins or antibiotics, whereas the second refers to modifying the physical topography of the titanium surface. The present thesis 4 focuses on the role of implant surface topography in determining the outcome of soft tissue healing. 3. Surface topography: methods used to alter surface texture The tissue response to an implant is in large part controlled by its surface texture. In contrast with smooth surfaces, textured surfaces offer more surface area for cell attachment as well as for tissue ingrowth (Pilliar, 1983). It can thus be inferred that surface roughness and mechanical interlocking play an important role in biologic performance. The role of surface topography is an important subject of investigation in implant dentistry. Endosseous dental implants are available commercially with many different surface configurations. These stem from the principle that bone tissue can adapt to surface irregularities in the 1-100 micron range, and that altering the surface topography of an implant can greatly improve its stability (Albrektsson et al, 2003). Creating a rough surface is one method to accomplish this goal. Textured titanium surfaces The increased surface area afforded by roughening provides greater bone-to-implant contact. It is this concept that has motivated the use of these topographies on commercial dental implants. The roughness of an implant surface can be quantified by studying the dimensions of its surface irregularities. Non-contact laser profilometry is one technique that involves the use of a laser beam to scan a surface and measure the vertical height of its irregularities. Roughness assessment is accomplished by moving the laser beam along the textured surface and measuring the 5 light that is reflected to photodiodes. As the light encounters surface irregularities, the reflected light will fall out of focus and be corrected by vertical movement of the objective (Vords et al, 2001). The movement is measured and used to calculate the average vertical amplitude, or R a value, for that surface (Wennerberg et al, 2003). It can be surmised that the greater the vertical height deviations, the rougher the surface. While amplitude parameters such as the R a value quantify the vertical dimensions of the surface irregularities, spacing parameters describe the horizontal space between the irregularities. The S m value represents the average horizontal distance between the peaks of the implant surface profile. The quantitative data are valuable in comparing the degree of surface roughness between different commercially available implants. A. Rough and porous surfaces Roughness may be created by rubbing the surface with an abrasive material, by blasting the surface with abrasive particles, by adding material to the surface, or by treating the surface with chemicals (Brunette et al, 2001). Surfaces are roughened in order to increase the tissue/implant contact and thus improve tissue integration. 1. Blasting: The titanium surface is bombarded at high velocity by rigid aluminum, glass or titanium particles. Upon contact, the particles remove material and create local deformations. The roughness of the blasted surface depends greatly on the size of particle used. For example, fine-blasting with small (150-230 um diameter) glass particles yields a relatively smooth surface with a R a value of 1.36 um. In contrast, coarse-blasting with larger alumina 6 particles (200-500 pm diameter) provides a much rougher surface with a R a value of 5.09 pm (Wieland et al, 2002). It should be noted that alumina particles used for blasting may become embedded into the implant surface, changing its chemical composition. The AstraTech dental implant system employs a surface blasted with titanium oxide particles (TiOBlast™) in order to sidestep this problem. 2. Acid etching: Etching refers to the treatment of a metal surface with an acid. The etching process dissolves the superficial oxide layer as well as part of the underlying metal, creating a rough surface. The most commonly used etchant is an aqueous mixture of nitric acid (HNO3) and hydrofluoric acid (HF), present in a ratio of 10:1 respectively. Alternatively, a 50:50 mixture of hydrochloric acid (HCI) and sulfuric acid (H2SO4) may be used. Acid etching results in a surface with a roughness value (Ra) value of 0.59 pm (Wieland et al, 2002). A commercial example of the acid-etched implant surface is the 31 dental implant (Osseotite™). 3. Plasma-spraying: The roughening of an implant surface may also be achieved by titanium plasma-spraying, a process where titanium powder is melted into liquid droplets and propelled onto the substrate in a concentrated and symmetrical spray. The droplets cool down and solidify into rigid particles, forming a rough coating. The plasma-spray process involves the use of an electrical current to create a jet of ionized, high-energy, high-temperature gas known as plasma. The gas jet acts as a heat source to melt the titanium particles fed into it which are subsequently accelerated and 7 deposited onto the implant surface (Gruner, 2001). The surface roughness obtained is dependent on the size, speed and temperature of the particles upon contact with the titanium surface. The titanium plasma-sprayed (TPS) surface carries a R a value of 5.85 urn (Wieland et al, 2002) and is found on the Straumann Institute's Bonefit™ dental implant. 4. Combination treatments: multi-cue surfaces Multi-cue surfaces are those subjected to multiple surface treatments in order to create secondary topographic features. For example, placing additional minor grooves within the walls of the major grooves of micromachined surfaces is one way to create a multi-cue surface. Another approach combines the use of "roughening" treatments, which is performed for the Straumann Institute's SLA™ implant surface. The SLA surface is produced by successive grit-blasting and acid-etching processes, yielding a R a value of 4.39 um (Wieland et al, 2002). In this case, any alumina surface contaminants introduced by the blasting process are removed by the etching process. It should be noted that the standard R a value may incompletely characterize the SLA surface. Even though the acid-etching process contributes additional "minor" surface roughness features to the previously blasted surface, these are overshadowed by the deeper irregularities created by the blasting process and therefore remain undetected by conventional roughness parameters such as R a. In other words, the blasted and SLA surfaces may have similar R a values despite their distinct surface topographies. The concept of roughness windows was introduced to enable 8 the identification of topographic features over various size ranges (Wieland et al, 2001). In brief, the raw data obtained from optical profiling is filtered into various wavelength ranges, allowing minor topographical features to be assessed independently. In the case of the SLA surface, the individual effects of the blasting and etching processes can be individually characterized (Voros etal, 2001). B. Machined surfaces The machining process modifies the titanium surface through cutting, milling or threading. Such manipulation produces an array of fine, parallel grooves and ridges oriented along the machining direction (Lausmaa, 2001). The machined surface carries a R a value of 2.15 pm (Wieland et al, 2002) and is found on the Nobel Biocare Branemark™ dental implant system. C. Micromachined surfaces A process known as micromachining is used to produce very precise micro-topographies. Grooves, pits or slots of specific shape, depth, and spacing can be chemically etched onto silicon surfaces. The microfabrication procedure is widely used in the fabrication of electronic components and entails several steps. First, the desired pattern is created on a glass template known as a mask plate. The glass plate consists of alternating transparent and opaque areas, dictated according to the desired pattern. As a result, light can pass through the plate at specific locations only. The silicon substrate is prepared as follows. Two layers are sequentially grown onto the silicon disk, a layer of silicon dioxide followed by a photosensitive material known as a photoresist. The chemical etching process 9 of the grooves then occurs in two stages. First, the mask plate is aligned with the substrate. By passing intense light through the glass mask plate, the photoresist is dissolved at specific sites according to the pattern dictated by the opaque areas, exposing the subjacent silicon dioxide at the planned groove spots. Using hydrofluoric acid, this selectively exposed silicon dioxide is chemically dissolved, which in turn selectively exposes the silicon substrate at the planned groove locations. The second stage of the etching process follows. After the remaining photoresist is removed, the selectively etched silicon dioxide layer represents a template in itself, and is known as an etch mask. Using potassium hydroxide, grooves are etched into the underlying silicon substrate according to the pattern dictated by the Si02 etch mask (Jaeger and Brunette, 2001). Finally, the patterned silicon disk can be coated with titanium for in vitro as well as in vivo experimental purposes. It should be noted that dental implants, along with all percutaneous devices, interface with epithelium and connective tissue. It is therefore of great interest to study the effect of implant surface roughening, machining and micromachining on soft tissue. II. I M P L A N T S IN SOFT TISSUE Teeth are natural examples of successful epithelium-penetrating structures and epitomize the desired relationship between implants and soft tissue. The soft gingival tissue surrounding teeth consists of an epithelial layer covering the subjacent connective tissue. The epithelium is attached to the tooth via hemidesmosomes and a basal lamina (Listgarten, 1966). Below this epithelial attachment, the tooth is 10 surrounded by a periodontal ligament, which consists of collagen bundles, known as Sharpey's fibers, inserting into the root cementum. It is stated in the periodontal literature that the intact connective tissue adhesion deters downward migration of the junctional epithelium (Stahl, 1977). The connective tissue attachment observed with teeth is often lacking around permucosal implants. Berglundh et al (1991) studied the interface between oral mucosa and implants placed in beagle dogs and reported that the collagen fibers within the connective tissue did not insert into the implant surface but instead were oriented parallel to the fixture. More recent studies by the Goteborg research group (Moon et al 1999, Abrahamsson et al 2002) studied the composition of the peri-implant connective tissue in greater detail. They reported that the highest proportion of fibroblasts are found within 40 pm of the implant surface and implied that this "cell rich zone" is involved in maintenance and repair of the peri-implant connective tissue. They also confirmed that connective tissue cells and libers are oriented parallel to the implant surface. One goal in improving implant-soft tissue integration is to determine how implant surfaces can be appropriately modified. It is therefore necessary to understand how epithelial cells and fibroblasts interact with titanium surfaces. 