三维培养条件下流体剪切力对人真皮成纤维细胞成骨分化的影响

Fluid shear stress affects osteogenic differentiation of human dermal fibroblasts in three-dimensional culture condition

目的通过准静态平面流场灌注系统,探讨不同流体剪切力对三维培养条件下人真皮成纤维细胞增殖和成骨分化的影响。方法将人真皮成纤维细胞按2×105/m L密度接种于胶原水凝胶支架中,同时设置低流速处理组(low flow velocity treatment group,LFV组)和高流速处理组(high flow velocity treatment group,HFV组)与三维静态培养的对照组(control group)进行比较。第3、6天和9天对各实验组进行HE染色并计数细胞;全程每隔3天测1次培养基中葡萄糖的消耗量;第21天行茜素红染色观察各组诱导矿化情况,同时采用FQ-PCR定量测定ALP和BMP-2基因的表达。结果 3组细胞均增殖明显,第6、9天LFV组细胞增殖最多,组间比较差异均有统计学意义(P<0.05);实验周期内各组培养基的葡萄糖含量均呈下降趋势,但组间比较差异无统计学意义;第21天茜素红染色3组均呈阳性,矿化结节面积大小统计显示HFV组最大,组间比较差异均有统计学意义(P<0.05);ALP和BMP-2基因表达量分别为LFV组:(1.780±0.233)/(2.788±0.241);HFV组:(2.262±0.306)/(4.576±0.253);对照组:(1.147±0.215)/(0.354±0.063),组间比较差异均有统计学意义(P<0.05)。结论流体剪切力能促进人真皮成纤维细胞的增殖和成骨分化,且流体剪切力较小时有利于细胞增殖,而较大时会在一定程度抑制细胞增殖。

Objective To determine the effect of different fluid shear stress on proliferation and osteogenic differentiation of human dermal fibroblasts in three-dimensional culture condition through a plane homogeneous flow field bioreactor. Methods Human dermal fibroblasts were pre-inoculated in collagen hydrogel scaffold with a density of 2 × 105/ m L and divided into 3 groups treated with different shear stress.The low flow velocity treatment group（ LFV group） was treated with fluid shear stress of 8 m Pa,the high flow velocity treatment group（ HFV group） was treated with fluid shear stress of 23 m Pa,and the control group was treated without any shear stress. HE staining and cell counting were performed on days 3,6 and 9. Glucose consumption in the medium was tested every 3 d. Alizarin Red S staining was used to measure mineralized nodule formation on day 21,and the expression of alkaline phosphatase（ ALP） and bone morphogenetic protein-2（ BMP-2） was determined by FQ-PCR. Results HE staining and cell counting assays showed that the cell proliferations were significant in all 3 groups,especially in the LFV group on days 6 and 9（ P 0. 05）. The glucose consumption of the 3 groups were all decreased along with time but showed no significant difference among them. On day 21,Alizarin red staining showed that the 3 groups were all positive,and the HFV group had the largest mineralized nodule area（ P 0. 05）. The expression level of ALP and BMP-2 were1. 780 ± 0. 233 and 2. 788 ± 0. 241 in the LFV group,2. 262 ± 0. 306 and 4. 576 ± 0. 253 in the HFV group,and 1. 147 ± 0. 215 and 0. 354 ± 0. 063 in the control group,respectively（ P 0. 05）. Conclusion Fluid shear stress promotes the proliferation and osteogenic differentiation of human dermal fibroblasts,and the cell proliferation is strengthened with smaller fluid shear stress and inhibited with larger shear stress.