Mineralized bone nodules formedin vitro from enzymatically released rat calvaria cell populations

Abstract

Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na beta-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and beta-glycerophosphate or ascorbic acid alone, three-dimensional nodules (approximately 75 micron thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3-4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II-V bore a linear relationship to the number of cells plated (r = .99). The results indicate that enzymatically released calvaria cells can form mineralized bone nodules in vitro in the presence of ascorbic acid and organic phosphate.

Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways

“…Osteoblastic cells in culture, when incubated in the presence of ascorbic acid, undergo a sequence of events that involve proliferation, differentiation and formation of a calcifying extracellular matrix over a two to three week period (8). Thus, the cells exhibit a more differentiated phenotype with time.…”

“…The murine osteoblastic cell line, MC3TC-E1, subclone 14 (CRL-2594) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in α-MEM supplemented as above. Neonatal osteoblastic cells were derived from the cells lining the calvarium of 2 day old rats as described in (8). In some experiments, neonatal calvarial osteoblasts or MC3T3-E1 cells were cultured in the presence or absence of 50 μg/ml ascorbic acid (Sigma) to promote osteoblast differentiation.…”

Statin-induced bone morphogenetic protein (BMP) 2 expression during bone regeneration: an immunohistochemical study

“…Osteogenesis induced by osteoblastic cells, is characterized by sequential events involving cell proliferation, followed by the expression of markers of the osteoblast phenotype and the synthesis, deposition, and mineralization of a collagenous matrix 12,13 Bone formation depends mainly on the number of osteoblastic cells rather than the activity of the osteoblasts 14 should not only be resorbed and nonimmunogenic, but it should also provide a three-dimensional structure as a scaffold for new bone formation. Polylactic acid/polyglycolic acid (PLGA) gelatin sponge (PGS) and collagen are considered to be suitable as carrier matrices for BMP and appear to facilitate the effects of BMP.…”

Characterization of a silencer element in the first exon of the human osteocalcin gene

“…Normal rat osteoblasts (ROB) were obtained by sequential enzymatic digestion of fetal rat calvaria as described (4). In brief, 21 day fetal rat calvaria were removed and digested with trypsin (2.5 mg/ml) and collagenase A (2.0 mg/ml).…”