| Bone defects which caused by different reasons are very common clinically. The demand for the bone repair materials is so large that no matter the patient's bone or the artificial substitudes can not satisfy the clinical demands owing to their inherent deficiencies.Hopefully,we can make sufficient ideal bone-repair-materials by the technique of tissue engineering which reveals good application prospects clinically.The mesenchymal stem cells (MSCs )in bone marrow are believed to be one of the best choices of seedling cells of bone tissue engineering for their strong potential and high activity of osteogenesis. Harvest enough seedling cells with superior quality and induce them to differentiate into osteoblasts efficiently play an important role in the researching works of bone tissue engineering.It has been verified that the Pulsed Electromagnetic Fields (PEMFs) clinical applications on the treatment of bone fractures,bone nonunions, delayed healing of the bone and pseudarthrosis is successfully. PEMFs with specific frequencies and field intensities could provoke the proliferation and differentiation of osteoblasts..increase the local growth factors to accelerate the mineralization of the bone matrix. However, the articles on biological effects of PEMFs on MSCs have not been reported until now. Yet the MSCs in the bone marrow is one of the important kinds of pre-ancestral cells in the bone repairing. So we presume that PEMFs' stimulation may result in a series of biological effects in hMSCs including promoting bone's formation. This research observed the process of PEMFs' acting on hMSCs' multiplication in vitro and the competence of PEMF's induction on the hMSCs to be osteoblasts. In this study new technique of obtaining top-quality MSCs clinically in vitro in a short time are provided and the main outcomes are as follows:1. Studies on the PEMFs' influence on the proliferation and the cellular cycle on hMSCs.The 3rd generation of hMSCs are devided into 4 groups: G1: The routine culture group; G2: The routine culture combine with PEMFs (12Hz,1.1mT,8h/d)group;G3: The routine culture combine with chemical inducers(desamethasone,β-Sodium Glycerophosphate,acidum ascorbicum)group;G4: The routine culture combine with PEMFs (12Hz,1.1mT,8h/d) and chemical inducers(desamethasone,β-Sodium Glycerophosphate, acidum ascorbicum)group.We detected the cellular growth by MTT colorimetry, drawed the growth curves of the cells and calculated the cellular doubling time. Flow cytometer was used to measure the cell cycle. Results: Cell proliferation in G1 is faster than that in G3,and we find that cell proliferation in G2 is improved a lot from the fact that the growth curve's antedisplacement compared with the controlled group.The cellular doubling time in G2 is 29.4h;in G3 is 36.2h;in G1 is 33.6h;and in G4 is 34.8h.The cells ratio in S period are as follows respectively:G1:(8.56+/-1.13)%,G2:(20.15+/-2.10)%(after 5d's stimulation by PEMFs ),G3:(5.91+/-0.43)%,G4:(7.73+/-0.96)%。Conclusion: From above ,We can see that an important aspect of PEMFs's biological effect on hMSCs is that it can affect cell proliferation and cellular cycle.2. Observations on the cell's differentiation from hMSCs to osteoblasts due to the induction of PEMFs.The 3rd generation of hMSCs are cultured in the routine subculturing medium with PEMFs (12Hz,1.1mT,8h/d).The cell's form and the ultrastructure were observed seperately by inverted microscope and transmission electronic microscope. The ALP Staining, Osteocalcin immune-histochemical staining, Collagen type-I immune-histochemical staining, Calcium staining for certain dyestuff, Von Kossa calcium staining, Tetracycline fluorescence labelling, Estimating the excretion of osteocalcin by radioimmunoassay and the activity of the cellular ALP were carried out separately.Results: The subcultured hMSCs after about 4 to 5 day's process by PEMFs changed their morphology. They aggregated into many calcification nodes something like a multi-storey structure after two weeks. Under the TEM we found that the ce... |
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