Diversity of bone matrix adhesion proteins modulates osteoblast attachment and organization of actin cytoskeleton.

Interaction of cells with extracellular matrix is an essential event for differentiation, proliferation and activity of osteoblasts. In bone, binding of osteoblasts to bone matrix is required to determine specific activities of the cells and to synthesize matrix bone proteins. Integrins are the major cell receptors involved in the cell linkage to matrix proteins such as fibronectin, type I collagen and vitronectin, via the RGD-sequences. In this study, cultures of osteoblast-like cells (Saos-2) were done on coated glass coverslips in various culture conditions: DMEM alone or DMEM supplemented with poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I). The aim of the study was to determine the specific effect of these bone matrix proteins on cell adherence and morphology and on the cytoskeleton status. Morphological characteristics of cultured cells were studied using scanning electron microscopy and image analysis. The heterogeneity of cytoskeleton was studied using fractal analysis (skyscrapers and blanket algorithms) after specific preparation of cells to expose the cytoskeleton. FAK and MAPK signaling pathways were studied by western blotting in these various culture conditions. Results demonstrated that cell adhesion was reduced with PL and VN after 240 min. After 60 min of adhesion, cytoskeleton organization was enhanced with FN, VN and Col-I. No difference in FAK phosphorylation was observed but MAPK phosphorylation was modulated by specific adhesion on extracellular proteins. These results indicate that culture conditions modulate cell adhesion, cytoskeleton organization and intracellular protein pathways according to extracellular proteins present for adhesion.

Influence of metal ion solutions on rabbit osteoclast activities in vitro.

The purpose of the present study was to compare the effects of various metal ions (aluminium, chromium, cobalt, gold, iron, strontium, titanium and vanadium) on rabbit osteoclast activities, with respect to their number, size, resorptive capacity and their capacity to release proteinases. Marked heterogeneous osteoclastic behaviour was observed early in culture with metal ions (24 h) in term of resorption parameters. In contrast, protease activities (cysteine-proteinase and metalloproteinase activities) were not modulated in our culture conditions. Aluminium, iron, gold and titanium reduced the number of osteoclasts significantly. Aluminium and gold had no effect on osteoclast-mediated resorption on dentin-slices, although aluminium induced a greater number of very small lacunae. Titanium reduced only the mean surface area per lacunae, cobalt reduced the mean surface area of lacunae and increased their number, and iron reduced both parameters. Strontium had no effect on osteoclast formation and on total dentin slice surface resorbed. However, strontium increased the number of small lacunae formed on dentin-slices by osteoclasts. Chromium had no effect on osteoclast activities. These findings indicate that metal ions induce very early effects on osteoclasts, which can contribute to periprosthetic pathologies via different cellular mechanisms.

Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces.

The influence of surface roughness and the presence of adhesion molecules in the culture medium were studied regarding cell adhesion, shape, and proliferation of osteoblast-like cells grown on two types of titanium disk. Type I disks were acid etched and type II disks were sandblasted and acid etched. Surface roughness was determined by contact profilometry and scanning electron microscopy. Chemical composition and oxide thickness of the superficial titanium layer were established with energy dispersive X-ray spectrometry, electron spectroscopy for chemical analysis and auger electron spectroscopy. Titanium release in the culture medium was assessed by inductively coupled plasma-optical emission spectrometry. Osteoblast-like cells (Saos-2) were cultured on both types of titanium disks (1) in standard conditions (DMEM culture medium supplemented with fetal calf serum), (FCS), (2) with the culture medium alone (DMEM alone), (3) in the presence of fibronectin or vitronectin (DMEM supplemented with fibronectin or vitronectin). Cultures were also performed in the presence of monoclonal anti-integrin (beta1, alphav) to test the cell adhesion molecules involved in the cell binding to the titanium surface. We found that sandblasting does not modify the chemical surface composition and that titanium represents only 5-6% (in the atom percentage) of surface elements. Release of titanium in the culture medium was found to increase from 24 to 72 hours. In the absence of FCS, fibronectin, or vitronectin, cells appeared scanty and packed in clusters. On the contrary, cells cultured in the presence of FCS, fibronectin, or vitronectin were flattened with large and thin cytoplasmic expansions. The addition of anti beta1 or alphav integrin subunit monoclonal antibody in the culture medium decreased adhesion and spreading of cells, particularly in the presence of fibronectin. Cell proliferation was significantly higher on culture plastic than on both types of disks, but was increased on rough but not on smooth surfaces. These results indicate that a high surface roughness and presence of fibronectin or vitronectin are critical elements for adhesion, spreading, and proliferation of cells on titanium surfaces.