Injectable Tissue Engineered Bone Based On Cell Sheet Technology Conbinded With Platelet-rich Fibrin(PRF)

【Background】Nonunion and bone defect repair treatment is still a very complex problems inclinical. Fortunately, the progress made in recent years in regenerative medicine can usecell therapy for bone regeneration. Tissue engineering refers to the application of themethods of engineering and life sciences and technology to build biological substitutes torestore or improve tissue function. However, the transplantation of tissue engineered boneand autologous bone often require open surgery. Compared with traditional surgicaltransplantation, the use of injection techniques to repair bone defect has its own obviousadvantages, including easy to operate, low risk, almost no surgery scar formation,minimally invasive and better repeatability, etc.In recent years, cell sheet technology has been proven to be a very effective method in the bone tissue engineering. Cell sheet induced by osteogenic has merits such as softand elastic, easy to change the shape and thickness can be controlled. Currently, there aresome experimental studies to build injectable tissue engineered bone by cutting the wholecell sheet to appropriate size fragments, but they have no use of growth factor in theexperiment, and its osteogenic capacity can be improved. This experiment aims to observeits osteogenic capacity whether can be increased or not by adding platelet-rich fibrinwhich can be release autologous growth factor gradually.【Objective】To explore the feasibility of building injectable tissue engineered bone by combiningapplications of bone marrow stromal stem cell sheet and platelet-rich fibrin, and fumblethe effect of platelet-rich fibrin on bone marrow stromal stem cell sheet in vitro and invivo.【Methods】1. Isolate and culture rabbit bone marrow stromal stem cells and build bone marrowstromal stem cells into cell sheet.2. First, draw the same donor rabbits’ arterial blood for preparation of PRF sheet, andthen do HE staining, TEM and SEM tests of the PRF sheet; Second, prepare the mediumcontaining different gradient concentration of PRF, to observe its influence on cell growth;Third, further prepare osteogenic induction medium containing the optimal concentrationof PRF to observe its influence on BMSC osteogenic sheet, RT-PCR was used to detectthe related osteogenic gene expression changes.3. In vivo: the mixtures of PRF fragments and BMSC sheet fragments, which havebeen prepared well in the above steps, were injected into subcutaneous of back of nudemice. BMSC sheet fragments alone without PRF fragments were also injected intosubcutaneous of back of nude mice as a control group. After6weeks of operation, take outthe transplants and then perform general observation, X-ray scanning, in vitro micro-CT,histological analysis, etc to evaluate the newly formation tissue.4. The experimental data was analyzed by SPSS18.0statistical software, using analysis of variance method or t-test of mean of random two groups.【Results】1. We can build BMSC sheet through the above methods and they have osteogenicdifferentiation characteristics.2. PRF prepared by drawing arterial blood has a concentration-dependent stimulativeeffect on BMSC growth. PRF prepared by each one ml arterial blood placed in two ml ofthe medium possesses the most suitable stimulative effect, and cells grow best under suchstimulative concentration.3. In vitro, PRF has a stimulative effect on the growth and osteogenic differentiationof BMSC sheet, RT-PCR results suggest that osteogenic induction medium containing asuitable concentration of PRF will promote the osteogenic related gene expression ofBMSC sheet, the result have a significant difference.4. Hard tissue formed after mixtures being transplanted into the body remains a goodshape, X-ray and Micro-CT scanning in vitro shows ectopic mineralization, the higherdensity of the experimental group than the control group, and the weight is also heavier inthe experimental group; Histology analysis proves it as a newly formation bone tissue, andexperimental group has a better osteogenic properties.【Conclusion】1. Cell sheet-like structure can be formed after BMSC being osteogenic induction,and has the characteristics of osteogenic differentiation.2. PRF can promote the growth and proliferation of BMSC, and PRF can enhanceosteogenic related gene expression of BMSC sheet in vitro.3. Mince osteogenic BMSC sheet and PRF sheet into pieces and mix well and theninject the mixtures into subcutaneous of back of nude mice, there are newly bone tissueformation, and PRF can increase the osteogenic ability of BMSC sheet, the results has asignificant difference. Our experimental study provides a novel methods and ideas forbone tissue engineering, has s broad application prospect, and also provides anexperimental basis for bone tissue engineering research and clinical repair of bone defects.