| ObjectiveAvascular necrosis of femur head is a common orthopedic disorders, which caused by increased pressure in the femoral head. The bone tissue of the femoral head can not get the normal blood supply of the nutrient vessels, so the necrosis of bone cells, bone marrow, hematopoietic cells the femoral head have occurred.. The treatment of femoral head necrosis is a major problem in today’s world. The necrosis of the femoral head was mainly due to the damaged blood supply of the femoral head, causing the death of bone cells combined with bone marrow composition and subsequent repair, which lead the femoral head structural changes, collapse of the femoral head, joint dysfunction disease. This experiment is the application of tissue engineering technology and construction of tissue-engineered bone to assess the process of blood vessels, which provide a feasible plan for the reconstruction of the blood supply of the femoral head. In this experiment, we extract the mesenchymal stem cells in the bone marrow of New Zealand white rabbits and apply platelet-rich plasma (PRP) to culture them which combined with the the good biocompatibility scaffold beta-TCP. The extending direction of the beta-TCP three-dimensional space can help control stem cell growth behavior and improve the units amplification rate, which is suitable for the growth of tissue cells in vivo microenvironment in simulated microgravity system to adapt to a shearing force to promote bonedifferentiation.. On the other hand, beta-TCP can be used as a good storage and controll the delivery of drug,which can have a gradual release of bone growth factor during the culture. Finally, the composite body is placed in the three-dimensional culture bioreactor to construct tissue engineered bone which will be implanted in animals. Three months later the animals will be killed,.We will use immunohistochemistry to observe specific markers of the blood vessels, which in turn is used to evaluate the vascularized tissue engineering bone.Materials and Methods1. We will use healthy New Zealand white rabbits approximately2-3months of age in this study. The New Zealand white rabbits were placed on the laboratory bench after anesthesia. Under sterile conditions with a puncture needle to extract5ml bone marrow from the the bilateral tibial platform of the rabbits. Purify the bMSCs by percoll separating medium and culture them primarily, and then generated. The bMSCs was identitied by flow cytometry.Collected fresh blood approximately10ml from the ear vein of the rabbits. Filled a15ml centrifuge tube with the blood which contain lml5%sodium citrate and gently shake it to prevent solidification.2. Blood in the centrifuge tube is placed in a centrifuge to produce platelet-rich plasma with the method of secondary centrifugation. First centrifuged at3300r/min, the second centrifugation at3000rpm/min, and then we will get the colorless transparent jelly-like substance called platelet-rich plasma. The platelet-rich plasma is added to the culture medium to culture BMSCs,which will be changed once3days later. The cell suspension made by BMSCs was added dropwise to (3-TCP to build cell scaffold which will be placed into a bioreactor.3. Implant composite into the New Zealand white rabbits,then we will use immunohistochemistry to observe specific markers of the blood vessels, which in turn is used to evaluate the vascularized tissue engineering bone.Results1.We will use the phase contrast microscope to observe the growth of the bMSCs. We will find that the adherent cells are small and round. The next day visible irregular cells adhere to the bottom of the bottle. After7days, the cell volume increased significantly and the clone cluster of the cells Appear. After12-14days, the cells can be basically covered the bottom of the bottle with uniform growth and cell full extension then the cells began to be transformed to polygonal cells.2. Bone marrow mesenchymal stem cells is an ideal seed cells for tissue engineering. Because of the rich and convenient source of bone marrow mesenchymal stem cells, it have stable culture performance in vitro,and ease of passaging.3.The platelet-rich plasma is rich in a variety of cytokines which can induce bone or cartilage and promote vascularization process.4.β-TCP scaffold has more advantages in tissue engineering scaffold material selection due to its good histocompatibility and three-dimensional structure so bone marrow mesenchymal stem cells are suitable for in β-TCP scaffolds adhesion and proliferation.5. Bioreactor can produce mechanical irritation to mesenchymal stem cells and make better adhesion and proliferation of cells in a three-dimensional scaffolds. |
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