组织工程口腔黏膜固有层修复皮肤缺损的初步研究

PRIMARY GRAFTING RESEARCH OF TISSUE ENGINEERED ORAL MUCOSA LAMINA PROPRIA ON SKIN FULL THICKNESS WOUNDS

目的探讨两种组织工程口腔黏膜固有层移植修复皮肤缺损的效果。方法分别以胶原凝胶和壳多糖-胶原凝胶为网架与体外培养的Wistar大鼠口腔黏膜成纤维细胞构建胶原凝胶口腔黏膜固有层(fibroblast-populatedcollagenlattice,FPCL)、壳多糖-胶原凝胶口腔黏膜固有层(fibroblast-populatedchitosancollagenlattice,FPCCL),用BrdU标记其中的成纤维细胞后,移植修复同种异体大鼠背部全层皮肤圆形缺损。将36只21～25周龄Wistar大鼠分为FPCL组、FPCCL组及创口仅覆盖纱布的对照组,每组12只。术后行大体观察创面愈合情况;4、7、14和21d3组创面直径测量;组织学及免疫组织化学染色观察其组织修复情况。结果术后大鼠创面均无感染,创面结痂在14及17d自然脱落,创面逐渐愈合,被新生表皮覆盖,术后21d新生皮肤光滑,颜色与正常皮肤接近,无体毛。术后各时间点3组创面直径均逐渐变小,差异有统计学意义(P<0.01),14d以后,创口大小较稳定。术后7d,FPCL组受植创面比FPCCL组受植创面和对照组创面小(P<0.01)。组织学观察:术后7d,3组创面未完全表皮化;14、21d,FPCL组、FPCCL组受植区完全表皮化,有钉突,对照组无明显钉突,表皮已分层,基底细胞层完整,最表面有角化物;真皮中新生胶原纤维细,含毛细血管。免疫组织化学染色显示,FPCL组、FPCCL组术后各时间点阳性成纤维细胞出现在肉芽组织的细胞密集处和新生真皮中,与新生肉芽组织共同参与了皮肤缺损的修复重建,未出现免疫排斥现象。结论口腔黏膜成纤维细胞作为修复皮肤缺损的种子细胞是可行和有效的,壳多糖-胶原凝胶在限制受植区创面收缩变小方面优于单纯胶原凝胶。FPCL和FPCCL两组新生皮肤的质量优于对照组,两种组织工程口腔黏膜固有层作为皮肤缺损区永久性真皮替代物是可行和有效的。

Objective To study the allograft effect of two kinds of tissue engineered oral mucosa lamina proprias on skin full-thickness wounds. Methods The cultured Wistar rat oral mucosa fibroblasts （OMF） were incorporated into collagen or chitosan collagen to construct the tissue engineered oral mucosa lamina proprias, and then the OMFs were labeled with BrdU. The full thickness round skin defects were made with a round knife （diameter, 0.8 cm） on the backs of 36 Wistar rats （21-25 weeks old）, which were divided into 2 experimental groups： the fibroblast-populated collagen lattices （FPCL） group （grafted by FPCLs） and the fibroblast populated chitosan collagen lattices （FPCCL） group （grafted by FPCCLs）, and the control group （only covered with gauges）. All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively. Results There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups ,which was replaced at 17 days. All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days, there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kiiad. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group （P〈0.01）. Under the light microscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar phenomenon in the control group. Each skin wound in the three groups was only partly keratinocyted at 7 days postoperatively. The recipient wounds were wholly keratinocyted with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocyted with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustrated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defects without any allergic reaction during the period of the wound healing. Conclusion The oral mucosa fibroblasts as the new seed cells can join in the repair of the skin defects effectively and feasibly. The fibroblast- populated collagen lattices and the fibroblast-populated chitosan collagen lattices can repair skin defects effectively and feasibly, too. And the quality of the new skins was better in the two experimental groups than in the control group.