Responses of human keratocytes to micro- and nanostructured substrates

We have previously shown that human corneal epithelial cells respond to synthetic topographic features with dimensions similar to those found in the native human corneal basement membrane. Epithelial cells integrated inputs from substrate topography and soluble factors in the culture medium to generate alignment responses to substrate topographic anisotropies. Human keratocytes are the main cellular components of the stroma, the tissue that underlies the corneal epithelium. Here we report that keratocytes aligned more strongly than epithelial cells along topographic patterns of grooves and ridges. On patterns with pitches of 800 nm and larger approximately 70% of keratocytes were aligned along the patterns compared to 35% for epithelial cells. On 70 nm-wide ridges on a 400-nm pitch, keratocyte alignment dropped to 45%, whereas epithelial cell alignment remained constant. Similarly to epithelial cells, focal adhesions and associated stress fibers in keratocytes were aligned mainly along the substrate topographies, although oblique orientations were also observed. Furthermore, keratocytes cultured on the nanoscale patterns had fewer stress fibers and focal adhesions than cells cultured on microscale patterns or on smooth substrates.     

A PLGA membrane controlling cell behaviour for promoting tissue regeneration

Barrier membranes are used in periodontal applications with the aim of supporting bone regeneration by physically blocking migrating epithelial cells. We report a membrane design that has a surface topography that can inhibit epithelial cell migration and proliferation on one side and a topography that guides osteoblast migration to a desired area. A PLGA copolymer (85:15) blended with MePEG, was cast to have surfaces with smooth, grooved or sandblasted-acid-etched topographies. Epithelial cells spread on smooth surfaces after 24 h, and cell numbers increased after 5 days. Cells on the smooth surface exhibited no preferred direction of migration. On the sandblasted-acid-etched topography epithelial cells spread but the cell number did not significantly increase after 5 days. Cell migration was inhibited on this surface. Osteoblasts spread on both grooved and smooth surfaces and cell number increased after 5 days on all surfaces. The cells that adhered in the grooves migrated preferentially in the direction of the grooves. Positive alkaline phosphatase staining was seen on all surfaces within 4 weeks and positive Von Kossa nodule staining within 6 weeks. These results suggest that surface topographies replicated on opposite sides of a biodegradable polymers membrane can inhibit proliferation and migration of the epithelial cells, and promote proliferation and directional migration of osteoblasts. Addition of appropriate surface topographies to membranes used in guided tissue regeneration has the possibility of improving clinical performance in periodontal tissue regeneration procedures.     

The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts

The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.     

Topographical control of human neutrophil motility on micropatterned materials with various surface chemistry

Controlling cell responses to an implantable material is essential to tissue engineering. Because the surface is in direct contact with cells, both chemical and topographical properties of a material surface can play a crucial role. In this study, parallel ridges/grooves were micropatterned on glass surfaces using photosensitive polyimide to create transparent substrates. The migratory behavior of live human neutrophils on the patterned surfaces was observed using a light microscope with transmitted light source. The width (2 microm) and length (400 microm) of the ridges were kept constant. The height (5 or 3 microm) and the repeat spacing (6-14 microm) of the ridges were systematically changed to investigate the effect of microgeometry on neutrophil migration. In addition, the effect of surface chemistry on neutrophil migration was studied by deposition of a thin layer of "inert", biocompatible metal such as Au-Pd alloy and titanium on patterned substrates. More than 95% of neutrophils moved in the direction of the long axis of ridges/grooves regardless of the topographical geometry and chemistry, consistent with a phenomenon termed "contact guidance". Therefore, cell migration was characterized using a one-dimensional persistent random walk. The rate of cell movement was strongly dependent on the topographical microgeometry of the ridges. The random motility coefficient mu, 9.8 x 10(-9) cm2/s, was the greatest at a ridge height of 5 microm and spacing of 10 microm, about 10 times faster than on smooth glass surface. The Au-Pd coating did not change neutrophil migratory behavior on patterned surfaces, whereas titanium decreased cell motility substantially. The results of this study suggest that optimization of both surface chemistry and topography may be important when designing biomaterials for tissue engineering. In addition, parallel ridges/grooves can be used to control the direction and rate of cell migration on the surface.     

Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface.     

Integration of basal topographic cues and apical shear stress in vascular endothelial cells

In vivo, vascular endothelial cells (VECs) are anchored to the underlying stroma through a specialization of the extracellular matrix, the basement membrane (BM) which provides a variety of substratum associated biophysical cues that have been shown to regulate fundamental VEC behaviors. VEC function and homeostasis are also influenced by hemodynamic cues applied to their apical surface. How the combination of these biophysical cues impacts fundamental VEC behavior remains poorly studied. In the present study, we investigated the impact of providing biophysical cues simultaneously to the basal and apical surfaces of human aortic endothelial cells (HAECs). Anisotropically ordered patterned surfaces of alternating ridges and grooves and isotropic holed surfaces of varying pitch (pitch = ridge or hole width + intervening groove or planar regions) were fabricated and seeded with HAECs. The cells were then subjected to a steady shear stress of 20 dyne/cm(2) applied either parallel or perpendicular to the direction of the ridge/groove topography. HAECs subjected to flow parallel to the ridge/groove topography exhibited protagonistic effects of the two stimuli on cellular orientation and elongation. In contrast, flow perpendicular to the substrate topography resulted in largely antagonistic effects. Interestingly, the behavior depended on the shape and size of the topographic features. HAECs exhibited a response that was less influenced by the substratum and primarily driven by flow on isotropically ordered holed surfaces of identical pitch to the anistropically ordered surfaces of alternating ridges and grooves. Simultaneous presentation of biophysical cues to the basal and apical aspects of cells also influenced nuclear orientation and elongation; however, the extent of nuclear realignment was more modest in comparison to cellular realignment regardless of the surface order of topographic features. Flow-induced HAEC migration was also influenced by the ridge/groove surface topographic features with significantly altered migration direction and increased migration tortuosity when flow was oriented perpendicular to the topography; this effect was also pitch-dependent. The present findings provide valuable insight into the interaction of biologically relevant apical and basal biophysical cues in regulating cellular behavior and promise to inform improved prosthetic design.     

Titanium-coated micromachined grooves of different dimensions affect epithelial and connective-tissue cells differently in vivo

A desirable feature of an implant surface which penetrates epithelium would be that the surface impedes epithelial downgrowth. Previous experiments have shown that the micromachined, horizontally oriented grooves on the percutaneous implant surface can impede epithelial downgrowth (Chehroudi et al., J. Biomed. Mater. Res., 22, 459 (1988) and 23, 1067 (1989)). However, little is known of the effect of varying groove parameters such as depth, spacing, and orientation on epithelial downgrowth and attachment of epithelial (E)-cells and fibroblasts (F) to percutaneous implants in vivo. Grooves were produced with a 30-micron pitch and depths of 22 microns, 10 microns, or 3 microns. In addition, 10-microns- and 3-microns-deep grooves were made with pitches of 39 microns and 7 microns, respectively. Implants with grooves oriented either horizontally or vertically to the long axis of the implant as well as smooth control surfaces were coated with 50 nm of titanium and placed in the parietal area of rats for a period of 7 days. Close attachment of E-cells was found on the smooth, 10-microns- and 3-microns-deep, horizontally or vertically aligned grooved surfaces; in contrast, E-cells bridged over the 22-microns-deep, horizontally oriented grooves. F formed a capsule on the smooth surface as well as the 10-microns- and 3-microns-deep horizontally oriented grooves, but F inserted obliquely into the 22-microns-deep, horizontally aligned grooved surface. Histomorphometric measurements indicated that the epithelial downgrowth was greatest on the vertically oriented grooved and smooth surfaces and was shortest on the 22-microns-deep and 10-microns-deep horizontally aligned grooved surfaces. These differences indicate that epithelial downgrowth was accelerated on the vertically oriented grooved surfaces and inhibited on the horizontally oriented grooved surfaces. Moreover, the mechanism of inhibition of the epithelial downgrowth may differ among these surfaces. E-cells bridged over the 22-microns-deep grooves and their migration appeared to be inhibited by the F that inserted into the implant surface. In the shallower horizontal grooves, however, epithelial downgrowth was probably inhibited by contact guidance because there was no evidence of F inserting obliquely into the implant surface.     

