Introduction: Supply of sufficient bone for reconstruction of the immature craniofacial skeleton is among the most challenging clinical scenarios encountered by craniofacial surgeons today. Repairing calvarial defects with autologous bone, the current gold standard, is limited to older patients, who have developed a diploic space; this marrow space enables split-thickness calvarial bone grafts to be harvested from the skull. Children aged two to seven do not possess a diploic space, and have lost the dural osteogenic potential present during infancy. Thus, development of a tissue-engineered autologous bone graft for these patients is paramount. Adipose-derived stem cells (ASCs) have been shown to have osteogenic potential and promote bone formation in vivo. This study seeks to establish the rabbit model of ASC osteogenesis for calvarial defect repair.

Methods: Rabbit ASCs were cultured in osteogenic media, +/- BMP-2, and were assessed for markers of osteoblastic differentiation: alkaline phosphatase activity, matrix mineralization, and osteonectin expression. Known numbers of ASCs were seeded on gelatin sponges, and quantified with Cell Titer Blue at t=0 (attachment) and over a five-day course (proliferation). For implant construction, 10^5 ASCs were seeded onto gelatin sponges, and cultured in osteogenic media (with BMP-2) for seven days, prior to placement in rabbit calvarial defects (8 mm). After six weeks, the calvaria were harvested, assessed radiographically for gross bony healing, and evaluated histologically.

Results: Alkaline phosphatase expression peaked during the third week of osteoinduction (no BMP-2); Alizarin Red staining for calcium became progressively stronger over four weeks. Male cells exhibited greater osteoblastic differentiation than did female cells. BMP-2 enhanced differentiation, with positive markers within the first 4 days. The optimal seeding density was 10^5 ASCs on the gelatin sponge, which became saturated within 36 hours (steady-state = 2.4 x 10^5 cells). Gelatin sponge +/- ASCs enhanced bony healing versus no treatment, as evidenced by classic bony morphology on radiograph. Statistical analyses are forthcoming, as animal studies are still in progress, and will be presented.

Conclusion: We have conducted several in vitro studies to optimize the success of calvarial defect repair with ASCs in a rabbit model. Rabbit ASCs were shown to exhibit osteoblastic differentiation, and BMP-2 enhanced this process. The significance and mechanism of the observed sex difference in osteogenic efficiency is unknown at this time, but is currently under investigation in our laboratory. We have demonstrated a quantitative approach to scaffold seeding density. Together, these data have directed the development of an osteoconductive implant for use in calvarial defect repair.