In Vivo Evaluation of Mixtures of Uncultured Freshly Isolated Adipose-Derived Stem Cells and Demineralized Bone Matrix for Bone Regeneration in a Rat Critically Sized Calvarial Defect Model

Abstract

Although many studies have suggested that human adipose tissue contains pluripotent stem cells, a few reports are available on stromal vascular fraction (SVF). In the present study, we evaluated the bone formation capacities of SVF. We implanted uncultured freshly isolated adipose-derived stem cells combined with demineralized bone matrix (DBM) to induce bone regeneration in a critically sized rat calvarial defect model. We used DBM (DBX (R)) and/or poly(70L-lactide-co-30DL-lactide) copolymer PLA as a scaffold. Fifty white rats were randomized to 5 different groups (n = 10): (1) control, (2) DBM, (3) DBM + SVF, (4) DBM + PLA, and (5) DBM + PLA + SVF groups. After acquiring SVF, an 8-mm critically sized calvarial defect was made in each rat. Specimens were harvested at 8 weeks postimplantation and evaluated radiographically and histologically. New bone formation was qualified by hematoxylin and eosin staining and anti-osteocalcin antibody (OC4-30) immunostaining of calvarial sections. Amounts of mineralization were determined by radiodensitometric analysis. In gross appearance, the DBM + SVF and DBM + PLA + SVF groups showed more abundant bone formation than the other groups. Radiodensitometric evaluations revealed that significant intergroup differences were observed according to the Kruskal-Wallis (rank) test (P = 0.030 < 0.05). The 5 groups show different amounts of filling of bone defects (control: 13.48%; DBM: 39.94%; DBM + SVF: 57.69%; DBM + PLA: 24.86%; DBM + PLA + SVF: 42.75%). Histological evaluation revealed that there was abundant new bone formation in the DBM + SVF and DBM + PLA + SVF groups. It was found that undifferentiated adipose-derived stem cells in the form of SVF induced new bone formation in rat calvarial defects. Accordingly, SVF offers a practical, promising candidate for regenerative tissue engineering or cell-based therapy.