A light and electron microscopic study of the effects of surface topography on the behavior of cells attached to titanium-coated percutaneous implants

Previous studies using light microscopy have demonstrated that micromachined grooved surfaces inhibit epithelial (E) downgrowth and affect cell orientation at the tissue/implant interface. This study investigates the ultrastructure of the epithelial and connective-tissue attachment to titanium-coated micromachined grooved, as well as smooth control, implant surfaces. V-shaped grooves, 3, 10, or 22 microns deep, were produced in silicon wafers by micromachining, replicated in epoxy resin, and coated with 50-nm titanium. These grooved, as well as smooth, titanium-coated surfaces were implanted percutaneously in the parietal area of rats and after 7 days processed for electron microscopy. The tissue preparation technique used in this study enabled us to obtain ultrathin sections with few artifacts from the area of epithelial and connective-tissue attachment. The histological observations demonstrated that E cells closely attached to, and interdigitated with, the 3-microns and 10-microns grooves. In contrast, E cells were not found inside the 22-microns-deep grooves and made contact only with the flat ridges between the grooves. As a general rule, fibroblasts (F) were oriented parallel to the long axis of the implants and produced a connective tissue capsule with 3-microns and 10-microns-deep grooved surfaces as well as smooth surfaces. On the 22-microns-deep grooved surfaces, however, F inserted obliquely into the implant. The attachment of F to the titanium surface was mediated by two zones; a thin (approximately 20 nm), amorphous, electron dense zone immediately contacting the titanium surface, and a fine fibrillar zone extending from the amorphous zone to the cell membrane. As oblique orientation of F has been associated with the inhibition of epithelial downgrowth, micromachined grooved surfaces of appropriate dimensions have the potential to improve the performance of percutaneous devices.     

The influence of substrate topography on the migration of corneal epithelial wound borders

Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.     

Variation in basement membrane topography in human thick skin

Samples of human plantar and palmar skin were excised and incubated in 20 mM EDTA after which the epidermis was gently separated from the dermis with the plane of separation occurring in the lamina lucida. Scanning electron microscopic examination of the dermal component revealed the classically described series of regularly spaced grooves and papillae that characterize the epidermal-dermal junction in thick skin. Primary dermal grooves exhibited evenly spaced tunnels that were originally occupied by sweat gland ducts. The basement membrane (basal lamina) in the primary grooves was relatively smooth but did exhibit a flattened, reticulated pattern at high magnifications. The basement membrane of secondary dermal grooves and papillae was in the form of numerous, elevated microridges off of which septae arose at roughly right angles. The surface appearance of the basement membrane in these areas was that of a honeycomb owing to the numerous compartments and recesses formed by the ridges and septae. Degradation of the basement membrane by trypsin demonstrated that the foundation for the highly folded and compartmentalized basement membrane was composed of dermal collagen fibrils, 60-70 nm in diameter, that were arranged in a series of variably sized, interconnected collagen bundles or walls. Epidermal basal cells extended cytoplasmic (foot) processes into two or more compartments, formed by the ridges and septae, which considerably amplified the basement membrane surface available for epidermal attachment. Scanning electron microscopic studies of the epidermal-dermal junction confirm the variable surface character of this interface previously reported by others using sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning electron microscopy of cell surfaces following removal of extracellular material.

The application of scanning electron microscopy to the study of cell surfaces is limited in intact tissues, because extracellular material may often obscure the details of nonluminal surfaces. To remove connective tissue elements we have treated human skin and both kidney, and an autonomic ganglion of the rat with hydrochloric acid and collagenase. Regional variations in the basal surface of the nephron are noted following removal of the basement membrane. The basilar interdigitations of the cells of the proximal tubule appeared as parallel ridges encircling the tubule. Ridges on the parietal epithelium of Bowman's capsule were randomly arranged and alternated with smooth surfaces. The dermal surface of the human epidermis has an alveolar or honeycomb appearance due to the elevation of the epidermal ridges and numerous pits for the dermal pegs. At higher magnifications the basal surface of cells of the stratum germinativum possessed numerous and irregular projections. Neurons with their processes are evident in the autonomic ganglion. The soma of the neurons are enclosed by flattened satellite cells. Irregular spaces between opposed satellite cells are interpreted as regions for the passage of processes related to the ganglion cells. Nodes of Ranvier were clearly seen along nerve fibers. Some pitting of the nerve fibers was also noted. The HCl-collagenase method has the advantage of the removal of collagen and basement membrane while preserving the structural integrity of the cell surface.     

