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Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity… Baharloo, Bahador 2004

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Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces By Bahador Baharloo B.Sc. University of British Columbia, 2001 A THESIS SUBMITTED I N P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biology) We accept this thesis is confirming To the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A August 2004 ©Bahador Baharloo  FACULTY OF GRADUATE STUDIES  THE UNIVERSITY OF BRITISH COLUMBIA  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the. University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Date (dd/mm/yyyy)  Name of Author (please print)  Title of Thesis:  Degree: Department of  ^JtSifyAy^M  A^f/Att^ fifoi  (?>/i>>fS  r  /  (&t>  ^(S^/UT^ ftS*//  l!U gstosx/pAj  Year: SUSST/SS*/  ^  STsSM^  3_0O  S2r,/>*rt-<e0  The University of British Columbia Vancouver, BC  Canada  grad.ubc.ca/forms/?formlD=THS  page 1 of 1  last updated:  20-Jtil-04  Abstract Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polishedTi (P) and rough grit blastedTi (B), acid etchedTi (AE), and grit blasted & acid !  etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the S L A surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher A E surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. The size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.  Table of Contents Abstract Table of Contents List of Figures Acknowledgment Chapter I- Introduction Overview Literature review I. Epithelial rests of Malassez A. Epithelial rests of Malassez in vitro and vivo II. Surface Topography III. Titanium IV. Surfaces V. Cell adhesion to the substratum A. Morphology B. Focal adhesions C. Vinculin D. Stress fibers i. Stress fibers ii. Actin E. Integrins  n. iii. v. vL-  F. Extracellular matrix => i. Collagen IV Laminin-5 ii. Fibronectin iii. The aim of this thesis Bibliography Chapter II- Manuscript Introduction  17  1  1 2 2 2.5 9 1 0  ^* ^ ^ 1 3 1 3  * 14 15 3  i f\ 1  7  '  1  1n  ' 22^ 1  34  ^  3  37  Methods and Materials I. Cell culture II. Surface topography III. Propidium Iodide staining IV. Scanning Electron Microscopy (SEM) determination of cell area V. Immunofluorescence VI. Immunogold staining and detection of vinculin in focal adhesions by B S E imaging on S E M VII. T E M evaluation of membrane Ti proximity VIII. Statistical analysis Results I. Epithelial growth II. Cell area III. Focal adhesions  3  7  9 ^  3 3  ^ 42  4  . . 43 44 r  4  4  5  -* 4  6  4  7  m  IV.  Immunogold staining and detection of vineulin focal adhesions using B S E imaging on S E M T E M evaluation of membrane Ti proximity  V. Discussion Figures Bibliography Chapter O - Conclusions and futures studies Conclusions Futures studies Bibliography  List of Figures Figure 1. S E M images of replica of rough surfaces. A) A E (Acid-Etched) surface. B) B (Blasted) surface, C) S L A (Acid-Etched & Blasted) surface.  (Page53)  Figure 2. B S E image of immunogold staining of vinculin in focal contacts in an E cell on P surface.  (Page 54)  Figure 3. PI staining of E cells, as indicated by arrow-heads, on day-1 and day-5 on surfaces of differing roughness.  (Page 55)  Figure 4. Effect of surface roughness on the growth of E cells.  (Page 56)  Figure 5. S E M images of three different classes of E cells on TCP. A ) Singlets (cells in isolation) B) Doublets (two cells in contact) C) Clusters (three or more cells in contact). (Page 57) Figure 6. Effect of surface roughness on E cells spreading.  (Page 58)  Figure 7. Visualization of vinculin in focal contacts. A) E cells on P B) E cells on AE.  (Page 59)  Figure 8. Effect of surface roughness on the distribution of focal contacts.  (Page 60)  Figure 9. Effect of surface roughness on the localization of focal contacts on the ridges (i.e.  flats).  (Page 61)  Figure 10. Effect of surface roughness on Ti membrane proximity.  (Page 62)  Figure 11. Effect of surface roughness on cell adhesion (distance in nm).  (Page 63)  Acknowledgments I would like to take this opportunity to extend my many thanks to the following people who have made the completion of this project possible: -  To Dr. Donlad M . Brunette, professor, Dentistry, U B C , for his guidance, patience, and support throughout this project.  -  To Dr. Babak Chehroudi, clinical assistant professor, Dentistry, U B C , for his expertise and continues academic support.  -  To Mr. Andre Wong for his E M expertise. To my parents who have made it possible for me to be where I am today. Koorush and Marry I thank you for all the sacrifices you have made throughout my life. This journey, without your love, support, and guidance would have never been possible.  -  To my siblings, Kianoosh, Baharak, Baharnaz, and Bahavar, for their unending friendship and love.  Chapter I- Introduction Overview The number of individuals receiving artificial devices to replace damaged or missing tissue or organs is increasing (Griffith and Naughton, 2002). Such devices include total or partial hip and other joint implants or prostheses, cardiovascular shunts and dental implants. The success of an implanted device depends upon the interaction of cells with artificial surface including those interactions involving cell adhesion and migration along the surface. These interactions are influenced by the physical and chemical composition of the substratum such as surface topography and surface energy (Brunette, 1986; Brunette, 1988; Boyan et al., 1996; Curtis and Wilkinson, 1997; Brunette and Chehroudi 1999; Curtis and Riehle, 2001; Brunette et al., 2003). The surfaces of commercially available implants vary greatly in their topography and physical/Characteristics (Esposito et al., 2003). Brunette (1986; Brunette et al., 1983; Chehroudi et al., 1990; 1995; Brunette and Chehroudi, 1999; Glass-Brudzinski et al., 2002; Chehroudi and Brunette, 2002; Teixeira et al., 2003; Karuri et al., 2004) investigated the effect of well-defined surface topographical features on cell behavior in vitro and in vivo. However, the effect of random surface topographical features on epithelial cell behavior has not been as extensively investigated (Hormia et al., 1991; Cochran et al., 1994).  1  Literature Review I.  Epithelial cells of rests of Malassez In general, epithelial tissue is one of the major types of tissues (others include:  nerve, muscle, and connective tissues) in vertebrates. In epithelial tissues or epithelium, cells are bound tightly together. Epithelium is a coherent cell sheet formed from one or more layers of cells covering external surfaces or lining cavities or surrounding glands (Alberts et al., 1994). Epithelial cells (E-cells) derived from rests of Malassez were used in this study. The epithelial rests of Malassez represent the fragmented remnants of an epithelial structure, Hertwig's epithelial root sheath, which in early tooth development delineates the shape of the roots (Wentz et al., 1950; Reeve and Wentz, 1962; Simpson, 1965; Listgarten, 1975) and persist as a network or as islands of epithelial cells. Some differences in the incidence of epithelial rests of Malassez cells between species have been reported. For instance, in pigs the rest cells are numerous (Grant and Bernick, 1969). Also, the prevalence of epithelial rest of Malassez in human periodontal ligament decreases rapidly in the first three decades of life, followed by a slower decrease thereafter (Simpson, 1965). The rest cells are completely surrounded by connective tissue cells (Ten Cate, 1972). Furthermore, the exact function of epithelial rest of Malassez cells in vivo is unknown.  A.  Epithelial cells of rests of Malassez in vitro and in vivo Hong and Brunette (1987) extended the primaryfindingsthat cell shape can  regulate proteinase secretion in fibroblasts (Aggelar et al., 1982) and investigated the relationship between cell proliferation, cell shape, and proteinase secretion using E-cells  2  derived from the epithelial rests of Malassez. Hong and Brunette (1987) altered the E cells' shape by physical means (growth on less adhesive substrata and grooved surfaces and mechanical stretching), application of cholera toxin and dibutyryl cyclic A M P (that induced cell proliferation and flatting), and 12-O-tetradecanoylphorol-13-acetate (that increased cell rounding). Cell shape influenced the proteinase secretion more than cell proliferation. Neutral proteinase and plamsminogen activator secretion were found to correlate with cell shape; the more round the cells, the greater the amount of proteinase secreted. Uitto et al., (1992) investigated the adhesion of E-cells derived from epithelial rests of Malassez to different extracellular matrix molecules, expression of fibronectin, and integrin receptors by means of immunoflorescent staining, in situ hybridization and time-lapse cinemicrography techniques. E-cells attached and spread well on different surfaces coated with fibronectin, vitronectin, and type I collagen and in some instances laminin-5. Immunofluorescent staining of fibronectin indicated that fibronectin was localized to tile extracellular matrix as well as intracellular granules and tracks left behind cells. Treatment with integrin binding peptide prior to culturing inhibited the attachment of receptors to the coated surfaces strongly indicating the involvement of integrin receptors in adhesion structures. Also, Pi integrin staining was localized to the cell-cell. contacts as well as leading cell projections. Abiko et al., (1994) using immunohistochemistry examined the localization of laminin, a marker protein for basement membrane, in E-cells derived from epithelial rests of Malassez grown on titanium. Gelatin zymography of conditioned medium confirmed the gelatinase activity of epithelial cells. The authors suggested that laminin contributes  3  to the E-cell attachment to titanium and that gelatinase may be mobilized for E-cell movement on titanium. Onishi et al., (2002) investigated the immunohistochemical localization of heat shock protein-25 of rat molar teeth (during root formation) and laminin. Heat shock proteins are a group of small molecules that are induced in certain types of cells in response to heat shock or other deleterious stresses, and enable cells to recover from