透明质酸促进人间充质干细胞复合磷酸三钙-胶原材料体外成软骨研究

Hyaluronan promote chondrogenesis of human mesenchymal stem cell in tricalcium phosphate-collagen scaffolds

目的 观察透明质酸对入骨髓间充质干细胞（hBMSCs）复合磷酸三钙-胶原材料体外构建组织工程软骨的影响.方法 将提取扩增的第3代hBMSCs体外与组织工程支架磷酸三钙-胶原复合,分两组进行三维培养：对照组和实验组（对照组培养液基础上加入100 mg/L透明质酸）.培养2周后进行苏木素-伊红（HE）染色、阿尔新蓝染色、Ⅱ型胶原（COL2）免疫组织化学染色,实时定量逆转录聚合酶链反应（RT-PCR）和糖胺多糖（GAG）定量检测,比较体外成软骨效果.结果 HE染色结果显示骨髓间充质干细胞能够均匀分布于材料中,实验组细胞基质的分泌大于对照组.实验组阿尔新蓝染色,COL2免疫组织化学染色,COL2、软骨特异性基SOX9、聚集蛋白聚糖（Aggrecan）的基因表达分别为8.954±2.612/1.062±0.459、3.512 ±0.638/1.111 ±0.595、4.588±1.964/1.042±0.335,糖胺多糖（GAG）定量检测结果（21.741±2.633/16.717±0.595）也都显著高于对照组,表明100 mg/L透明质酸能够有效促进hBMSCs复合磷酸三钙-胶原体外成软骨.在COL10和COL1的基因表达上实验组也大于对照组（21.741 ±2.633/16.717 ±0.595）,说明透明质酸除了能体外诱导hBMSCs成软骨分化外,还能进一步诱导其成熟发展.结论 100 mg/L透明质酸能够在三维组织工程支架中促进hBMSCs体外成软骨分化,并且能够进一步诱导其成熟发展.

Objective To investigate the effect of hyaluronan （HA） on engineered cartilage formation.Methods Human mesenchymal stem cell （hBMSCs） were isolated by density gradient centrifugation and were seeded onto tricalcium phosphate-collagen （TCP-COL） scaffolds.hBMSCs were cultured on the scaffold for 2 weeks in two groups：（1） control group ; （2） HA group （control group culture medium with 100 mg/L HA）.The effect of hyaluronan on chondrogenesis was assessed by hematoxylin-eosin （HE） staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,Real-time polymerase chain reaction （RT-PCR） and glycosaminoglycans （GAG） quantification assay.Results The data of hematoxylin-eosin （HE） staining,alcian blue staining,anti type Ⅱ collagen immunohistochemical staining,RT-PCR [Collagen Ⅱ （COL2） （P 〈 0.01）,SRY-related high mobility group-box gene9 （SOX9） （P 〈 0.01）,Aggrecan （P 〈 0.05）] （8.954 ± 2.612/1.062 ± 0.459,3.512 ± 0.638/1.111 ± 0.595 and 4.588 ± 1.964/1.042 ± 0.335,respectively） and GAG quantification assay （21.741 ± 2.633/16.717 ± 0.595） （P 〈 0.05） shows HA （100 g/L） could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffold.In addition,in the HA group,a significant up-regulation of COL1 （P〈0.05） （3.258 ±1.038/1.127 ±0.678） andCOL10 （P〈0.05） （5.961 ±2.598/1.080 ±0.458） gene were detected compared with the control group,indicating that HA may also promote robust maturation of tissue-engineered cartilage.Conclusion HA not only could significantly induce the chondrogenic differentiation of hBMSCs on the TCP-COL scaffolds,but also could promote maturation of the tissue-engineered cartilage.