Problems in osteogenic differentiation of rat bone marrow stromal cells

ABSTRACT

rat bone marrow stromal cells (BMSC) isolated with standard flushing method and investigate the plasticity of transdifferentiation between osteoblastic and adipocytic lineage of cultured BMSC. Unlike aspiration method in human, rat bone marrow was extracted by means of irrigation with culture media that elevates the possibility of co-extraction of committed osteoprogenitor, or preosteoblast or other progenitor cells of several types present inside bone marrow. The cultured stromal cells showed high ALP activity which is representative marker of osteoblast without any treatment. Osteogenic inducers such as Dex and BMP-2 were examined for the evaluation of their effect on osteogenic and adipocytic differentiation of stromal cells, because they function as osteoinductive agent in stromal cells, but simultaneously induce adipogenic differentiation. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity or mRNA expression of osteoblast markers such as osteopontin, bone sialoprotein, collagen type I and CbfaI, and in vitro matrix mineralization by von Kossa staining. Oil red staining method was used to detect adipocyte and adipocytic marker, aP2 and PPARgamma2 expression was examined using RT-PCR. It can be supposed that irrigation procedure resulted in high portion of already differentiation-committed osteoprogenitor cell showing elevated ALP activity and strong mineralization only under the supplement of 100microM ascorbic 2-phosphate and 10mM beta-glycerophosphate without any treatment of osteogenic inducers such as Dex and BMP-2. Dex and BMP-2 seemed to transdifferentiate osteoprogenitor cells having high ALP activity into adipocytes temporarily, but continuous treatment redifferentiated into osteoblast and developed in vitro matrix mineralization. This property must be considered either in tissue eng ineering for bone regeneration, or in research of characterization of osteogenic differentiation, with rat BMSC isolated by the standard irrigation method.