stressful conditions (Lavoie et al., 1993). Onishi et al., (2002) observed heat shock protein-25 immunoreactivity in odontoblasts and cells in close proximity to the acccelular cementum tliat lacked laminin immunoreactivity. Furthermore, the transmission electron micrographs of heat shock-25 immunoreactive cells showed that cells along the surface of the cementum were round and rich in organelles such as mitochondria and rough endoplasmic reticulum. In contrast, cells at the cervical region were oval and contained few cell organelles. Onishi et al., (2002) suggested that heat shock protein-25 might be involved in shape alteration of epithelial rests of Malassez cells, cementoblasts, and odontoblasts during differentiation. Furthermore, Onishi et al., (2002) confirmed the in vitrofindingsof Abiko et al., (1994) showing that most of the cells of rests of Malassez had expressed laminin. Uitto et al., (2002) examined the expression and localization of matrilysin, a matrix metalloproteinase, upon exposure to an oral bacteria in gingival epithelium and E cells derived from epithelial rests of Malassez. Supra-basal cells of junctional epithelium facing the teeth expressed matrilysin. As well, the bacteria exposure elevated the expression of matrilysin by epithelial rests of Malassez in vitro. The authors suggested  4  that the matrilysin might have an active role in the physiology of periodontal ligament in defence as well as normal physiology of junctional epithelium. Yoshihiro et al., (2003) using reverse transcription polymerase chain reaction compared the profiles of alkaline phosphatase (ALPase), osteopontin (OPN), and bone morphogenetic protein 2 & 4 (BMP-2 & BMP-4) in fibroblasts and E-cells derived from human periodontal ligament. Epithelial rests of Malassez expressed lower levels of ALPase and BMP-4 compared to the fibroblasts. However, the expression level of O P N in E-cells was higher than fibroblast cells with no differences in expression level of BMP-2 between E-cells and fibroblast cells. Yoshiro et al., (2003) suggested that epithelial rests of Malassez in the periodontal ligament may be involved in cementum repair. Talicet al., (2004) investigated the effect of morphological changes/ shape of periodontal ligament in rats and the effect on the expression of integrins using immunofluorescent staining. The periodontal ligaments were either compressed or tensed using an elastic band placed around molars. £b integrin was strongly localized at the membrane of epithelial rests of Malassez within the ligaments suggesting a mechanical (adhesion) or signaling role in response to the conditions of stress or remodeling.  II.  Surface topography There is sufficient published evidence to convince even the most skeptical  newcomer to implantology that surface topography and roughness strongly influence cell behavior. Over the years implant materials have been designed with different surface topographies. For example, and of relevance to this work, surfaces with horizontal grooves have been applied to prevent epithelial down-growth (Brunette, 1986; Chehroudi  5  et al., 1990; Chehroudi and Brumette, 2002). Structural features respectively in the range of 0.1-100 urn and 1-100 nm define micro- and nanotopography (Curtis and Wilkinson, 1997; Teixeira et al., 2003; Karuri et al., 2004). In general, cells are extremely sensitive to microstructures of biomaterials and their behavior and function can be modified by different topographies (Chehroudi and Brunette, 1995; Curtis and Wilkinson, 1997; Brunette and Chehroudi, 1999; Curtis and Riehle, 2001; Brunette et al., 2003). Topographical features can promote adhesion and spreading of different cell types. Osteoblast cells preferentially attach to roughened surfaces (Abe et al., 1983) as do macrophages (Rich and Haris, 1981, Salthouse T.N., 1984); whereas, gingival fibroblast cells prefer tp adhere to smooth polished surfaces (Bowers et al., 1992). Therefore, it has been postulated that surface topography can be designed to select for specific cell populations. Moreover, topographical features can act as directional cues to influence cell orientation and direction of migration of population of cells. Directional cues can be chemical (adhesive) or physical (topographic or mechanical) as reviewed by Curtis and Clark, (1990), Cutis and Wilknison (1997), Brunette and Chehroudi (1999), and Brunette et al., (2003). Population pressure and the phenomena of contact inhibition of locomotion cause cells to move away from densely populated areas towards more sparsely populated areas. In particular contact between epithelial cells results in contact induced spreading as first observed by Middleton C A . (1977). Upon contact epithelial cells increase their area, form stable lateral contacts between adjacent cells and assemble into sheets (Brown and Middleton, 198f).-  6  Weiss (1934; 1945) was the first to coin the term contact guidance following the classical work done by Harrison (1914) who investigated the reaction of tissues from embryos of frog chick of various ages on a fibrin networks of the clotted plasma, spider webs, cover-glasses and the surface film of the fluid drops. Weiss (1945) described the orientation response of neural cells in culture along thin glass fibers, scored surfaces or grooves. By contact guidance (also called topographical guidance) Weiss referred to the tendency of a cell to be oriented and guided in its direction of migration by substratum topography (Brunette et al., 1983; Chehroudi et al., 1990; 1995; Curtis and Clark, 1990; Brunette and Chehroudi, 1999; Glass-Brudzinski et al., 2002; Chehroudi and Brunette, 2002; Teixeria et al., 2003; Karuri et al., 2004). In vitro and in vivo, contact or topographic guidance has been observed in different cell types, such as epithelium, on a variety of substrata including surfaces with grooved features (Brunette et al., 1983; Chehroudi et al., 1988; Chehroudi and Brunette, 2002; Teixeria et al., 2003; Karuri et al., 2004). Oakley and Brunette (1995) using epifluorescence and confocal microscopy examined the effect of titanium coated grooved substrata on;the shape, orientation, direction, and distribution of actin filaments and microtubules on single, double, and clusters of E-cells derived from epithelial rests of Malassez. They showed that cell contact increased the spreading. Furthermore, actin filaments and microtubule staining showed the initial alignment of the filaments along the walls and the ridge-groove edges. The data presented also confirmed that the clusters of epithelial cells were less susceptible to the topographical modulation as local topographic effects on the cytoskeleton could be overridden by adjacent cell contacts as suggested by Clark et al., (1990; 1991).  7  Grooved substrata can be obtained by a variety of techniques and can be defined by groove width, depth, and geometry. On substrata with multiple parallel sets of identical grooves, the groove width, depth, and the number of adjacent grooves is important. The response of cells to grooves depends on factors such as the type of cells, the size of cells, and the dimensions, density, and spacing of the groove features (den Braber et al., 1996; Curtis and Wilkinson, 1997; Teixeira et al., 2003; Karuri et al., 2004) However, the effects of random topographical features on the behavior of epithelial cells have not been investigated so extensively. Hormia et al. (1991) investigated the effect of random topographical roughness of titanium surface on the growth and adhesion of gingival epithelial cells in vitro. Based on observation of immuno-fluorescent staining of actin, vinculin, and components of desmosomes (DP 1&2) they suggested that epithelial cells on titanium prefer smooth to rough titanium surfaces. Also, they proposed modes of adhesions such as close contacts, extracellular matrix contacts, and hemidesmosomes as obserrved previously by Gould et al., 1981. Hormia et al., (1991) did not observe staining of vinculin in focal adhesion like structures and instead observed vinculin in cell-cell contacts of epithelial cells grown on titanium surfaces of varying roughness. Cochran et al., (1994) investigated the effect of finely and coarsely blasted titanium surfaces on the growth of gingival epithelial and fibroblast cells as well as fibroblasts from periodontal ligaments. Growth on tissue culture plastic, smooth titanium surface and the two rough surfaces was monitored up to day-9 post seeding. The smooth tissue culture plastic and titanium surfaces supported the growth of epithelial cells in contrast to the blasted surfaces that demonstrated no statistically significant differences in cell numbers between day-land day-9. However, the growth of  8  periodontal ligament fibroblasts was unaffected by the surface topography of the growth substratum i.e. all surfaces supported the growth to the same extent.  III.  Titanium Although there is no agreement on what constitutes the ideal topography, there is  some agreement on which materials are biocompatible (Brunette D . M . , 1988; Esposito et al., 2003). Titanium and its alloys, Ti6A17Nb and Ti6A14V, are among the most commonly used implant materials. Titanium, as the choice for biomaterial purposes, is known to have a passive surface, low rates of metal ion release, low specific weight, low modulus of elasticity, high tensile strength, poor heat conduction, and minimal affinity to cause adverse cell or tissue responses (Ratner B.D., 2001). Native titanium is a very reactive metal and has a very high affinity for oxygen. Therefore, when titanium and its alloys are exposed to air or moisture a thin (3-7 nm) layer of titanium oxide (TiCh, Ti2C«3 or TiO) spontaneously covers the surface. The thickness of this natural oxide layer can vary according to the conditions and environments under which the metal has been processed. However, the excellent corrosion resistance of titanium and its alloys results from the formation of this stable, continuous, and highly adherent natural barrier. The solubility of this oxide layer is considered to be negligible in water. In general, different processes that influence the adsorption of biomolecules are dependent on the surface properties of the biomaterial and the general mechanisms of protein adsorption have been reviewed (Wahlgren and Arnebrant, 1991). Titanium oxide has an affinity for proteins, glycoproteins, and glycolipids and other matrix molecules  9  that in turn promote the adhesion of cells and tissues to the surface of the implanted devices (Brunette, 1986; Winkelmann et al., 2003; Scotchford et al., 2003).  