Healing of Tibial and Calvarial Bone Defect using Runx-2-transfected Adipose Stem Cells

Abstract

The purpose of this study was to test the effect of Runx-2-transfected hASCs to heal the defect created on proximal tibiae and calvaria of immunosuppressed rats. Three kinds of hASCs (untransfected, pECFP-transfected ASCs or Runx-2-transfected ASCs) were cultured under osteogenic medium. Osteoblastic differentiation was measured by ALP staining on day 7 and osteoid matrix formation was observed by alizarin red staining on day 14 after osteogenic induction. Osteogenic potential in long bone defects were tested via 6 mm-sized circular defect on proximal tibiae of 9 immunosuppressed rats. Untransfected ASCs, pECFP-transfected ASCs or Runx-2-transfected ASCs embedded in fibrin scaffold were implanted in the defect (N=3 in each group). In order to assess the in vivo osteogenic capability of Runx-2-transfected ASC in intramembanous ossification, two critical size bone defects were created on parietal bone of 12 immunosuppressed rats. The defects were filled with fibrin scaffold containing pECFP-transfected ASCs, Runx-2-transfected ASCs or no cell (N=4 in each group). Runx-2 transfected ASCs showed much stronger activity of ALP and greater formation of osteoid matrix compared with untransfected ASCs or pECFP-transfected ASCs 7 and 14 after osteo-induction, respectively. When the volume of regenerated bone was compared from gross examination and radiographs after 5 weeks in the proximal tibial defect model, the defects treated with Runx-2-transfected ASCs had the greatest area of healed bone compared with other groups. In the calvarial defect model, Runx-2-transfected ASCs had significantly increased area healed with bone (p < 0.05) as well as better quality of regenerated bone compared with defects which was treated with untransfected ASCs from gross and micro-CT examination 8 weeks after implantation. The implanted human cells persisted in the newly regenerated bone in defects treated with pECFP-ASCs and Runx-2-transfected ASCs. In conclusion, Runx-2-transfection significantly increased the osteogenic potential of ASCs in the in vivo orthotopic models.