Study On Repairing Critical-Sized Bone Skull Defect With Long-Term Cryopreserved Allogeneic BMSCs

Study on Repairing Critical-Sized Bone Skull Defect with Long-Term Cryopreserved Allogeneic BMSCsObjective To establish methods of the isolation, proliferation, culture and osteoinduction of canine bone mesenchymal stem cells (BMSCs). And to evaluate the use of osteo-induced allogeneic mesenchymal stem cells cryopreserved for six months along withβ-TCP scaffold in the treatment of critical-sized bone defect in dogs'skull bone without the use of immunosuppressive therapy.Methods Bone marrow of Beagles was separated by density gradient centrifugation with 1.077g/ml Ficoll solution. BMSCs was cultured and proliferated in vitro, osteoinducted with conditioned medium for osteogenic induction. Alkaline phosphatase staining, Alizarin Red staining, and immunofluorescence test or immunocytochemistry staining of OPN, OCN, Col I were performed. After six months of cryopreservation, osteo-induced allogeneic BMSCs was resuscitated for treatment. Two critical-sized segmental bone defect,20mm in diameter was created at the parietal bone of every 10 adult dogs with an average weight of 11.9 kg. The defects at the experimental sides (left) of six dogs were repaired with the osteoinduced allogeneic BMSCs/β-TCP complex, while the defects at the control sides (right) were treated withβ-TCP alone (group Allogeneic). Two dogs (the two donors) were repaired with the osteoinduced autogeneic BMSCs/β-TCP complex, while the defects at the control sides (right) were treated withβ-TCP alone (group Autogeneic). The rest two dogs were repaired withβ-TCP alone, while the defects at the control sides (right) were untreated (group Blank). The wounds were then closed in layers with 1-0 silk sutures. All animals survived the surgical procedure, no one got infection.The healing response was evaluated histologically and radiographically at sixteen, and twenty-four weeks after implantation. The radiographic and histological results at 24th weeks were compared. The systemic immune response was evaluated by the analysis of T lymphocyte cells. Results Ficoll of 1.077g/ml can seperate BMSCs with high purity, inoculated cells grew well, with an average doubling time of 24h, the cultured BMSCs can be induced to differentiate into osteoblasts. For defects treated with osteo-induced allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically and Radiographically, by 16th weeks, there are obvious signs of osteogenesisno in the group with allogeneic BMSCs/β-TCP complex implants.And immunological analysis showed no significant difference in the number of CD4+and CD8+cells, the CD4+/CD8+value between the above three groups..Conclusions The results of this study demonstrated that 1.077g/ml Ficoll separating solution can isolate Beagle's BMSCs very well, isolated cells can be induced to differentiate into osteoblasts, and long-term cryopreserved osteo-induced allogeneic mesenchymal stem cells loaded onβ-TCP scaffold implants enhanced the repair of a critical-sized segmental defect in the canine skull without the use of immunosuppressive therapy.