IV.  Surfaces One of the main differences among the commercially available dental implants is  their surface topography. The quest for practical and functional implant materials has resulted in a number of commercially available osseointegrated dental implants that differ in materials, body shapes, diameters, lengths, and surfaces properties. Examples include Astra® (Tio2 blast titanium screws with a homogenous structure), Branemark® (Titanium screws with orientation in form of screw threads), Steri-Oss® (acid-etched titanium screws), ITI® (hollow TPS titanium screws with a rough inhomogeneous structure), and ITI® (coarsely blasted and acid-etched surfaces "SLA") (Esposito et al., 2003). In recent years, the S L A surfaces have been extremely investigated in a number of in vitro and in vivo studies. S L A surfaces have produced a high rate of success clinically and appear tp promote bone production adjacent to the implant (Cochran et al., 2002; Boyan et al., 2003; Wieland et al., in press; Buser et al., 2004). Therefore, it was of interest to this thesis to evaluate the effect of rough surfaces such as acid-etched, coarsely blasted, and SLA, on epithelial cells in vitro. All original surfaces were prepared and analyzed in Institute Straumann and were a generous gift from Dr. M . Wieland. Wieland et al., (2002) evaluated the surfaces using non-contact laser profilometry, SEM, stereoSEM, and X-ray photoelectron spectroscopy. (For the complete evaluation of surface properties refer to Wieland PhD thesis, 1999). Non-contact laser profilometry and stereo-SEM were used for the quantitative evaluation of surface-roughness values (Wieland et al., 2002). The roughness values were  10  recorded in form of Ra, Rq, Rt, RZDIN, S , SR, and Lr. The latter parameters values are m  described as following". R : The arithmetic average of the absolute values of all points of profile a  Rq! The root mean square of the values of all points of profile Rt: The maximum peak to valley height of the entire measurement trace RZDIN- The arithmetic average of the maximum peak to valley height of the roughness values of five consecutive sampling sections over the filtered profile S : The arithmetic average spacing between the falling flanks of peaks on the m  mean line Sic: The amplitude distribution skew Lr: The relation of stretched length of the profile L to the scanned length L 0  m  X-ray photoelectron spectroscopy analysis indicated that Ti, O, C, and N were present on all Ti surfaces. Ti and O were found to be the dominant elements. The TiC»2 thickness of 4.5-5.5 nm protected the Ti surfaces (Wieland et al., 2002).  V. A.  Cell adhesion to the substratum Morphology The attachment of cell membrane to the substratum is a complex process. This  interaction has been described on the basis of the appearance of the attachment sites in light and electron microscopy. Abercrombie et al. (1971) used transmission electron microscopy technique to reveal electron dense plaques that were associated with actin filaments. In 1976, Izaard and Lochner applied the technique of interference reflection microscopy to distinguish three types of regions depending upon the distance between the substrate and the plasma membrane, extracellular matrix contacts (a distance separation  11  of 100-140 nm), close contacts (a distance separation of ~ 30 nm), and focal adhesions (a distance separation of 10-15 nm). Heath and Dunn (1978) used the combined techniques of electron microscopy and interference reflection microscopy to confirm that adhesion plaques (reported by Abercrombie et al., 1971) and focal adhesions (reported by Izzard and Lochner, 1976) described the same structures.  B.  Focal adhesions FocaJ adhesions evolve from small dot-like adhesion sites. Transition of these  small dot-like structures into the mature focal adhesions is accompanied by transition of the associated actin mesh into densely packed straight bundles of filaments known as stress fibers (Heath and Dunn, 1978). Bershadsky et al. (1987) showed that accumulation of vinculin in focal adhesions parallels the transition of dot-like adhesion sites to the dash-like mature focal adhesions. Focal adhesions are usually located under peripheral sites of leading lamellae and near edges of non-spreading regions of the cell margins in moving and stationary cells. Focal adhesions play a critical role in the adhesion mediated signaling (through focal adhesion kinase pathway) as well as the transfer of mechanical force (through the stress fibers). However, the small dimensions of individual adhesion sites, their spatial proximity, and the small magnitude of the forces involved, make it challenging to accurately measure and map these sites (Bershadsky et al., 2003). Among the proteins that are concentrated on the cytoplasmic side of focal adhesions are talin, paxillin, vinculn, cc-actinin, tensin, filamin, and the signaling molecule focal adhesion kinase (Liu et al., 2000; Zamir and Geiger, 2001).  12  C.  Vinculin Vinculin is a 130 Kda cytoskeletal protein localized in focal adhesions that has  been proposed to be involved in the attachment of microfilaments to the receptors within the plasma membrane (Geiger, 1979; Geiger et al., 1980). The above attachment comprises a complex, and possibly orderly, assembly of proteins at the adhesion plaques. For example, the cytoplasmic domain of Pi can bind to talin (review by Beckerle and Yeh, 1990; Marti et al., 2003) and a-actinin. In turn, talin can bind to vinculin (Otto, 1990) or nucleate actin assembly and self-associate. Vinculin can bind paxillin and aactinin that in turn the latter can bind and crosslink actin. Also, tensin can bind to vinculin. Woods and Couchman (1992) reported that vinculin is a late addition of focal adhesions and others (Herman and Pledger, 1985) investigated the persistence of focal adhesion structures that lack vinculin. Avnur et al. (1983) showed that the association of vinculin with focal adhesions is largely actin independent. Furthermore, Herman and Pledger (1985) and others (Feltkamp et al., 1991) showed that presence of vinculin in focal adhesions could precede the presence of stress fibers. Finally, Samuelsson et al. (1993) investigated the direct aggregation and association of integrin heterodimers with stress fibers independent of vinculin and talin.  D.  Stress-fibers i.  Stress-fibers- Stress-fibers are actin containing contractile structures that  also contain a number of actin-associated proteins such as myosin I and II. Myosin I is a single molecule with one globular head and tail that attaches to another molecule and in this way the attachecLmolecule get carried relying on the motor activity of myosin head.  13  However, Myosin II is composed of a pair of identical myosin molecules thus having two globular heads with tail twisting around each other length. ii.  Actin - Actin filaments are found in all eukaryotic cells and are essential  for many of their functions including cell shape, movement, contractile activities, phagocytosis of large particles, intracellular transport, cell division, and trans-membrane signaling (Ridley et al., 2003; Engqvist-Goldsrein and Drubin, 2003). Although actin filaments are dispersed throughout the cell, they are most highly concentrated in the cortex, just beneath the plasma membrane (Bray and White, 1988; Ridley et al., 2003). Actin filaments are helical polymers of the protein actin. They appear as flexible structures, with a diameter of about 7 nm, that are organized into a variety of linear bundles, two-dimensional networks, and three-dimensional gels (Alberts et al., 1994). Individual dumbbell-shaped actin subunits polymerize to form globular actin /G-actin microfilaments with an intrinsic polarity, the heads of actin monomers all pointing to the same direction (Bremer and Aebi, 1992; Alberts et al., 1994; Ridley et al., 2003). Actin polymerization in lamollipodia is mediated by Arp 2/3 complex that binds to the sides or tip of a preexisting actin filamnt and induces the formation of new daughter filament that branches off the mother filament (Pollard and Borisy, 2003). Addition of each monomer of actin to the microfilament is ATP dependant. However the current opinion in the actual event of protrusive, pushing of the membrane, is that the process occurs not by elongation of the actin filaments but by "elastic Brownian ratchet" that means the thermal energy bends the short filaments, storing elastic energy. Unbending of an elongated filament against the leading edge would then provide the force for protrusion (Pollard and Borisy, 2003). The rate and organization of actin polymerization is controlled through the  14  availability of free available monomers. For example, profilin prevents self-nucleation by binding to actin monomers (reviewed by Ridley et al., 2003). Also, filament elongation is restricted by the action of capping proteins. In addition, A D F / cofilin family assists the disassembly of older filaments that releases actin monomers that are needed for the polymerization process at the front end (Welch and Mullins, 2002; Ridley et al., 2003). There are a number of other supporting/stabelzing proteins (cortactin, filamin A, and aactinin) involved in the process of polymerization and de-polymerization (reviewed by Welch and Mullins, 2002). The interactions between the stress fibers and the extracellular matrix molecules (collagen, laminin, fibronectin, and etc.) on the substratum are regulated by transmembrane integrin receptors.  £.  