J.E. de Ruijter

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.

The effect of poly-L-lactic acid with parallel surface micro groove on osteoblast-like cells in vitro

In this study we evaluated the behavior of rat bone marrow (RBM) cells on microgrooved poly-L-lactic acid (PLA) and polystyrene (PS) surfaces. The applied groove depth was 0.5, 1.0 or 1.5 microns, with a groove and ridge width of 1, 2, 5 or 10 microns. Scanning electron microscopical examination showed that a collagen-rich mineralized layer of extracellular matrix (ECM) was deposited. Alignment of the cells and matrix to the surface grooves was observed as described before. Quantitative evaluation, using a tetracycline labeling assay, revealed that more mineralized ECM was formed on the PLA than on the PS. Further, PLA surfaces with a groove depth of 1.0 micron and groove widths of 1 and 2 microns induced most mineralized ECM. Finally, alkaline phosphatase activity was also higher on most microgrooved PLA surfaces, compared with the other materials. On the basis of these observations, we concluded that microtextured surfaces are able to influence the differentiation of osteoblast-like cells and the deposition of mineralized matrix. Probably, this phenomenon can be used to increase the bone regeneration around oral implants.

Histological evaluation of a biodegradable polyactive®/ hydroxyapatite membrane

Guided tissue regeneration (GTR) is a technique which is used for the treatment of bone defects associated with periodontal disease or enossal dental implants. In most experimental studies on GTR, non-degradable membranes are used. A drawback inherent to such devices is that at the end or in the course of the wound healing they have to be removed. Therefore, the aim of the present study was to investigate a new biodegradable membrane material for use in GTR, which also has excellent mechanical properties and is biocompatible. The material is a composite consisting of poly(ethyleneglycol terephthalate) and poly(butylene terephthalate) segmented copolymer (PEG/PBT), which for the experiments was used in pure form and also mixed with hydroxyapatite (HA) grains. Subcutaneous and subgingival implantation studies in goats were performed to determine the biocompatibility and biodegradability characteristics of several of these materials. Differences between materials were introduced in the production process, PEG/PBT ratio, material thickness and presence of HA. Implantation periods were 3, 6 and 12 wk. The histological results indicated that all investigated materials were biocompatible with the surrounding tissue. Degradation of the membranes was attended by a mild cellular reaction. The degradation process was mainly influenced by the PEG/PBT ratio. A higher PBT content resulted in a decreased degradation.

Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol-gel-derived titania.

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol-gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated body fluid prior to the experiment. Overall, variable effects between experimental runs were seen. Apparently, this was due to the heterogeneous nature of the used cell population. Therefore, only careful conclusions can be made. Initial cell adhesion and growth rates between 3 and 5 days of culture--analyzed by cell numbers--were in general comparable for the two titanium substrates, while initial growth up to day 3 is suggested to be higher in Ti c.p. compared to Ti sol. Although initial cell adhesion on the S53P4 glass discs was lower than the titanium substrates, cell growth rates appeared to be higher on the silica gel compared to the two titanium substrates. Further, there were some indications that the early and late osteoblast differentiation markers, alkaline phosphatase and osteocalcin, monitored up to day 24, were elevated in Ti c.p cultures compared to Ti sol cultures. There were no differences observed in in vitro mineralization between the titanium groups. S53P4 seemed to display a substantially higher differentiating capacity for both osteogenic cell markers as well as in vitro mineralization compared to the two titanium substrates.

Evaluation of tricalciumphosphate/hydroxyapatite cement for tooth replacement; an experimental animal study.

A calciumphosphate cement, consisting mainly of tricalciumphosphate (85% α-TCP and 15% β-TCP), was inserted in 16 surgical defects created in the tibia of goats. X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) showed that after 3 months of implantation the α-TCP was transformed to hydroxyapatite (HA). Histological evaluation revealed that the presence of cement stimulated the ingrowth of bone compared with unfilled cavities. Active resorption and remodelling of cement particles was observed. The cement did not evoke an inflammatory reaction. At 6 months after implantation no further changes in the composition of the cement occurred. All remaining material was surrounded by mature bone.

Tissue reaction to Dacron velour and titanium fibre mesh used for anchorage of percutaneous devices.

Dacron velour is often used to anchor a percutaneous device, like the catheter used in peritoneal dialysis. However, exit-site infections complicate this method of dialysis and are supposed to be related to the design of the catheter. In animal experiments, a percutaneous device provided with a titanium fibre mesh to anchor the implant was not affected by infectious complications. The purpose of this study was to compare the differences in soft tissue reaction to Dacron velour and titanium fibre mesh under the same experimental conditions. Therefore, we placed implants, provided with either Dacron or titanium mesh, subcutaneously in the dorsum of goats. The implants were left in situ for 4 months. Histological and histomorphological evaluations were performed. It was found that the soft tissue response inside the Dacron was mainly inflammatory, while the titanium mesh evoked good biocompatible behaviour. We concluded that the limited fibrous tissue ingrowth into the Dacron cuff has to be the reason for the observed high failure incidence of a percutaneous device.

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.

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.