Quantitative analysis of osteoblast-like cells (MG63) morphology on nanogrooved substrata with various groove and ridge dimensions

Nanotextured silicon substrata with parallel ridges separated by grooves with equal width from 90 to 500 nm, were fabricated by electron beam lithography and dry etching techniques. Osteoblast-like cells, MG-63, were cultured on the sterilized nanopatterned substrata for 4 or 24 h, and then imaged by scanning electron microscopy. The influence of substrate topography on cell morphology was analyzed by image software. We found the initially cells spread faster on the nanopatterned surfaces than on the flat surface, suggesting that surface anisotropic feature facilitates initial cell extension along its direction. However, because of inhibition of cell lateral expansion across nanogrooved surfaces, the cells on the nanogrooved surface did not further expand laterally, and cell spreading area was less than that on the flat surface after 24 h of incubation. Cells elongated and aligned along the direction of grooves on all the nanopatterned substrata. Furthermore, fluorescence staining of cell nuclei indicated that the nuclei of the cells cultured on the nanopatterned surfaces also displayed a more elongated and aligned morphology along the direction of the grooves. Since cell shape and orientation influence cell functions and alignment of extracellular matrix secreted by cells, our results may provide the information regarding responses of osteoblasts to the nanostructure of collagen fibrils, and benefit bone tissue engineering and surface design of orthopedic implants.     

Scanning electron microscopy of cell surfaces following removal of extracellular material

The application of scanning electron microscopy to the study of cell surfaces is limited in intact tissues, because extracellular material may often obscure the details of nonluminal surfaces. To remove connective tissue elements we have treated human skin and both kidney, and an autonomic ganglion of the rat with hydrochloric acid and collagenase. Regional variations in the basal surface of the nephron are noted following removal of the basement membrane. The basilar interdigitations of the cells of the proximal tubule appeared as parallel ridges encircling the tubule. Ridges on the parietal epithelium of Bowman's capsule were randomly arranged and alternated with smooth surfaces. The dermal surface of the human epidermis has an alveolar or honeycomb appearance due to the elevation of the epidermal ridges and numerous pits for the dermal pegs. At higher magnifications the basal surface of cells of the stratum germinativum possessed numerous and irregular projections. Neurons with their processes are evident in the autonomic ganglion. The soma of the neurons are enclosed by flattened satellite cells. Irregular spaces between opposed satellite cells are interpreted as regions for the passage of processes related to the ganglion cells. Nodes of Ranvier were clearly seen along nerve fibers. Some pitting of the nerve fibers was also noted. The HCl-collagenase method has the advantage of the removal of collagen and basement membrane while preserving the structural integrity of the cell surface.     

脱细胞猪角膜表面基底膜成分的检测

背景：前期实验显示脱细胞猪角膜具有良好的组织相容性,可以支持角膜细胞和皮肤上皮细胞的生长。 目的：检测脱细胞猪角膜是否保存了利于角膜上皮细胞生长的重要组织结构—基底膜。 方法：利用荧光抗体对脱细胞猪角膜表面的基底膜成分(层粘蛋白和Ⅳ型胶原)进行免疫组织化学检测,荧光显微镜下观察脱细胞猪角膜表面是否保存了基底膜成分。 结果与结论：免疫荧光染色显示脱细胞猪角膜前基质表面层粘蛋白和Ⅳ型胶原呈阳性表达,与新鲜猪角膜表面基底膜的荧光表达相同,表明脱细胞猪角膜保存了利于角膜上皮细胞生长的基底膜。     

