Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosy nthetic activity as assessed by expression of the osteoblastic phenotype an d mineral deposition. At intervals, samples were harvested and analyzed his tologically, biochemically, and by Northern hybridization for type I collag en, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (C bfa1), Histologic evaluation showed greater viability, more alkaline phosph atase (ALP)-positive cells, and more mineralized tissue in the perfused spo nges after 21 days. Immunohistological assessment of proliferating cell nuc lear antigen revealed 5-fold more proliferating cells in the perfused spong es compared with the controls (p = 0.0201), There was 3-fold more ALP activ ity in the perfused sponges than the controls at 6 days and 14 days (p = 0. 0053). The perfused sponges contained twice the DNA and eight times more ca lcium than the nonperfused controls after 21 days (p < 0.0001 for both). No rthern hybridization analysis revealed more mRNA for collagen type I (2-fol d) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA e xpression remained unaffected by the medium perfusion. These results show t hat medium perfusion had beneficial effects on the proliferation and biosyn thetic activity of this osteosarcoma cell line. This system mimics the 3D g eometry of bone tissue and has the potential for revealing mechanisms of re gulation of osteogenesis.