Analysis of gene expression in osteogenic cultured marrow/hydroxyapatite construct implanted at ectopic sites: a comparison with the osteogenic ability of cancellous bone

Abstract

We investigated the in vivo osteogenic ability of cultured marrow cells subcultured in porous hydroxyapatite. This osteogenic ability was compared with that of cancellous bone grafts. Fresh marrow cells were obtained from young adult rat femora and cultured in a standard medium for 10 days, then trypsinized and used to make constructs of porous hydroxyapatite (HA) and cultured marrow cells. An additional 2-week culture (subculture) was performed for the construct in standard medium with and without the addition of dexamethasone (Dex). The 2-week subcultured constructs then were implanted into subcutaneous sites of syngeneic rats. These implants and the rat cancellous bone were harvested and prepared for gene expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) as well as for histological analysis. ALP and OC mRNAs could be detected in Dex-treated subcultured constructs 1 week after implantation with an increase at 2 weeks, comparable to the observation in cancellous bone. Histological analysis showed active bone formation even 1 week postimplantation. In contrast, the subcultured constructs without the addition of Dex did not show bone formation, and only small levels of ALP and OC mRNAs were found. These results indicate that the bone tissue formed by grafting the Dex-treated construct of cultured marrow cells and hydroxyapatite possesses a high osteoblastic activity comparable to that of viable cancellous bone. Thus the prefabricated osteogenic subcultured marrow/HA construct may be applicable in bone reconstructive surgery.