The Effects Of Simvastatin On Proliferation And Osteogenic Differentiation Of Human Fracture Hematoma Cells In Vitro

Objective:1.Isolation,culture and phenotype identification of human fracture hematoma cells(hFHCs)2.To observe the effects of Simvastatin on proliferation and osteogenic differentiation of human fracture hematoma cells(hFHCs) in vitro.3.To study the possible mechanisms of Simvastatin on bone formation by human fracture hematoma cells.Methods:1.Expose the fracture site,harvest the organized haematoma and isolate human fracture haematoma by tissue culture.2.The primary hFHCs were cultured in ordinaryα-MEM containing 10% heat-inactivated fetal bovine serum (FBS),2mmol/L L-glutamine and antibiotics (penicillin G,100 units/ml and streptomycin 100μg/ml) in 37℃,5% CO2.Passage it in vitro until the culture dish was full.3.Identification of hFHCs:(1)Morphology observation:Cell morphology observed with phase-contrast microscope. (2)Flow cytometry tests CD29, CD14, CD34, CD45 and CD133.4.Experimental group:hFHCs were respectively treated with different concentrations of Simvastatin:A group (0 mol/L),B group (1.0×10-9mol/L), C group (1.0×10-8mol/L), D group (1.0×10-7mol/L),E group(1×10-6mol/L).5.Osteogenesis induce:hFHCs were cultured in an osteogenic medium consisting of the original medium, plus 10 nmol/L dexamethasone (Dex),10 mmol/Lβ-glycerophosphate and 50μg/ml ascorbic acid. At the same time hFHCs were respectively treated with different concentrations of Simvastatin (A group (0 mol/L), B group(1.0×10-9mol/L), C group(1.0×10-8mol/L), D group(1.0×10-7mol/L), E group(1.0×10-6mol/L)).6.The proliferation of hFHCs was evaluated by MTT assay.7.The alkaline phosphatase (ALP) activity was measured.8.Statistical analysis:Data are presented as x±s. All statistical analysis was performed with PASW Statistics 18.0. The differences among the groups were determined by One-way ANOVE. Differences were considered significant at a value of P<0.05.Results:1.hFHCs were isolated and cultured. The characteristics of hFHCs:cells appearance presented fibroblasts-like. It is up to 90% confluence about 8 days. The primary culture of adherent hFHCs showed the formation of colonies of fibroblast-like cells. Passage cells were attached after 2 to 4 hours, spread and restored to fibroblast-like.It took about 24 hours to adhere completely. The cells uniformly grew and show no colony.After 5 to 7 days,the disk was full at the bottom. There was no contact inhibitation. The size of the third passage was uniform and fibroblast-like.After 5 to 7 days, the disk was full at the bottom and they were ordered swirlingly.2.Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells.3.MTT:As the concentration increases, the promotion of hFHCs proliferation gradually increased, and its maximal effect concentration is D group (1.0×10-7mol/L). compared with A group,proliferation of hFHCs of B group was increased by Simvastatin,but the difference is not significant(P>0.05); compared with A group and B group,treatment of cells with high concentration of Simvastatin (1.0×10-8, 1.0×10-7,1.0×10-6mol/L) led to a significant increase in proliferation(P＜0.05); the difference between C group and E group is not significant(P>0.05); compared with C group and E group, proliferation of hFHCs of D group was significantly increased by Simvastatin (P<0.05).4.ALP activity:As the concentration increases, the promotion of hFHCs ALP activity gradually increased, and its maximal effect concentration is D group (1.0×10-7mol/L). compared with A group, ALP activity of hFHCs of B group was increased by Simvastatin, but the difference is not significant(P>0.05); compared with A group and B group,treatment of cells with high concentration of Simvastatin (1.0×10-8, 1.0×10-7,1.0×10-6moI/L) led to a significant increase in ALP activity (P<0.05); the difference between C group and E group is not significant(P>0.05); compared with C group and E group, ALP activity of hFHCs of D group was significantly increased by Simvastatin (P<0.05)Conclusion1.hFHCs were isolated from orgnized haematoma and cultured.The lineage is useful for many pharmacologic experiments. It is a new experimental lineage model for studying local human fracture2.The modest concentration of Simvastatin may increases the proliferation and ALP activity of hFHCs.This is maybe the mechanism of promoting bone formation.