Fluid Shear Stresses Affected The Osteogenic Differentiation Of Human Dermal Fibroblasts In Three-dimensional Culture Condition

Objective Designed a new plane homogeneous flow flied bioreactor independent to research the effects of different fluid shear stresses on proliferation and osteogenic differentiation of human dermal fibroblasts in three-dimensional culture condition.Methods To design and construct a new plane homogeneous flow flied bioreactor and carry on the theory with CFD analysis.Human dermal fibroblasts were isolationed and characterized by vimentin staining,then pre-inoculated in collagen hydrogel scaffold with a density of 2×105/mL and separated into three groups treated with different shear forces,low flow velocity treatment group(LFV group)which was treated with a fluid shear stresses of 8mPa,high flow velocity treatment group(HFV group)which was treated with 23 mPa and control group which was treated without any shear stresses.HE staining and cell counting were performed at 3,6d and 9d.Hochest33342/PI staining were performed at 3,9,15 and 21 d.Glucose consumption in the medium were tested every 3 days.Alizarin Red S staining was used to measure mineralized nodule formation at 21 d and the ALP and BMP-2 gene expression levels also were determined by FQ-PCR.Results HE staining and cell counting assays showed the cell proliferations were significant in all three groups,and the largest degree of proliferation was in LFV group at 6d and 9d(versus the other two groups,P<0.05).Each group of cell activity ratio were decreased dured the experiment,HFV group decreased the most(versus the other two groups,P<0.05).The glucose consumption in the three groups all decreased with time and showed no significant difference between each other(P>0.05).At the 21 d,Alizarin red staining in three groups were positive and the results of mineralization area calculation by Image Pro Plus 6.0 software displayed the largest degree of mineralization area was in HFV group(versus the other two groups,P<0.05).ALP and BMP-2 gene expression assays showed clear tangles in all three groups and the most significant tendency was detected in HFV group(versus the other two groups,P<0.05),and the ALP and BMP-2 gene expression levels were: LFV group: 1.780 ± 0.233 / 2.788 ± 0.241;HFV group: 2.262 ± 0.306 / 4.576 ± 0.253;control group: 1.147 ± 0.215 / 0.354 ± 0.0631,.Conclusion Our results revealed that fluid shear stress promoted the proliferation and osteogenic differentiation of human dermal fibroblasts,while the cell proliferation effect was strengthened with smaller fluid shear force and inhibited with larger.