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目的探讨第3代软骨细胞在生物反应器中经载荷培养的软骨生成效应,为自体软骨移植临床应用提供实验依据。方法取3～4月龄西门塔尔小牛新鲜膝关节软骨,采用酶消化法分离培养软骨细胞。将第3代软骨细胞与多孔聚氨酯支架复合制备细胞-支架复合体。实验分为5组,分别为空载培养2周组(A组)、直接载荷培养2周组(B组)、空载培养4周组(C组)、直接载荷培养4周组(D组)、空载培养2周后载荷培养2周组(E组)。空载培养时将细胞-支架复合体置于培养箱中孵育;载荷培养是在生物反应器中模拟体内关节微环境,对细胞-支架复合体进行垂直加压和界面旋转的复合载荷运动。各组于各时间点取材,行糖胺聚糖(glycosaminoglycan,GAG)/DNA定量分析,实时定量PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖、软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)和表层蛋白(superficialzone protein,SZP)mRNA表达,并行甲苯胺蓝组织学观察和免疫组织化学染色观察。结果各组细胞-支架复合体分泌的DNA含量、GAG含量以及GAG/DNA比率比较差异均无统计学意义(P>0.05)。载荷培养后,大量GAG从支架释放至培养液中,且随载荷时间增加GAG释放相应增加(P<0.05)。Ⅰ型胶原mRNA相对表达量各组间比较差异均无统计学意义(P>0.05)。经不同载荷培养后,B组Ⅱ型胶原mRNA相对表达量显著高于A组(P<0.01),D、E组显著高于C组(P<0.01);D、E组蛋白聚糖mRNA相对表达量显著高于C组(P<0.01),E组显著高于D组(P<0.01);B组COMP mRNA相对表达量显著高于A组(P<0.01),E组显著高于C组(P<0.01);E组SZP mRNA相对表达量显著高于C、D组(P<0.05)。甲苯胺蓝染色和免疫组织化学染色示,载荷培养能增强软骨细胞合成分泌GAG;各组Ⅰ型胶原和Ⅱ型胶原免疫组织化学染色无变化,但D、E组表现较强的蛋白聚糖免疫染色增强作用。结论不同载荷均能促进以传代软骨细胞为基础的软骨再生,空载培养后载荷培养可能是最佳的软骨再生培养模式,但载荷诱导第3代软骨细胞的软骨生成效应弱化。
Objective To investigate the chondrogenic effect of the 3rd generation of chondrocytes cultured in a bioreactor under load to provide experimental evidence for the clinical application of autologous cartilage transplantation. Methods Fresh knee cartilage of Simmental calves aged 3 ~ 4 months was collected. Chondrocytes were isolated and cultured by enzyme digestion. The third generation of chondrocytes and porous polyurethane scaffold complex preparation of cell-scaffold complex. The experiment was divided into five groups: group A (no load), group B (group B), group C (no-load group C), group D ). After 2 weeks of no-load incubation, the cells were cultured for 2 weeks (group E). The cell-scaffold complex was incubated in an incubator under no-load culture. The load culture was performed in a bioreactor simulating the in vivo joint microenvironment, and the composite load movement of the cell-scaffold complex under vertical pressurization and interfacial rotation. At each time point, the rats in each group were drawn for GAG / DNA quantitative analysis. The expression of collagen type Ⅰ, type Ⅱ collagen, proteoglycan, cartilage oligomeric matrix protein COMP) and superficialzone protein (SZP) mRNA were detected by immunohistochemical staining and toluidine blue staining. Results The DNA content, GAG content and GAG / DNA ratio secreted by the cell-scaffold complex in each group showed no significant difference (P> 0.05). After loading culture, a large amount of GAG was released from the scaffold into the culture medium, and the release of GAG increased with the loading time (P <0.05). There was no significant difference in the relative expression of type Ⅰ collagen between groups (P> 0.05). The expression of type Ⅱ collagen mRNA in group B was significantly higher than that in group A (P <0.01), but significantly higher in group D and E than that in group C (P <0.01) The expression of COMP mRNA in group B was significantly higher than that in group C (P <0.01), while that in group E was significantly higher than that in group D (P <0.01) (P <0.01). The relative expression of SZP mRNA in group E was significantly higher than that in group C and D (P <0.05). Toluidine blue staining and immunohistochemical staining showed that the loading culture could enhance the synthesis and secretion of GAG in chondrocytes. There was no change in type Ⅰ collagen and type Ⅱ collagen in each group, but strong proteoglycan immunization in groups D and E Dyeing enhancement. Conclusion Different loadings can promote chondrocyte regeneration based on passaged chondrocytes. After no-load culture, loading culture may be the best model for cartilage regeneration, but chondrogenic effect induced by load-induced third generation chondrocytes is weakened.