1. The interface between titanium implants and soft tissues Relatively few studies have described the soft tissue-implant interface in vivo. A major impediment to studying this interface at the cellular level has been the difficulty in obtaining thin histological sections (Rosengren et al, 1996) when solid metal implants are used in the experimental animals. Different techniques for 11 circumventing this problem have been presented over the years. The saw-and-grind technique entails cutting the implant along with its surrounding tissues into 100-150 um portions. The cutting procedure is followed by machine grinding to produce 5-10 jam sections (Donath et al, 1982). The saw-and-grind technique can introduce artifacts into the specimen due to the abrasive and vigorous nature of the manipulations. The thickness of the sections obtained may also limit the resolution of microscopic study. Later, another elaborate and time-consuming technique was introduced that involved electrochemical dissolution of the bulk metal, leaving the metal-tissue interface intact prior to sectioning (Bjursten et al, 1990). Finally, a third method uses plastic coated with a thin titanium layer for experimental implantation (Gould etal\9%\, Chehroudi et al 1989, Chehroudi et al 1992, Listgarten et al 1992). The use of plastic implants presents several advantages. Thin (1-2 urn) sections can be readily obtained since the plastic-bodied implants permit ideal sectioning. Artifacts are also reduced to a minimum. Moreover, it has been demonstrated that the surface topographies described in Section 1-3 can be reproduced faithfully onto epoxy resin replicas that can be coated with titanium. Furthermore, the surface chemistry of the titanium-coated replicas is similar to that of the pure titanium samples, allowing study of surface topography under constant chemical conditions (Wieland et al, 2002). Despite the relative paucity of morphological studies, data have been gathered to characterize the cells and tissues residing alongside titanium implants. 12 A. Epithelium-titanium interface Past studies have examined the epithelium adjacent to oral implants retrieved from dogs (McKinney et al, 1988), monkeys (Schroeder et al, 1981) and humans (Hansson et al 1983, Gould et al 1984). It has been found that epithelial cells attach to titanium through hemidesmosomes and basal lamina, very similar to the manner in which they attach to natural teeth. Although epithelium fulfills an important role by sealing teeth and implants from their harsh external environments, its tendency to proliferate along implanted devices can lead to their demise. The epithelial attachment around teeth is stabilized by an underlying connective tissue adhesion and periodontal ligament. Unfortunately, obtaining this stabilizing attachment to titanium implants has been largely unpredictable (Holgers et al, 2001). B. Connective tissue-titanium interface Connective tissue may be organized around implants in two ways. Typically, collagen organizes into a fibrous capsule, with fibers oriented parallel to the implant surface in a nonfunctional manner. This is commonly observed around implants with a polished titanium surface (Berglundh et al, 1991). It is also observed around subcutaneous implants. For example, fibrous capsule formation is one of the most common complications associated with breast implants (Kasper, 1994). A more desirable connective tissue organization would have collagen fibers and cells proliferating onto the implant surface forming a perpendicular or oblique orientation. A connective tissue attachment similar to Sharpey's fibers around natural teeth has been observed 13 around implants with a rougher, plasma-sprayed surface. A light and scanning electron microscopic study revealed that fibers inserted perpendicularly into the plasma-sprayed surface. Moreover, tensile forces at the tissue-implant interface avulsed titanium particles along with the connective tissue, suggesting a particularly firm attachment (Schroeder et al, 1981). The nature of the connective tissue attachment around percutaneous and permucosal implants can contribute to their long-term biologic success or failure. 2. Failure at the implant-soft tissue interface A major challenge facing percutaneous or permucosal implants is achieving long-term stability at the soft tissue interface (von Recum 1984, Jansen et al 1994, Heaney et al 1996). Permucosal or percutaneous devices integrated into bone, such as dental implants and some auditory prostheses, are more stable and are reported to have higher long-term success rates than those devices placed only in soft tissue (Jansen et al 1988, Jansen et al 1992, Gerritsen et al 2000, Parker et al 2002). Indeed, certain percutaneous implants, such as catheters for peritoneal dialysis, are not in the vicinity of bone. Such implants rely solely on their interface with soft tissues and fail more frequently for a number of reasons, including infection, avulsion, marsupialization and permigration (von Recum 1984, Knabe et al 1999, Holgers et al 2001). The latter two result from the aggressive proliferative behavior of epithelial cells. Marsupialization is caused by proliferation of the epidermis from each side of the percutaneous device. Since epithelial cells require a vital connective tissue bed for 14 locomotion, they penetrate the tissue near the implant, but do not contact its surface directly. The epithelium proliferates and migrates downwards, forming a sinus tract around the implant and excluding it from the body. Heaney et al (1996) also investigated the phenomenon of marsupialization. Using a percutaneous mouse model, they implanted smooth polyethylene implants that were not conducive to epithelial or connective tissue attachment. While notable epithelial downgrowth was observed adjacent to the implants, the authors found that the deep muscular tissue acted as a barrier to epithelial proliferation. Heaney et al argued that exploiting the deep connective tissue is more desirable for inhibiting marsupialization than modifying the implant surface. The views of Heaney et al stand in clear contrast to the majority view in the literature that favors the formation of an epithelial seal against the implant. For example, many authors modify surface texture in order to discourage epithelial downgrowth by allowing connective tissue infiltration into the implant (von Recum 1984, Jansen et al 1994). Pores that are less than 40 pm in diameter may be too small to foster optimal connective tissue anchorage, allowing epithelial cells to migrate into the pores and replace the connective tissue, a phenomenon known as permigration. The epithelial cells may eventually undermine the implant and produce a force vector that extrudes the implant outside the body. Previous research suggests that integration of connective tissue at the implant interface plays an important role in inhibiting downward epithelial proliferation (Chehroudi et al, 1992). A biocompatible material should thus be conducive to functional connective tissue organization at its surface. 15 Accordingly, the principles that govern fibroblast behavior on titanium substrata must be identified and understood at the cellular level. III. C E L L B E H A V I O R O N T I T A N I U M S U R F A C E S Several phenomena have been identified in the study of cell response to surface topography that play an important role in tissue organization at the implant interface (Brunette et al, 2003). Such phenomena include cell attachment, cell shape, haptotaxis and contact guidance. 1. Cell attachment Successful implant surfaces should encourage cell attachment. Fibroblasts do not appear to adhere directly to the implant surface and tend to attach indirectly through proteins that are adsorbed onto the surface upon implantation. In other words, host cells "see" the titanium surface through this layer of adsorbed proteins that ultimately serve as the adhesive medium for cell attachment (Brunette, 2001). Fibronectin and vitronectin are two major molecules involved in mediating cell behavior (Hakkinen et al, 2000). Therefore, it of great interest to know if altering the titanium surface would change the network of proteins adsorbed onto it. For instance, a surface topography that was more receptive to fibronectin adsorption might be more conducive to cell adhesion. Cellular protein expression at the interface appears to be affected by surface topography-induced cell shape changes (Chou et al, 1995). 16 2. Cell shape It has been proposed that surface topography induces cytoskeletal changes in cells that in turn alter cell shape. For example, epithelial cells cultured on grooved surfaces have demonstrated a rounder shape than when cultured on smooth surfaces (Hong and Brunette, 1987). A computer-assisted, three-dimensional survey of epithelial cells on a percutaneous implant found that they assume a relatively flat morphology and are more spread on smooth substrata (Chehroudi et al, 1995). For their part, human gingival fibroblasts also demonstrate topography-related shape changes. For example, they show a greater cell height when cultured on grooved surfaces than on smooth surfaces (Oakley and Brunette 1993, Chou et al 1995). They also tend to be more flattened on machined titanium than on plasma-sprayed surfaces. Alterations to cell shape are known to affect cell growth, cell differentiation, gene expression and enzyme secretion. The effect of cell shape on molecule expression has been studied in several cell types, namely epithelial cells (Hong and Brunette 1987, Watt et al 1988), chondrocytes (Newman and Watt, 1988) and human gingival fibroblasts (Chou et al 1995,1996, 1998). Newman and Watt (1988) used chemical means to alter the shape of chondrocytes and found that this stimulated their proteoglycan synthesis, a process integral to cartilage formation. Chou et al (1995) found an increased expression of fibronectin, an important attachment protein, by human gingival fibroblasts when cultured on grooved substrata, compared to a smooth control. The increase in fibronectin expression was attributed to altered cell shape, as the fibroblasts assumed a more elongated shape on the grooved surfaces. 17 Therefore, the ideal topographic modifications should alter fibroblast shape in vivo in a way that maximizes their expression of adhesion molecules, increasing their probability for attachment. 3. Haptotaxis Haptotaxis refers to preferential cell movement in an adhesive gradient (Carter, 1967). When an implant is placed into tissue, this adhesive gradient should ideally attract cells to the implant surface, rather than to each other. An adhesive gradient away from the implant surface would result in cells aggregating together, forming a thick fibrous capsule separating the implant from the surrounding tissue. As with the other cell behavior phenomena, exploiting the principle of haptotaxis can be approached in two ways. First, the titanium surface can be chemically treated prior to implantation so as to optimize the adsorption of adhesion molecules. Sauberlich et al (1999) found that coating titanium surfaces with fibronectin prior to culturing with human gingival fibroblasts enhanced their adhesion and growth. In an in vivo study, Rosengren et al (1996) found fibronectin and fibroblasts closer to titanium implant surfaces when compared to PTFE controls. The second type of surface modification involves its topographic features. In addition to inducing appropriate cell shape changes, the ideal surface topography would maximize fibroblast affinity by favoring the adsorption of proteins such as fibronectin from the extracellular environment. 18 4. Contact guidance The principle of contact guidance states that cells on a substratum respond to the underlying surface topography, first described by Weiss and Taylor (1956). Indeed, epithelial cells and fibroblasts cultured on grooved surfaces become oriented with the grooves (Rovensky et al 1971, Dunn and Heath 1976, Brunette et al 1983, Dunn and Brown 1986, Curtis et al 1990, Abiko and Brunette 1993, Curtis et al 1998). In addition, the cells do not usually move across these grooves. For example, Brunette et al (1983) found that cultured epithelial cells from human gingival explants did not bend around the ridges of the underlying grooved substrata. Contact guidance can be exploited to help restrict epithelial downgrowth, a major cause of percutaneous and permucosal implant failure (Chehroudi et al, 2002). It has been demonstrated that horizontally oriented grooves on percutaneous implants are in fact capable of inhibiting vertical epithelial proliferation in vivo (Chehroudi et al, 1989). Moreover, Chehroudi et al (1992) suggest that grooved surface topography may promote connective tissue attachment, which can also inhibit downgrowth of the overlying epithelium. The grooves may encourage the interlocking and perpendicular orientation of fibroblasts with the implant surface, analogous to the periodontal ligament around teeth. Organized in this way, the connective tissue may help impede epithelial downgrowth along the implant surface, analogous to the role played by Sharpey's fibers around natural teeth. 