Repair Of Large Segmental Bone Defect By Tissue-Engineered Periosteum And Deproteinized Bone Scaffold In Rabbits

Objective (1) To isolate the rabbit bone marrow mesenchymal stem cells (BMSCs) by density gradient centrifugation and induce them to osteoblasts;(2) To observe the adhesion and growth of osteogenetically induced BMSCs in the SIS, and to confirm that it is feasible to construct tissue engineering periosteum in vitro;(3)To evaluate the effect of tissue-engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) served as supporting scaffolds of tissue engineering periosteum.Methods (1)Rabbit bone marrow mesenchymal stem cells (BMSCs) were cultured and osteogenetically induced into osteoblasts.(2)Porcine small intestine submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue-engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then observed by scanning electron microscopy (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineering periosteum/DPB complex was constructed by tissue engineering periosteum and DPB. Tissue engineering periosteum was "coat-like" package the DPB, and bundled with absorbable sutures.(3)48New Zealand white rabbits (4-month-old,2.0-3.Okg, no gender limitations) were randomly divided into4groups (group A, B, C and D). A rabbit model of3.5cm length bone defect in the radius was created. In group A, the defect area was occupied by tissue engineered periosteum;In group B, the defect area was implanted with DPB; In group C, the defect area was implanted with tissue engineered periosteum and DPB; In group D, the defect was untreated. At4w,8w and12w after operation,4rabbits in each group were observed by X-ray, after X-ray examination, the rabbits of each group were sacrificed and examined by histological examination.Results (1)3days after primary culture, the rabbit bone marrow mesenchymal stem cells are fusiform morphology, short rod, crescent-shaped and irregular-shaped;7-9days after primary culture,the cells overgrow the cultured bottom.2-3days after subculture cultures, the cells can overgrow the cultured bottom can be Confluent bottom, the shape of the cells are fusiform, fish-like arrangement; Osteogenetically induced for8days, the size of cells become larger, the cells change into triangles, squares and polygons short,12days after,there gradually appear some high-refraction round clumps, cells are surrounding the clumps radially; Osteogenetically induced for12days, ALP staining showe that there are light brown to brown-black fine particles the cytoplasm; Alizarin red staining showe the purple calcium nodules, cytoplasm also be dyed purple:DPB is white, hard but brittle, hollow intramedullary; SIS is white, membranous shap, the thickness is about100μm; Tissue engineering is soft, good toughness, and has the similar appearance to the physical periosteum;(2)SEM showed abundant se eding cells adhered on tissue engineered periosteum. At4w,8w and12w post-operation, X-ray showed the newly formed bone was much abundant in group A and C than group in B and D. The radiographic score was significantly higher in group A and C than in the other two groups, and in group A was much abundant than in group C (P (0.05). Histological staining:in group A, there are a lot of newly formed bone in the defect space4weeks after operation, newly formed bone connect osteotomy ends and have the similar density to normal bone;8weeks after operation, newly formed medullary cavity are abundant among the new bone tissue,12weeks after operation,the marrow cavity has been through; While in group B, the DPB fill with the defect space,the degradation of DPB are not obvious.In group C, there are some newly formed bone in the defect space4weeks after operation, newly formed bone connect osteotomy ends8weeks after operation, island-like DPB was seen in newly formed bone, the degradation of DPB are obvious.12weeks after operation, there are irregular medullary cavity among the new bone tissue, and we can not see the DPB; In group D, the defect space only replaced by some connective tissue.Conclusion (1) It can successfully obtain a lot of high-purity BMSCs by using density gradient centrifugation, bone marrow mesenchymal stem cells can be induced into osteoblasts.(2) SIS is an ideal membranous scaffolds for tissue engineering. Osteogenetically induced BMSCs can adhere and grow in the SIS. Construct tissue engineering periosteum in vitro is feasible.(3)Tissue-engineered periosteum has the feasibility to repair the large diaphysis defect in rabbit. The supporting scaffolds of tissue engineering periosteum need further exploration. Tissue engineered periosteum is worthy of the further investigation and exploitation for clinic.