Integrins Integrins are a family of heterodimeric, transmembrane glycoproteins that act as  cell-surface receptors for extracellular matrix molecules including fibronectin, different types of collagen, and laminins and cooperate with growth factor receptors to promote cell survival, cell cycle progression, and cell migration (Anderson and Springer, 1987; Buck and Horwitz, 1987; Hynes, 1987; Gumbiner, 1996; Giancotti and Ruoslahti, 1999; Giancotti, 2003). Intergins are composed of a single a and a p subunit. There are several subfamilies of integrins defined by their common P chains. At least 18 different a and 8 P subunits are currently known. These subunits can combine to form at least 22 different cell surface receptors that have distinct ligand binding specifities (Larjava et al., 1996; Giancotti and Ruoslahti, 1999; Giancotti, 2003). Integrins are responsible for most cell  15  extracellular matrix interactions in various cell types including epithelial cells (review by Watt and Jones, 1993). Integrins are important in many aspects of tissue organization and cell migration during development. The only cell types that lack expression of integrins are red blood cells. A singje cell may express multiple integrins some of which have similar binding specifity. For example, Kovisto et al. (1999) showed that a human epithelial keratinocyte can express three different fibronectin-binding integrins namely asPi, a Pi, and v  a P6v  Some integrins are expressed in only one cell type; however, many integrins are expressed by more than one cell type. For example, leukocytes are the only cell type expressing  oiifii  integrin; however, many cell types express variousTli integrins (review  by Halddnenet al., 2000).  F.  Extracellular matrix Extracellular matrix or basal lamina is a mat of bio-molecules supporting the  epithelium. Mechanical stability between epithelium and the under-lying basal lamina is brought about by the interaction between the basal cells and the specialized extracellular matrix molecules forming a thin bio-mat otherwise known as basement membrane zone. The basement membrane zone provides structural support, partitions tissue into specific divisions, and influences cellular behavior (Paulsson, 1992). Basement membrane components are involved in cell migration and influence stratification as well as differentiation (Fine, 1994). The basal cell layer is responsible for the synthesis of a number of basement membrane zone components including different types of collagen (collagen IV), laminins (Laminin-5), fibropectin, and etc. (Burgeson and Christianson, 1997).  16  i.  Collagen IV- Collagen I V is the major structural component of the  basement membrane that possibly influences epithelial adhesion (Reviewed by BouGharious et al., 2004). Collagen IV is a hetero-trimer consisting of two a i chains and one 012 chain, and in large parts it adopts the collagen characteristic triple-helical fold. (Fraser et al., 1979). The triple-helical fold is known to be essential for higher affinity binding of otiPi integrin to collagen type IV. (KuhnEble, 1994). ii.  Laminin-5- Laminin-5 is characterized as a component of anchoring  filaments in the basement membrane (Carter et al., 1991). Laminin-5 is composed of three subunits called ct3, 03, Y2 (Matsui et al., 1995). Both 013P1,  0^4  integrins interact  with laminin-5 (Carter et al., 1991). Previous investigations have shown that laminin-5 is expressed by migrating keratinocytes during re-epithelialization of human full-thickness wounds (Larjava et al., 1993). In vitro, laminin-5 promotes the formation of hemidesmosomes (Hormia et al., 1995). iii.  Fibronectin- Fibronectin is a critical early component of the clot and the  forming granulation tissue (Review by Hakkinen et al., 2000). Fibronectins are a family of closely related adhesive glycoproteins, and are key components of the basement membrane and the provisional wound matrix. Fibronectin is also an integral extracellular matrix that serves as the substratum during cell migration in wound healing (Yamada et al., 2003). Since fibronectin contains multiple binding sites, it is capable of linking various components of basement membrane to each other as well as to the receptors on the cell surface. Furthermore, fibronectin molecules can bind to each other, further enhancing their capacity to form a stable network of basement membrane components and their anchorage to.the cell surface (Alberts et al. 1994; Yamada et al., 2003).  17  Plasma fibronectin is the soluble form of the fibronectin found in blood. Fibrin is a blood clotting protein that interacts with plasma fibronectin. The ability to bind fibrin makes fibronectin an important component of the plasma clot found in wounds. However, the blood clot initially contains plasma fibronectin that is later replaced by cellular fibronectin produced by keratinocytes, fibroblasts, and macrophages (Clark, 1996). A fibronectin molecule consists of two large polypeptides linked near their carboxyl ends by a pair of disulfide bonds consisted of three types of homologous repeating modules termed I, II, and III (Peterson et al., 1983). Each polypeptide contains about 2500 amino acid residues and has a mass of approximately 250Kda. Polypeptides are non-identical and each of the polypeptides is folded into a series of globular domains connected by short, flexible segments of the polypeptide chain. The capability of some of the domains to recognize various components of the basement membrane, including several types of collagen (1,11, and IV) and specific proteoglycans allows specific binding interactions between fibronectin and basement membrane. Other domains recognize and bind cell surface receptors via a specific tetrapeptide sequence, RGDS (arginine-glycineaspartate-serjne). The RGDS sequence is a common motif recognized by integrins such as a B i , "fibronectin receptor" (Alberts et al., 1994; Yamada et al., 2003). 5  The capacity for the large number of variants of a gene to exist is based on differential splicing pattern of the mRNA. There are more than 20 different isoforms of fibronectin recognized. Fibronectin can vary structurally in a tissue specific manner by alternative splicing of three regions namely EIIIA, EIHB, and VI20 (Yamada and Clark, 1996). For example, wound healing in adult rat skin is accompanied by a re-expression of cellular fibronectin that contains both EIIIA and EIHJB (Buck and Horwitz, 1987).  18  The Aim of the thesis M y hypothesis is that titanium implant surfaces with different surface roughness will differ in their effects on epithelial cell behavior. The specific aims of this thesis are to compare and evaluate the effects of random surface topographical roughness features on the growth, area, focal adhesion formation and proximity of epithelial cells to the titanium surfaces. In particular: The primary purpose of rough surfaces in dental implants was meant to optimize the behavioral response of bone cells in respect to growth and adhesion. Epithelial cells can also come in contact with rough surfaces (Deporter et al., 1986); therefore, it is of interest to determine the effect of these rougher surfaces, such as SLA, on the proliferation of epithelial cells. As a working hypothesis based on the data of Cochran et al., (1994) one would expect smooth surfaces to promote the growth of E-cells to a greater extent than rough S L A surfaces. The above hypothesis could be tested by monitoring the growth of epithelial cells on surfaces of varying roughness as a function of time. -  Contact-induced spreading (increase in projected cell area upon contact with another cell of same origin) of epithelial cells in vitro has been shown to be characteristic of epithelial cells. Oakly and Brunette (1995) and Clark et al., (1991) evaluated the contact-induced spreading behavior of epithelial cells on smooth surfaces and grooved surfaces. Besides other findings Oakly and Brunette (1995) as suggested by Clark et a l , (1991) reported a less responsive behavior of epithelial cells in clusters to the topographical cues i.e. the  19  topographical effects on the cytoskeleton could be overridden by adjacent cell contacts on the groove surfaces. Therefore, a working hypothesis would be that isolated epithelial cells would spread well on smooth surfaces and exhibit contact-induced spreading whereas spreading and contact-induced spreading would be less evident on rough S L A surfaces. In addition one would expect that clusters of epithelial cells on rough surfaces to be less responsive to the topographical cues. The above hypotheses could be tested by collecting morphological data on the projected area of epithelial cells in singlets, doublets, as well as clusters on smooth and rough S L A surface using S E M microscopy. A critical aspect of cell behavior on the biomaterial surfaces for many applications is the adhesion of cells to the surfaces. Previously it has been observed that epithelial cells adhere and spread well on smooth surfaces. However, the response of epithelial cells on the rough S L A surfaces in terms of adhesion has not been studied. As a working hypothesis one could expect that epithelial cells develop more and/or larger focal adhesions on the smooth surfaces compared to the rougher surfaces. The latter hypothesis could be tested by quantitative assessment of area of focal adhesions/ area of cell using immunofluorescence staining of vinculin in focal adhesions. To resist bacterial invasion a tight seal between the soft tissue, in particular epithelial tissue, and the dental implants is considered to be important in the long-term success. As a working hypothesis one expects that epithelial cells adhere more closely to the smooth surfaces compared to the rough S L A  20  surfaces. 