Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue

Enzymatic digestion of bone tissue potentially releases a mixture of precur sor, differentiating, and mature cells. Conceptually, early fetal osteogeni c tissue should provide a more uniform population of cells than late embryo nic or newborn bone in which cells have already differentiated. In this con text, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where de position of bone matrix has not yet started at this site. These cultures we re compared with those of osteogenic cells isolated from newborn rat calvar iae and grown under similar conditions. Matrix production and composition w ere examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated ce lls formed mineralized nodules by day 14. The presence of mineral was deter mined by von Kossa staining and backscattered electron imaging (BEI), and t he accumulation of calcium and phosphorus within the nodules was demonstrat ed by X-ray microanalysis and elemental mapping At early time intervals, ce lls were generally cuboidal in shape and showed a well-developed Golgi appa ratus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was al so associated with electron-dense portions of the bone-like matrix. These d ata demonstrate the potential of presumptive fetal rat calvarial cells to f orm a bone-like matrix in vitro and suggest that the assembly and mineraliz ation pattern show similarities to the process of intramembranous ossificat ion. Such a culture system is of interest not only for studying cellular an d matrix events of bone formation, but also factors which influence mesench ymal cells in committing themselves to the osteogenic pathway. Anat. Rec. 2 52:554-567, 1998. (C) 1998 Wiley-Liss, Inc.