Cell-cycle control in cell-biomaterial interactions:Expression of p53 and Ki67 in human umbilical vein endothelial cells in direct contact and extract testing of biomaterials

Current biocompatibility testing involves the demonstration of cell proliferation, which is usually interpreted as a sign of positive biocompatibility when the materials sustain cell proliferation. As the field of biomaterials research is rapidly moving toward tissue-engineered devices and hybrid organs, control of cell function has become a main topic. Cell function, which involves specific differentiation pathways, cannot be separated from cell-cycle control. The study of cell-cycle control is an important extension of routine proliferation assays and has extensive roots in developmental and tumor biology. We studied the expression of the tumour suppressor gene p53 and the proliferation-associated antigen Ki67 of endothelial cells in response to biomaterial contact. Cells were seeded in six- or 24-well plates, in which one or three 12-mm-diameter biomaterial disks were laid down. After 48- and 72-h incubation periods, cells were processed for flow cytometry, immunofluorescence, or Western blotting. The following materials were used: titanium, NiCr alloy, and CoCr alloy. Cells were also exposed to 24-h (ISO-norm) extracts in 25-cm(2) culture flasks (600,000 cells) for 24 and 48 h. For extract testing, serially diluted Ni-ion suspensions were also used. Human umbilical vein endothelial cells adhered to metal surfaces and started forming a monolayer within 3 days. Ki67 expression was positive in more than 60% after 2 days and decreased markedly after 3 days of adhesion. During this time cells developed focal contacts and produced a fibronectin matrix. p53 expression could be demonstrated with Western blotting and flow cytometry, but not with immunofluorescence. Differences due to both culturing time and material were found in expression patterns with both methods. Inverse correlations between Ki67 and p53 expression were detected, which are probably based on culture kinetics. The results indicate that expression of p53 and also Ki67 is clearly influenced by biomaterials in direct contact testing, despite the absence of obvious morphological differences. The p53 marker can be used for defining cell function in more detail, although the correlation with specific physiological function has still to be clarified. (C) 2000 John Wiley & Sons, Inc.