Study On The Flow Rate Of Perfusion Bioreactor On The Proliferation And Osteogenic Differentiation Of ?-TCP Mesenchymal Stem Cells

Background:Severe trauma,infection,and bone tumor resection often lead to serious bone tissue defects,especially large bone defects,which not only increase the difficulty of treatment,but also easily lead to disability and later treatment difficulties.Repairing large bone defects and rebuilding their functions has always been an orthopedic problem and a research hotspot.At present,autologous bone and allogeneic bone are mostly used in the treatment of large bone defects in clinic.Vascular autogenous bone transplantation repairs large segments of bone defects.The operation is complicated and the patient has a heavy surgical burden.Moreover,autogenous bone is limited.Although allogenic bones come from a wide range of sources,there is a risk of immune rejection and disease transmission.With the rapid development of bone tissue engineering,tissue engineering bone may become an ideal way to repair large bone defects.Tissueengineered bone has sufficient sources,biological functions similar to or better than autogenous bone,and shows unprecedented superiority in the repair of bone defects.The research of bone tissue engineering is to combine seed cells and bone scaffold materials in a pre-designed bioreactor.After they are vascularized and osteogenesis,they are transplanted into the bone defect in the body to regenerate the defect tissue.This topic is divided into three parts:(1)Using soluble polymethyl methacrylate(PMMA)ball mold grouting technology to prepare degradable connected porous bioceramic artificial bone material(ie ?-tricalcium phosphate,?-tricalcium phosphate,?-TCP),and detect its Performance characterization;(2)Bone marrow mesenchymal stem cells(BMSCs)are extracted and cultured from rabbit bone marrow using the whole bone marrow adherent method,and passaged and identified as seed cells;(3)Using perfusion bioreactor as a carrier,inoculate BMSCs into ?-TCP to form cell/material complexes,perfusion culture the cell/material complexes with different perfusion flow rates,and evaluate their effect on BMSCs proliferation and osteogenic differentiation influences.Part ? Preparation and characterization of biodegradable connected porous bioceramic artificial bone materialsObjective:Prepare and test the physical and chemical properties of degradable porous?-TCP scaffold materials,optimize the preparation specifications of artificial bone materials,study the physical and chemical properties of artificial bone materials in vitro,and evaluate the feasibility of their application in biological materials for bone tissue engineering.Methods:(1)Observe the surface and internal morphology of porous ?-TCP material by scanning electron microscope;(2)Use scanning electron microscope to measure the actual diameter of the material and the diameter of the inner diameter of the hole;(3)Use X-ray energy spectrometer to analyze the surface,cross section and longitudinal section elements of porous ?-tricalcium phosphate bioceramic scaffold;(4)The contact angle measuring instrument(Kruss,Germany)is used to detect the contact angle of the cross-section and longitudinal section of the surface of multiple porous ?-tricalcium phosphate bioceramic scaffolds;(5)Use the electronic universal material mechanics testing machine to measure the compressive strength of the degradable porous ?-TCP scaffold material;(6)Calculate the porosity of porous ?-tricalcium phosphate bioceramic scaffolds by weight-volume method and Archimedes drainage method,and calculate the water absorption rate.Results:The pore diameter of ?-TCP material is uniform,and the pore size fluctuates around 500?m.The pores are connected to each other.The connected diameter is about 120?m.The porosity is greater than 60%.The microstructure of the surface of the stent is granular and uneven.It is a weak hydrophobic material.The water absorption between the stents is relatively uniform,and the Ca/P ratio=1/0.67 is consistent with natural bone.Biomechanical results show that ?