Surface properties, including topography and chemistry, are of prime importance in establishing the response of tissues to biomaterials. Microfabrication techniques have enabled the production of precisely controlled surface topographies that have been used as substrata for cells in culture and on devices implanted in vivo. This article reviews aspects of cell behavior involved in tissue response to implants with an emphasis on the effects of topography. Microfabricated grooved surfaces produce orientation and directed locomotion of epithelial cells in vitro and can inhibit epithelial downgrowth on implants. The effects depend on the groove dimensions and they are modified by epithelial cell-cell interactions. Fibroblasts similarly exhibit contact guidance on grooved surfaces, but fibroblast shape in vitro differs markedly from that found in vivo. Surface topography is important in establishing tissue organization adjacent to implants, with smooth surfaces generally being associated with fibrous tissue encapsulation. Grooved topographies appear to have promise in reducing encapsulation in the short term, but additional studies employing three-dimensional reconstruction and diverse topographies are needed to understand better the process of connective-tissue organization adjacent to implants. Microfabricated surfaces can increase the frequency of mineralized bone-like tissue nodules adjacent to subcutaneously implanted surfaces in rats. Orientation of these nodules with grooves occurs both in culture and on implants. Detailed comparisons of cell behavior on micromachined substrata in vitro and in vivo are difficult because of the number and complexity of factors, such as population density and micromotion, that can differ between these conditions.
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.
Ideally, the surface of epithelium-penetrating implants should impede apical epithelial migration. Previous studies have shown that micromachined grooved surfaces can produce connective-tissue ingrowth, which inhibits epithelial downgrowth on percutaneous implants [Chehroudi et al., J. Biomed. Mater. Res., 24, 9, (1990)]. However, in those studies, connective tissue and epithelium interacted with the same surface so that the effects of the surfaces on each population could not be determined separately. The objectives of this study were (a) to examine cell behavior on implants in which connective tissue contacted surfaces of various topographies and epithelium encountered only a smooth surface, and (b) to compare one-stage and two-stage surgical techniques. Implants had a base component (BC) which was either smooth or had a surface with 19-micron- or 30-micron-deep grooves or 120-micron-deep tapered pits, and a skin-penetrating component (SPC) which was smooth. In the two-stage technique, the BC was implanted subcutaneously for 8 weeks, which permitted the healing of the peri-implant connective tissue. In the second stage the SPC was connected to the BC. For one-stage implants, BC & SPC were connected and implanted percutaneously. Implants (BC & SPC) were removed 1, 2, or 3 weeks after percutaneous implantation and histological sections were measured for recession, connective tissue and epithelial attachment as well as capsule thickness. Light microscopy indicated that both grooved and tapered pitted surfaces encouraged connective tissue ingrowth. On the grooved surfaces, the orientation of fibroblasts changed from an oblique to a more complex pattern which included cells having round nuclei within the grooves, as well as cells oriented oblique or perpendicular to the grooves. In the tapered pits a hammock-like arrangement of fibroblasts was observed. In some cases, foci of mineralization and formation of bonelike tissue were found on the grooved and pitted surfaces. The apical migration of the epithelium was significantly (p less than 0.05) inhibited by those micromachined surfaces which produced connective tissue ingrowth to the BC. This study found that placing the implants in two stages improved the performance of percutaneous devices, and that a further improvement was achieved if the implant had a surface promoting connective tissue ingrowth.
A two-stage replica technique with a subsequent titanium (Ti)-coating treatment was used to faithfully replicate topographies of polished, acid-etched, machined-like, finely blasted, coarsely blasted, coarsely blasted and acid-etched, and Ti plasma-sprayed Ti surfaces. The replicas were used to study the influence of different rough surface topographies on the response of human fibroblasts in vitro under conditions of constant surface chemistry for all surfaces. The surface topographies of the replicas were characterized using non-contact laser profilometry, scanning electron microscopy (SEM), and stereo-SEM, whereas surface chemistry was examined using X-ray photoelectron spectroscopy. Fibroblasts were trypsinized and plated onto the Ti-coated epoxy-resin replica surfaces for 24 h and observed with SEM. Fluorescein-5-thiosemicarbazide was used to stain the cell components including cell membrane, and the stained cells were optically sectioned using epifluorescent microscopy. The optical sections were computationally reconstructed to obtain three-dimensional images and cell volume and cell thickness determined. The different surface topographies were found to alter cell thickness and cell morphology. However, cell volume as computed from three-dimensional reconstructions was not affected by surface features. The results suggest that cells distort themselves to accommodate to rough surfaces but their volume is not significantly altered.
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.
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.
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