离心应力下构建组织工程髓核

The effects of centrifugal force on constructing of tissue engineered nucleus pulposus

目的:探讨离心应力对髓核细胞功能表达和组织工程髓核结构的影响。方法:取兔胸腰段椎间盘髓核组织,经胰蛋白酶,Ⅱ型胶原酶消化分离出细胞,在含15%灭活胎牛血清的DMEM/F12培养基中体外扩增至第3代,以3×107/ml的细胞悬液种植于PLGA无纺网支架上。实验组在离心力下培养(实验1组相对离心力为134.16g,实验2组相对离心力为301.86g,每天2次,每次离心20min),对照组静态培养,每组4个标本,培养4周后取出髓核细胞与支架复合物,行大体标本,扫描电镜,组织学和免疫组织化学染色观察离心应力对组织工程髓核结构及Ⅱ型胶原分泌情况的影响。结果:离心应力下构建的组织工程髓核的厚度和直径大于对照组,细胞外基质明显多于对照组,Ⅱ型胶原免疫组化染色,实验1组阳性染色面积为(14.36±2.82)%,实验2组阳性染色面积为(18.71±2.14)%,对照组为(9.13±1.58)%,实验1组、实验2组与对照组差异有统计学意义(P<0.01),实验2组与实验1组差异有统计学意义(P<0.05)。结论:一定范围的离心应力刺激髓核细胞增加Ⅱ型胶原的分泌,有利于在体外构建组织工程髓核。

Objective：To study the effect of the centrifugal force on the constructing of tissue engineering nucleus pulposus. Methods：Nucleus pulposus were isolated by trypsin and type Ⅱ collagenase from rabbit thoracic and lumbar intervertebral discs.Cells were proliferated to the third generation and seeded into PLGA scaffolds（3 × 107/ml）. Experimental groups were subjected to the centrifugal force（134.16g and 301.86 g of relative-centrifugal force,twice per day,for 20 min） while the control group was cultured under normal condition,each group containing 4 specimens. The complex of nucleus pulposus and scaffolds were observed after 4 weeks. The structure of nucleus pulposus and typeⅡ collagen secretion were assessed by HE,immunohistochemistrical stain and the scanning electron microscope. Results：After 4 weeks,all groups formed the similar of complex of nucleus pulposus and scaffolds. The tissue engineered nucleus pulposus under centrifugal force were morphologically thicker than that in control,the area of immunohistochemistrical stain was（14.36 ± 2.82）% in experimental group 1,（18.71 ± 2.14） in experimental group 2,（9.13 ± 1.58）% in control group respectively. There were statistical significance between experimental group and control group（P 〈 0.01）,also between experimental group 1 and experimental group 2（P 〈 0.05）. Conclusion：The centrifugal force promotes nucleus pulposus cells to secrete typeⅡ collagen and the centrifugal force might benefit the construction of tissue engineered nucleus pulposus.