脱细胞跟腱复合人成纤维细胞共培养的实验研究

EXPERIMENTAL STUDY ON CO-CULTURE OF HUMAN FIBROBLASTS ON DECELLULARIZED Achilles TENDON

目的探讨脱细胞跟腱的制备及其与人成纤维细胞复合培养效果,为组织工程韧带的构建提供一种支架材料。方法取10只5月龄雄性新西兰大白兔(体重4～5 kg)双后肢跟腱,经胰蛋白酶、Triton X-100、SDS脱细胞处理后,行大体、组织学及扫描电镜观察。将脱细胞跟腱与人成纤维细胞复合培养,于培养1、3、5、7、9 d行细胞相容性检测,并于4周后取材行组织学、扫描电镜观察以及生物力学检测,并与脱细胞前、后跟腱进行比较。结果跟腱经脱细胞处理后腱膜完整,质柔软、韧性好,体积较处理前跟腱显著增大;跟腱腱束间未见疏松结缔组织及腱细胞,胶原纤维排列疏松,呈三维网状分布,腱束间残留较多孔隙;且细胞相容性良好。复合培养4周后,细胞迁移至纤维束间的孔隙内,三维网状结构消失。生物力学测试示,与脱细胞前跟腱组及细胞-支架复合组比较,脱细胞跟腱组拉伸强度及杨氏弹性模量均显著降低(P<0.05),细胞-支架复合组与脱细胞前跟腱组比较差异无统计学意义(P>0.05);各组间断裂伸长率比较,差异均无统计学意义(P>0.05)。结论兔脱细胞跟腱与人成纤维细胞生物相容性良好,可作为支架材料用于组织工程韧带构建。

Objective To investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Methods Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits （5-month-old; weighing, 4-5 kg）. The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate （SDS）, and then gross observation, histological examination, and scanning electron microscope （SEM） observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. ResultsAfter decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young’s elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group （P 〈 0.05）, but no significant difference was found between normal Achilles tendons group and cell-scaffold composite group （P 〉 0.05）. There was no significant difference in elongation at break among 3 groups （P 〉 0.05）. ConclusionThe decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.