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

The differentiation a nd maturation of osteoprogenitor cells into osteoblas ts are processes which are thought to be modulated by transforming growth f actors-beta (TGF-R) as well as by bone morphogenetic proteins (BMPs). Osteo genic 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 embryog enesis 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 cond itions, survival of the bone marrow cell population was dependent on the pr esence 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, de xamethasone (10(-8) nn) and IS-glycerophosphate (2 mM) were added to potent iate osteoinduction. Concomitant with an up-regulation of cell proliferatio n, DNA synthesis levels, colony number and size were determined. Chondro-os teogenic 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 dif fusion chambers or inactivated demineralized bone matrix (DBM) cylinders an d implanted subdermically into the backs of old rats for 28 days. Biochemic al, histological and immunocytochemical analyses provided evidence of carti lage and osteoid tissue inside the diffusion chambers, whereas bone was als o observed inside the DBM implants. In conclusion, this experimental proced ure is capable of selecting a cell population from bone marrow which, in th e presence of rhOP-1, achieves skeletogenic potential under in vitro as wel l as in vivo environments.