离心力下构建组织工程纤维环的实验研究

目的 探讨离心力对体外构建组织工程纤维环的影响. 方法 取兔胸腰段椎间盘纤维环组织,经胰蛋白酶和Ⅱ型胶原酶消化后细胞原代培养,体外扩增至第3代,5×10<'7>/mL的细胞悬液种植于聚乳酸-羟基乙酸(PLGA)无纺网支架上.实验组在离心力下培养(相对离心力:85.86×g,每天2次,每次离心15min),对照组静态培养.每组5个标本,培养4周后取出纤维环细胞与支架复合物,行大体标本观察,组织学和免疫组织化学染色,检测Ⅰ型胶原分泌情况. 结果 体外培养4周后,实验组与对照组均为类纤维环样组织,实验组纤维环厚度和组织弹性均优于对照组,Ⅰ型胶原免疫组织化学染色,实验组阳性染色面积为(45.39±6.78)%,对照组为(33.53±4.58)%,两者差异有统计学意义(P<0.01).实验组细胞排列较对照组更具方向性. 结论 离心力刺激纤维环细胞增加Ⅰ型胶原的分泌,有利于在体外构建组织工程纤维环.

Effects of centrifugal force on construction of tissue-engineered fibrous annulus

Objective To study the effect of centrifugal force on construction of tissue-engineered fibrous annulus in vitro. Methods The fibrous annuli were isolated by trypsin and type Ⅱ collagenase from thoracic and lumbar intervertebral discs of rabbits. Cells were proliferated to the third generation in vitro and seeded into PLGA scaffolds (5 × 10<'7>/mL). The experimental group was subjected to the centrifugal force(85.86 × g of relative-centrifugal force, twice per day, for 15 min) while the control group was cultured under normal condition. Each group contained 5 specimens. Both groups were cultured in vitro. The complex of fibrous annuli and scaffolds were observed 4 weeks later. Tissue texture and type Ⅰ collagen secretion were assessed by HE and immunohistochemistrical stain. Results After 4 weeks, in both groups similar complex of fibrous annuli and scaffolds formed. The tissue-engineered fibrous annuli under centrifugal force were morphologically thicker. The area of immunohistochemistrical stain was (45.39±6.78) % in the experimental group, and (33.53±4.58) % in the control group. The difference was statistically significant ( P < 0.01).The structural arrangement of the tissue-engineered fibrous annulus in the experimental group showed more regularity than in the control group. Conclusion The centrifugal force may promote chondrocytes to secrete type Ⅰ collagen, which benefits the construction of tissue-engineered fibrous annulus.