Injectable Tissue Engineered Bone Based On Cell Sheet Technology Combined With Particulate Decellularized Cartilage Matrices And Platelet-rich Plasma

[Background]Bone defects are always caused by trauma,tumors and some congenital genetic diseases.So the bone regeneration has been an important issue for clinic especially the patients who are suffering the sever bone defects or irregular bone resorption of maxillofacial.Recently,stem cells' therapeutic has become the focus of study.Since the researchers discovered the BMSCs and successfully acquired bone regeneration with BMSCs,more and more break through has been obtained.With the development of the standard of bone regeneration,researchers find the bone' quality that simply uses the collection of single BMSCs can't be as good as use the BMSCs sheet.Because during the formation of the cell sheet much more extracellular matrices are accumulated,in which there are abundant of growth factors that promote the vascularization and ossification.Based on the results,researchers discovered more stem cells like adipose-derived stem cells,dental pulp stem cells,periodontal ligament stem cells and so on.During the processes of endochondral ossification,stem cells form the growth template firstly and then undergo the hypertrophy,vascularization,calcification and ossification.Stem cells secrete the factors about vascularization and home the cells of host to join the bone reconstruction.A mass of researches prove that chondroblasts from BMSCs have better bone formation capacity after cloning.Besides,the ability of bone formation of chonroblasts is enhanced by the added growth factors.Up till now,scientists usually use hydroxyapatite,three tricalcium phosphate as the bone substitute.But these biomaterials have low degradation rate in vivo and can't fuse perfectly with the tissue of the host,what's more,they are easily leading to inflammatory response which finally results in the failure of transplant.So we need a low immunological rejection,micro-invade and figurability support material.The PDCM comes from the organic system which ensures the supplement of the materials,besides,after decellularization process,the PDCM still possess the most col and GAG of matrices which is the main strength of support.The growth factors remained in the matrices of PDCM can improve the bone formation quality.In addition,the porous constructs of PDCM facilitate the attachment of stem cells and the growth factors and the invasion of the blood vessels.As the basic factors of tissue engineering,growth factors are necessary.Most previous studies prefer to use the BMP to promote the bone regeneration.And the BMP never let the researchers disappoint and it has been successfully applied to clinic treatment.But with the restriction of the separation and purification technology,it is too expensive to bear for patients.The hydrogel always company with central necrosis and inflammatory response.Afterwards,we discover the PRP,which contains more than 600 different growth factors.It not only promotes the vascularization and ossification of transplants but also comes from the autologous that avoids the immunology rejection.So we mix the chondrogenic induced BMSCs bricks,PDCM and PRP together to form the bone through endochondral ossification,in order to provide a new method for clinic treatment of bone defect prosthetic.?Objective?To explore the feasibility of building injectable tissue engineered bone by combining applications of bone marrow stromal stem cell sheet and platelet-rich plasma and decellularized cartilage matrices in vitro and in vivo.?Methods?1.Isolate and culture rabbit bone marrow stem cells and build bone marrow stem cells into cell sheet and chondrogeic inducted the cells,For further examination,part of BMSCs derived chondrocyte macroaggregate are histologicalliy stained with safranin-O staining,scanning electronic microscopy(SEM),and PCR examination for collagen II and X,as well as VEGF.2.Prepare the particulate decellularized cartilage matrices and for further examination,part of PDCM are histologicalliy stained with HE staining,safranin-O staining,immunohistochemistry staining for collagen II,scanning electronic microscopy(SEM),and quantification test of collagen and GAG.3.Draw the same donor rabbits' arterial blood for preparation of PRP through high-speed centrifugation.4.In vivo test in subcutaneous of nude mice: the mixtures PDCM and chondrogenic induced BMSCs sheet bricks and PRP,which have been prepared well in the above steps,were injected into subcutaneous of back of nude mice.PDCM and PRP complex was also injected into subcutaneous of back of nude mice as a control group.After 4 and 12 weeks of operation,take out the transplants and then perform general observation,histological analysis,Biochemical analysis(collagen and GAG content),Histological and immunohistochemical assay,immunofluorescence assay to evaluate the newly formation tissue.5.Injected the PDCM-CB-P mixture and PDCM-P mixture into the bone defect around the implant in rabbit tibia,after 12 weeks of operation,take the sample and perform general observation,histological analysis,Histological and immunohistochemical and Micro-CT to evaluate the newly formation tissue.6.The experimental data was analyzed by SPSS20.0 statistical software,using analysis of variance method or t-test of mean of random two groups.?Results?1.We can build BMSCs sheet through the above methods and they have chondrogenic differentiation characteristics after chondrogenic induce.2.PDCM has no cells left after decellularization processes and keeps the most of collagen and GAG.3.Angiogenesis occurred quickly inside PDCM-CB-P constructs after implantation,which improved tissue survival and bone formation.4.Faster replacement of PDCM in rabbit tibia bone defect around implant in PDCM-CB-P group than in PDCM-P group.?Conclusion?1.BMSCs bricks,PDCM,and PRP compound maintained the morphology and tissue volume;2.The compound enabled the rapid vascularization and prevented the necrosis;3.The compound enabled rapid endochondral ossification.More importantly,the peri-implant bone regeneration experiment performed in rabbit tibia demonstrated well implant osseointegration between titanium implant and transformed bone,thus indicates their clinically translational significance in oral surgery.