19 5. Fibroblast organization into connective tissue Fibroblast organization can be affected by numerous factors such as the adhesive gradient of their environment, mechanical stimuli and chemical cues. Since fibroblasts and myofibroblasts show significant contractility, it is plausible that they be capable of stretching and arranging their extracellular collagen matrix (Harris, 1999). It has been demonstrated that in this process, fibroblasts produce tracts of cells and fibers around them. Weiss described this phenomenon as the two-center effect to describe the cellular bridges formed between two tissue explants cultured in plasma. Fibroblast contraction causes the alignment of fibers between the explants, allowing other cells to migrate along the fibers. Cellular contraction is important for rearranging and realigning collagen fibers during the development of ligaments, tendons and muscles (Harris et al 1981, Stopak et al 1985, Harris, 1987). Since connective tissue is organized along the direction of fibroblast traction, implant surfaces should encourage a fibroblast orientation that is oblique or perpendicular to the implant. Controlling cell orientation thusly would favor functional connective tissue organization and ingrowth, in contrast to the commonly observed fibrous encapsulation with cells and fibers oriented parallel to the implant surface. In summary, surface topography may guide fibroblast orientation and thus the direction in which they organize their extracellular matrix. Moreover, their expression of important adhesion molecules such as fibronectin (Abiko et al 1993, Chou et al 1995) may also differ between smooth and textured surfaces. Surface topography may thus affect cell adhesion, cell orientation and the direction of the 20 cells' fractional forces, ultimately affecting the overall tissue organization (Brunette et al, 2003). These cellular events occur as part of the wound healing process. I V . W O U N D H E A L I N G : G e n e r a l p r i n c i p l e s 1. O v e r v i e w Implant placement is an invasive procedure that inevitably creates tissue injury. After any such injury, a host response takes place to contain the damage and reconstitute wounded tissues. A blood clot forms immediately after the surgical insult, followed by an inflammatory period where blood vessels become more permeable to fluid and host immune cells remove dead matter by phagocytosis. The clot is replaced by an immature and highly vascular granulation tissue, which undergoes contraction and remodeling with collagen deposition. The healing process ends with the formation of a fibrous scar in the case of dermal wounds, or the formation of a fibrous capsule around implanted materials. While dermal scar formation and fibrous encapsulation of implants typically occur following the inflammatory phase of normal wound healing, scar tissue should be regarded as a distinct entity. For instance, scar tissue may form in the absence of inflammation. In such cases, excessive accumulation of extracellular matrix components is believed to cause the scar tissue (Le et al, 2004). Furthermore, a histological distinction can be drawn between fibrous capsules and scar tissue. While the fibrous capsule habitually consists of collagen fibers and fibroblasts elongated parallel to the implant surface, the histological presentation of scar tissue can vary considerably. Collagen fibers may be oriented randomly in a "basket-weave" pattern or be oriented into thick parallel bundles, depending on the 21 severity of the scarring response. This range of histological features forms part of the criteria used to assess the degree of scarring (Beausang et al, 1998). 2. The phases of wound healing The entire wound healing process may be categorized into 3 overlapping phases of events (Clark et al, 1996). 1. Blood clot formation and inflammation The first response to a surgical wound is the formation of a blood clot rich in fibrin and fibronectin (Bartold et al, 1998). The clot provides stability to the healing wound and is a scaffold for migrating cells. The ensuing inflammation represents a key phase. It involves the movement of fluid and leukocytes from the bloodstream to the wound site. Their function is the removal of bacteria and damaged tissue via phagocytosis. The inflammatory phase is mediated by polymorphonuclear leukocytes, monocytes, lymphocytes and platelets. Meanwhile, epithelial cells from the wound margins migrate underneath the fibrin clot to cover the underlying connective tissue (Bartold et al, 1998). 2. Formation of granulation tissue and neovascularization The initial fibrin clot serves as a framework for the subsequent healing phase, the formation of an intermediate connective tissue matrix known as granulation tissue. Three to five days after injury, the fibrin clot is destroyed in favor of a highly vascular granulation tissue. New blood vessels are formed through a process known as angiogenesis, where endothelial cells from 22 existing vessels bud into new vessels. The new vessels are highly permeable, allowing fluid and blood exudation leading to pronounced erythema and edema at the wound site (Bartold et al, 1998). 3. Remodeling and collagen synthesis The later stage of wound healing is characterized by matrix deposition and tissue remodeling, often creating fibrous scar tissue. Undifferentiated mesenchymal cells as well as fibroblasts migrate into the wound granulation tissue. Cell migration is mediated by integrins, molecules located on the cell surface that allow communication between the cell and the extracellular matrix. Integrin expression by cells appears to increase during wound healing (Larjava et al, 1993) and likely plays a key role in matrix deposition. Fibroblasts may undergo transformation into myofibroblasts, which exhibit features of both fibroblasts and muscle cells (Darby et al, 1990). Myofibroblasts are responsible for collagen synthesis and wound contraction. Following this contraction, the wound tissue shrinks to 5-10% of its original size. The granulation tissue becomes less vascular and is transformed into mature fibrous tissue. Collagen synthesis ensues, marked by the deposition of hyaluronic acid, chondroitin sulfate and dermatan sulfate. Type III collagen predominates during the early stages, type I collagen prevails later. Collagen synthesis peaks at 7-14 days after wounding but is an ongoing process that continues until tensile strength of the wound is restored to its original state (Bartold et al, 1998). 23 V . S O F T T I S S U E H E A L I N G A R O U N D T I T A N I U M I M P L A N T S After titanium is implanted into soft tissues, the healing response follows a pattern analogous to that described above: inflammation followed by matrix deposition and remodeling. Initially, acute inflammation and fluid accumulation are observed, with protein adsorption on the implant surface. The inflammatory period is followed by the formation of granulation tissue around the implant, which is a scaffold for macrophages, fibroblasts and endothelial cells. Finally, this granulation tissue is remodeled into a fibrous capsule consisting of fibroblasts, macrophages and collagen. A. Early healing (1-5 weeks') The acute inflammatory phase involves increased vascular permeability and an exudation of proteins, fluid and inflammatory cells from the blood vessels into the tissue. A fluid space is created adjacent to the implant surface (Johanssen et al, 1992), containing proteins such as albumin, complement factor 3, immunoglobulins, fibrinogen and fibronectin (Rosengren et al, 1994). Polymorphonuclear cells (PMN's) make up most of the leukocyte population during the first 24 hours, with their numbers gradually decreasing in favor of lymphocytes and macrophages (Eriksson et al, 1994). Macrophages perceive the implant as a foreign body and attempt to engulf it, however the size of the implant makes this impossible. The "frustrated phagocytosis" incites the macrophages to fuse and form giant cells. Since the giant cells will also be unable to engulf the implant, fibroblasts will be attracted to the implant site at approximately one week after implantation. Macrophage-derived growth factors likely play a role in fibroblast proliferation and collagen synthesis (Zeller 1983, Song et al 2000, 24 Thomsen and Gretzer 2001), and before the third week of healing, the fibroblasts will finally succeed at surrounding the implant with a fibrous capsule (Anderson 1996, Rosengren et al 1998, Jansson et al 2001). The peri-implant connective tissue consists of fibrin strands parallel to the implant surface and distinctly bordered from the fluid space. The fibrin strands serve as an attachment scaffold for macrophages and other fibroblasts. B. Later healing (6 weeks) After 6 weeks of implantation, the fluid space is absent (Rostlund et al, 1990). The formation of the fibrous capsule is established. Fibroblasts are the predominant cells in the capsule (Holgers et al, 2001). Closer to the implant, macrophages predominate and are in contact with the implant surface (Johanssen et al, 1992). The macrophages may persist along the interface for years after implantation (Anderson 1988, Thomsen and Gretzer 2001). The thickness of the fibrous capsules has been reported to range from 50 to 250 pm (Tengvall 2003), yet there is contention in the literature concerning changes in capsule width over time. Some studies report a decrease in capsule width over time (Rosengren et al, 1997), others report an increase (Jansson et al, 2001), while still others report no change (Rosengren etal, 1998). VI. T H E FIBROUS C A P S U L E 1. Significance of capsule width The dimensions of the fibrous capsule are significant in that they may reflect upon the implant's biocompatibility and long-term success. The thickness of this capsule is 25 inversely related to biocompatibility (Jansen et al, 1994) and is also a reflection of the degree of chronic inflammation surrounding the implant (Eltze et al, 2002). Such a relationship is understandable given that the capsule represents the host's attempt to exclude the implant (Ungersbdck et al, 1994). Indeed, excessive peri-implant fibrosis is associated with three major problems. Firstly, thick capsules have been implicated in foreign body-related carcinogenesis in rodents. Since thick capsules are highly cellular and densely populated with collagen bundles, they are associated with insufficient blood supply. As a result, altered cell growth may occur due to insufficient nutrition and hypoxia, especially for those cells closest to the implant surface (Kordan 1967, James et al 1997, Kirkpatrick et al 2000). Secondly, the implant's mechanical stability may be compromised. A thick, nonadherent fibrous capsule may separate from the implant surface, resulting in implant loss (von Recum, 1992) . It should be noted that there may a two-way relationship between mechanical stability and capsule formation. Some studies have suggested that micromovement propagates a continued inflammatory response to soft tissue implants (Meyle et al, 1993) and contributes to capsule formation (Rosengren et al 1999, Ungersbdck et al, 1994) . Holgers et al (1995) microscopically examined human biopsies of percutaneous auditory prostheses. The lack of fibrous capsule formation around these implants was attributed to the firm fixation of the implant into bone. In another study, Jacob et al (1996) reported increased cellular ingrowth and decreased fibrous encapsulation around porous versus solid ophthalmic implants. The authors reported that cellular ingrowth into the porous implants probably improved implant fixation, whereas the absence of ingrowth into the solid implants allowed continued 26 micromotion, resulting in a prolonged inflammatory period. Rosengren et al (1999) studied polyethylene implants placed in rats and found increased cell necrosis in areas of mechanical shear. The cell necrosis was correlated to increased capsule formation around the implants. The potential mechanical instability caused by thick capsules is a suspected etiological factor for a third problem: impaired host defense. Motion between a thick, non-adherent capsule and the implant may allow the implant to be surrounded by a liquid film, which in turn can enhance the spread of bacteria and impair the body's ability to mobilize immune cells to the implant surface (Richards, 1996). On the other hand, when a thin, tightly adherent capsule is present, no such fluid space is present. Moreover, host blood vessels are more likely to exist close to the implant surface, suggesting that both vascularization and host defense are improved (Ungersbock et al, 1994). Beyond the quantitative assessments of width, the biocompatibility of a capsule can also be evaluated qualitatively in terms of its constituents. 