Soft tissue and epithelial models.

The applicability of a biomaterial for the manufacturing of oral implants is determined by its physicochemical and geometric surface properties. Research, therefore, is concerned with the cellular reactions that occur when an implant material comes into contact with body tissues. For permucosal oral implants, this involves both the reaction of bone and gingival cells. In vitro cell culturing--including the use of various analytical techniques like light microscopy, scanning and transmission electron microscopy, confocal laser scanning microscopy, and digital image analysis--is a good tool whereby investigators can obtain more insight into the relevant components of implant-tissue adhesion. In the current overview, the role of cell models in oral implant research is discussed, specifically with reference to responses of epithelial cells and fibroblasts.

Scanning electron microscopic, transmission electron microscopic, and confocal laser scanning microscopic observation of fibroblasts cultured on microgrooved surfaces of bulk titanium substrata.

During this study, microtechnology and plasma etching were used to produce gratings 1.0 (TiD01), 2.0 (TiD02), 5.0 (TiD05), and 10.0 microns wide (TiD10) into commercially pure titanium wafers. After incubation of rat dermal fibroblast (RDFs) on these surfaces for 3 days, the cells were observed with scanning electron (SEM), transmission electron (TEM), and confocal laser scanning microscopy (CLSM). Results showed that the RDFs as a whole and their stress fibers oriented strictly parallel to the surface pattern on the TiD01 and TiD02 surfaces. On the TiD05 and TiD10 surfaces, this orientation was not observed. In addition, TEM and CLSM demonstrated that the focal adhesion points (FAP) were located mainly on the surface pattern ridges. TEM revealed that FAP were wrapped occasionally around the edges of the ridges. Only the RDFs on both the TiD05 and TiD10 surfaces protruded into the grooves and possessed FAP on the walls of the grooves. Attachment to the groove floor was observed only on the TiD10 textures. Comparison of these results with earlier observations on microtextured silicone rubber substrata suggests that material-specific properties do not influence the orientational effect of the surface texture on the observed RDF cellular behavior. The proliferation rate of the RDFs, however, seems to be much higher on titanium than on silicone rubber substrata.

Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces.

The microfilaments and vinculin-containing attachment complexes of rat dermal fibroblasts (RDF) incubated on microtextured surfaces were investigated with confocal laser scanning microscopy (CLSM) and digital image analysis (DIA). In addition, depositions of bovine and endogenous fibronectin and vitronectin were studied. Smooth and microtextured silicone substrata were produced that possessed parallel surface grooves with a groove and ridge width of 2.0, 5.0, and 10.0 microns. The groove depth was approximately 0.5 micron. CLSM and DIA make it possible to visualize and analyze intracellular and extracellular proteins and the underlying surface simultaneously. It was observed that the microfilaments and vinculin aggregates of the RDFs on the 2.0 microns grooved substrata were oriented along the surface grooves after 1, 3, 5, and 7 days of incubation while these proteins were significantly less oriented on the 5.0 and 10.0 microns grooved surfaces. Vinculin was located mainly on the surface ridges on all textured surfaces. In contrast, bovine and endogenous fibronectin and vitronectin were oriented along the surface grooves on all textured surfaces. These proteins did not seem to be hindered by the surface grooves since many groove-spanning filaments were found on all the microgrooved surfaces. In conclusion, it can be said that microtextured surfaces influence the orientation of intracellular and extracellular proteins. Although results corroborate three earlier published hypotheses, they do not justify a specific choice of any one of these hypotheses.

The effect of a subcutaneous silicone rubber implant with shallow surface microgrooves on the surrounding tissues in rabbits.

It has been suggested that during wound healing microtextured surfaces can alter events at the interface between implant surface surface and surrounding tissues. To investigate this phenomenon, smooth and microtextured silicone rubber implants were implanted subcutaneously in rabbits for 3, 7, 42, and 84 days. The textured implants possessed parallel surface microgrooves and ridges with a width of 2.0, 5.0, and 10.0 microns. All grooves had a depth of approximately 0.5 microns. SEM observation showed fibroblasts, erythrocytes, lymphocytes, macrophages, fibrin, and collagen on all implant surfaces after 3 and 7 days. After 42 and 84 days only little collagen, a small number of fibroblasts, but no inflammatory cells were seen on the implant surfaces. The fibroblasts were not oriented along the surface grooves on all textured surfaces. Three-dimensional reconstruction of CLSM images and LM images showed no significant differences between the thickness of the capsules surrounding the smooth and those surrounding the microgrooved implants. In contrast LM did show a significantly lower number of inflammatory cells and a significantly higher number of blood vessels in the capsules surrounding the microgrooved implants. Differences between the 2.0, 5.0, and 10.0 microns grooved implants were not detected. Our results concerning the capsule thickness suggest that the depth of our grooves was not sufficient to facilitate mechanical interlocking, but the cause for the observed differences in inflammatory response and number of blood vessels remains unclear.

