CHANGES IN OSTEOBLAST PHENOTYPE DURING DIFFERENTIATION OF ENZYMATICALLY ISOLATED RAT CALVARIA CELLS

Osteoblasts enzymatically isolated from newborn rat calvariae show var ious phenotypes including formation of mineralized bone nodules in cul ture. We investigated the temporal changes in osteoblast phenotype in these cells up to day 20 in culture. These cells formed unmineralized nodules by day 5, Mineralization was observed at the center of nodules by day 10, and nodules became larger on day 15, The nodules were surr ounded by numerous alkaline phosphatase (ALP)positive cells, ALP activ ity gradually increased by day 20, Parathyroid hormone (PTH) responsiv eness increased with time in culture. Osteoblasts produced no osteocal cin by day 10, but its synthesis was detected from day 15, These cells expressed substantial levels of ALP and PTH/PTHrP receptor mRNAs as e arly as day 5 in culture, but very weak expression of osteocalcin mRNA on day 5, The levels of expression of these transcripts increased wit h time in culture. In situ hybridization demonstrated that PTH/PTHrP r eceptor and osteocalcin mRNAs were strongly expressed in nodules, but the former appeared much earlier than the latter, BMP-2 and BMP-4 mRNA s also appeared in the cells forming nodules, Immunohistochemical anal ysis demonstrated that cells expressing either BMP-2/4 or their recept ors (BMPR-IA, BMPR-IB, and BMPR-II) preferentially appeared in nodules , These observations suggested that BMPs play an important role in the formation of mineralized hone nodules in an autocrine and/or paracrin e fashion in these cells. The present study confirmed that osteoblasts enzymatically isolated from newborn rat calvariae are a useful tool f or studying the differentiation process of osteoblasts. (C) 1998 by El sevier Science Inc. All rights reserved.