Study Of Erythropoietin’s Effect On Bone Formation

Bone defect is a common disease which is induced by trauma, tumor,genetic factors and so on. Presently, the main treatments of bone defect areAutogenous Bone Transplantation, Allogenic Bone Transplantation, TissueEngineering Technology, Gene Therapy and Adjunctive Therapy of GrowthFactor. Because of the limitation of different method and the inconsistency oftreatment, the treatment of bone defect serves as a difficult problem which isurgently to be solved. EPO, a drug for anemia, also exerts multiple non-hematopoietic functions, such as anti-inflammation, anti-apoptosis and tissueprotection. The latest research shows that EPO also stimulates bone healing,but the mechanism is unclear, considered as the following two aspects: On theone hand, EPO promotes bone healing directly by acting on both osteoblastsand osteoclasts; On the other hand, EPO, acting as an anti-inflammatory,anti-apoptotic and tissue protective factor, can alleviate inflammation andprotect the cells linked to bone formation and bone healing, which advances theprocess of bone healing indirectly.Objective: To investigate the function and mechanism of EPO of boneformation by the research of EPO’s effect on osteoblasts and bone mesen-chymal stem cells (MSCs). Meanwhile, to show the mechanism of EPO’s effecton bone healing indirectly, so as to provide evidence for bone defect repair.Method: C3H10T1/2and ROS cell lines were used in this study. Thereare four groups in the experiment: DMEM/10%FBS,100U/L penicilin，100U/L streptomycin were added into C3H10T1/2group and ROS group,DMEM/10%FBS,50μmol/L vitamin C，10mmol/L β-glycerophosphate，100U/L penicilin，100U/L streptomycin were added into another two C3H10T1/2and ROS group. On this basis, the medium was replaced every day till the cells overgrow80%-90%of cell culture hole,then different concentrations of EPO(0.3,3,30,100,300U/L) were added into each group. C3H10T1/2and ROS cellin four groups were detected by morphological observation and ALP stainingor Von-kossa staining at the2nd,4th and8th day.Result: Morphological observation of osteoblast: As soon as the passagehave been done, we observed that the ROS cells were spherical due to theywere not adhered to the bottom of plate.After one day culture, the cells whichhave been adhered to the bottom of plate were spindle-shaped or triangle-shaped and have many processes. ROS cells overgrew the bottom of plate aftertwo days, then administrating EPO. After four days, we can observed the cellfusion and cobblestone-like by overlapping, a part of cells aggregated mutuallyand formed opaque mineralized nodules. The number of mineralized nodulesare increased and cell growth inhibition appeared at the8th day which led to anumber of cells died.Morphological observation of MSCs: We observed that the C3H10T1/2cells were spherical after the passage due to they were not adhered to thebottom of plate.After one day culture, the cells which have been adhered to thebottom of plate were round-shaped、spindle-shaped or polygon-shaped, andthe ration of nucleus and plasma increased apparently. C3H10T1/2cellsovergrew the bottom of plate after three days, then administrating EPO. Afterfour days, closely arranged cells and triangular cells were found undermicroscope, but didn’t find mineralized nodules. The proportion of triangularcells were increased and cell growth inhibition appeared at the8th day whichled to a number of cells died, mineralized nodules were not found.ALP staining: After administration of the first two days，there was nosignificant difference between the general group of ROS and ROS different-iation group, which is the same between the different concentrations of EPOgroups and control group. The same results appear in C3H10T1/2cells. Von-kossa mineralized nodules staining: In the4th day and8th day afterthe administration of EPO, the Von-kossa staining was taken to ROScells(differentiation group) and C3H10T1/2cells(differentiation group). Thefourth day, after the administration of EPO, No mineralized nodules in neitherROS group nor C3H10T1/2group were stained. At the8th day, mineralizednodules were stained in ROS group, but there was no difference between anyconcentrations of EPO groups and control group. C3H10T1/2group stillshowed no stained mineralized nodules.Conclusion: EPO has no significant effect on proliferation, differentiationand maturation of osteoblasts and MSCs. The mechanism of EPO’s stimula-ting bone healing needs further study.