The Investigation Of Tissue Engineering Using Rapidly Proliferated Bone Marrow Mesenchymal Stem Cells (BMSCs) By Microcarrier In Rotated Bioreactor In Vitro

ObjectiveRotary Cell Culture System (RCCS) & Microcarrier Suspension Culture System is one new technique that connected with microcarrier and bioreactor, now is widely used in tissue engineering. Tissue engineering bioreactor system could make the cell culture in vitro similar to in vivo. Rotary Cell Culture System could make cell to suffer minimum shearing force in rotation and easily for cell material to exchange. So it was better for attached cell to grow. To utilize microcarrier Suspension Culture System, we had Large Scale Amplification of Tissue Engineering Seeding Cells in rotary cell culture system. To break through the bottleneck of traditional cell culture, it was necessary for clinical application. RCCS was a bioreactor which was engineered for simulating space micro-gravitational effect by National Aeronautics and Space Administration (NASA) .It was a cell culture system that gas exchange was used membrane to diffuse. It could simulate space miro-gravitation and cultivate high density histiocyte. It could be used for large scale cell culture with microcarrier, also could be used to form three-dimension compounds with cultural cell and bracket material.We utilized the principle to manufacture RCCS , and microcarrier technique to culture BMSCs. The BMSCs were seeded into polyhydroxybutyrate (PHB), and then implanted to the rabbit mandible defects. This reseach was going to explore the methods for rapidly proliferated bone cell. This might have clinical applications in Tissue EngineeringThe empirical study includes 3 sections:Section 1: Construction of Rotary Cell Culture System Rotary Cell Culture System contains: fresh media vessel, oxygenator, peristaltic pump, reactor and control panel.They could be placed in incubator after connected. Fumigation by epoxyethane before using.empirical method: to different particle diameter ,in any position of the container ,the greatest fluid shearing force is detected.experimental result: The system could provide proper low-shear force mechanics circumstance for particle culture.Section 2: Comparison of different methods for mesenchymalstem cell from rabbit bone marrow.To evaluate the efficiency of different methods of detaching and cultivating mesenchymal stem cells from rabit bone marrowempirical method: To take out rabit bone marrow first and then to cultivate these cells directly , or to detach the cells with Ficoll - Hypaqueor(1.077g/ml) or Percol(1.073g/ml) before cultivation ,then to harvest those cells and measure the proportion of mesenchymal stem cells .experimental result: the proportion of mesenchymal stem cell was different: to cultivate directly is 67.3% , isolation with Ficoll-Hypaqueor before cultivation is 83.6% and isolation with Percol is 93.4% .Conclusion Application of Percol to isolate cells from bone marrow before cultivation is the most effective way to get mesenchymal stem cells.Section 3: The investigation of tissue engineering using rapidly proliferated bone marrow mesenchymal stem cells (BMSCs) by microcarrier in rotated bioreactor in vitro and repair mandible defectsempirical method: BMSCs of rabbit were seperated and purified in vitro, and rapidly proliferated by microcarrier in rotated bioreactor in a short period of time. The BMSCs were seeded into polyhydroxybutyrate (PHB), and then implanted to the mandible defects, with simple mandible defect group and PHB alone group as control. Two rabbits each group were killed after 3, 8,14,42,84 days, and specimen were evaluated by histological examination, BMP immunohistochemistry, x-ray and measurement of the ash weight of the implants.experimental result: Most PHB were replaced by bone tissue in the BMSCs and PHB group after 84 days implantation, and the reconstruction of defects were superior to the control groups.Conclusion1 In the study, we did not induce BMSCs in vitro. We placed Experimental Studies on the complexed of Cytocompatibility of Polyhydroxybutyrate (PHB) and bone marrow stromal cells (BMSCs) .We utilized microcarrier cell culture technique to induce BMSCs ,complex with PHB ,then implanted to rabbits mandibular defect to repair rabbits mandibular defects.2 Rotary Cell Culture System (RCCS) could provide proper low-shear force mechanics circumstance for particle culture; In RCCS, We utilized microcarrier cell culture technique to induce BMSCs which could fit for bone engineering; By microcarrier cell culture technique, the cell amplification velocity was higher than normal. The BMSCs amplificated by microcarrier cell culture technique had high ability to repair bone defect through implanted onto PHB matrix in vivo. It shortened bone forming time. BMSCs could synthesis and secretev BMP, also have bone forming ability.The osteoblast-like cells were implanted on PGA matrix immediately can secrete BMP.3 Polyhydroxybutyrate can be one of tissue engineering materials which can repair bone defect. In material, bone morphogenetic protein colored obviously. BMP was promoter in the process that BMSCs induced to bone cells. To utilize tissue engineering technique, we confined endogenous BMP to bone defect area, raised density of endogenous BMP, and improved its distribution.This may be one of the reasons of tissue engineered bone intergreting new bone regeneration by increasing the concentration of endogenic BMP and improving its distribution.4 In the study, the variant BMSCs passaged and amplificated in vitro complexing with PHB ,which imbed to rabbit mandible defect to repair bone defect. Obviously rejection was not observed.