2. Capsule composition and biocompatibility Qualitative methods have been proposed to evaluate the quality of the fibrous capsule (Jansen et al 1994, Parker et al 2001). The biocompatibility of soft tissue implants was assessed based on two major criteria. The first parameter, capsule quality, was evaluated based on its histological resemblence or dissemblance to the original tissue. The "better" capsules were described as thin, mature and histologically similar to the 27 connective tissue in the non-injured regions. Conversely, poorer quality capsules were described as thick, immature, and heavily populated with inflammatory cells. The second parameter assesses the characteristics of the cells that are in direct contact with the implant surface. According to Jansen (1994), the most biocompatible implants allow for direct fibroblast contact without the presence macrophages or giant cells. The quality of the tissue interface is assessed as poor when high numbers of inflammatory cells are observed. The thickness and quality of the capsule formed is a function of the entire wound healing process, including inflammation, cell-cell interactions, cell-matrix interactions and tissue organization. It is hypothesized that cellular and molecular events at the implant surface during the early phases of healing control the subsequent tissue manifestations (Tengvall, 2003). In the pursuit of the best implant surface for soft tissue healing, the effects of surface texturing on fibroblast activity and fibrous capsule formation should be addressed. VII . S U R F A C E T O P O G R A P H Y A N D SOFT TISSUE R E S P O N S E 1. In vitro studies of surface topography and fibroblast behavior Surface texture is a decisive factor in controlling cell attachment and orientation. Different surfaces have been studied in terms of their in vitro effects on fibroblast behavior, including randomly roughened (Inoue et al 1987, Lowenberg et al 1987, Cochran et al 1994) and microfabricated substrata. The use of the latter allows precise control over the shape and dimensions of the surface features. Microfabricated 28 grooves appear to favor fibroblast adhesion in vitro. Abiko and Brunette (1993) assayed tracks of fibronectin left by cultured fibroblasts on a grooved surface. The fibronectin tracks were aligned with the grooves, presumably reinforcing the cells' alignment as well. In another study, fibroblasts demonstrated extension of their cellular processes into the microgrooves, leading to mechanical interlocking (Meyle et al, 1993). Moreover, Jain et al (2003) assessed fibroblast attachment by measuring the distance between the titanium substrate and apposed cells. They found that this cell-substrate distance was smaller (0-0.4 nm) on microgrooved titanium when compared to smooth titanium, suggesting that fibroblasts are in more intimate contact on grooved surfaces. Microgrooved substrata are also strong tools for determining cell orientation, as cultured fibroblasts tend to become oriented along the grooves (Brunette 1986, den Braber et al 1996, Walboomers et al 1999). Others have studied fibroblast orientation to randomly roughened surfaces. Studies by Inoue et al (1987) and Lowenberg et al (1987) examined the orientation of human gingival fibroblasts cultured on porous versus smooth or machined titanium discs. In these studies, porous surfaces were produced by sintering titanium-aluminum-vanadium alloy particles to solid Ti discs. Cellular bridges formed between the cultured fibroblasts and the titanium discs, typically oriented parallel to the smooth-surfaced substrate, forming capsule-like structures. On the porous surface, the cellular bridges formed at right angles to the substrate, suggesting that the rough surface is more conducive to a ligament-like attachment. However, while in vitro studies of implant surfaces in cell 29 culture suggest beneficial effects of surface texturing, they do not necessarily predict tissue response to these surfaces in vivo. 2. Surface topography and fibrous capsule formation Capsule formation: role of the macrophage The formation of the fibrous capsule around an implant involves matrix deposition and organization by fibroblasts. In this process, macrophages are thought to act as messenger cells for the fibroblasts, as they are among the first cells to populate the implant surface. In addition, the macrophage produces many biologically active molecules, including interleukin-1 (IL-1), tumor necrosis factor (TNF), transforming growth factor (TGF-p), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), all of which may act as signals for the fibroblasts (Anderson, 2000). It is known that macrophages have an affinity for rough surfaces, first demonstrated by Rich and Harris (1981) who used roughened glass rods as their experimental substrata. The findings of Rich and Harris were confirmed for titanium by Soskolne et al (2002) who reported that more monocytes adhered to rough, blasted Ti surfaces than to smoother, machined surfaces. It is thus reasonable to suggest that the surface topography may affect capsule formation through its interactions with macrophages (Thomsen 2000, Thomsen and Gretzer 2001). However, the macrophage's specific roles in fibrogenesis remain unclear. Macrophages can exert both positive and negative effects on fibrogenesis (Song et al, 2000). Growth factors such as TGF are 30 believed to encourage fibroblast proliferation and matrix production while matrix metalloproteinases have the opposite effect (Bartold et al, 1998). Therefore, the question is how surface topography changes the profile of cytokines secreted by the macrophages. Recently, Refai et al (2004) found that cultured macrophages expressed greater amounts of IL-lp\ IL-6 and TNF-a on an etched and blasted surface (SLA) than on smoother substrata, implying that surface topography influences cytokine production. However, the exact role of the macrophage in peri-implant fibrosis in vivo is presently unknown, although several studies have compared the degree of fibrous encapsulation observed around implants with different surface topographies. 3. In vivo studies of surface topography and fibrous capsule formation Materials such as silicone, polyethylene and titanium have been textured to investigate their integration in connective tissue in vivo. However, there are several aspects of these studies that are problematic. Comparisons of surface topography should be under conditions of constant surface chemistry. However, the exact surface chemistry is often unknown even when textures of the same material are being compared. A solution to this problem was offered when Wieland et al (2002) demonstrated that titanium-coated epoxy resin replicas of various surface topographies had nearly identical surface chemistries. A second problem is that histomorphometric measurements of capsule width are difficult to perform when the boundaries of the fibrous capsule are unclear (Holgers et al, 2001). Moreover, many studies employ non-specific staining methods that do 31 not provide insight into all of the constituents of the fibrous capsule (Thomsen et al 1986, Rostlund et al 1990, Chehroudi et al 1992, Ungersbock et al 1994). Finally, comparisons among the various studies can be difficult because they utilize different species (mice, rats, rabbits, dogs, sheep) and different implantation sites (subcutaneous, intramuscular, abdominal wall, intraperitoneal) (Holgers et al, 2001). Comparisons of fibrous capsule formation between surfaces As is the case for in vitro research, the surfaces implanted for in vivo study have typically employed different types of textured surfaces. The first type uses random roughening processes found on commercially available implants, such as grit-blasted (Ungersbock et al 1994, 1996) or plasma-sprayed (Schroeder et al 1981, Listgarten et al 1992). Ungersbock et al (1994, 1996) reported that blasted titanium surfaces were surrounded by thinner, more adherent fibrous capsules than smooth surfaces. Their findings are consistent with studies that report a perpendicular, ligament-like orientation of fibroblasts on rough substrata, both in vitro (Inoue et al, 1987) and in vivo (Schroeder et al, 1981). In addition, Rosengren et al (1999) found thinner fibrous capsules around randomly roughened polyethylene surfaces and fewer inflammatory cells at the interface. They suggested that the chronic inflammatory response that leads to thick capsule formation is somewhat attenuated around roughened implant surfaces. A second type of textured surface is microfabricated. Microfabricated surfaces have been treated with regular patterns of grooves (Chehroudi et al 1992, Parker et al 32 2001, den Braber et al 1997, Walboomers et al 1998), pillar-like structures (Picha et al 1996) or pores (Campbell et al 1989). Microfabricated substrata are not available on commercial implants, but are used for research purposes owing to the precision of their surface features. Studies by Chehroudi et al (1992) report thinner fibrous capsule formation and ingrowth of fibroblasts on grooved titanium substrata when compared to smooth implant surfaces. In contrast, when den Braber et al (1997) and Walboomers et al (1998) examined, in two separate studies, microgrooved polystyrene and silicone implants, no differences in capsule thickness were noted compared to smooth surfaces. The discrepancy could be attributed to their use of 1-um grooves that may not have been sufficiently deep to achieve mechanical tissue interlocking (den Braber et al, 1997). The importance of feature size is also addressed by von Recum et al (1995). Their studies propose that capsule thickness is reduced on porous surfaces when the pores are of 1-2 jam in diameter, whereas larger or smaller pore sizes show a greater inflammatory response with thick fibrous capsule formation. The results lead to speculation that there may be an optimal size of surface irregularities for cell attachment and tissue organization to occur (von Recum et al, 1995). It may be argued that such optimal surface features may be obtained through random roughening surface treatments, such as acid etching, blasting or a combination thereof. 33 A i m s of this project The ideal connective tissue attachment to an implant would be associated with a fibrous capsule of minimal thickness and with fibroblasts organized in a manner suggestive of a ligament-like attachment. It has been demonstrated by Chehroudi et al that surface grooves deeper than 19 (am are capable of allowing this over a short period (2-3 weeks). In this thesis, our interest is to examine the connective tissue response to well-defined surface topographies, including some found on commercial implants. In light of the previous findings by Schroeder et al (1981) and Ungersbdck et al (1994) that used plasma-sprayed and blasted substrata respectively, our hypothesis is that other roughened surfaces such as SLA will also foster an improved connective tissue attachment with thinner fibrous encapsulation. Using subcutaneous implantation in a rat model, titanium-coated surfaces with varying degrees of roughness were compared in terms of (a) their ability to foster soft tissue attachment, (b) the thickness of the fibrous capsule formed around them and (c) the relative degree of tissue-implant separation in areas where detachment occurred. 