适宜组织工程化骨构建的流体剪切力和物质转运速度的研究

目的 探讨适宜组织工程化骨构建的流体剪切力和物质转运速度.方法 对接种人骨髓间质干细胞的多孔β-磷酸三钙支架进行灌注培养.灌流量相同时,对支架上的细胞分别施加1×、2×及3×的流体剪切力;流体剪切力相同时,分别给予3ml/min、6 ml/min和9 ml/min的灌注.细胞增殖采用MTT法,通过检测碱性磷酸酶(alkalilinphosphatase,AKP)活性、骨桥蛋白(osteopontin,OP)、骨钙素(osteocalcin,OC)分泌及细胞外基质的矿化评价组织工程化骨的构建.通过计算流体动力学得出流体剪切力和物质转运速度.结果 灌流量相同时,剪切力2×组细胞增殖活性最高;2×和3×组AKP活性及OC分泌高于1×组;第28天时,3×组矿化基质最多.流体剪切力相同时,6 ml/min组AKP活性最高;第28天时,3 ml/min组OC分泌最高,矿化基质最多.灌流量相同时,流体剪切力分别为0.004～0.007 Pa、0.009～0.013 Pa和0.013～0.018 Pa;流体剪切力相同时,物质转运速度分别为0.267～0.384 mm/s、0.521～0.765mm/s和0.765～1.177 mm/s.结论 利用接种人骨髓间质干细胞的β-磷酸三钙支架构建组织工程化骨时,0.013～0.018 Pa的流体剪切力及0.267～0.384 mm/s的物质转运速度是适宜的.

Abstract：

Objective To explore the optimum flow shear stress and mass transport for the construction of tissue-engineered bone.Methods The β-tricalcium phosphate (β-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (HBMMSCs) were cultured in perfusion bioreactor.When the same flow rate was applied,the flow shear stress was separately 1×,2× and 3×.When the same flow shear stress was applied,the flow rates were separately 3 ml/min,6 ml/min and 9 ml/min.Cell proliferation was measured by MTT method.The construction of tissue-engineered bone was evaluated by measuring alkaline phosphatase (AKP) activity,secretion of osteopontin (OP) and osteocalcin (OC),and the mineralization of extracellular matrix (ECM).The flow shear stress and the mass transport were obtained using computational fluid dynamics.Results When the flow rate was same,the most cell proliferation was found in 2× group.The AKP activity and secretion of OC was higher in 2× and 3× groups than in those in 1× group.After 28days,the highest amount of mineralization of ECM was found in 3× group.When the flow shear stress was same,the AKP activity was highest in 6 ml/min group.After 28 days,secretion of OC and formation of mineralized ECM was highest in 3 ml/min group.When the flow rate was same,the flow shear stress was separately 0.004-0.007 Pa,0.009-0.013 Pa and 0.013-0.018 Pa.When the flow shear stress was same,the flow rate was separately 0.267-0.384 mm/s,0.521-0.765 mm/s and 0.765-1.177 mm/s.Conclusion When the tissue-engineered bone was constructed,0.013-0.018 Pa flow shear stress and 0.267-0.384 mm/s mass transport velocity could improve the construction of the tissue-engineered bone in vitro.     

灌注式生物反应器中流体剪切力对大段组织工程化骨构建的作用

目的　结合流体力学模型研究灌注式生物反应器中大段组织工程化骨的构建与多孔支架内流体剪切力的关系。方法　利用灌注式生物反应器对复合骨髓基质干细胞的多孔磷酸三钙支架进行灌注培养。培养基的黏度分别为1.12mPa?s, 2.23mPa?s及3.35mPa?s。通过细胞增殖、成骨分化及组织形态学评价组织工程化骨的构建,建立流体力学模型,求解支架内的流体剪切力。结果　培养基黏度2.23mPa?s组,细胞增殖高于其他组。培养基黏度2.23mPa?s及3.35mPa?s组第28d的碱性磷酸酶活性及第7d后的骨钙素分泌高于1.12mPa?s组。培养基黏度越高,骨桥蛋白的分泌高峰出现越早。28d后,黏度3.35mPa?s组的钙化基质最多。流体力学模型分析,培养基黏度1.12mPa?s, 2.23mPa?s及3.35mPa?s组中,支架内的平均流体剪切力分别为5mPa, 11mPa和15mPa。结论　在利用复合人骨髓基质干细胞的多孔磷酸三钙构建大段组织工程化骨的过程中,15mPa的流体剪切力最有利于组织工程化骨的构建。