Bone formation by rat calvarial cells grown at high density in organoid culture

Calvarial cells from day 21 rat fetuses were isolated by enzymatic digestion and grown at high density in an organoid culture system at the medium/air interface. In this type of culture, mineralization occurred as early as 7 days in vitro, as revealed by light and electron microscopic means. After about 18 days in vitro, most of the culture consisted of mineralized tissue. Mineralization was also achieved without β-glycerophosphate, but it was delayed by 2 to 3 days. Maximal alkaline phosphatase activity occurred at days 8 to 12 in vitro and then declined continuously during further cultivation. Two types of mineralization could be observed: (1) mineralization of a collagen-rich osteoid by typical apatite crystals; (2) mineralization of a nearly collagen-free matrix by amorphous material which was possibly secreted by the cells. The importance of higher cell densities for cell differentiation and formation of histotypic tissue in vitro is apparent, and it is indicated that cell-cell contacts and cell-matrix interactions may be prerequisites for the development of histotypic conditions similar to the in vivo situation.