L.A. Ginsel

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.

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.

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.

Contact guidance of rat fibroblasts on various implant materials

Providing a substrate surface with micrometer-sized parallel grooves influences the behavior of cells growing on such substrates in vitro. Cells elongate in the direction of the groove and migrate guided by the grooves. It has been suggested that cellular alignment on microgrooves is predominantly dependent on groove dimensions and that surface chemical variation of the substrate material has little effect. Therefore we seeded primary rat dermal fibroblasts (RDF) on smooth and microgrooved (groove width 1-10 microm, depth 0.5 microm) polystyrene (PS), poly-L-lactic acid (PLA), silicone (SIL), and titanium (Ti) substrates. The production process was found to be more accurate for PS and PLA than for SIL and Ti substrates. A proliferation study, scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed differences between RDF behavior on the materials. Our conclusions are (1) the accuracy of microtexture production by casting depends greatly on the material used; (2) even if no sharp discontinuities are present, microtextures still are potent tools for inducing contact guidance; and (3) besides surface texture, surface chemistry has a definitive influence on cell morphology.

Growth behavior of fibroblasts on microgrooved polystyrene.

We investigated the contact guidance phenomenon of rat dermal fibroblasts (RDF) on microgrooved polystyrene substrates. Grooves were 1 microm deep, and between 1 and 10 microm wide. Light microscopy and digital image analysis (DIA) showed that RDF were oriented on all microgrooved substrates. Scanning electron microscopy showed that RDF cultured on 1 or 2 microm wide grooves were positioned on top of the ridges. On the wider 5 and 10 microm grooves, the cells were able to descend into the grooves. In confocal laser scanning microscopy, focal adhesions were lying in the same direction as the actin filament where they attached to. DIA confirmed an orientational behavior of focal adhesions and actin filaments on microgrooves. There were no differences in the measured orientation between the different grooves. Besides, no obvious preference was found for focal adhesions to lie along edges of the surface ridges. Transmission electron microscopy showed that focal adhesions were able to bend along the edges of ridges. On the basis of our observations, we suggest that the breakdown and formation of fibrous cellular components, especially in the filopodium, is influenced by the microgrooves. The microgrooves create a pattern of mechanical stress, which influences cell spreading and cause the cell to be aligned with surface microgrooves.

Early spreading events of fibroblasts on microgrooved substrates

We investigated the contact guidance phenomenon, shortly after cell attachment. For this purpose polystyrene substrates were produced, either smooth, or equipped with micogrooves (depth 0.5 micrometer, width 1-10 micrometer). On these substrates, fibroblasts were cultured for 15, 30, 45, 60, 120, or 240 min. Subsequently, they were studied with light microscopy, scanning electron microscopy, confocal laser scanning microscopy, and digital image analysis. Up to 1 h, cell attachment on the grooved substrates was impaired. Further, cells oriented to the direction of the microgrooves. This orientation was established fastest on the narrow grooves. After 30 min, cells showed abundant membrane extensions in all directions. Well-formed actin filaments were not present in the cell body at timepoints before 4 h. Furthermore, cells on smooth surfaces exhibited less filaments. The addition of cytochalasin-B only caused a delay of cell attachment and spreading. From these experiments, we conclude that a well-formed cellular actin cytoskeleton is no prerequisite for the occurrence of contact guidance. Actin microfilaments in the lamellipodia, and the interplay between the lamellipodium and extracellular matrix molecules seem to be the determining factor in the establishment of contact guidance.

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.

Microgrooved subcutaneous implants in the goat

We investigated the behavior of microgrooved implants in soft tissue using polystyrene implantable disks, either smooth or microgrooved (1-10 microm) on both sides. The implants were placed subcutaneously in a goat for 1, 4, or 12 weeks. Light and transmission electron microscopy showed that fibrous capsule formation around the implants was fairly uniform. After 1 week the implants were covered with a fibrous capsule about 80 microm thick. The collagen matrix was loose, and many inflammatory cells were present. After 4 weeks the matrix was more dense and contained many newly formed blood vessels. At the implant surface a layer of inflammatory cells about 10 microm thick had accumulated. Finally, after 12 weeks the matrix had densified. One cellular layer of inflammatory cells was present at the implant surface. We carried out histomorphometric measurements of capsule thickness, inflammatory layer thickness, and the number of blood vessels. Capsule thickness appeared not to decrease with time. Further, these measurements showed that there were no differences in tissue reaction between smooth and microgrooved implants. On the basis of our observations, we suggest that 1 microm deep and 1-10 microm wide microgrooves do not influence tissue response around polystyrene implants in soft tissue.