34 Chapter One Bibliography: 1. Abiko Y, Brunette D M . Inimunohistochemical investigation of tracks left by the migration of fibroblasts on titanium surfaces. Cells Mater 1993 ;3:161-168. 2. 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Titanium for hip and knee prostheses. In: Brunette D M , Tengvall P, Textor M , Thomsen P (eds) Titanium in Medicine (2001):703-746. Springer-Verlag, Berlin Heiderberg. 128. ZellerJMS. Surgical implants: physiological response. AORN Journal 1983 ;37(7): 1284-1291. 51 Chapter Two - Manuscript I. Introduction The use of implanted devices to replace organs or to restore their function has increased over the years on account of the continuing occurrence of injury and disease as well as an aging population. It is therefore desirable to thoroughly understand the principles that govern the tissue-implant interface. After implant placement, a wound healing response occurs that is characterized by blood clotting and inflammation in its early stages, followed by matrix deposition and tissue remodeling in its later stages (Bartold et al, 1998). Ideally, this wound healing process should lead to integration of the implant into the body tissues. Among metallic implant materials, titanium and its alloys have garnered a great deal of interest for orthopedic and dental applications on account of their mechanical strength, durability and relative biological safety. Most studies of titanium have focused on integration with bone (Branemark et al 1969, Deporter et al 1988, Buser et al 1999) but the in vivo soft tissue response to titanium has received comparatively less attention, despite the requirement that titanium devices such as dental implants interface with gingival connective tissue and epithelium. Epithelium fulfills an important role in implant function by forming a seal for percutaneous and dental implants from their harsh external environments (Schlegel et al, 1978). However, epithelium's tendency to proliferate along an implanted device can create sinus tracts that undermine the implant and can lead to its loss. Aggressive epithelial downgrowth can be impeded by a firm attachment between the soft connective tissue and the implant, with cells and fibers attached to the implant surface, as is the case for 53 Sharpey's fibers around natural teeth (Chehroudi et al, 1992). In health, collagen bundles insert into the root cementum; the connection deters downward migration of the overlying epithelium (Stahl, 1977). Unfortunately, connective tissue does not usually attach to titanium substrata in this manner (Sauberlich et al 1999, Holgers et al 2001). Instead, connective tissue typically becomes organized into a fibrous capsule, with collagen fibers oriented parallel to the implant surface. The presence of this fibrous capsule often denotes a failure of the implant to integrate with the host tissue. In addition to compromising local blood supply, thick fibrous capsules are associated with a persistent fluid space between the tissue and the implant, precluding stable implant fixation (Ungersbdck et al, 1994). A promising approach to optimizing tissue-implant integration involves modification of the implant surface. Specifically, altering the topographical features of the implant surface is known to modulate cell behavior. The ideal implant surface would therefore promote maximal connective tissue attachment while minimizing fibrous capsule formation. The effects of surface texturing on fibroblast attachment have been documented both in vitro (Inoue et al 1987, Lowenberg et al 1987, den Braber et al 1996) and in vivo (Schroeder et al 1981, Chehroudi et al 1992, Ungersbdck et al 1994, Rosengren et al 1999). Moreover, a multitude of surface modifications have been studied, including microfabricated grooves of varying dimensions. The results suggest that there may be an ideal type and size of surface irregularity for cell attachment and tissue organization to occur (von Recum et al, 1995). It is also possible that treatments that 54 roughen the surface, including those that are presently used on commercially available dental implants, may be effective for soft tissue integration. These include, but are not limited to processes such as sintering, plasma-spraying, acid etching, grit blasting or a combination of the preceding. The aim of the present in vivo study was to evaluate the soft tissue response to commercially available implant surfaces in a subcutaneous rat model using the histomorphometric parameters of attachment, capsule thickness and tissue-implant separation. The greatest attachment and thinnest fibrous encapsulation were found around the roughest surfaces, whereas the smoothest substrata were associated with the poorest attachment and the thickest fibrous capsules. 55 II. Materials and Methods 1. Preparation of the titanium surfaces Seven of the eight surface topographies used in this investigation were replicated from disks (15 mm diameter, 1 mm width) of grade 2 commercially pure titanium. The topography of the surfaces was defined and manufactured as follows: (1) Machined-like surface: The Ti disks were treated with 60-grit SiC grinding paper to produce a striated surface that resembled a machined surface. (2) Polished surface: The Ti disks were treated with 60-grit SiC grinding paper, then polished to a mirror finish with a 10-um diamond paste in oil and a 0.06 um silicon dioxide suspension. (3) Finely-blasted: The Ti surface was blasted with 150-230 um diameter glass beads. (4) Coarsely-blasted: The Ti surface was blasted with 200-500 urn diameter alumina particles. (5) Acid-etched: The Ti surface was treated in a hot solution of HCI/H2SO4. (6) Coarsely-blasted and acid-etched (SLA) surface: Obtained by blasting the Ti surface with 250-um diameter alumina particles prior to etching with HCI/H2SO4. (7) Titanium plasma-sprayed (TPS) surface: Obtained by blasting the Ti surface with 250-um diameter alumina particles and plasma-spraying the surface with Ti hydride powder. The roughness of the above surfaces was quantified by non-contact laser profilometry as reported by Wieland et al (2002). Briefly, optical instruments are used to measure 56 the height deviations, or amplitude, of the surface irregularities. They are then are averaged to determine the R a value for that surface. Table 1 shows the R a values of the surfaces used in this experiment. 2. Micropatterning of the silicon surface The eighth surface was produced by etching parallel grooves onto silicon substrata using a technique first described by Camporese et al (1981). In brief, following sequential protection of the silicon substrata with a layer of silicon dioxide and a photosensitive agent, the surface is exposed to intense light through a computer-generated photomask. The light dissolves the photosensitive coating at the planned groove sites, selectively exposing the underlying SiC^-coated silicon substrata. Next, the exposed portions of the Si02 coating are chemically dissolved, in turn selectively exposing the silicon according to the desired pattern. Finally, the silicon surface is anisotropically etched to form V-shaped grooves (Jaeger et al, 2001). The array of grooves obtained were 30-um deep, 35-um wide, with a pitch of either 40 or 45 pm (Figure 1). 3. Surface replication and titanium coating All topographies were replicated onto epoxy resin to form exact duplicates of the original surfaces. The method of replication has been shown to accurately reproduce the original surface textures (Chehroudi et al 1992, Wieland et al 2002). In brief, impressions were made of the titanium and silicon surfaces using vinyl polysiloxane impression material (PVS). Epoxy resin was then poured into the impressions to 57 obtain the replicas. Replicas were cleaned with 7X detergent (ICN Biomedicals) and washed with distilled water twenty times. After 30 minutes of ultrasonication, the epoxy replicas were sputter-coated with 50 nanometers of titanium using the Randex 3140 Sputtering System and glow-discharged in an argon gas chamber for 3 minutes. From the titanium-coated replicas, U-shaped devices were fabricated for subcutaneous implantation. Each implant consisted of two vertical posts connected at their base by a pedestal (Chehroudi et al, 1988). Each post had two opposing Ti-coated implant surfaces. Therefore, a single implant exhibited four test surfaces (Figure 2 ) . 4. Implantation procedure The surgical procedure used in this study has been described previously (Chehroudi et al, 2002). Male Sprague-Dawley rats weighing 350-500 g were intubated and anesthetized with halothane. The fur in the parietal area was shaved and treated with a depilatory. The underlying skin was scrubbed with Betadine for 1 minute, then with 70% ethanol for 1 minute. An access incision was made between the two ears. The parietal bone was smoothed with a file to ensure that the implant rested firmly on the surface. The implant was fixed to the bone with miniature titanium screws. The access incision was sutured with 5-0 silk and animals received a prophylactic antibiotic combination of penicillin G and streptomycin (Pen-Di-Strep). 58 Animals were sacrificed at weekly intervals up until 11 weeks post-implantation. After perfusion with 2.5% glutaraldehyde, the implants were removed together with the parietal bone and placed in Karnowsky's fixative for 24 hours at 4°C, followed by 2% buffered osmium tetroxide for 4 hours. The specimens were dehydrated in a graded water miscible resin (Aquembed), then infiltrated with graded Aquembed/Epon, and finally embedded in Epon. 5. Histology and histomorphometric analysis Using a Sorvall MT2 microtome, 2-um thick sections were cut parallel to the long axis of the implant, stained with 1% toluidine blue and examined under a light microscope. Photomicrographs were taken using a Canon EOS D60 digital camera. Measurements were made using NIH Image 1.63 software. A. Percent attachment Low-magnification (32x) photographs were used to capture the entire implant length. The length of the implant surface and the area of attached tissue were measured. The attachment was expressed as a percentage of the total implant length (Figure 3a). % attachment = length of attached tissue / total implant length B. Capsule thickness and tissue-implant separation The entire length of the implant was divided into three zones, each representing one-third of the implant length: (1) the base (pedestal) of the implant, (2) the middle third of the implant and (3) the apex (area closest to the skin). For each zone, a photomicrograph was obtained at higher 59 magnification (lOOx) in order to measure capsule thickness and tissue-implant separation (Figure 3a). In each zone, the thickness of the fibrous capsule was measured at 5 points along the implant length, 200 um apart. Measurements of capsule thickness were therefore performed 15 times per section. In areas of tissue detachment, measurements of the distance between tissue and implant were performed and repeated in the same manner as for capsule thickness (Figure 3b). 6. Statistical analysis The data were grouped into two time periods: (1) the initial healing stage (weeks 1-5) and (2) the late healing stage (weeks 6-11). A multivariate analysis of variance (MANOVA) and Bonferroni post-hoc multiple comparison tests were used to assess the parameters of percent tissue attachment, capsule thickness and degree of tissue-implant separation as functions of surface type and time. The null hypothesis was rejected at p<0.05. 