Cell mediated calcification in collagen gel cultures of fetal rat calvaria cells. Loss of gap junctions precedes calcification

Abstract

To analyze the mechanism of initiation of cell-mediated calcification in hard tussue and its relationship to the frequency of gap junctions, enzymatically isolated cells from fetal rat calvaria cultured in collagen gels were observed ultrastructurally over a time course. Calcification was observed at 2–3 weeks after the initiation of culture when the seeding cellularity and the concentration of β-glycerophosphate were sufficiently high. In the collagen gels, round cells (R), spindle or stellate cells (S), and fat cells (F) were characterised morphologically. The ultrastructural features of initial calcification could be classified into 4 subtypes: 1) a large mass greater than 10 µm in diameter (Type I), 2) deposition associated with dead R cells or matrix vesicles (Type II), 3) intracellular deposition (Type III), and 4) other than Types I–III (Type IV). Type II was the most frequent (44.5%) and Type III was the least (6.8%). Gap junction was observed frequently between 1) R cells, 2) S cells, 3) between R cells and S cells. The frequency of gap junctions in collagen gels decreased statistically (X2-test; p<0.001), when calcification was initiated. This cell culture system can be regarded as a useful model to analyze the initiation of cell mediated calcification in hard tissue. Gap junctions might function in cell communication and a decrease in their numbers could lead to cell death and, subsequently to calcification.