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The quality of the seal between the dental implant and the surrounding gingival tissue, among other things, contributes to the success of an oral implant (Cranin et al., 1977; Brunette and Chehroudi, 1999; Brunette D M . , 1986) because a tight seal between the epithelium and the surface is required to protect the underlying tissues from bacterial invasion. However, a tight seal itself is not a sufficient condition for success of an implant device that penetrates the oral mucosa. The down-growth of epithelial tissue along an implant can result the failure of devices penetrating epithelium (von Recum A.F., 1990). Surface topography has been used to directly influence the response of connective tissue and epithelium and to inhibit the down-grpwth of epithelium at the device interface (Brunette D . M . , 1986; Chehroudi et al., 1995). However, surface topography can also affect cell adhesion (Harris A.K., 1984). One important class of cell adhesion structures are focal adhesions that are specialized regions of the cell membrane and are separated from the substratum by 10-15 nm. Bundles of actin filaments terminate at focal adhesions where they attach to integrins. Integrins coniprise a large family of cell-surface matrix receptors with an extra-cellular domain that binds to a protein component of the extra-cellular matrix and an intra-cellular domain that indirectly binds to actin filaments via a complex of attachment proteins, including talin, a-actinin, and vinculin (Anderson and Springer, 1987; Buck and Horwitz, 1987; Hynes R.O, 1987). Localization of vinculin by immunofluorescence microscopy is often used to demonstrate the presence of focal adhesions (Geiger 1979; Geiger et al.,  35  1983). Richards et al., (2001) and others (Tan et al., 2003) have argued that the area of vinculin staining is an indication of the strength of cell adhesion. Titanium (Ti) coated acid etched (AE), grit blasted (B), and grit blasted & acid etched (SLA) surfaces decreased the growth and the area of epithelial (E) cells compared to smooth tissue culture plastic (TCP), tissue culture plastic Ti coated (TCP-Ti), and polished Ti coated (P) surfaces. Moreover, E cells formed more and larger focal adhesions on P than on rougher A E surface. T E M measurements of membrane proximity to the Ti surface indicated that membrane-Ti distance increased as the Ti surface roughness increased. In summary the findings in this study indicated that rough surfaces decreased the growth, spreading, membrane-Ti proximity, and adhesion of epithelial cells on Ti surfaces indicating that epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.  36  Methods and Materials I.  Cell culture The epithelial (E) like cells were derived from epithelial rests of Malassez as  described in Brunette et al., (1976). E cells between the 4 and 6 subculture were th  th  maintained in the alpha Minimal Essential Medium ( a M E M ) , plus 15% fetal bovine 1  serum (FBS) , and antibiotics (penicillin G , 100 pg/ml; gentamicin , 50 pg/ml; 2  3  3  amphotericin B , 3 pg/ml) at 37°C in a humidified atmosphere of 95% air and 5% CO2. 4  The media was changed on day one and subsequently at 48 hours intervals. For counting, cells were detached from the surfaces using 0.25% citrate saline trypsin (1:250) solution 3  (pH 7.8) containing 0.1% glucose and then numbers were determined using a Z l 3  Coulter® cell counter instrument. 5  1  2  3  4 5 6  7 8  9 1 0  StemCell Technologies Inc., Vancouver, B.C. MEDICORP, Montreal, Quebec Sigma, St. Louis, M o . Fungizone, Gibco, Grand Island, N Y Coulter Electronics Limited, Luton, Beds (Thermonax), Nalge Nunc Int., Rochester^ N . Y . (PROVIL®novo Light), Heraeus Kuzler GmbH & Co., Hanau (EPO-TEK 302-3), Epoxy Technologies, Billerica, M A (METASERVE), Buehler U K Ltd., Coventry I C N Biomedicals Inc., Aurora, Ohio  37  II.  Surface topography E cells were cultured on T C P , TCP-Ti, P, A E , B , and S L A Ti coated replicas. 6  Replicas of these surfaces were prepared according to Wieland et al., (2002). In brief, impressions of P, A E , B , and S L A surfaces were made with vinyl polysiloxane  7  impression material. Vinyl polysiloxane negative replicas were then used to cast epoxyresin positive replicas. The epoxy replicas were cured at room temperature for 24 hours 8  followed by a 36 hours curing in the oven at 58°C. Surfaces were then ground on the back (i.e. flat) side of the replicas to final even thickness of 1mm on a grinder-polisher machine . All surfaces were cleaned by ultrasonication in 7X-detergent and sputter 9  10  coated with 50 nm of Ti. Final, cleaning and sterilization was accomplished by glow 11  discharge treatment for 4 minutes in an argon chamber prior to culturing of the E cells. S E M images of the rough surfaces are shown (Figure 1). The Ra, average roughness, values of the epoxy-resin replica surfaces follow: P: 0.06 pm, A E : 0.58 pm, B: 5.09 pm, and SLA: 4.33 pm (complete review characterization of these surfaces is given in Wieland et al., 2002).  11  1 2  1 3  Randex 3140 Sputtering System, Palo Alto, C A (Axioskop 2), Zeiss, Jena Empix Imaging Inc., Mississauga, O N  38  III.  Propidium Iodide (PI) staining To determine the effect of surface roughness on cell proliferation, cells were  seeded at an initial density o f - 35 cells/mm and cell numbers assayed on days 1,2,3,4, 2  and 5 post seeding. Also, high-density (~ 500 cells/mm ) cultures were seeded and 2  observed on days 1,3,7, 14, and 28 post seeding. Modification of the protocol described by Mazzini et al., (1980) was used to stain E cells with the PI. In brief, cells were rinsed thoroughly with warm (37°C) plain a M E M prior to fixation. Cells were then fixed in absolute ethanol for 2 minutes. Cells were hydrated in graded (95%, 90%, 70%, 50%, 30%) ethanol series, 2 minutes each, followed by a final hydration in distilled water. Each surface was then inverted on a 50 pi drop offreshlydiluted PI dye at the final concentration of lg/L in phosphate saline buffer (PBS), p H 7.3, for 20 minutes in dark. Cells were rinsed thoroughly in distilled water followed by a final rinse in 100% ethanol in order to remove any unbound dye. Glass coverslips were mounted on the surfaces with immersion oil and examined using epifluorescence microscope equipped with 12  rhodamine filter set  IV.  (Xerni sion  =  S  540nm) and image analysis software, Northern Eclipse . 13  Scaning electron microscopy ISEM) determination of cell area To determine cell morphology, E cells were seeded at an initial density of ~ 100  cells/mm on the surfaces of differing roughness. Cell morphology was prepared for 2  examination in the scanning electron microscope (SEM) on day-2 post seeding according  39  to Wieland et al., (2002). In brief, cells were rinsed thoroughly with warm (37°C) a M E M , fixed for 1 hour in 2.5% gluteraldehyde in PBS at 4°C, and rinsed in 0.1M PBS. 3  Che subsequent treatment steps were performed using a microwave technique . Cells 14  were irradiated in the microwave at 37°C with a power of 750 W for post-fixation (2% osmium in PBS, followed by 1% tanic acid enhancement of osmium fixation) and at 45°C with 750 W for dehydration (50%, 70%, 90%, 95%, 100% ethanol, 1 minute each). Cells were further critical point dried with C O 2 in a semi-automatic critical point dryer , 15  and sputter-coated with 10 nm of gold. Cells were observed with a Cambridge 16  Stereoscan 260 at an accelerating voltage of 12 KeV. Singlets (single cell), doublets (2 cells in contact), and clusters of E cells (3 and more cells in contact) were examined separately. Micrographs of ten cells in each class on each surface were collected. Cells were traced individually using a PC computer station equipped with a W A C O M drawing plate and analyzed for cell area using software ImageJ from NTH.  V.  Immunofluorescence To eliminate the background fluorescence caused by the epoxy replicas, original  metal surfaces were used in these experiments. Focal adhesion formation was visualized on day-2 post seeding at which time the cells were prepared for incubation with fluorescent hydrazide to stain cell components including the cell membrane and vinculin in focal adhesions using anti-Vinculin antibody. Samples were prepared for immunofluorescence observation according to Oakley and Brunette (1993) with minor  1 4  15  16  (PELCO™ 3470 Hornet Microwave System), Pelco International, Redding, C A (SAMADRI®-795), Tousimis, Rockville, M D (Hummer V I Sputtering System), Technics 40  modifications. In brief, cells were rinsed twice in 37°C plain cdVLEM and 37°C cytoskeleton-stabilization (CS) buffer, fixed for 10 min in 3.7% formaldehyde at 37°C and rinsed in 37°C CS buffer twice, oxidized in 4.2mM sodium periodate  in PBS for 30  minutes at 4°C and rinsed in 0. I M PBS. Samples were inverted on a 50 pi drop of 10 m M fluorescein-5-thiosemicarbazide , filtered with 0.45 pm syringe filter, for 2 hours in 18  37°C to stain the cell membrane and rinsed in PBS with 0.1% bovine serum albumin (BSA) 5 times. This was followed by 3 min extraction in 0.5% Triton X-100 at 4°C in CS buffer and 5 rinses in CS buffer at room temperature. Samples were then quenched in fresh 0.05% sodium borohydride in PBS for 10 min at room temperature and 5 rinses in 17  CS buffer. All the rinses were performed on a rotator for 2 min each in dark. 19  Nonspecific binding was blocked with 3% B S A in PBS at 37°C for 30 min. Incubation of the mouse monoclonal primary anti-Vinculin antibody was performed overnight at 4°C 20  in a humidified sealed chamber in the dark. Cells were then rinsed five times with 1% B S A in PBS and then inverted on a 50 pi drop of Texas-Red goat anti-mouse IgG secondary antibody for 1 hour at room temperature in the dark, rinsed in 0.1% B S A in 18  PBS for 5 times. Glass coverslips were mounted onto the surfaces with 1:1 glycerol: PBS solution containing 0.02% sodium azide and 1,4-diazabicyclo [2.2.2] octane. Twenty randomly selected cells that were not in contact with any other cell on each of the surfaces (P and AE) were examined with 40x objective on a confocal laser-scanning microscope ( C L S M ) . The outer boundary of the cell membrane, as visualized with 12  1 7  1 8 1 9 2 0  B D H Inc., Toronto, Ont. Molecular Probes Inc., Eugene, Oreg. (Rotomix 48200), Thermolyne, Dubuque, Iowa Chemicon International Inc., Temecula. C A 41  fluorescent hydrazide staining was traced using a PC computer station equipped with a W A C O M drawing plate and analyzed for cell area using software ImageJ from NTH for each cell. The total area of focal adhesions as assessed by anti- Vinculin staining was analyzed in the same manner.  VI.  Immunogold staining and detection of vinculin in focal adhesions using back-scatter ed electron ( B S E ) imaging on S E M To visualize vinculin in focal adhesions at a higher resolution and to evaluate the  relationship between topography and the focal adhesion sites an immunogold labeling 21  technique using Alexa Flour-488 Fluoronanogold- Fab' 1.4 nm gold particle conjugate was developed. Cells were seeded at an initial density of ~ 100 cells/mm on P, A E , B , and S L A epoxy replicas. The staining protocol was identical to that of immunofluorescence staining of vinculin in focal adhesions prior to the application of secondary antibody incubation. At this time, cells were incubated with 5% goat serum in PBS to block nonspecific binding sites for 30 min at 37°C. Cells were incubated with Alexa Flour-488 Fluoronanogold- Fab' 1.4 nm gold particle conjugate (diluted 1: 200 in 0.1% B S A in PBS) for 1 hour at room temperature and rinsed in 1% B S A in PBS 5 times. Cells were then fixed in 1% gluteraldehyde in PBS for 10 minutes at room temperature, 3  and rinsed 5 times with 50 m M glycine in PBS to remove aldehydes. Samples were then 22  rinsed in 1°/ B S A in PBS with 0.5% Tween-20 5 times and rinsed with distilled water. 