Geometric control of fibroblast growth on proton beam-micromachined scaffolds

Circular three-dimensional (3D) micropatterns with grooves and ridges of various sizes on the circumference of the structure were micromachined in polymethylmethacrylate, using proton beam micromachining. Fibroblasts were seeded in the center smooth nonpatterned surface of the circle. The circumference grooves could retard the outward spreading of cells after they became confluent in the central smooth surface. The fibroblasts eventually migrated across the grooves and ridges several days later. Wider and deeper grooves were more effective in retarding fibroblast spreading. Our results indicate that groove structures in cellular dimensions can effectively retard fibroblasts invasion. Proton beam micromachining, which has the unique advantage of being the only technique capable of manufacturing direct-write precise 3D microstructure at cellular dimensions, has great potential in generating 3D microscaffolds for studying cell behavior in a 3D microenvironment, which is important for tissue engineering.     

Scanning electron microscopy of epithelia prepared by blunt dissection

Simple dissection techniques of samples to be examined in the scanning electron microscope allow one to visualize easily the three-dimensional shape of epithelial cells in situ. Such preparations reveal a complex system of ridges and folds on the lateral surface of the cells whose intricacy can best be appreciated with SEM. In many epithelia there is a smooth apical band which corresponds to the region occupied by the junctional complex previously identified with conventional EM techniques. The secretion of chylomicra that result from a fatty meal can be observed. It is possible to study the distribution of concanavalin A binding sites on the lateral surfaces of the cells utilizing hemocyanin as a marker. In the case of the proximal tubule epithelium, the apical cell surface has many more binding sites than the lateral cell surface and there is a sharp demarcation at the level of the apical band. After blunt dissection the relationship of the basal surface of the cells with the basement lamina and the basement membrane can be appreciated as well. Possible physiological meaning of the morphological features observed is briefly discussed.     

Effects of a grooved epoxy substratum on epithelial cell behavior in vitro and in vivo

The effects of grooved epoxy substrata on epithelial (E) cell behavior were studied in vitro and in vivo. V-shaped grooves, 10 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of microelectronic components. The grooved substrata were replicated in epoxy resin. More E cells attached to grooved surfaces than to adjacent smooth surfaces. Clusters of E cells were markedly oriented by the grooved surfaces in comparison to the adjacent smooth surfaces where the orientation was random. Grooved and smooth epoxy implants were placed percutaneously in the parietal area of rats. One week after implantation E cells were found to adhere tightly to the implant surfaces. In the grooved portion of the implant E cells interdigitated into the grooves and had rounded nuclei. Histomorphometric measurements indicated that there was a shorter length of epithelial attachment and a longer length of connective tissue attachment in the grooved, compared to the smooth, portion of implants. After 10 days the epithelial attachment had migrated down the length of the protruding smooth portion of the implant and was located on the base of the implant. However, epithelium remained attached to the grooved portion of the implant. These observations indicate that grooved surfaces have the potential to impede epithelial downgrowth on percutaneous devices.     

Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues

A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400 nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.     

Effects of a grooved titanium-coated implant surface on epithelial cell behavior in vitro and in vivo

The effects of a grooved titanium-coated substratum on epithelial (E) cell behavior were studied in vitro and in vivo. V-shaped grooves, 10 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of microelectronic components. The grooved substrata were replicated in epoxy resin and coated with 50 nm of titanium. More E cells were found attached to the grooved titanium surfaces than to adjacent smooth surfaces. In comparison to the smooth surfaces where clusters of E cells were randomly oriented, on the grooved surfaces, clusters of E cells were markedly oriented along the long axis of grooves. Grooved and smooth titanium-coated epoxy implants were placed percutaneously in the parietal area of rats. Electron and light microscopic observations indicated that E cells were tightly attached to the implant surfaces and this attachment is through basal lamina-like and hemidesmosome-like structures. In the grooved portion of the implant, E cells interdigitated into the grooves and had rounded nuclei. Histomorphometric measurements indicated that there was a shorter length of epithelial attachment, longer length of connective tissue attachment, and less recession in the grooved, compared to the smooth portion of implants after 7 and 10 days. These results indicate that horizontal grooves produced by micromachining can significantly impede epithelial downgrowth on titanium-coated epoxy implants.     

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and 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 SLA 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 AE 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. Therefore, 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.