不同脱细胞方法对猪尾纤维环生物力学特性及组织结构的影响

Effects of different decellularization methods on biomechanical properties and histological structure of annulus fibrosus in pigtail

目的探讨不同脱细胞处理方法对猪尾椎间盘纤维环生物力学特性及组织结构的影响,为构建组织工程纤维环提供实验依据。方法取新鲜猪尾纤维环60个,随机分为4组,每组15个。Triton X-100组(A组):先将纤维环放入Tris-HCl低渗缓冲液中振荡48 h,再用TritonX-100、DNaseⅠ和RNase A对纤维环脱细胞处理;SDS组(B组):将纤维环冻融3次,接着用SDS、DNaseⅠ和RNase A对纤维环脱细胞处理;胰蛋白酶组(C组):用含胰蛋白酶、DNaseⅠ和RNase A的Tris缓冲液对纤维环脱细胞处理;对照组(D组):纤维环不做任何处理。采用HE染色与扫描电镜方法,观察各组脱细胞情况以及纤维环超微结构的变化;采用生物化学和生物力学方法检测各组纤维环的胶原含量、氨基葡聚糖(GAG)含量及力学参数。结果与对照组比较,HE染色及扫描电镜可见A、B、C 3组均无细胞残留;A组纤维环超微结构未见破坏,C组纤维环超微结构可见轻度破坏,B组纤维环超微结构可见明显破坏。A、B、C 3组纤维环胶原含量与对照组比较无统计学差异(P>0.05),但GAG含量均明显低于对照组(P<0.05)。A、C两组的力学参数(极限载荷、极限应力、韧度、弹性模量、断裂功耗)与对照组无统计学差异(P>0.05),B组上述力学参数均低于对照组(P<0.05)。结论 Triton X-100组处理后的猪尾椎间盘纤维环无细胞残留,结构无破坏,基质成分及力学性能保存良好,适合于构建组织工程纤维环。

Objective To investigate effects of different decellularization methods on biomechanical properties and histological structure of annulus fibrosus in pigtails and provide experimental evidence for the construction of tissue engineering annulus fibrosus. Methods Sixty fresh annulus fibrosus were dissected from caudal disks of pigs and randomly assigned to 4 groups with 15 in each group. Triton X-100 group（Group A） ： annulus fibrosus were treated with hypotonic Tris-HCI buffer for 48 hours and de-cellularized with Triton X-100, DNase I and RNase A. SDS group （Group B） ： annulus fibrosus were subjected to 3 cycles of freeze-thaw and subsequently de-cellu- larized with SDS, DNase [ and RNase A. Trypsin group （Group C） ~ annulus fibrosus were de-cellularized with Tris buffer containing trypsin, DNase I and RNase A. Control group： fresh annulus fibrosus underwent no treat- ment. After the decellularization process was completed, hematoxylin-eosin （HE） staining was conducted to ex-amine the efficacy on cell removal, and the ultrastructure of annulus fibrosus were observed by scanning electron microscopy. The collagen content, glycosaminoglycan （GAG） content and biomechanical parameters in each group were also detected. Results HE staining and scanning electron microscopy showed that no residual cells were found in Group A, B and C. The structure of annulus fibrosus in Group A was not disturbed, while that in Group B and C was damaged severely and slightly, respectively. There was no statistical difference in collagen content among Group A, B and C, as compared to the control group （P 〉 0.05）. But the GAG content was sig- nificantly more lower in Group A, B and C than in the control group （ P 〈 0.05）. There was no statistical differ- ence in ultimate load, ultimate stress, toughness, elastic modulus and mechanical work to fracture between Group A, C and control group （ P 〉 0.05）, while these parameters of Group B were lower than those in the con- trol group （ P 〈 0.05）. Conclusions The Triton X-100-treated annulus fibrosus retained the major extracellular matrix composition after cell removal and preserved the major structure and mechanical strength, which is prefer- able for the construction of tissue engineering annulus fibrosus.