体外构建组织工程化肌腱的初步研究

Preliminary study on in vitro tendon engineering using tenocytes and polyglycolic acids

目的 探讨利用可吸收生物材料聚羟基乙酸 (polyglycolicacids ,PGA)和肌腱细胞在体外构建组织工程化肌腱的可能性。 方法 取肌腱细胞经体外培养、扩增至第 2代 ,与PGA混合培养形成细胞 生物材料复合物 ,体外培养 1周后分组 ,用U形弹簧给细胞 PGA复合物持续张力后体外培养 (n =5 )为实验组 ;没有外加张力的作为对照 1组 (n =4 ) ;单纯PGA作为对照 2组 (n =3) ,每2天换液 1次。分别于体外培养 2、4、6周取组织做组织学、免疫组化检测 ,同时对第 6周的标本进行透射电镜和生物力学检测。 结果 第 2周时 ,3组标本在大体、组织学等方面无明显差异 ,组织学上主要是未降解的PGA纤维。第 4周时 ,实验组和对照 1组均有新生肌腱组织形成 ,HE染色和免疫组化检测均提示胶原纤维形成 ,而对照 2组的PGA已大部分降解。第 6周时 ,对照 1组形成的肌腱组织比实验组的粗 ,透射电镜检测两组标本形成的胶原纤维有周期性横纹 ,与对照 1组相比 ,实验组形成的胶原纤维沿纵轴排列有序更接近正常肌腱组织 ,生物力学检测显示实验组的最大应力 (1 10 7± 0 32 7N/mm2 )明显强于对照 1组 (0 2 94± 0 138N/mm2 ) (P <0 0 5 )。 结论 应用组织工程技术在体外可以构建肌腱组织 ,施加周期性的应力可能更有利于肌腱组织的形成?

Objective To find out the feasibility of tendon engineering in vitro using expanded tenocytes and polyglycolic acids (PGA). Methods Tenocytes were isolated using tissue explant method and expanded in vitro. Tenocytes (20×10 6) at the second passage were collected and then seeded onto PGA unwoven fibers to form a cell-scaffold construct in a shape of tendon. The constructs were cultured in DMEM with 20% FBS for 1 week. The cell-scaffold constructs were then cultured under constant tension generated by a U-shaped spring ( n =5) ,which served as experimental group,or cultured without tension ( n =4) ,which served as control group 1. PGA fibers alone were cultured ( n =3) ,which served as control group 2. Small fragments at the end of the constructs were harvested at 2,4 and 6 weeks respectively for histological and immunohistochemistry (IHC) analysis. Six-week samples were also evaluated by transmission electron microscope (TEM) and mechanical test. Results No obvious difference was observed among the three groups at 2 weeks grossly and histologically as the constructs remained to be mainly undegraded PGA fibers. By 4 weeks,a neo-tendon was formed in the experimental group and control group1 grossly,and histology and IHC revealed the formation of collagen fibers. In contrast,PGA fibers alone in control group 2 were mostly degraded. At 6 weeks,tendons of control group 1 were much thicker [(2.55±0.18)mm in diameter] than those of experimental group [(1.44±0.13)mm in diameter]. Periodical striae were observed in collagen fibers of experimental group and control group1 by TEM. However,histology of tendons in experimental group revealed longitudinally aliened collagen fibers,which resembled the structure of normal tendon more closely than that of control group1 tendons. Furthermore,the maximum tensile stress (N/mm 2) of experimental group (1.107±0.327)was greater than that of control group1(0.294±0.138)( P <0.05). Conclusion It is possible to use an engineering to construct tendon tissue in vitro. Periodical strain generated by bioreactor may be the optimal mechanical stimulation,which is currently under investigation.