60 Table 1. Ra values of the surfaces used in this investigation SURFACE R a value (um) Polished 0.06 Acid-etched 0.59 Finely-blasted 1.36 Machined 2.15 SLA 4.39 Coarsely-blasted 5.09 TPS 5.85 35 nm 40/45 um 4 H Figure 1. Schematic representation of the micromachined grooved surface. The V-shaped grooves were 30-pm deep, 35-pm wide, with a pitch of either 40 pm or 45 pm. 62 Figure 2. Schematic representation of implant. There were four (4) test surfaces per implant. 63 Figure 3. Schematic representation of morphometric parameters. Fig. 3a (left): the attached tissue was assessed as a percentage of the total implant length. Note the 90° angle between the test surface and the implant pedestal (P). The implant was divided into 3 zones for study at higher magnification. Fig. 3b (right): View of a single implant zone. The thickness of the fibrous capsule was measured 5 times per zone (15 times per implant), as was the distance between tissue and implant in areas of separation. 6 4 III. Results Obtaining histological sections from the implant-tissue interface proved to be a challenging task. The different hardness values of the embedding resin, epoxy implant and titanium coating resulted in wrinkles, tissue separation and scratch marks on many sections, making them unusable for histological and morphometric analysis. These problems were addressed by adjusting the speed of the sectioning and the angle of the glass knife. Nevertheless, numerous histological samples had to be discarded on account of such artifacts. Another difficulty arose from the large and thick tissue samples which frequently prevented adequate resin infiltration. Excessive volumes of tissue were meticulously trimmed under a dissecting microscope prior to embedding. Some samples were also re-embedded. Table 2 shows the number and fate of all test surfaces. 1. Histological observations A great number of the samples showed connective tissue in contact with the surface along part of the implant length. The healing pattern appeared to be similar with all surfaces. Early healing was characterized by the accumulation of fibrin-rich tissue seeded with undifferentiated mesenchymal cells, neutrophils, lymphocytes and monocytes. By the first week of healing, elongated fibroblasts appeared near the implant surface, along with numerous blood vessels. On the micromachined grooved surface, fibroblasts were often inserted into the grooves (Figure 4a). On the other substrata, fibroblasts were typically elongated parallel to the implant surface and organized into a multilayered fibrous capsule (Figure 4b). The thickness of the 65 capsule appeared to decrease with time on most surfaces (Table 5). The etched, grooved, SLA, coarsely-blasted and TPS surfaces had thinner capsules whose dimensions did not change appreciably during the study period. Areas of tissue detachment from the implant surface were consistently observed, often where the implant body connects to the base at a 90° angle (Figure 3a). It could not be ascertained as to whether the detachment occurred in situ or during histological processing. In either case, detachment of tissue from the implant indicates that an implant surface may be undesirable as it failed to maintain attachment either in vivo or during tissue processing. For the SLA, TPS, grooved and coarsely-blasted surfaces, remaining cells could be seen on the implant surfaces even in areas of tissue detachment (Figure 5). Residual tissue was not observed for any of the other four test surfaces. 2. Morphometric results The histomorphometric analysis was based on a total of 5236 measurements collected from 154 samples representing the eight test surfaces. The multivariate analysis of variance (MANOVA) revealed that both surface type and time had a statistically significant effect on each of the three dependent variables: (1) attachment, (2) capsule thickness, and (3) degree of tissue-implant separation. Bonferroni post-hoc multiple comparison tests were used to elucidate individual differences among the eight surface types. Statistical significance was determined at p<0.05. 66 A. Attachment Attachment was determined as the percentage of the implant length in contact with the contiguous soft tissue. The substrata with the higher R a values, namely the SLA, TPS, coarsely-blasted and grooved surfaces, demonstrated the highest levels of attachment during both time periods, representing a statistically significant difference when compared to the smoother machined, polished and finely-blasted surfaces (Table 3). By the later stage of healing (weeks 6-11), the mean attachment on the three roughest test surfaces (coarsely-blasted, SLA, TPS) was approximately four times that of the polished surface and approximately ten times that of the finely-blasted surface. Comparisons between the two time periods show that attachment remained constant on the polished, coarsely-blasted and grooved surfaces, while increasing on the machined, finely-blasted, etched, SLA and TPS surfaces. The machined surface showed a three-fold increase in mean attachment levels between the two time periods. B. Complete attachment and detachment Complete tissue attachment was deemed to have occurred when soft tissue contacted the implant along its entire length, with no areas of separation (Figures 6a, 6b, 6c). The coarsely-blasted and TPS surfaces showed the highest incidence of complete attachment, on 50% and 38% of samples, respectively (Table 4). Complete attachment was not observed on any of the polished or finely-blasted samples and was seen on only 8% of the machined samples. Conversely, complete detachment refers to an implant surface that 67 was completely devoid of tissue contact (Figure 6d). Complete detachment was observed on 83% of the finely-blasted surfaces and on 31% of the polished surfaces. Furthermore, complete detachment was not observed on any of the SLA, TPS or coarsely-blasted samples. C . Capsule thickness The fibrous capsule was delineated from the surrounding tissue based on differences in cell orientation. In addition, the toluidine blue stained the fibrous capsule more intensely than it did the peripheral tissue. For both time periods, the thickest capsules were found around the polished, finely-blasted and machined surfaces. There was a statistically significant difference when the mean capsule thickness of the polished and finely-blasted surfaces was compared to that of other six test surfaces (Table 5). For example, during the early healing stage (weeks 1-5), the polished and finely-blasted surfaces harbored capsules that were approximately twice as thick (122-144 um) as those around the etched, grooved, SLA, TPS and coarsely-blasted surfaces (50-64 um), while the machined surface presented an intermediate capsule thickness (76 urn). The effect of time on capsule width varied between surface types. The capsule thickness of the polished surface during the second time period (75 um) was approximately half of its width during the first time period (123 um). Capsule thickness also decreased for the finely-blasted surface, albeit less dramatically. Decreases in capsule thickness for the SLA 68 and coarsely-blasted surfaces were not statistically significant. No other surface type exhibited a time-related decrease in capsule thickness. D. Tissue-implant separation distance The polished and finely-blasted surfaces demonstrated the greatest degrees of tissue-implant separation, with no statistically significant difference between them (Table 6). In contrast, the TPS, SLA, coarsely-blasted and etched surfaces showed the least separation (1 pm - 45 pm). There was a statistically significant difference in tissue-implant separation between the polished surface and all other test surfaces with the exception of the finely-blasted. The data show a general trend towards decreasing separation from one time period to the next, implying that the tissue approached the implant surface over time. The most striking observation in this regard was with the polished surface, where the tissue-implant distance underwent a four-fold decrease between the two time periods, from 214 pm to 57 pm. E. Relationship between measured parameters The data indicate a relationship between surface roughness, attachment, capsule thickness and separation. The polished and finely-blasted surfaces have the lowest R a values of all the surfaces employed and demonstrate the greatest capsule thickness, the least amount of attachment and the greatest degree of implant-tissue separation. In contrast, the rougher SLA, TPS and coarsely-blasted surfaces display significantly thinner capsules, greater attachment and less tissue-implant separation. 69 Table 2. Number and fate of each surface type tested SURFACE Number of Number of Number of Percentage samples samples usable (%) of usable processed discarded samples samples Polished 38 22 16 42 Finely-blasted 19 13 6 32 Machined 55 29 26 47 Etched 55 27 28 51 Grooved 71 33 38 54 SLA 19 9 10 53 Coarsely-blasted 19 6 13 68 TPS 19 3 16 84 70 Figure 4 . Photomicrographs of the (a) grooved and (b) machined implant surfaces. Note the oblique orientation of the fibroblasts onto the grooved surface (arrow 1). Fibroblasts are organized into a fibrous capsule parallel to the machined surface (arrow 2). Ti=titanium coating. 71 Figure 5. Area of tissue separation (S) from a coarsely-blasted implant. Note the residual tissue (R) on the implant surface. Ti=titanium coating, Ocapsule. 72 Table 3. Effect of surface type and time on attachment SURFACE R a value Mean % attachment Mean % attachment (um) (weeks 1-5) (weeks 6-11) Polished 0.06 22.46 ± 10.35 22.28 ± 6.94 Finely-blasted 1.36 8.88 ± 8.88 Machined 2.15 18.71 ± 7 . 6 7 60.88 ± 9 . 5 7 Etched 0.59 43.66 ± 13.04 65.24 ± 10.19 Grooved n/a 62.55 ± 7.96 58.02 ± 7.93 SLA 4.39 70.49 ± 6.84 90.44 ± 7.93 Coarsely-blasted 5.09 100 ± 0 98.51 ± 1 . 4 9 TPS 5.85 75.48 ± 15.35 94.80 ± 2.67 *Mean % attachment ± SEM ** Only one sample available for the finely-blasted surface at this time period 73 Figure 6. Photomicrographs of (a) titanium plasma-sprayed, (b) SLA and (c) coarsely blasted implants showing complete tissue attachment along the implant lengths. Note the complete absence of tissue attachment adjacent to the (d) polished implant. S=separation, Ti=titanium coating, C=capsule. 74 Table 4. Effect of surface type on the incidence of complete attachment/detachment SURFACE R a value % samples % samples % samples with (um) with complete with partial complete attachment attachment detachment Polished 0.06 0 69 31 Finely-blasted 1.36 0 17 83 Machined 2.15 8 73 19 Etched 0.59 21 54 25 Grooved n/a 10 77 13 SLA 4.39 18 82 0 Coarsely-blasted 5.09 50 50 0 TPS 5.85 38 62 0 75 Table 5. Effect of surface type and time on capsule thickness SURFACE R a value Mean capsule thickness Mean capsule thickness (urn) (weeks 1-5) (weeks 6-11) Polished 0.06 122.55 ± 19.12 75.00 ± 22.04 Finely-blasted 1.36 144** 114.65 ± 1 1 . 9 2 Machined 2.15 76.03 ± 8.38 90.46 ± 11.22 Etched 0.59 50.81 ± 7 . 8 3 50.94 ± 4 . 3 1 Grooved n/a 62.04 ± 6.99 62.96 ± 5.60 SLA 4.39 64.19 ± 8 . 6 9 54.70 ± 7.97 Coarsely-blasted 5.09 64.12 ± 2 6 . 0 5 52.58 ± 4 . 8 0 TPS 5.85 59.37 ± 1 1 . 1 0 64.39 ± 9.23 *Mean capsule thickness (nm) ± SEM ** Only one sample available for the finely-blasted surface at this time period Table 6. Effect of surface type and time on tissue-implant separation SURFACE R a value (pm) Mean separation (weeks 1-5) Mean separation (weeks 6-11) Polished 0.06 213.94 ± 7 0 . 0 3 57.25 ± 18.15 Finely-blasted 1.36 252.76** 104.29 ± 2 8 . 8 2 Machined 2.15 71.12 ± 19.16 50.46 ± 20.50 Etched 0.59 44.62 ± 15.70 41.26 ± 12.89 Grooved n/a 50.14 ± 13.60 66.45 ± 20.48 SLA 4.39 43.20 ± 14.57 20.52 ± 11.17 Coarsely-blasted 5.09 29.26 ± 2 1 . 7 0 0.89 ± 0.88 TPS 5.85 27.31 ± 1 1 . 3 4 18.88 ± 9 . 