Proliferation and differentiation of osteoblasts on Biocement D modified with collagen type I and citric acid

In this study, the proliferation and differentiation of rat calvarial osteoblasts cultured on either (1) calcium-phosphate bone cement Biocement D, (2) Biocement D with 2.5% (w/w) mineralized collagen type I, or (3) Biocement D with 2.5% (w/w) mineralized collagen type I and 3% (w/w) citric acid were investigated. Incubation of the composites in cell-culture medium resulted in a fast decrease of pH and calcium concentration as well as in an increase of phosphate concentration. Although these effects occurred with all investigated materials, the lowest extent could be observed for the citric-acid-containing composites. As shown by scanning-electron microscopy, osteoblasts adhered to the composite surfaces. Proliferation and differentiation of the cells grown on the composites were found to be reduced compared to cells grown on tissue-culture polystyrene. Cells cultured in the vicinity of the composites but without direct contact also exhibited a reduced rate of proliferation, reduced alkaline phosphatase activity, and reduced mineralization. Simulating the changes in calcium and phosphate concentration occasioned by the composites through exposing cells to EGTA and phosphate gives rise to the same effects of reducing proliferation, ALP activity, and mineralization. No indication for apoptosis in cells exposed to low calcium and high phosphate concentrations was found. The number of necrotic cells, however, increased after incubation with EGTA and phosphate. For assessment of cell-composite interactions and the success of the composites in vivo, as well as for more effective material development, it seems to be important to know how changes in microenvironmental pH and ion composition of the material affect cellular proliferation and differentiation.