| Objective To compare proliferation and osteogenic differentiation ability between cultures of hMSCs obtained from the different age donor in vitro and bone formation in nude mice.Methods HMSCs cultures were obtained by using density centrifuge method from 39 donors between 7 and 79 years of age, these donors were divided into four different groups: younger than 20 year old; 20 year old to 39 year old; 40 year old to 60 year old; elder than 60 year old. The adhesive cells were preserved to passage culture, 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. The first passaged non-induced cell cycle of hMSCs were investigated by flow cytometer.We detected the cellular growth at the different time point by MTT colorimetry, drawed the growth curves of the cells and calculated the cellular doubling time. The induced cells' activity of alkaline phosphatease(ALP) and the density of osteocalcin at diffrent time point were investigated. Immunodeficient 6-week-old nude mice were used as subcutaneous transplant recipients. At each timepoint the nude mice photographed and the three-dimensional (3D) cultures in nude mice were explanted, fixed, included, sectioned, and analyzed by histochemistry and immunohistochemistry.Results The total culture time was positively increased with donor age. The cell's form of young donors was more regular than aged donors. We observed more colony forming units of primary passage from young donors than aged donors. Under the TEM, there was no salient difference of any different age donor, we found that the non-induced cell were immature and the included cells were maturer comparatively, the ratio of included cells' N/P became lesser and the Endoplasmic reticulum a little dilatated. All the staining of induced cell were positive.The growth rates of hMSCs from aged donors were less than the growth rates of hMSCs from young donors. The cellular doubling time of each group was 32.3h, 34.2h, 37.4h, 39.8h. Flow cytometer was used to measure the cell cycle: Cell ratio in G0+G1(%) of <20 was less than other groups, cell ratio in S+G2+M (%) of <20 was more than other groups.Furthermore, included hMSCs from aged donors showed lower ALPase activity and osteocalcin concentration than included hMSCs from young donors. we noticed that the concentration of ALP increased strikingly after 4 day's and reached its peak on the 8th to 12th day, descended slightly on the 14th day. Mineralized and vascularized in the explants of 3D cultures after 4,8,12 weeks. The development of transplant in nude mice on X-ray photograph of young donor' exponent express higher density than aged donor' exponent.HMSCs of younger donors showed a higher differentiation and mineralization capability in nude mice than older donors.Conclusion The proliferation and osteogenic differatiation capability of hMSCs decreases with age. Our study has highlighted the significant effect of donor age on hMSCs quality. These findings have significant implications for the proposed clinical application of hMSCs.
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