Leporine-derived adipose precursor cells exhibit in vitro osteogenic potential

Adipose-derived stem cells (ASCs) possess osteogenic potential and have been shown to undergo in vitro osteoblastic differentiation and promote bone regeneration in vivo. In this study, we describe the isolation and osteoblastic differentiation of rabbit ASCs and their behavior on a gelatin foam scaffold. These studies will form the basis of future in vivo studies of the osteogenic potential of rabbit ASCs for calvarial defect repair. Adipose-derived stem cells were isolated from New Zealand White rabbits and cultured in osteogenic medium +/- bone morphogenetic protein 2. Osteoblastic differentiation was assessed via histochemical stains for alkaline phosphatase (AP) and extracellular matrix (ECM) calcification. Reverse transcriptase polymerase chain reaction was performed to evaluate the expression of AP and the osteogenic transcription factor Runx2. Adipose-derived stem cells were seeded onto gelatin foam scaffolds at various densities, and cell proliferation was measured fluorometrically. Cells isolated from rabbit adipose tissue exhibited classic ASC morphology. Adipose-derived stem cells cultured in osteogenic medium exhibited more robust staining for AP and ECM calcification compared with ASCs in control medium. Furthermore, this staining was more marked in male ASCs versus female ASCs and also enhanced by bone morphogenetic protein 2. mRNA for AP and Runx2 were also increased in the osteoinduced cells. The optimal seeding density was 1 x 10(5) ASCs on an 8-mm gelatin foam scaffold. We have shown that rabbit ASCs have in vitro osteogenic potential and are compatible with a gelatin foam scaffold. Characteristic features of osteoblasts, such as ECM mineralization and expression of osteogenic genes, were demonstrated in this cell population. In vitro osteoblastic differentiation and scaffold studies are necessary before in vivo trials. The mechanism underlying the sex-based variation in osteoblastic differentiation is unknown but may involve signaling via factors such as estrogen.