Experimental Study Of Tissue-engineered Bone Constructed By Osteoinduction Active Material With Hunman Bone Mesenchymal Stem Cells

ObjectiveThe treatment of bone defects, especially segmental bone defects, was still one of the problems faced in clinical orthopedics. Conventional graft materials, lacking biological activity, their degradation rate varied a lot, and their roles in promoting osteogenesis were often limited. Mesenchymal stem cells, derived widely and conveniently, possessed the capability of self-renewal and multipotent differentiation. These mesenchymal stem cells, with a strong potential for osteogenesis, had been confirmed by in vivo and in vitro experiments, and were ideal cell sources for the research on bone tissue engineering. Osteoinduction active material (OAM), with a three-dimensional structure, was biocompatible and biodegradable in vivo, which were in line with the requirement for biological scaffold uesed in bone tissue engineering. Currently, the combination of seed cells and scaffold materials were facing problems, including inefficient inoculation of seed cells, loss of capacity, and the uneven distribution of cells in the scaffold. This research, using osteoinduction active materials as carriers, human bone marrow-derived autologous mesenchymal stem cells cultured three-dimensionally in vitro were used to build individually constructed tissue engineered bones. At the same time, the use of autologous platelet-rich plasma (PRP) as a two-phase cell inoculated seed medium, increased cell adhesion and osteogenesis, and could be used to build tissue engineered bones suitable for clinical application. These results provided useful experimental data for clinical individual transplantation for the treatment of bone defects, and had great application prospects.Methods1. The isolation, cultivation and identification of the human marrow mesenchymal stem cells (hBMSCs)Cells were isolated from human marrow via density gradient centrifugation and bone marrow adherent culture.Bone marrow cells were cultured in fetal calf serum and autologous serum. Cell morphology was observed and flow cytometry was used to examine the expression of cell surface antigens. The growth curve of cell culture was obtained based on assay of the proliferation status of passage 3 and passage 5 cells by MTT method.2. Study of differentiation potentiality and osteogenesis capability of the hBMSCs in vitroCultured passage 3 cells were utilized for osteoblasts differentiation and adipogenic differentiation. Cell morphology was observed. Oil red staining was used for lipogenesis testing, whilecollagen I, osteocalcin (OCN) and calcium node were used for osteogenesis testing. Alkaline phosphatase activities in inductive and non-inductive groups were tested. And the expression of ALP and OPN mRNA were tested by RT-PCR. 3. Study of bone tissue engineering of hBMSCs and OAM-PRP in vitroCultured passage 3 cells were labeled cells with BrdU, and the traditional inoculation method and PRP two-phase cell inoculation method were used to construct cell-scaffold complex. Cell adhesion ablitity and ALP concentration were compared. RT-PCR detection of ALP, OPN mRNA expression both in osteogenic induction group and non-induced group. The growth, proliferation and matrix secretion of hBMSCs in scaffold were observed by scanning electron microscope.4. In vivo study of tissue engineered bones constructed by hBMSCs and OAM-PRPFemoral defected animal model was established using the New Zealand white rabbits. Rabits were divided into 3 groups, groupⅠOAM, groupⅡhBMSCs and OAM, groupⅢhBMSCs and OAM-PRP. Tissue engineered bones were transplanted into the animals to repair bone defect. Animals were observed generally; X-ray examination for the observation of bone formation at the site of bone defect; HE staining of tissue sections for the detection of scaffold degradation and new bone growth.Results1. Both bone marrow adherent method and density gradient centrifugation method could successfully separate hBMSCs, and both autologous serum and fetal bovine serum could be used to culture fibroblast-like cells. Growth curve made by MTT method showed strong proliferation in cells of passage 3 and passage 5. Flow Cytometry detected passage 5 cells derived by density gradient centrifugation and cultured with autologous serum, which were CD44, CD90 positive and CD34, CD45 negative.2. Passage 3 hBMSCs induced 21 days for osteogenesis, were positively stained for collagen I, OCN and calcified nodules; 21 days after adipogenesis induction were oil red O positive staining; in the process of bone induction, ALP levels increased gradually, and had a significant difference compared with non-induction groups (P<0.05); RT-PCR showed ALP expression both in induction group and non-induction group, the non-induction group was weaker; and OPN expression in induction group and not in non-induction group.3. Passage 3 hBMSCs were BrdU labeled and used for construction of cells-biological scaffold complex by the traditional standing inoculation and PRP two-phase inoculation. Cell adherent percentage using the two methods 4h,8h,12h,24h later after inoculation was significantly higher using the latter method (P<0.05); ALP level in the cells seeded by two methods increased graduallyon, and was significantly higher using the latter method (P<0.05); RT-PCR showed that both methods had ALP and OPN expression either in induction or non-induction group, but their expression was weaker in the non-induction group; scanning electron microscope showed that cells grew on the surface of tissue engineered bones inoculated by PRP two-phase method and could extend into the pores of material with matrix secretion.4. Tissue engineered bones of different groups were implanted in the site of femoral defect rabits, the wound grew well and healed with no adverse reactions. X ray showed that groupⅢbasically reached to a healing after 12 weeks, groupⅡhad a relatively worse healing, while groupⅠhardly healed. HE staining displayed that 8 weeks later OAM was almost absorbed in groupⅠ, while groupⅡand groupⅠcould see scaffold material degradation and newly generated bone tissue 8 weeks later, and 12 weeks later groupⅢhad stronger osteogenesis.Conclision1. Density gradient centrifugation and autologous serum could produce human bone marrow mesenchymal stem cells as seed cells with uniform composition and high proliferation ability.2. Platelet-rich plasma duplex-mediated method could promote the adhesion of human bone marrow mesenchymal stem cells with osteoinduction active materials and osteogenesis, useful for the construction of tissue engineered bones.3. Constructed tissue engineered bones grafted to the bone defected rabbits could increase osteogenesis at the site of the bone defect and promote bone healing.