23  P  Nanoprobes Inc., Stony Brook, N . Y . E M Sciences, Gibbstown, N J Fisher Scientific, Edmonton, A B 42  The 1 4nm gold particles were enhanced to ~ 15 nm in diameter using the gold enhance kit . Speciniens were then carbon coated with 10 nm of carbon using a vacuum 21  evaporator . Specimen examination was performed on a Hitachi S-4700 S E M field 24  emission microscope with modifications to parameters set by Richards and ap Gwynn (1995). B S E image of vinculin in focal adhesions in an E cell on P is shown in Figure 2.  VII.  Transmission electron microscopy (TEM) evaluation of membrane-Ti proximity To investigate the effect of surface roughness on the proximity of E cells' basal  membranes with the Ti surface, cells were seeded at an initial density of - 500 cells/mm  2  on P, A E , B , and S L A epoxy replicas. Samples were prepared for T E M evaluation on day-2 post seeding using a modification to the technique of Gould et al., (1981). In brief, samples were treated in the same manner as described above for S E M preparation prior to the application of critical point drying. At this time samples were infiltrated in a mixture of ethanol: pjiain Araldite epon-resin without accelerator in a 2: 1, 1: 1, 1:2 ratio for 1 25  hour each at room temperature on the rotator and 100% resin over night at room temperature. Further, samples were infiltrated with resin-accelerator, DMP-30 , mixture for 1 hour at room temperature on the rotator, and embedded in vinyl polysiloxane impression molds. The resin was cured for 24 hours at 37°C, 24 hours at 45°C, and 24 hours at 58°C. Ultrathin sections perpendicular to the surface were cut using a diamond knife from each of the surfaces. The sections were then stained with alcoholic uranyl acetate and aqueous lead citrate, and five cells from each surface were viewed under an 2 4 2 5  Mikros Inc., Portland. Oreg. Canemco Inc., St. Laurent, Quebec 43  electron microscope . Five randomly selected locations on each cell were imaged at 26  12000x magnification and 10 different measurements at each location were taken. The micrographs were developed and scanned into a P C computer station equipped with a W A C O M  drawing plate and analyzed for membrane Ti proximity using software ImageJ  from N T H .  VIII.  Statistical Analysis A  one-way analysis of variance  (ANOVA)  difference) test was employed using J M P I N  2 7  . A  with Tukey H  S D  (honesty significant  level of P<0.05 was accepted as  statistically significant.  ( E M 300), Philips, Holland S A S Institute Inc. 44  Results i.  Epithelial Growth E cell interactions with surfaces of differing roughness were studied using low  population density cultures that were assessed on days 1,2,3,4, and 5 and high population density cultures that were observed on days 1, 3, 7, 14, and 28. Figure 3 shows the PI staining of E cells at low population density on day-1 and day-5 on surfaces with different roughness. The cell numbers were plotted against the time for the different roughness topographies (Figure 4). A tissue culture plastic (TCP) surface was included as a standard for comparison with other studies. To investigate the effect of Ti coating relative to this standard, a TCP surface coated with Ti (TCP-Ti) was also employed. It was found that there was a 10-fold increase in E cell numbers on day-5 (~ 100 cells/mm ) compared to day-1 (~ 10 cells/mm ) on TCP whereas there was a 52  2  fold increase in the cell number on day-5 (~ 50 cells/mm ) compared to day-1 (-10 2  cells/mm ) op TCP-Ti. Although there were no significant differences in E cell numbers 2  on different surfaces on day-1 (P>0.05), on day-2 there was a significant increase, 2-fold increase, (p<0.05) in E cell numbers on TCP (~ 30 cells/mm ) compared to the TCP-Ti (~ 2  15 cells/mm ). The same trend was observed on day-3, day-4 and day-5. Furthermore, on 2  day-3 there was a significant increase in E cell numbers on TCP-Ti and P (smooth topography) compared to the A E , B , and S L A (rough topography). The same trend was observed on day-4 and day-5. There were no statistically significant differences (P>0.05) in E cell numbers on the rough surfaces (AE, B , and SLA) on day-1 through day-5. High population density cultures were used to assess the effects of the rough surfaces on sheets of E cells that could be considered analogous to epithelial tissue. On 45  day-1 E cells were uniformly distributed on all the surfaces and had formed a confluent layer o f cells. O n day-3 E cell cultures on T C P , T C P - T i , and P surfaces had formed multi-layers whereas the cells on the A E , B , and S L A surfaces remained a confluent monolayer. However, on day-7 some o f the E cells cultured on B and S L A had detached and patchy areas were evident that lacked any cells. O n day-14 the growth of E cells on A E surface had increased so that small densely packed islands o f E cells could be observed in the confluent monolayer; however, on B and S L A E cells formed distinct colonies with large gaps between the colonies. The cultures were followed until day-28 at which time E cells grown on T C P , T C P - T i , and P surfaces had undergone exfoliation cycles, in which confluent sheets of E cells detached from the surface leaving a few cells behind which multiplied to re-establish confluence. O n A E surface the highly densely packed islands of E cell islands had grown in area and increased in numbers. E cells on B and S L A surfaces in colonies grew in area but still did not contact other colonies.  II.  Cell area As previous studies (Middleton C . A . , 1977; Oakley and Brunettte 1995) have  shown that epithelial cells exhibit contact-induced spreading, it was necessary to measure the cell area o f three different categories (singlets, doublets, and clusters). Images comparing the three different classes o f E cells are shown (Figure 5). Area^ of E cells on different surfaces were measured and are plotted by surface (Figure 6). In agreement with previous reports E cells in contact with other E cells exhibited a threefold increase in cell area. There were no significant difference (P>0.05) in area/cell for each cell category between T C P and T C P - T i surfaces. However, the area/cell o f isolated E cells on T C P , T C P - T i , P, A E , B surfaces was significantly  46  (P<0.05) greater than on the SLA surface. Furthermore, the area/cell of E cell doublets was significantly higher (P<0.05) on TCP and TCP-Ti compared to those on the A E , B , and SLA. The area/cell of doublets was significantly (P<0.05) higher on P compared to the SLA and no significant (P>0.05) difference in area/cell was observed among P, AE, and B. Finally, the area/cell of E cells in clusters was significantly (P<0.05) higher on TCP, TCP-Ti, P, and A E compared to the B and SLA. In general, E cells were more flattened and well spread on TCP, TCP-Ti, P, and A E in contrast to their more cuboidal morphology on the B and SLA surfaces.  III.  Focal adhesions There were more and larger focal adhesions on the P surface compared to the A E  surface (Figure 7). The % area of focal adhesion/cell on the P (smooth surface) was 10.1 ± 4.52 ( S D ) , which was significantly (P<0.05) higher than the A E (rough surface) of 3.38 ± 1.51 (S.D.).  IV.  Immunogold staining and detection of vinculin in focal adhesions using BSE imaging on S E M An immunogold labeling technique of vinculin in focal adhesions using Fab'-gold  (Au) probes was developed to investigate relationships of the focal adhesions with topographic features at higher resolution. BSE images of vinculin in focal adhesions on different surfaces are shown (Figure 8). As found earlier by fluorescence microscopy the focal adhesions were larger and more prominent on the P surface compared to the rough surfaces (AE, B , ancLSLA). Moreover, the focal adhesions on the rough surfaces (AE, B ,  47  and SLA) tended to be localized to the ridges rather than the valleys of these surfaces (Figure 9).  V.  T E M evaluation of membrane-Ti proximity In agreement with the BSE imaging on S E M immunogold labeling, cell contact  with the surfaces was most frequently found on the protruding portions of the rough surfaces (Figure 10). The distance in nm between the basal membrane and the Ti surface topography is given in Figure 11. On average, E cells were significantly (P<0.05) closer to the Ti on the P and A E surfaces compared to the B and SLA surfaces.  