体外构建组织工程化肌腱的初步研究

目的探讨利用可吸收生物材料聚羟基乙酸(polyglycolic acids ,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(1.107±0.327 N/mm2)明显强于对照1组(0.294±0.138 N/mm2)(P<0.05). 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.

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

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 x 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 group 1 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 group 1 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 group 1 tendons. Furthermore, the maximum tensile stress (N/mm(2)) of experimental group (1.107 +/- 0.327) was greater than that of control group 1 (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.