应用人皮肤成纤维细胞体外构建组织工程化肌腱

In vitro tendon engineering using human dermal fibroblasts

目的探讨应用国产可吸收生物材料聚羟基乙酸（PGA）和人皮肤成纤维细胞在体外构建组织工程化肌腱的可行性。方法酶消化法获得人皮肤成纤维细胞经体外培养、扩增至第2代，接种于PGA材料（将材料固定于U形弹簧）并给予持续张力作为实验组（n=15）；接种成纤维细胞但不给予任何张力作为对照组1（n=15），即无张力组；给予张力但未接种细胞的单纯PGA为对照组2（n=3），即无细胞组；给予张力并接种肌腱细胞的作为对照组3（n=5，仅限第9周时间点），即肌腱细胞组。体外培养后分别于第2、5、9、14和18周取材进行组织学、生物力学和电镜检测。结果第2周时，实验组与无张力组大体观察未见明显差异，组织学上主要是未降解的PGA纤维，电镜观察显示细胞在材料上黏附伸展良好。第5周时，除无细胞组外均有新生肌腱样组织形成，组织学检查提示胶原纤维形成。第9周时，无细胞组PGA发生断裂；实验组形成的肌腱组织直径为（1．18±0．25）mm，明显细于无张力组[（2．43±0．49）mm，P=0．017]；实验组和肌腱细胞组除后者细胞数量略少外，在大体和组织学上较为相似。实验组形成的胶原包括I型和Ⅲ型，细胞和胶原纤维沿受力方向排列，类似正常肌腱；无张力组则杂乱排列，且残留PGA较多。实验组的抗张强度为（2．75±0．59）MPa，接近肌腱细胞组[（3．08±0．30）MPa，P=0．439]，明显强于无张力组[（0．824±0．21）MPa，P=0．006]。第14周时，无细胞组的PGA基本降解；实验组胶原纤维直径增粗，死亡细胞增多，出现中空现象，但抗张强度比同组第9周时增加。第18周时，实验组中空现象更为明显，抗张强度下降，余与同组第14周时相似。结论应用人皮肤成纤维细胞在体外可以构建出与肌腱细胞所构建的相类似的人肌腱样组织，施加一定张力可能更有利于组织形成，但体外培养时间不宜过长。

Objective To examine the feasibility of using human dermal fibroblasts （DFbs）and polyglycolic acids （PGA） to engineer tendon in vitro. Methods Human dermal fibroblasts （DFbs） were isolated from the foreskin tissues of children obtained during operation with collagenase and cultured in vitro. Human tendon was obtained from a patient undergoing amputation during operation to isolate tenocytes. The DFbs of second passage were seeded on PGA fibers to form cell-scaffold constructs in shape of tendons.Those constructs were divided into 4 groups： experimental group （n = 15） with the DFbs inoculated on PGA scaffold under constant tension generated by a U-shaped spring, control group 1 （n = 15 ） with the DFbs inoculated on PGA scaffold without tension, control group 2 （n = 3 ） , i. e. , cell-free pure PGA scaffolds under tension, and control group 3 （ n = 5 ） , i. e. , tenocyte-scaffold constructs under tension that was harvested only at the ninth week. Samples were harvested 2, 5, 9, 14, and 18 weeks later to undergo histological examination and biomechanical test. Results Two weeks later histological examination showed that the constructs were mainly composed of PGA fibers in both the experimental group and the group without tension. Transmission electron microscopy showed fine cell attachment and stretching on the scaffold. By the 5th week, a neo-tendon was formed in all groups except for the cell-free group, and histology revealed the formation of collagen fibers. At the 9th week, the PGA fibers of the cell-free group were broken and partially degraded, the neo-tendon＇s diameter of the experimental group was （ 1.18 ± 0.25 ） ram, significantly thinner than that of the group without tension [ （2.43 ± 0.49 ） mm, P = 0. 017 ]. The gross morphology of tendons of the experimental group and tenocyte group were similar to each other except for more cells in the experimental group. In experimental group, immunohistochemistry revealed the production of fibers of collagen type Ⅰ ＆ Ⅲ that were aligned longitudinally along the force axis like the normal tendon pattern. An irregular collagen pattern was observed in the group without tension. The maximum tensile stress of the experimental group was （2.75 ±0.59） MPa, similar to that of the tenocyte group [ （3.08 ±0.30） MPa, P=0.439], and significantly greater than that of the group without tension [（0. 82 ± 0. 21） MPa, P =0. 006]. At the 14th week the PGA fibers of the cell-free group were mostly degraded. In addition, more dead cells and tissue atrophy were observed in the experimental group, and the tensile stress was higher than that of the same group by the 9th week. In the 18th week the number of hollow fiber of the experimental group was more obvious, the number of dead cells increased, and the tensile stress was lower, however, there was no significant difference in other characteristics compared with those in the 14th week. Conclusions DFbs can be used for in vitro tendon engineering as tennocytes. Mechanical stimulation by statistic strain is beneficial for tissue formation, but the effect may not be optimal if the tension is applied for too long.