48  Discussion This study examined the effect of Ti surface roughness on the growth, spreading, adhesion, and membrane-Ti proximity of E cells. Our results demonstrated that surface roughness is an important determinant of these epithelial cell behaviors. In general, the smoother surfaces of TCP, TCP Ti, and P best supported the growth, spreading, and attachment of E cells. Our results agree with those of Hormia et al., (1991), who used immunofluorescence techniques to observe that gingival epithelial cells attached and spread more readily on smooth than on rough, sandblasted Ti surfaces. Furthermore, Cochran et al., (1994) investigated the attachment and growth of periodontal cells on smooth and rough Ti surfaces and observed that after a lag period of attachment human gingival epithelial cells proliferated on control and smooth Ti, but not on rough Ti surfaces. The growth studies reported here extend those findings by examining the growth in more detail and for high population density at times up to 28 days. As expected the smooth surfaces supported growth to a greater extent than the rough surfaces. In the long term, 28-day, cultures of E cell on TCP, TCP-Ti and P surfaces underwent cycles of exfoliation in which sheets of cells were sloughed off but E cells recolonized the underlying denuded surfaces. On rougher surfaces some E cell exfoliated but the cells that remained were much slower to grow and growth limited to patches of isolated E colonies. A novel aspect of this study is that we quantitatively evaluated the effect of roughness on cell area as a measure of cell spreading in three categories of isolated E cells, doublets, and clusters of E cells. The S E M analysis of E cell areas on different  49  surfaces indicated that E cells were in general more spread and flattened on the smoother surfaces (TCP, TCP-Ti, and P) and A E as compared to the roughest surfaces (B and SLA). Isolated E cells spread more on TCP, TCP-Ti, P, A E , B compared to the S L A surface, that is the result of both etching and blasting. The same trend was observed for E cells in doublets. As well, spreading of E cells was enhanced on contact with other E cells on smooth surfaces (TCP, TCP-Ti, and P) a phenomena first observed and named contact-induced spreading by Middleton C.A. (1977). Contact-induced spreading was not evident on the A E , B , and S L A surfaces as on the smooth surfaces. These findings along with the growth data suggest that epithelial coverage of rough surfaces will be difficult to attain if such surfaces become exposed because the growth of E cells as well as spreading is decreased on rough surfaces. Although Hormia et al., (1991) did not observe the localization of vinculin in focal adhesions, several other laboratories have reported vinculin stained in focal adhesions in other types of epithelial cells on a variety of substrata (Teixeria at al., 2003; Shiraiwa et al., 2002). Focal adhesions can be classified by areas to immature (area<2 pm ) (Galbraith et 2  al., 2002), mature (area 2-6 pm ) (Tamariz and Grinnell 2002), and super-mature (area>6 2  pm ) (Dugina et al., 2001). Immunofluorescence staining of vinculin in focal adhesions 2  showed that E cells expressed more and larger focal adhesions on the P surface compared to the rougher A E surface. The quantification of adhesion by means of vinculin immunofluoresence staining specific to focal adhesions indicated that the total % area of focal adhesions/cell was greater on the P compared to A E surface. As the area of focal adhesions, is thought to be related to the strength of cell adhesion (Lotz et al., 1989; Hinz  50  and Gabbiani, 2003; Richards et al., 2001), it seems likely that epithelial tissue attaches more strongly to the smooth Ti surfaces by promoting the formation of mature to supermature focal adhesions in comparison to the rougher A E surface. These observations suggest that epithelial tissue could be more susceptible to mechanical removal from a rough surface compared to a smooth surface. This study involved the development of an immunogold labeling technique using Alexa Flour-488 Fluoronanogold- Fab' 1.4 nm gold particle conjugate to visualize vinculin in focal adhesions. The technique is novel in that it involves smaller size 1.4 nm gold probes (nanogold) and application of Triton-X post fixation in contrast to that of Richards et al., (2001), who used larger gold particles in an application of conventional immunogold staining of vinculin in focal adhesions in fibroblasts and Triton-X extraction prior to the fixation. In agreement with the data using immunofluorescence to visualize vinculin in focal adhesions the immunogold method revealed fewer and smaller focal adhesions on the rough surfaces. The focal adhesions on rough surfaces tended to be localized to ridges (i.e. protruding structures) and were less evident in the valleys. The ultrastructural analysis of the proximity of the cell membrane to the Ti showed that E cells were significantly closer to the smooth Ti surface (P) and A E compared to the rougher B and S L A surfaces. However, there was a high variation in membrane-Ti proximity on B and S L A as the protuberances on the rougher surfaces were in close contact with the cell membrane while the cells bridged over valleys. These observations are similar to those of Rakhi and von Recum (2003) who evaluated the proximity of fibroblasts on Ti surfaces using T E M , and also observed the bridging of cells between surface protrusions. The bridging behavior has the consequence that both  51  the mean and the standard deviation increase on the rough surfaces; nevertheless, statistically significant differences in membrane-Ti proximity were observed between rough and smooth surfaces. Overall this study supports the view that smooth Ti surfaces are better suited for interfacing devices with epithelium than rough surfaces with random features for the smooth surfaces promote better adhesion, growth, and close adaptation of E cell membrane to the Ti surface. However, this conclusion is dependant on size and shape of the features and is limited to sand blasted and acid etched features that were examined in this study. Microfabricated surfaces with grooves of varying dimensions, whose walls, ridges, and floors are smooth, are also well suited for interfering with epithelial tissue although their Ra values may be high (Chehroudi et al., 1990). 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International Journal of Oral and Maxillofacial Implants. 1994; 9: 289-297.  25)  Teixeira A.I,, Abrams G.A., BerticsP.J., Murphy C.J., NealeyPF. Epithelial contact guidance on well-defined micro-and nanostructured substrates. Journal of Cell Science. 2003; 116: 1881-1892.  26)  Shiraiwa M . , Goto T., Yoshinari M . , Koyano K., Tanaka T. A study of the initial attachment and subsequent behavior of rat oral epithelial cells cultured on titanium. J Periodontal. 2002;73:852-60.  27)  Hinz B., Gabbiani G. Mechanisms of force generation and transmission by myofibroblasts. Curr Opin biotechnology. 2003; 14(5): 538-46.  28)  Galbraith C.G., Yamada K . M . , Sheetz M.P. The relationship between force and focal complex development. J Cell Biol. 2002; 159(4): 695-705.  29)  Tamariz E., Grinnell F. Modulation of fibroblast morphology and adhesion during collagen matrix remodeling. M o l Biol Cell. 2002; 13(11): 3915-3929.  30)  Dugina V., Fontao L , Chaponnier C , Vasiliev J., Gabbiani G. Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J Cell Sci. 2001; 114(18): 3285-96.  31)  Lotz M . , Burdsal C A , Erikson H P . , McClay D R . Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response. J Cell Biol. 1989; 109(4 Pt 1): 1795-1805.  67  32)  Rakhi J., Recum A.F. Fibroblast attachment to smooth and microtextured PET and thin cp-Ti films. Journal of Biomedical Materials Research Part A. 2003; 68A: 296-304.  33)  Chehroudi B., Gould T.R.L., Brunette D . M . Titanium-coated micromachined grooves of different dimensions affect epithelial and connective-tissue cells differently in vivo. J. Biomed. Mater. Res. 1990; 24: 1203-1219.  68  Chapter III- Conclusions and Future studies Conclusions In contrast to orthopedic implants, which are fully enclosed in the body, dental implants are exposed to the environment of a germ-laden oral cavity. The problem of integrating a foreign body in bone has largely been solved; but a second major problem, the maintenance of peri-implant soft tissue around the implant has not been addressed adequately. Jn my thesis, I addressed one aspect of the latter problem through evaluation of the effect of surface roughness on the growth, area, focal adhesion formation, and proximity of epithelial cells to titanium surfaces. E cells responded to surface topography by decreasing their growth on the rougher surfaces of A E , B , and S L A compared to the smooth surfaces. During a 28-day culture period, cultures of E cells on smooth surfaces of TCP, TCP-Ti, and P underwent cycles of exfoliation, in which confluent sheets of cells detached from the surfaces and underlying remaining cells covered the exposed surfaces, whereas on the rougher A E , B , and S L A surfaces growth was much slower and limited to the isolated colonies spaced relatively far apart. Furthermore, projected cell area was measured using S E M in the three classes of isolated, doublets, and clusters of E cells indicated that E cells were in general more spread and flattened on the smoother surfaces (TCP, TCP-Ti, and P) and A E compared to the rougher surfaces of B and SLA. Contact-induced spreading was observed on smooth surfaces (TCP, TCP-Ti, and P) but not on A E , B, and SLA. Also, E cells in isolation and doublets spread the least on S L A surface compared to the other surfaces.  69  E cells appeared to adhere more tightly to the smooth P surface as compared to the A E surface using "mature to super mature" focal adhesions (terminology used to classify focal adhesions by their size by Galbraith et a l , 2002 and Tamariz and Grinell, 2002). The data in my thesis showed that focal adhesion numbers and total area were decreased on rough surfaces. As the number and size of focal adhesions is thought to be related to the strength of adhesion, these observations suggest that epithelial tissue could be more susceptible to mechanical removal from a rough surface compared to a smooth surface. Another novel aspect of my thesis was the development of an immunogold labeling technique using Alexa Flour-488 Fluoronanogold- Fab' 1.4 nm gold particle conjugate to visualize vinculin in focal adhesions using B S E imaging on SEM. The spatial visualization of vinculin, which is one of the 50 proteins found at focal adhesion sites (reviewed by Zamir and Geiger, 2001), in relation to the underlying substratum topography at high magnification with the resolution of E M was an interesting and promising application of the technique. Focal adhesions on rough surfaces were concentrated to the ridge tops i.e. the protrusions of the rough surfaces. This finding will be more fully explained in the future studies section. Finally the ultrastructural analysis of the E cells proximity to the Ti showed that epithelial tissue adhered much more closely to the smooth P surfaces and the A E surface compared to the rough B and S L A surfaces. On rougher surfaces of B and S L A E cells bridged between surface protrusions. These findings suggest that it would be problematic to use rough surfaces to form a successful soft tissue seal to the implant neck.  70  Future studies The application of 1.4 nm nanogold particles in visualization of vinculin was demonstrated for the first time in my thesis (chapter-2, submitted for publication). Other applications of nanogold include