3 6 •Mean implant-tissue separation (u.m) ± SEM ** Only one sample available for the finely-blasted surface at this time period 76 IV. Discussion The topographical features of an implant surface are known to affect soft tissue response in vivo (Schroeder et al 1981, Chehroudi et al 1992, Ungersbock et al 1994). Most in vivo studies of surface topography and soft tissue have employed microfabricated or porous substrata as opposed to the randomly roughened titanium surfaces commonly used on dental implants. Moreover, many in vivo studies have employed polymeric implants (Mohanty et al 1992, Picha et al 1996, Rosengren et al 1999) whereas fewer studies have examined the soft tissue reaction to roughened titanium substrata (Holgers et al 2001). Most studies of rough titanium surfaces investigated their effects on bone integration (Buser et al 1991, Cochran et al 1996, Wennerberg et al 1995, 1996, 1997). The aim of the present investigation was to examine, in a subcutaneous rat model, the in vivo soft tissue attachment to surface topographies of varying roughness including titanium-coated replicas of commercially available dental implant surfaces. The implant-soft tissue interface was assessed histologically and quantitatively using the morphometric parameters of attachment, fibrous capsule thickness and where applicable, the degree of tissue separation from the implant. The smoothest substrata used in this investigation demonstrated the thickest fibrous capsule formation, the least amount of attachment and the greatest degree of tissue-implant separation. In contrast, the roughest surfaces displayed significantly thinner fibrous capsules, greater connective tissue attachment and the least tissue-implant separation. The machined surface, representing an intermediate degree of roughness, 77 presented capsule thickness, attachment and separation values that were approximately midway between the smoothest and roughest surfaces. An exception to this was the acid-etched surface. It had the second-lowest R a value among the test surfaces, yet its capsule thickness and separation data were comparable to those of the SLA, TPS and coarsely-blasted surfaces. Acid etching is a widely-used method for implant surface treatment. It may be used as a sole treatment or in conjunction with other methods such as blasting (Buser et al 1 9 9 8 , 1 9 9 9 ) . The current study indicated that the etched surface, despite its low roughness value, promoted connective tissue integration comparable to that of much rougher surfaces. It is possible that the unique geometry created by the etching procedure can play a dominant role in promoting connective tissue integration. Fibroblasts and the extracellular matrix tend to interdigitate into the rough surfaces and secure the implant in position (Smahel et al 1 9 9 3 , Tarpila et al, 1 9 9 7 ) . It would be expected that such an implant be mechanically stable and resistant to dislodgement forces. In contrast, smoother and polished surfaces tend to promote a thick, nonintegrated fibrous capsule, which is less likely to support and secure the implant in its location (Morehead, 1 9 9 4 ) . A more secure implant will reduce the micromotion at the interface, which could indirectly promote healing with a thinner capsule. These findings are in agreement with those of Ungersbdck et al ( 1 9 9 4 ) who cited mechanical stability and intimate tissue adhesion as the cause for thinner fibrous capsule around blasted ( R a = 1 . 5 0 ) as compared to polished ( R a = 0 . 1 9 ) titanium implants after a three-month implantation period in rabbits. 7 8 Schroeder et al (1981) reported that collagen fibers inserted perpendicularly into rough titanium-sprayed implant surfaces placed in primates. In their study, the application of tensile stress at the implant interface tore out particles of the rough surface, suggesting firm tissue anchorage. In our study, evidence of firm attachment was often present on the SLA, coarsely-blasted and TPS surfaces where either no tissue separation was noted or separation occurred within the tissue and not at the implant surface. The lack of separation indicates a tight and strong connective tissue adhesion with the rough surfaces. Chou et al (1995) reported that cell shape is affected by the surface topography of the substratum which could in turn promote the expression of adhesion proteins such as fibronectin. The preferential expression and adsorption of fibronectin to the rough surfaces could provide a partial explanation along with mechanical interlocking for the greater attachment found with the SLA, TPS and coarsely-blasted implants. The time periods used in this study were based on past studies comparing the early and late stages of implant healing. It has been demonstrated that the sixth week represents the time at which the peri-implant fluid space disappears and that cells contact the implant surface directly (Rostlund et al 1990, Johansson et al 1992, Holgers et al 2001). The machined, etched, SLA and TPS surfaces showed a statistically significant increase in the attachment between the early and late stage of healing. The time-related increase in attachment was not observed for the polished or finely-blasted surfaces, implying that the connective tissue attachment to the rough surfaces continues to improve during the late stage of healing. It is possible that 79 following early tissue detachment, the rough implant surfaces became repopulated with new tissues originating from cells remaining on the implant surface. The residual tissue found on rough surfaces in areas of separation may represent detachment that occurred during histological processing and not in situ. By the same token, the absence of residual tissue on smooth implants suggests that they never fostered attachment. In any event, data from our study indicate that the greatest tissue detachment from the implant surface occurred at the polished, finely-blasted and machined implants. One explanation for this observation could relate to the tendency of the fibrous capsule to contract, leading to tissue detachment away from the implant surface. Capsular contracture around implants is a major complication in reconstructive and esthetic breast surgery. There is abundant literature focused on the effect of breast implant texturing on capsule formation and contraction. Several studies indicate that textured breast implants are associated with less capsule contraction than implants with smooth, polished surfaces (Hakelius et al 1992, Smahel et al 1993, Batra et al 1995, Hakelius et al 1997, Wyatt et al 1998, Rubino et al 2001). Histological observations around smooth breast implants have noted that fibroblasts and collagen fibers align parallel with the implant surface, whereas textured implants foster a multidirectional collagen fiber orientation (Wyatt et al, 1998). These histological findings are supported by our observations of oblique fibroblast orientation on surfaces with 30-pm grooves, and the parallel arrangement seen on the polished control surface. A multidirectional collagen organization directs contractile forces in different paths, resulting in the neutralization or reduction of the 80 magnitude of forces that cause tissue separation (Hakelius et al, 1992). Rubino et al (2001) also reported that the capsule tissue immediately adjacent to textured breast implants had a random collagen fiber orientation and was non-contractile. In contrast, the outer capsule layers not affected by the surface texture were comprised of fibers with a parallel orientation. The authors speculated that this is the only layer where contractile forces could be generated by the capsule. The increased capsular contraction observed around smooth silicone implants is consistent with our observations of smooth titanium-coated implants. It can thus be inferred that the thicker capsules of parallel collagen fibers formed around polished and finely-blasted implants create a greater contractile force than that generated by the capsules around the rougher surfaces. The contractile force is likely generated by the myofibroblasts within the parallel oriented fibers (Baker et al 1981, Lossing et al 1993, Coleman et al 1993). In our study, the capsule was typically attached at two locations: on the test surface and on the pedestal. The alignment and contraction of the collagen fibers between these two points would retract the tissue away from the implant surface, analogous to the straightening of a bow upon release of the arrow. The greater tissue-implant separation observed with the smoother surfaces is also suggestive of a weak attachment, although detailed quantitative assessments of tissue attachment strength would be required to directly substantiate this hypothesis. The coarsely-blasted, TPS and SLA surfaces promoted the greatest connective tissue attachment that improved significantly with time, which bodes well for their clinical performance in permucosal or percutaneous applications. For example, while great 81 emphasis has been placed on the integration of dental implants into bone, the integration of the overlying soft tissue around transmucosal components has been studied less extensively in vivo (Holgers et al 2001). The dental implant is anchored in bone and connected to the prosthetic tooth via an abutment that projects through the mucosa. The transmucosal abutment links the bone-integrated fixture to the prosthetic tooth. It should thus be conducive to a firm soft tissue seal between the implant and the harsh oral environment. The most commonly used implant abutments have either a machined or polished surface (Quiryinen et al 1996, Sawase et al 2000). Recent studies have examined the mucosal response around implant abutments with differing surface topographies in dogs (Abrahamsson et al 2002, Zitzmann et al 2002) and in humans (Wennerberg et al 2003). These studies compared machined, blasted and acid-etched surface topographies and found no significant difference in the nature of the connective tissue attachment to the abutments. Our findings suggest that roughened surfaces can improve the connective tissue attachment to the transmucosal portions of dental implants. However, it should be noted that epithelial cell attachment and proliferation are markedly reduced on rough titanium (Cochran et al 1994, Baharloo et al, in press). It should also be noted that rough surfaces exposed to the oral cavity have a propensity to accumulate dental plaque (Quirynen et al, 1993). Although canine biopsies of implants obtained by Zitzmann et al (2002) showed no difference in the size or composition of the plaque-induced inflammatory lesion, this potential problem of plaque accumulation can nonetheless be addressed by a differential texturing of the abutment. Specifically, the abutment could be composed of a TPS or SLA-like surface in its lower portion to 82 maximize the fibroblast adhesion and promote a stable connective tissue seal, whereas its upper portion, closest to the oral cavity, could feature a smoother surface to favor epithelial adhesion while minimizing plaque retention. The implant design used in this study has a sharp, 90° angle between the test surface and the pedestal (base). The abrupt boundary between the test surface and the pedestal has the potential to hinder a stable contact between tissue and implant, creating a dead space at the base. Sanders et al (2003) placed polymer fiber implants subcutaneously at varying angles to the skin. They found that the likelihood of fibrous capsule formation was dependent on the degree to which the implant was placed parallel to the skin surface. In their study, fibrous encapsulation was avoided when implants were placed parallel to the skin surface. When the implant was placed at an angle relative to the skin surface, a dead space was created, attracting inflammatory cells and leading to the formation of a fibrous capsule. The observations of Sanders et al are consistent with the lack of attachment and thicker fibrous capsules frequently observed at the base of implants in our study, especially with the smoother surfaces. However, despite the potential dead space at the implant pedestal and obvious difficulty in promoting soft tissue adhesion in this zone, complete attachment in this area was nonetheless observed with the SLA, TPS and coarsely-blasted samples. Clinical