To Study The Ossification In Vitro Of Tissue Engineered Periosteum Constructed By Bone Marrow Mesenchymal Stem Cells And Human Acellular Amniotic

BackgroundBone defects caused by trauma, tumor, infection is a common clinical disease.The treatment of bone defects are autogenous bone graft, allograft, periostealtransplantation and artificial bone transplantation, and periosteum plays an importantrole in bone repair, intramembranous bone is one of the important mechanisms of bonerepair. But autologous transplantation is limited source of funding for the District, anddamage for the District to bring some pain to the patient. Allograft may cause somecomplications,such as immune rejection reaction and the spread of disease. Therefore,we assume that applicating the tissue engineering methods, to build a true sense of theperiosteum to repair bone defects. To build a bionic the periosteum must have thestructure and function of physiological periosteum, which should not only containosteoblasts could ossify, but also need biodegradable scaffold materials make good cellattachment, proliferation, differentiation. Therefore, choosing the similar physiologicalperiosteal seed cells and scaffold materials is excellent important. Currently, tissueengineering periosteum made some progress, many scholars at home and abroadapplicating into bone cells and bone marrow mesenchymal stem cells in vitrocomposite chitosan small intestinal submucosa acellular matrix, achieved good resultson repairing bone defect. Biodegradable scaffolds such as chitosan, still can not becompared with the natural membrane material, small intestinal submucosa acellularmatrix is a natural structural materials, but there is still the risk of immune rejectionfor applied small intestinal submucosa acellular matrix to a person’s bone defect repair.It has been confirmed that bone marrow mesenchymal stem cells can be compositedhuman acellular amniotic membrane well. Human amniotic have rich source,excellenthistocompatibility, containing multiple collagen, which could be an ideal scaffold. Theother hand, there has been applied to bone marrow mesenchymal stem cells combined with human amniotic acellular matrix successfully repaired rat full-thickness skindamage, but the two composite need further verify the feasibility of buildingbiomimetic periosteum.ObjectiveTo discuss HAAM as the scaffold materials loaded the osteoinductive bonemarrow mesenchymal stem cells to construct tissue engineered periosteum, as well asthe feasibility of ossification in vitro. Provide a theoretical basis for making bionicperiosteum ossify in the animal or human body in future.MethodsIn this study, first, isolated SD rats (5-7days of suckling mice) double tibial，using syringe needle rinse marrow and made into single-cell suspension，successfully isolated and cultured SD rat BMSCs and fulfill its histologicalidentification; Second, the preparation of human acellular amniotic membrane, fix thehuman acellular amniotic membrane and HE staining to confirmed the cellularcomponent removal clean; observe the structural features of HAAM by scanningelectron microscopy; And cultured bone marrow mesenchymal stem cells combinedwith the HAAM as a scaffold, constructed in vitro tissue engineering periosteum, HEstaining and scanning electron microscope significantly on HAAM,BMSCs wereobserved under a microscope for their composite,growth and proliferation, Andproliferation of MTT assay bone marrow mesenchymal stem cells on acellularamniotic membrane (experimental group) and unmanned acellular amnioticmembrane (control group). The experiment was divided into two groups that bonemarrow mesenchymal stem cells combined with acellular amniotic without inducer(control group), bone marrow mesenchymal stem cells combined with acellularamniotic plus inducer group (experimental group). Results1.After HAAM was treated with the physical and chemical methods, thesurface of HAAM have no residual cells, HAAM have many lacunas, collagen fibersare not damaged.2. With osteogenic induction culture conditions, BMSCs weremultilayer growth, without contact inhibition, and have the morphologicalcharacteristics and growth characteristics similar to osteoblasts, Alizarin red stainingtest is positive for calcium nodules. With adipogenic induction culture conditions,BMSCs have no contact inhibition, gradually appear vacuoles and lipid droplets, andhave morphological and growth characteristics similar to adipocyte, oil red staining ispositive. With chondrogenesis induction culture conditions, BMSCs have no contactinhibition, gradually appear vacuoles, and have the morphological characteristics andgrowth characteristics similar to chondrocyte, the new blue staining is positive.3.Under the same culture conditions, the cells of experimental group(HAAM group)than the control group(no materials) were detect by absorbance value through theanalysis of MTT of two groups at different time points, the experiment results showthat at different time points, at the first four days the cell proliferation rate of the twogroups was no significant difference (p>0.05); at the5d,6d the cell proliferationquantity in both groups, there was significant difference between groups by” F” test(p <0.05), the proliferation cells in experimental group were more than the controlgroup; at the7d,8d the cell proliferation quantity in both groups, there wassignificant difference between groups by”F” test (p <0.01), the proliferation cells inexperimental group were more than the control group.4. The experimental group wereobserved that in vitro cells cultured composite with material show excellent adhesion,secrete large amounts extracellular matrix,and Active cell differentiation andproliferation; By general observation, the color of composites turned white andstiffness increased; by histology and electron microscopy, a lot of bone tissue was formed; The control group were observed that in vitro cells cultured composite withmaterial show excellent adhesion and growth, by general observation,the compositeshave the same color and stiffness compare to HAAM without cells.Conclusion1.HAAM have excellent cell compatibility, do not affect the morphology ofBMSCs, do not restrain the growth, proliferation and function expression ofBMSCs.2.BMSCs could express osteoblast phenotype in vitro by osteogenic inductionculture is an ideal bone tissue engineering seed cells.3.HAAM have excellentbiocompatibility, can be used as a scaffold for bone tissue engineering to constructtissue engineered periosteum.