labeling of antigens (CAV-lct in capillary endothelial cells of placenta (Takizawa and Robinson, 2003); actin layer in frozen rabbit testis sections (Guttman et al., 2001)). The Alexa Fluor- FluoroNanogold is a dual-purpose probe that consists of an affinity-purified Fab' fragment conjugated covalently through a hinge thiol to 1.4 nm gold. The Fab' fragment carries amine groups that carry positive charges. Also, the gold itself is positively charged during the process of enhancement. During the development of the technique, one of the difficulties I faced was the high background resulting from non-specific adhesion of the secondary probe. The background could be seen both on the titanium oxide surface (hydrophilic and negatively charged) as well as E cells. Four different methodological modifications were introduced to decrease the unspecific background staining: a) addition of a secondary blocking step i.e. goat serum, b) an increase in duration of blocking incubation of both primary blocking agent as well as secondary blocking agent, c) an increase in concentration of the BSA-PBS (primary) blocking agent, and d) an increase in washing time with PBS buffer. The modifications helped in reducing the non-specific background staining on Ti surfaces as well as reduction in non-specific staining observed in cells. The remaining nonspecific staining can possibly be attributed to the positive charge on the gold particles (personal communication with Nanoprobes Inc.) or free vinculin in the cytoplasmic pool. Addition of pon-fat milk powder at 5% concentration to the bathing buffer (PBS) has shown promise in quenching the interaction between gold and cells (personal  71  communication with Nanoprobes Inc.). More recently others have used Alexa fluor streptavidin FluoroNanogold particles (Takizawa and Robinson, 2003). Streptavidin has been added as it is thought to carry the required negative charge to suppress the positive charge on the gold particles. Therefore, the next logical step in enhancement of the immunogold staining procedure discussed in my thesis would be the use of milk proteins as well as use of streptavidin conjugated Nanogold probes to see if the non specific staining caused by the secondary antibody can be further reduced. Another approach would be, staining of actin filaments (using mild osmium fixation (Owen et al., 2001)) that are associated with focal adhesions as means to distinguish free vinculin from vinculin specific to focal adhesions. The magnification and resolution power of an E M as well as successful and specific staining of vinculin in focal adhesions is of potential interest because other antigens pertaining to the adhesion sites (paxillin, talin, a-actinin, ARP 2/3, integrins and etc.) could be investigated in terms of spatial distribution in respect to the surface roughness characteristics, as a function of time and in relation to one another (in terms of the order of arrival at the adhesion sites). The latter could possibly be accomplished by sequential enhancement. In brief enhancement of Nanogold particles was performed using a goldTenhance kit. The 1.4 nm Nanogold particles could be developed to the particle sizes of up to 50 nm in diameter as the function of time (the longer the particle is enhanced the larger the particle size would be). Therefore, it is possible to form a standard curve relating the enhancement time to the particle size. The manufacturer states that " The time period for optimum gold enhancement varies with application; but 3-20 minutes has been found to be optimal for enlarging the 1.4 nm Nanogold particles to 3-20  72  nm or larger in size... and larger development times will give larger particles up to 50 nm in size" (Nanoprobes Gold-enhance kit, Cat. #2113). Thus one could stain antigens sequentially with enhancement procedures between each step. The first antigen stained would be associated with the largest gold particles and the last antigen stained with the smallest gold particles. In this way one could study the sequence of addition of specific proteins in focal adhesions. Therefore, one could possibly propose a mechanism for the formation of focal complexes, dot shape structures maturing to focal adhesions, (reviewed by Wozinal et al., 2004) as well as fibrilar focal adhesions, responsible for matrix organization, (reviewed by Wozinal et a l , 2004), and the regular focal adhesions, ranging from immature to super mature, (Galbraith et al., 2002; Tamariz and Grinell, 2002; Dugina et al., 2001). A further possible, application of the technique could be directed towards the investigation of adhesion structures expressed by E cells in 3-D, culturing the cells in collagen gels, (following the classical work done by Emerman and Pitelka, 1977; Cukierman et al., 2001; and more recently Glass-Brudzinski et al., 2002;). Integrin expression of osteoblasts on the biomaterial surfaces has been investigated previously (Sinha and Tuan, 1996; Krause et al., 2000; and recently reviewed by Paine and Wong, 2003). It has been shown that profile of integrin expression by primary osteoblasts is dependent on underlying substratum topography and the type of material (Sinha and Tuan, 1996). Sinha and Tuan showed that hetro-dimeric functional integrin receptors are specific in terms of preference for proteins found in matrix on biomaterial surfaces. Findings relating to osteoblasts could be further investigated in terms of epithelial cells. Profile of integrin expression of epithelial cells on smooth and rough titanium surfaces could be studied by coating these surfaces with fibronectin,  73  vitronectin, laminin and collagen. Comparison of integrin expression of epithelial cells on different topographies coated with different matrix proteins as well as control surfaces could help elucidate the putative the role of surface features on differential expression of integrins.  '  Previously it has been shown that surface topography can modulate cell shape and secretion of proteins by epithelial cells (Hong and Brunette, 1987). Also, the data in my thesis showed that rough surface topographies modulate the morphology of epithelial cells. Therefore, it would be interesting to further investigate the effect of surface topography on production of different matrix proteins such as fibronectin, laminin, and collagen with which epithelial cells come into contact in vivo. One means to quantify the amount of protein produced by epithelial cells as influenced by underlying surface topography would be to use Western blots. However, the choice of the antibodies, monoclonal or polyclonal, used for the detection of the antigens and the commercial availability of the antibodies could be limiting. The major advantage of monoclonal antibodies is their specificity; however, the denaturation of antigens that can occur during the preparation can potentially make monoclonal antibodies useless in an application of Western blotting. Also, the specific cross-reactions between proteins can produce false positive bands when monoclonal antibodies are used. Polyclonal antibodies are widely used in immunoblotting as they bind to a number of denaturation-resistant epitopes on the antigen; however, polyclonal antibodies could also produce false positive bands due to presence of contaminating antibodies. Another frequently encountered problem in immunoblotting is the presence of diffuse background staining due to the nature of the secondary antibody (when indirect immunoblotting is performed). However, serial  74  dilution of the secondary antibody, usage of other alternative secondary antibodies as well as direct immunoblotting could reduce the diffuse background. In any case, it would be of interest to determine whether surface topography can stimulate epithelium to alter their microenvironment of attachment proteins. In summary there are several possibilities to extend the findings of this thesis from the current base of morphological characterization to the molecular level of protein production and specific mechanisms of binding to Ti surfaces.  75  Bibliography 1) Cukierman E., Pankov R., Stevens D.R., Yamada K . M . Taking cell-matrix adhesions to the third dimension. Science. 2001; 294(5547): 1708-1712. 2) Dugina V., Fontao L., Chaponnier C , Vasiliev J., Gabbiani G. Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J Cell Sci. 2001; 114(18): 3285-3296. 3) Emerman J.T., Pitelka D.R. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro. 1977; 13(5): 316-328. 4) Galbraith C.G., Yamada K M . , Sheetz M.P. The relationship between force and focal complex development. J Cell Biol. 2002; 159(4): 695-705. 5) Glass-Brudzinski J., Perizzolo D B r u n e t t e D . M . Effects of substratum surface topography on the organization of cells and collagen fibers in collagen gel cultures. J Biomed Mater Res. 2002; 61(4): 608-618. 6) Guttman J.A., Janmey P., Vogl A.W. Gelsolin- evidence for a role turnover of junction-related actin filaments on Sertoli cells. J Cell Sci. 2002; 115: 499-505. 7) Hong H.L., Brunette D . M . Effect of cell shape on proteinase secretion by epithelial cells. J Cell Sci. 1987; 87(2): 259-267. 8) Krause A., Cowles E.A., Gronowicz G. Integrin-mediated signaling in osteoblasts on titanium implant materials. J Biomed Mater Res. 2000; 52(4): 738-747. 9) Osteoblast response to pure titanium and titanium alloy. In Bio-Implant Interface. 2003; 123-140.  76  10) Owen G.R., Meredith D O . , Ap Gwynn I., Richards R.G. Enhancement of immunogoid-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions. Cell Biol Int. 2001; 25(12): 1251-1259. 11) Sinha R.K., Tuan R S . Regulation of human osteoblast integrin expression by orthopedic implant materials. Bone. 1996; 18: 451-457. 12) Takizawa T., Robinson J.M. Ultrathin cryosections: an important tool for immunofluorescence and correlative microscopy. J Histochem Cytochem. 2003; 51(6): 707-714. 13) Tamariz E., Grinnell F. Modulation of fibroblast morphology and adhesion during collagen matrix remodeling. Mol Biol Cell. 2002; 13(11): 3915-3929. 14) Wozniak M . A . , Modzelewska K., Kwong L., Keely P.J. Focal adhesion regulation of cell behavior. Biochim Biophys Acta. 2004; 1692(2-3): 103-119. 15) Zamir E., Geiger B. Components of cell-matrix adhesions. J Cell Sci. 2001; 114 3583-3590.  

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