Quantitative analysis of fibroblast morphology on microgrooved surfaces with various groove and ridge dimensions.

Fibroblasts have been shown to respond to substratum surface roughness. The change in cell size, shape and orientation of rat dermal fibroblasts (RDF) was therefore studied using smooth and microtextured silicone rubber substrata. The microtextured substrata possessed parallel surface microgrooves that ranged in width from 1.0 to 10.0 microns, and were separated by ridges of 1.0 to 10.0 microns. The grooves were either 0.45 or 1.00 microns deep. Prior to incubation, the substrata were cleaned and given a radio frequency glow discharge treatment. After surface evaluation with scanning electron microscopy and confocal laser scanning microscopy, RDF were incubated on these substrata for 5 days. During this period of incubation, the RDF were photographed on days 1, 2, 3, 4, and 5, using phase contrast microscopy. Digital image analysis of these images revealed that on surfaces with a ridge width < or = 4.0 microns, cells were highly orientated (< 10 degrees) and elongated along the surface grooves. Protrusions contacting the ridges specifically could be seen. If the ridge width was larger than 4.0 microns, cellular orientation was random (approximately 45 degrees) and the shape of the RDF became more circular. Furthermore, results showed that the ridge width is the most important parameter, since varying the groove width and groove depth did not affect the RDF size, shape, nor the angle of cellular orientation.

Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves.

In order to quantify the effect of the substrata surface topography on cellular behaviour, planar and micro-textured silicon substrata were produced and made suitable for cell culture by radio frequency glow discharge treatment. These substrata possessed parallel surface grooves with a groove and ridge width of 2.0 (SilD02), 5.0 (SilD05) and 10 microns (SilD10). Groove depth was approximately 0.5 micron. Rat dermal fibroblasts (RDFs) were cultured on these substrata and a tissue culture polystyrene control surface for 1, 2, 3, 5 and 7 days. After incubation the cell proliferation was quantified with a Coulter Counter, and RDF size, shape and orientation with digital image analysis. Cell counts proved that neither the presence of the surface grooves nor the dimension of these grooves had an effect on the cell proliferation. However, RDFs on SilD02, and to a lesser extent on SilD05 substrata, were elongated and aligned parallel to the surface grooves. Orientation of the RDFs on SilD10 substrata proved to be almost comparable to the SilD00 substrata. Finally, it was observed that the cells on the micro-textured substrata were capable of spanning the surface grooves.

Effect of parallel surface microgrooves and surface energy on cell growth.

To evaluate the effect of surface treatment and surface microtexture on cellular behavior, smooth and microtextured silicone substrata were produced. The microtextured substrata possessed parallel surface grooves with a width and spacing of 2.0 (SilD02), 5.0 (SilD05), and 10 microns (SilD10). The groove depth was approximately 0.5 microns. Subsequently, these substrata were either left untreated (NT) or treated by ultraviolet irradiation (UV), radiofrequency glow discharge treatment (RFGD), or both (UVRFGD). After characterization of the substrata, rat dermal fibroblasts (RDF) were cultured on the UV, RFGD, and UVRFGD treated surfaces for 1, 3, 5, and 7 days. Comparison between the NT and UV substrata revealed that UV treatment did not influence the contact angles and surface energies of surfaces with a similar surface topography. However, the contact angles of the RFGD and UVRFGD substrata were significantly smaller than those of the UV and NT substrata. The dimension of the surface microevents did not influence the wettability characteristics. Cell culture experiments revealed that RDF cell growth on UV-treated surfaces was lower than on the RFGD and UVRFGD substrata. SEM examination demonstrated that the parallel surface grooves on the SilD02 and SilD05 substrata were able to induce stronger cell orientation and alignment than the events on SilD10 surfaces. By combining all of our findings, the most important conclusion was that physicochemical parameters such as wettability and surface free energy influence cell growth but play no measurable role in the shape and orientation of cells on microtextured surfaces.