situations may arise where anatomic limitations preclude placement of the endosseous portion of the dental implant in line with the crown. The transmucosal abutment, therefore, must be angulated in order to reconcile the directional difference between the implant and prosthetic tooth. Such a 83 compromise creates a dead space between the transmucosal abutment and the endosseous implant surface, similar to that encountered in our experimental design. The roughened surface texture could facilitate soft tissue adhesion to the abutment despite its atypical and unfavorable geometric configuration. All titanium-coated surfaces used in this study have been shown to have identical chemical composition (Wieland et al, 2002). It was thus possible to study the effect of surface texture on soft tissue attachment under conditions of constant surface chemistry. The application of a titanium coating to epoxy implants has also enabled thin (2-um) histological sectioning. Previous animal studies have examined the soft tissue response to endosseous SLA implants (Cochran et al 1997, Hermann et al 2000). In their studies, the SLA surfaces were placed in bone, with the soft tissues primarily contacting components with a machined surface. Our study is among the first to examine the in vivo effects of SLA components placed entirely within soft tissue. Our study is one of the few to provide a detailed quantitative comparison of connective tissue attachment (percent attachment) between implants with well-defined surface topographies. Although statistically significant results were obtained, a limitation of the study is the relatively few samples of polished and finely-blasted implants. Many of the retrieved smooth surfaces contained extremely supple tissue that did not provide good sections. It is possible that extensive fibrous encapsulation around these implants prevented proper resin infiltration during processing. Another problem was that the tissue 84 around many finely-blasted and polished samples became detached from the implant surface immediately upon sectioning, rendering them unsuitable for morphometric analysis. These problems limited the numbers of the polished and finely-blasted surfaces, but they also provide further evidence that such smooth surfaces are less amenable to a stable connective tissue attachment. This study was carried out in a subcutaneous rat model. However, it would be of interest to conduct subsequent quantitative and qualitative research on connective tissue attachment to transmucosal implants in animal models so that the role of surface topography on epithelial seal formation could be evaluated. 85 Chapter Two Bibliography: 1. Abrahmasson I, Zitzmann NU, Berghlundh T, Linder E , Wennerberg A, Lindhe J. The mucosal attachment to titanium implants with different surface characteristics: an experimental study in dogs. J Clin Periodontol 2002;29:448-455. 2. 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The influence of time on human breast capsule histology: smooth and textured silicone-surfaced implants. Plast Reconst Surg 1998; 102(6): 1922-1931. 55. Zitzmann NU, Abrahamsson I, Berglundh T, Lindhe J. Soft tissue reactions to plaque formation at implant abutments with different surface topography. An experimental study in dogs. J Clin Periodontal 2002;29:456-461. 93 Chapter Three - Conclusions and Future Directions 94 Chapter Three - Conclusions and Future Directions I. Conclusions There are currently a number of commercially available implant surfaces prepared by various roughening processes, such as acid-etching, grit-blasting, or a combination thereof. In vitro research has suggested that these surfaces are capable of supporting fibroblast attachment and growth (Cochran et al 1994, Mustafa et al 1998). The present investigation was designed to evaluate titanium surfaces in terms of their ability to promote connective tissue attachment in vivo. Histological and histomorphometric analysis of subcutaneous titanium-coated implants in a rat model resulted in the following conclusions: 1. As a general rule, implant surfaces with the highest R a values, namely the TPS, SLA and coarsely blasted surfaces, promote the greatest degree of connective tissue attachment, the least fibrous encapsulation and the least tissue-implant separation. 2. The geometry of the surface irregularities can be as important as their size, since the acid etched surface presented comparable capsule thickness and separation data despite its relatively low R a value. 3. The polished surface allows the least connective tissue attachment and is associated with thick fibrous capsules and significantly greater tissue-implant separation. 4. The roughest surfaces show increasing attachment over time, whereas smoother surfaces do not exhibit significant time-related improvement in this regard. 95 5. Rough surfaces may allow connective tissue adhesion in areas where anatomic dead spaces are otherwise likely to form, such as zones where the implant undergoes abrupt geometric change. 6. The greater tissue-implant separation distances observed for the polished and finely-blasted substrata may be in part due to increased capsular contraction associated with smooth surfaces. II. Future directions The present investigation elucidated differences between smooth and textured substrata in terms of their effects on soft tissue in vivo. There are several ways in which this theme can be further explored in basic science and clinical applications. 1. The subcutaneous model used in this study allowed the observation of connective tissue organization around the various test surfaces. The ability of these surfaces to inhibit epithelial downgrowth can be tested directly by attaching a percutaneous component to the subcutaneous implant (Chehroudi et al 1992, 2002). Histomorphometric analysis would include measurements of both epithelial and connective tissue attachment length. In light of the favorable connective tissue attachment observed with the SLA, TPS and coarsely-blasted surfaces in the present study, one would also expect them to effectively inhibit epithelial downgrowth in a percutaneous or transmucosal model. Recent studies by Abrahamsson et al (2002) and Wennerberg et al (2003) reported that roughening transmucosal oral implant abutments by either acid etching or blasting did not affect fibroblast orientation nor the length of the connective tissue attachment alongside the abutments. However, 96 the surfaces used in their studies did not possess average height deviations of greater than 1.87 pm, which is in contrast to the height deviations of the rougher surfaces used in the present study (4.39-5.85 um). Moreover, since the unique combination of blasting and etching treatments has not been tested on transmucosal implant components, the SLA surface would be an interesting test abutment for oral implants in future large animal or human studies. For example, SLA abutments along with machined controls, could be attached to dental implants placed in canine jaws. Histomorphometrics may then be used to assess the relative length of the connective tissue attachments, which would be expected to be greater on the SLA surface. These results would provide more direct evidence to support the role of rough surfaces in promoting connective tissue adhesion to transmucosal components. The hypothesis that cell attachment is stronger on roughened substrata could not be directly measured by the data in this study. Measuring the tensile force required to detach tissue from the various surfaces could provide a quantitative assessment of adhesion strength. For example, an in vivo assay of connective tissue adhesion strength was described by Bundy et al (2000): following removal of the implant and surrounding soft tissue, a "peel tester" apparatus is used to separate the tissue from the implant, and measure the force required to do so. The data obtained from these methods could be used to compare tissue adhesion strength to substrata with varying degrees of roughness, allowing one to test the hypothesis that cells and tissues adhere more tenaciously to rough surfaces. 97 3. The present study was primarily histomorphometric in nature and employed a non-specific stain (toluidine blue). Future studies that stain for specific molecules can provide insight into the constituents of the tissue around the various surface topographies. One approach could involve staining for alpha-smooth muscle actin (a-SMA), a protein expressed by myofibroblasts. The expression of alpha smooth muscle actin has been positively correlated with fibroblast contractile activity (Hinz et al, 2001). Its detection in the tissues surrounding polished implants would support the hypothesis that smooth surfaced implants are more prone to capsular contracture, especially if a-SMA was found to be absent around the rougher implant surfaces. 4. In implant dentistry, angulated dental implant abutments are required when anatomic limitations do not allow placement of the bone-integrated fixture in line with the prosthetic tooth. The dead spaces created by the abutment's atypical geometry could compromise the connective tissue attachment, which could be evaluated in two ways. Using an animal model, one approach would be to compare the histological characteristics of the soft tissues around transmucosal abutments with varying angulations and surface topographies. Such a study would likely show that connective tissue adhesion is hindered in areas of angulation change, but that a rough abutment texture may compensate for this problem by enhancing the cells' affinity for the surface. A second approach would involve a clinical evaluation of the peri-implant mucosa of patients who received angulated abutments as part of their treatment. A deficient mucosal seal around an implant abutment would be characterized by 98 migration of the epithelial attachment and increased soft tissue inflammation. Appropriate comparisons could then be drawn between soft tissues around straight and angulated abutments. In summary, although the results of these experiments indicate that roughened titanium substrata are superior in sustaining connective tissue attachment, there are still a number of open questions. Identification of the cellular mechanisms that drive tissue organization at the implant interface, and extension of the current approach to well-controlled large animal studies, would allow us to select the most biocompatible surface modifications for clinical use on a more rational basis. 99 Chapter Three Bibliography: 1. Abrahamsson I, Zitzmann NU, Berghlundh T, Linder E, Wennerberg A, Lindhe J. The mucosal attachment to titanium implants with different surface characteristics: an experimental study in dogs. J Clin Periodontol 2002;29:448-455. 2. Bundy K, Schlegel K, Rahn B, Geret V, Perren S. An improved peel test method for measurement of adhesion to biomaterials. J Mater Sci Mater Med 2000; 11(8):517-521. 3. Chehroudi B, Gould TRL, Brunette D M . The role of connective tissue in inhibiting epithelial downgrowth on titanium-coated percutaneous implants. J Biomed Mater Res 1992;26(8): 493-515. 4. Chehroudi B, Brunette D M . Subcutaneous microfabricated surfaces inhibit epithelial recession and promote long-term survival of percutaneous implants. Biomaterials 2002;23:229-237. 5. Cochran DL, Simpson J, Weber HP, Buser D. Attachment and growth of periodontal cells on smooth and rough titanium. Int J Oral Maxillofac Implants 1994;9:289-297. 6. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Molecular Biology of the Cell 2001;12:2730-2741. 7. Mustafa K, Silva Lopez B, Hultenby K, Wennerberg A, Arvidson K. Attachment and proliferation of human oral fibroblasts to titanium surfaces blasted with T i 0 2 particles. Clin Oral Impl Res 1998;9:105-207. 100 8. Wennerberg A, Sennerby L, Kultje C, Lekholm U. Some soft tissue characteristics at implant abutments with different surface topography. A study in humans. J Clin Periodontol 2003;30:88-94. 101 

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