A.F. von Recum

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 influence of micro-topography on cellular response and the implications for silicone implants

Tissue attachment to substratum surfaces is of central importance to the in vivo performance of prosthetic implant materials. It is not yet understood why connective tissue does not attach to the surface of silicone or any other polymeric material. Recently the authors have conclusively demonstrated that micro-range surface roughness modifies cellular responses in cell culture and modifies biocompatibility and tissue attachment in vivo significantly. In order to better understand the basic interactions between living cells or tissues on one hand and man-made substratum surfaces on the other hand, the germane literature is reviewed here. Cells adhere to substratum surfaces mainly through focal adhesions which are a complex of intracellular, transmembrane and extracellular proteins. Adhesion is facilitated and modified by proteins adsorbed to the substratum surface. Protein adsorption in turn is modified by the underlying substratum surface properties including surface chemistry, charge, and free energy. Wh...

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.

Texturing of polymer surfaces at the cellular level

A technique to surface-texture polymer sheets with any conceivable surface morphology is described. It uses technology developed for semiconductor device fabrication. It allows the definition and control of implant surface texture at subcellular levels, down to 2 micron dimensions, and the cellular response to those textures to be studied in vitro and in vivo. The results of a pilot study are presented and the biological significance of a microscopic surface texture is discussed.

Semi-quantitative and qualitative histologic analysis method for the evaluation of implant biocompatibility.

An adequate histologic evaluation is a prerequisite for a good understanding of the behavior of tissue to implant materials. However, despite improvements in histologic sectioning techniques, few studies have used histomorphometric methods for the quantification of the tissue response. This paper discusses new simple histologic grading scales, which can be used for the fast standardized light microscopic analysis of the biocompatibility of hard and soft tissue implants. Two examples of the application of the grading scales are demonstrated.

Surface micromorphology and cellular interactions

Contact guidance induced by the topographical properties of the underlying substratum is of great importance in morphogenesis and also in- fluences the interaction of tissue cells with implanted material. A large body of evidence has accumulated since the first detection of this phenomenon in 1910. Several major hypotheses have been developed to explain the observed cell behaviour. The technological progress enabled researchers to produce pure substrata with a defined and controlled surface microgeometry. Based on these specimens, it could be demonstrated that cytoskeletal structures and receptors forming focal adhesions most likely are involved in contact guidance. In a study using human gingival fibroblasts, the reaction of these cells to a regular surface microstructure of 1 μm pitch and 1 μm depth was tested. After two days on the microstructured samples, all the cells showed a strong alignment to the topography of the surface. Transmission electron microscopy revealed that the cells either bridged the grooves or conformed to the surface structures. The latter confirms earlier investigations with porous subcutaneous implants, where the inflammatory reaction and the formation of a fibrous tissue capsule was reduced due to enhanced tissue adhesion.

Soft tissue response to different types of sintered metal fibre-web materials

Recently, it has been demonstrated that the soft tissue response to polymeric filter implants was predominantly dependent on the implant surface topography and that variation in the implant material had little effect. The purpose of this study, therefore, was to compare histologically the soft tissue response to sintered fibre-web implants made from different materials and with varying web porosity. Three different fibre-web materials with two different weights and two different porosities were used. The implants were inserted subcutaneously in the dorsum of rabbits. The implants were left in situ for 4 and 12 wk. Histological and tissue compatibility evaluations were performed. It is found that all the tested fibre-web materials show a good biocompatible behaviour. In addition, the results appear to indicate a relation between flexibility of an implant material and tissue behaviour.

Types I and III collagen in the tissue capsules of titanium and stainless-steel implants.

Collagen plays an important role in wound healing and as such is present in connective tissue capsules around implanted materials. The proportion of type I collagen to type III collagen is lower during wound healing than that found in normal dermis, but the amount of type I collagen gradually increases and type III decreases as healing continues. The intent of this research was to investigate a possible correlation between implant metals and type I and type III collagen formation in the ensuing soft-tissue capsules. Smooth implants of grade 4 commercially pure titanium and 316L stainless steel were placed via injection needle into the subcutaneous tissue of 12 rats for 6 weeks. The soft-tissue capsule that formed around each implant was evaluated histologically and by immunofluorescence. Although the two metal implant types differ in many respects, they were chosen for their differences in surface conductivity. Titanium oxide is semiconducting, whereas the oxide of stainless steel is conducting. Impedance spectroscopy was used for conductivity analysis of the surface. No qualitative differences were found in amount of type I or type III collagen in the capsules of the two metals. However, the capsules around titanium stained for type I collagen were thicker than capsules around stainless steel. In addition, type I collagen showed evidence of a greater association with densely packed, highly orientated collagen fibers.

Electron microscope investigation of soft tissue ingrowth into Dacron® velour with dogs

Electron microscopic and light microscopic analyses were performed on Dacron velour covered percutaneous devices implanted in the dorsum of canines for periods between 1 week and 9 months. The results indicated that although the capsular region around the implant was mature collagen after the 1st week of implantation, the tissue ingrowth region remained mostly immature collagen through 9 months of implantation. It appears that increasing the diameter of the fibres, which would decrease the chronic inflammatory response, and increasing the average distance between fibres should increase the amount of mature collagen between the velour fibres. In view of species differences between canines and humans, further investigations are needed before the relevancy of these findings can be extended to possible clinical applications with humans.