-TCP material is similar to natural cancellous bone.Conclusion:The use of soluble polymethyl methacrylate(PMMA)ball mold grouting technology to prepare biodegradable connected porous bioceramic artificial bone materials has well-characterized properties,and many indicators are close to natural bone,which is completely in line with bone tissue engineering materials for pore porosity and mechanics The performance requirements can be used as an ideal biomaterial for the construction of tissue engineering bone research.Part ? Extraction,culture,passage and identification of rabbit bone marrow mesenchymal stem cellsObjective:BMSCs were used as seed cells for bone tissue engineering,and were extracted,cultured,passaged and identified to evaluate their purity and their ability to differentiate into osteoblasts and adipocytes.Methods: Bone marrow extracted from New Zealand white rabbit femur and tibia bone marrow cavity was isolated and purified by whole bone marrow adherence method.Observe the morphology and growth pattern of BMSCs,use osteogenic and fat-inducing kits to induce osteogenic and adipogenic differentiation,stain them with alizarin red and oil red,and detect the multi-directional differentiation ability of BMSCs.The expression of surface markers CD29,CD34,CD44,CD45,CD90 was detected by flow cytometry.Results: The whole bone marrow adherence method is convenient and fast for the extraction and cultivation of BMSCs.Under the microscope,BMSCs are long fusiform,adhere to the wall,and grow in a vortex,fish or radial shape.After 3 weeks of osteogenic induction,alizarin red staining showed calcified nodules,and after 2weeks of adipogenic induction,oil red staining showed that the cytoplasm was filled with round lipid-like droplets.Results of BMSCs surface markers: CD29+,CD44+,CD90+,CD34-,CD45-.Conclusion:The BMSCs extracted,isolated and cultured by the whole bone marrow adherence method have excellent proliferation,good morphology,and multidirectional differentiation ability such as bone formation and fat formation,and are ideal seed cells for bone tissue engineering.Part ? Design and construction of perfusion bioreactor and the effect of flow rate in perfusion bioreactor on the proliferation and osteogenic differentiation of?-TCP mesenchymal stem cellsObjective:Design and construct a perfusion bioreactor to evaluate the stability and reliability of its perfusion flow rate;use the perfusion bioreactor to culture the cell/material composite at different flow rates,and observe the morphology and BMSCs in ?-TCP at different perfusion flow rates.In the case of proliferation and osteogenic differentiation,different perfusion flow rates are evaluated to screen the optimal perfusion flow rate,which provides theoretical parameters for the design of in vivo bioreactors and the cultivation of tissue engineering bone.Methods:(1)Use cell counting kit-8(CCK-8)to detect cell proliferation and biocompatibility between different groups after perfusion culture for 1,4,7 days after inoculation;(2)7 days after inoculation and culture,observe the cell morphology between different groups using scanning electron microscopy(SEM);(3)Calculate the cell survival rate of artificial bone materials between different groups using trypan blue staining method after perfusion culture for 7 days;(4)Alkaline phosphatase kit was used to measure alkaline phosphatase activity of cells on artificial bone materials between different groups after perfusion culture for 7 days;(5)After inoculation and culture for 7 days and 10 days after inoculation,real-time quantitative RT-PCR was used to detect the osteogenic effect between different groups;(6)7 days after inoculation,the expression of osteogenic protein in different groups was detected by Western blot detection technology.Results: The results of CCK-8 in each group showed that on the 4th and 7th days of culture,the cell proliferation in the 0.01ml/min perfusion flow rate group was faster than that in the other groups(P<0.05).The trend of cell proliferation showed that the cell and the material had good biocompatibility.Scanning electron microscope observation revealed that the cells in the static culture group(0ml/min)were sparsely distributed and grew in loose clusters;In the 0.01ml/min perfusion flow rate group,there was a membrane-like aggregation growth,and most of the cells protruded pseudopodia distributed around the connecting hole;0.05ml/min perfusion flow rate group was partly in the form of a diaphragm;0.1ml/min mostly in the form of a diaphragm Grow.The cell survival rate was the highest in the 0.01ml/min perfusion flow rate group.The perfusion flow rate of 0.01ml/min constitutes the expression levels of bone-related genes(Runx2,ALP,OPN,Col-I)and osteogenic proteins(Runx2,OPN,Col-I)are significantly higher than other groups(P<0.05),It was also observed that the alkaline phosphatase activity was the highest in the 0.01ml/min perfusion flow rate group.Conclusion:?-TCP material can induce BMSCs to differentiate into osteoblasts.With the decrease of perfusion flow rate of bioreactor,it can promote the synthesis and distribution of extracellular matrix,and can increase the expression of osteoblastrelated genes and related proteins.Among them,the low perfusion flow rate of0.01ml/min is most conducive to the proliferation and differentiation of BMSCs.