生理水平流体剪应力对三维多孔支架中成骨细胞力学敏感性及黏附、分化的影响

Effects of shear stress at physiological level on mechanical sensitivity, adhesion and differentiation of MC3T3-E1 cells in three-dimensional porous scaffold

目的 构建可以达到生理剪应力水平的三维流动模型，研究流体剪应力对成骨细胞黏附、分化及力学敏感性的影响。方法 利用灌注式流动腔对生长在β-磷酸三钙（β-TCP）多孔支架内的MC3T3-E1成骨样细胞施加不同强度的流体剪应力6 h，比较加载组和静态组的细胞活性表征细胞黏附；一氧化氮（NO）和碱性磷酸酶（ALP）表征力学敏感性和细胞分化。采用流固非线性耦合的数值计算，获得支架内各流量下的剪应力分布。结果 平均剪应力小于0.4 Pa，细胞的黏附率为74% - 81%；0.41 Pa时，黏附率为60.22%。NO的产生率在加载后5 min达到最大，15 min显著降低，30 min后产生率趋近于0。在0.232 - 0.304 Pa平均剪应力强度范围，ALP水平随着剪应力的升高显著增强（P〈0.01）；而在0.304 - 0.412 Pa范围，剪应力增加对ALP水平的改变无显著影响（P〉0.05）。结论 生理水平剪应力条件下，支架内大部分细胞可以维持正常黏附。三维条件下细胞力学敏感性与剪应力变化率成正比，与二维条件的规律相同。支架内平均剪应力小于0.304 Pa，剪应力显著促进细胞分化；大于这一剪应力，细胞分化水平不再明显变化。该研究有望加快骨组织工程的实现。

Objective To construct the three-dimensional （3D） fluid model at the physiological level of shear stresses and study the effects of fluid shear stress （FSS） on adhesion, differentiation and mechanical sensitivity of osteoblasts. Methods The MC3T3-E1 osteoblasts cultured on β-tricalcium phosphate （β-TCP） scaffolds were subjected to various FSSs in the perfusion flow chamber for 6 hours to compare cell adhesion in FSS-loading groups and control group. Nitric oxide （NO） and alkaline phosphatase （ALP） were detected to compare mechanical sensitivity and cell differentiation. The FSS magnitude and distributions corresponding to various fluid rates were calculated with nonlinear fluid-structure coupling analysis. Results Cell adhesion rate was up to 74%-81% when the average FSS magnitude was lower than 0.4 Pa, but reduced to 60.22% when the average FSS was 0.41 Pa. The NO production rate reached the maximal concentration after loading for 5 min, then significantly reduced at 15 min, and gradually diminished to none at 30 min. ALP level significantly increased （P〈0.01） at the shear stress range of 0.232 - 0.304 Pa, but maintained at the range of 0.304 - 0.412 Pa （P〉0.05） with the increase of shear stress. Conclusions Majority of the cells kept a normal adherence to the scaffold at the physiological level of shear stresses. The mechanical sensitivity of the cells under 3D condition was dependent on the FSS rate, which was consistent with two-dimensional （2D） condition. When the average FSS was lower than 0.304 Pa in the scaffold, FSS could significantly promote cell differentiation, but no significant change in cell differentiation could be found when FSS was higher than 0.304 Pa. The present study is expected to accelerate the realization of bone tissue engineering.