| In this study, 3 new biomaterials of different composition were fabricated: nHA/PCL1,nHA/PCL2and PCL. The effect of these nHA/PCL materials on osteoblast proliferation, cell cycle, alkaline phosphatase ctivity,especially bone-related gene (ALP,BSP,Col I,OC,ON,OP ) expression were explored to provide valuable molecular biological information for the development of these materials.In this study, primary osteoblasts from calvaria of 2-3 days old mice were isolated and purified. Result showed that purified primary osteoblasts were observed to be in proper morphology and their alkaline phosphatase activity is maintained. Alkaline phosphatase activity in primary mouse osteoblast steadily increased in the first 6 days .These osteoblasts were good for further application in this study.An established osteoblast-like cell line MC3T3-E1 was the most suitable to obtain reproducible data in thestudy of the cell cycle. MTT assay was used to detect osteoblast proliferation rate on nHA/PCL composite materials. Result demonstrated that osteoblast proliferation rate on nHA/PCL1 and nHA/PCL2 composites extracts was higher than PCL, which suggests that nHA in nHA/PCL composites can enhance osteoblast proliferation rate. In the same time, Flow Cytometry (FCM) was applied to detect effects of nHA/PCL composites extrasts on cell cycle of osteoblast MC3T3-E1 at 24h, 48h and 72h. Results demonstrated that osteoblast PI (proliferative index) on nHA/PCL composites extrasts (PI) was lower than that of control(cuiture in DMEM medium), while that of nHA/PCL1 and nHA/PCL2 composites extrasts PI was higher than that on the PCL, which also suggests that nHA in the nHA/PCL composites can enhance osteoblast proliferation activity.This study also revealed that alkaline phosphatase activity in primary mouse calvaria osteoblast steadily increased in all materials extrasts in the first 6 days and kept at a high level. After that, grown in nHA/PCLcomposites extracts has a markable change. it decreased with culture time, which suggests that osteoblasts developed into a more matured phenotype. Especiliiy alkaline phosphatase activity of osteoblasts which grown in nHA/PCL2 composites extracts has a more change than others, This suggested that increased nHA can markable mighty alkaline phosphatase activity.In this study, gene expression of osteoblasts was evaluated at 2, 6 and 12 days by using semi-quantitative reverse transcriptional PCR (SemiRT-PCR). Results demonstrated that OP and ON mRNA decreased with culture time on all materials. Compare to OP and ON expression on PCL, OP and ON expression in osteoblasts grown on the nHA/PCL1 and nHA/PCL2 composites decreased quicker with culture time. OC mRNA expression in MC3T3-E1 increased with culture time on all material surfaces, but its expression on nHA/PCL1 and nHA/PCL2 composites increased quicker than that on PCL with culture time. This suggests that nHA component in the composites may affect the bone-related gene express of osteoblast.In this study, the effect of nHA/PCL with different phase composites on osteoblast prolifition, cell cycle, ALP activity, and bone-related gene expression was studied. Result showed that nHA/PCL2 composite is a potential bone substitute. By optimizing their phase composition and structure, a novel nHA/PCL composite may be developed to be used in clinics.
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