| It is very common of bone defects in department of orthopaedics, but the available therapeutic methods on this disease still have deficiency. The technique in tissue engineering provides a new approach for the reparation of bone defects. The advantages include: ⑴ the grafts posses biological activity, ⑵ the source of material is extensive, ⑶ the shape of grafts can be changed for adaptation, ⑷ the antigenicity isn't obvious if the seeding cells come from oneself, ⑸ it avoids some complications of obtaining bone from oneself. American scientist Vacanti obtained calf artificial bone with certain morphology, structure and function by bioengineering technology in 1994. Nowadays, there are many initial problems to be solved, such as optimal accumulation of seeding cells, preparation of biodegradable scaffold composites, action of growth factors, three-dimensional culture of cell-scaffold complex, efficiency of the tissue-engineering bone to repair bone defects. It predicts that the times of ' from organ transplant to organ copy ' is coming as the research of tissue engineering has been developed quickly. Many important problems must be solved for repairing bone defects in clinic by bioengineering technology. Our study attempts to research human mesenchymal stem cellS as the seeding cells and construct tissue engineering bone in vitro.Contents ⑴ the amplification culture of hMSCs in vitro under optimal conditions, ⑵ the culture under osteogenic differentiation conditions with any chemicals or growth factors, and investigation of the biological characteristics, ⑶ preparation of novel biodegradable scaffold composites, construction cell-scaffold complex, ⑷ embed tissue-engineering bone into subcutaneouspart of nude mouse and preliminary assessment the biological behavior on earlier period.Result1. The method of primary culture hMSCs is reasonable that firstly human bone marrow of iliac bone is separated by gradient centrifugation, secondly seeding density is 1×105~3×105cell/cm2, thirdly half of medium is changed after 48 hour and all medium is changed every 3 days.2. There are 3.2×107 mono-nuclear cells in 4ml human bone marrow, it can obtain 6.4×106~8.1×106 hMSCs after 12~14 days primary culture, 2.5×107~4.3×107 hMSCs after 6~8 days first passage culture, and time of six passage culture is 9~12 days.3. With Dexamethasone, β-Glycerphosphoric sodium, Vitamin C or rh-BMP2 or BMPS under optimal concentration, hMSCS may be expressed alkaline phosphatase,collagen I, collagen III and osteocalcin, and localized regions of mineralization is deteced.4. Positive regions of alkaline phosphatase is detected in any cells masses during first passage culture. Cells masses can form regions of mineralization while existing Dexamethasone, β-Glycerphosphoric sodium, Vitamin C, etc.5. Induced hMSCs show relatively slow proliferation, doubling time changes from 36 hours to 41 hours. After hMSCs were seeded, adhesive rate was 60% in 4 h, 92% in 24 h. Adhesive rate of induced hMSCs is little low.6. It is reasonable to produced the novel scaffold composites for tissue-engineering bone with poly-L-lactic acid (PLLA),calcium polyphosphate fiber (CPPf) and collagen I. Induced hMSCs can attach well with the novel scaffold composites and maintenance the phenotype of osteoblast.7. After tissue-engineering bone embed into subcutaneous part of nude mouse, lots of cells appear in the novel scaffold composites, collagen Ι andosteocalcin increase gradually, the scaffold composites degrade gradually and appear vascularization.Conclusion1. It can obtain lots of hMSCs that human bone marrow of iliac bone is separated by gradient centrifugation and then amplification culture.2. It is reasonable to use induced hMSCs and the novel scaffold composites with poly-L-lactic acid, calcium polyphosphate fiber and collagen I for construction tissue-engineering bone.3. The methods of osteogenic differentiation for hMSCs are similar to the methods for animals. The phenotype and ability of pr...
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