An Experimental Study Of BMSCs As Seed Cells For Bone Tissue Engineering In Allogeneic Fetal Rabbit

The repair of bone defects poses a great challenge in the reconstructive surgery.Autogenous bone cause patients donor sites morbidity. The bone substitutes used in clinic all have limitation, such as individual differences of demineralized bone matrix and long-term foreign-body sequestration of hydroxyapatite. Tissue-engineered technique maybe afford a ideal therapy method in mandibular repair.Purposes: Explore the feasibility of establishing an inter gestation period of rabbits allogeneic bone marrow mesenchymal stem cells as seed cells combined with demineralized bone matrix build bone tissue engineering.Methods : Onepregnant rabbits(pregnancy=3 weeks) were treated with artificial fertilization, and 7 fetal rabbits were obtained with cesarean section. Fetal rabbit bone marrow was harvested and cultured in vitro. The cell morphology, growth curve, cloning efficiency and trilinear differentiation of BMSCs of passage 3 were observed. Cryopreserved primary cells were recovered after 30 days, the growth curve of passage 3 was measured to compare with that non-cryopreserved. With scanning electron microscope, the fetal cells in DBM cell-material compound were observed. Finally, after osteogenic induction, the BMSCs-DBM compound was implanted into the nude mice subcutaneously and the histological results of tissue-engineered bone at 1, 3, 6 months were observed. Rabbit radius defects(1.2cm) were built at right side and repaired with induced BMSCs-DBM constructs. New bone tissues were examined by X-ray, Micro-CT, gross.Results: The BMSCs from fetal rabbit demonstrated a spindle-shaped morphology. After several passages, the morphology and growth curve remained almost the same. The calcium, fat and mucopolysaccharide were witnessed in trilinear differentiation. After cryopreserved for 30 days, the growth curve of passage 3 showed few changes compared with non-cryopreserved. According to SEM, the fetal cells tightly attached to the materials and extended well after seeded on DBM for 7 days. The fresh bone tissue were all observed at different time point by histological staining. X-ray displayed the radiopacity increased of induced BMSCs-scaffolds post-operatively. At 12 weeks, bony healing was found from gross view and Micro-CT images.Conclusion:1. The bone mesenchymal stem cells(BMSCs) derived from rabbits gestation period, after a series of observations and experiments, proved to be a readily available, the ability to induce a strong differentiation, combined with biomaterials good seed cells for bone tissue engineering. 2. Pure demineralized bone matrix(DBM) as mere biological material can not fix the rabbit radial standard segmental bone defects.3.Gestational age of theallogeneic BMSCs combination of demineralized bone matrix(DBM) can build bone tissue engineering to repair rabbit bone defect models.