Selection and amplification of a bone marrow cell population and its induction to the chondro-osteogenic lineage by rhOP-1: an in vitro and in vivo study

Abstract

The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by bone morphogenetic proteins (BMPs). Osteogenic protein-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and bone healing. Rat bone marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the bone marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and 10% FBS. During the last 2 days, dexamethasone (10(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized bone matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas bone was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from bone marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.