动态培养环境下透明质酸与国产多孔钽复合对软骨细胞功能的影响

Effects of hyaluronic acid combined with domestic porous tantalum on chondrocyte function under the dynamic environment

背景:支架材料与生物因子在不同条件下复合培养对软骨细胞功能的影响,是组织工程材料研究的热点。目的:探讨动态环境下透明质酸与国产多孔钽复合对大鼠软骨细胞功能的影响。方法:分离培养SD大鼠膝关节软骨细胞,分5组培养:A组单纯细胞静态培养;B组接种于国产钽片上静态培养;C组接种于国产钽片上,加入含透明质酸的培养基,静态培养;D组接种于国产钽片上,置于动态转瓶中(双向交替正反转,旋转角度为40°,40 r/h)培养;E组接种于国产钽片上,加入含透明质酸的培养基,置于动态转瓶中(双向交替正反转,旋转角度为40°,40 r/h)培养,5组均连续培养7 d。扫描电镜下观察材料上的细胞形态,采用ELISA法检测细胞蛋白聚糖、Ⅱ型胶原、SOX9的分泌情况,Western Blot实验检测细胞蛋白聚糖、Ⅱ型胶原、SOX9蛋白的表达。结果与结论:①扫描电镜下可见,随着静态培养时间的延长,C组软骨细胞数量逐渐增多,并逐渐覆盖钽材料表面;②培养第5天,C组蛋白聚糖、Ⅱ型胶原分泌量均高于A、B组(P<0.05),D组SOX9分泌量低于E组(P<0.05),B组蛋白聚糖、Ⅱ型胶原分泌量低于D组(P<0.05),C组蛋白聚糖、Ⅱ型胶原分泌量低于E组(P<0.05);③培养第5天,C组Ⅱ型胶原蛋白表达量高于B组(P<0.05),D组蛋白聚糖、Ⅱ型胶原蛋白表达量高于B组(P<0.05),E组蛋白聚糖、Ⅱ型胶原蛋白表达量高于C组(P<0.05);④结果表明,国产多孔钽与透明质酸复合后可促进软骨细胞蛋白聚糖、Ⅱ型胶原的分泌和蛋白表达,动态环境培养下蛋白聚糖、Ⅱ型胶原的分泌及蛋白表达优于静态环境培养,SOX9的分泌及蛋白表达不受动、静态培养环境影响。

BACKGROUND:The effect of composite culture of scaffold materials and biological factors on chondrocyte function in different conditions is a hot topic in the research of tissue-engineered materials.OBJECTIVE:To investigate the effect of hyaluronic acid combined with domestic porous tantalum on the function of rat chondrocytes under the dynamic environment.METHODS:Knee chondrocytes of SD rats were extracted and cultured.Five groups of experiments were established as static culture group(group A),tantalum static group(group B),tantalum hyaluronic acid static group(group C),tantalum dynamic group(in the dynamic bottle rotation,two-way alternating forward and reverse rotation;the rotation angle is 40°,40 r/h;group D),and tantalum hyaluronic acid dynamic group(in the dynamic bottle rotation,two-way alternating forward and reverse rotation;the rotation angle is 40°,40 r/h;group E).The samples were continuously cultured for 7 days in the five groups.Cell morphology on materials was observed under a scanning electron microscope.The secretion of proteoglycan,type Ⅱ collagen and SOX9 was detected by ELISA.The expression levels of proteoglycan,type Ⅱ collagen and SOX9 protein were detected by western blot assay.RESULTS AND CONCLUSION:(1)Scanning electron microscope observation showed that with the prolongation of static culture time,the number of chondrocytes in group C gradually increased,and gradually covered the surface of tantalum material.(2)On the 5th day of culture,the secretion of proteoglycan and type Ⅱ collagen in group C was higher than that in groups A and B(P<0.05).The secretion of SOX9 in group D was lower than that in group E(P<0.05).The secretion of proteoglycan and type Ⅱ collagen in group B was lower than that in group D(P<0.05).The secretion of proteoglycan and type II collagen in group C was lower than that in group E(P<0.05).(3)On the 5th day of culture,the expression of type Ⅱ collagen in group C was higher than that in group B(P<0.05),and the expression of proteoglycan and type Ⅱ collagen in group D was higher than that in group B(P<0.05).The expression levels of proteoglycan and type Ⅱ collagen in group E were higher than those in group C(P<0.05).(4)These findings suggest that the combination of domestic porous tantalum and hyaluronic acid can promote the secretion and protein expression of proteoglycan and type Ⅱ collagen in chondrocytes.Under dynamic environment culture,the secretion and protein expression of proteoglycan and type Ⅱ collagen are better than in static culture.SOX9 secretion and protein expression are not affected by the dynamic and static culture environments.