Low Oxygen Tension Enhances Chondroinduction by Demineralized Bone Matrix in Human Dermal Fibroblasts in vitro

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Abstract
Endochondral bone formation is induced by demineralized bone powder (DBP) when DBP is implanted subcutaneously in rodents. Previously, we developed an in vitro model of this process, wherein human dermal fibroblasts (hDFs) differentiate to chondrocytes when cultured in a three-dimensional porous collagen sponge containing DBP. In other studies, medium perfusion was beneficial in maintaining phenotype and viability of many cell types in plain porous collagen sponges, including fibroblasts, bone marrow stromal cells, osteoblasts, and epidermal cells. In contrast, medium perfusion inhibited chondrogenesis by articular chondrocytes; reduction of oxygen tension to 5%, however, restored chondrogenesis. These observations are consistent with the fact that in vivo cartilage is avascular and relatively hypoxic compared with other vascularized tissues. In this study, we tested the hypothesis that low oxygen tension (hypoxia, 5% oxygen) would enhance induced chondrogenesis in hDFs cultured with DBP. As expected, hypoxia upregulated hypoxia-inducible factor-1alpha in hDFs in all conditions (i.e. +/- perfusion, +/- DBP). Hypoxia increased accumulation of cartilage-specific matrix chondroitin 4-sulfate in hDFs, but only in the presence of DBP (165%, compared to normoxia, p < 0.05). Hypoxia did not appear to have detrimental effects on cell viability and proliferation. In sum, hypoxia enhanced cartilage matrix accumulation by hDFs cultured with DBP. These defined conditions can optimize the use of dermal fibroblasts for cartilage tissue engineering.