Angiogenic Effects of Different Cultured Skin Substitutes in Vivo

培養式人工皮膚活體內血管增生效應之比較

鄭秀婷(Hsiu-Ting Cheng) ； 蔡錡函(Chi-Han Tsai) ； 劉士豪(Shih-hao Liu) ； 馬旭(Hsu Ma)

《臺灣整形外科醫學會雜誌》 21卷3期 (2012/09) Pp. 212-221

https://doi.org/10.7096/TJTSPS.201209.0212

摘要

背景：組織工程已經是臨床上開始應用的一門科技，它結合了材料力學及細胞學而發展出的一個新的研究領域，也提供了因先天或後天因素造成器官或組織缺損另一方向的移植治療方法。對於皮膚廣泛受損而缺乏足夠供皮區的病人，人造真皮提供形成真皮的良好環境，改善了重建皮膚的品質；然而，即使是人造真皮，也需要二到三週才能更換成自體組織。人工合成皮膚對於感染抵抗能力差，因此希望能縮短人工合成皮膚更換成自體組織的時間。目的及目標：加速血管新生不僅可提前植皮手術的進行，同時可以避免感染的攻擊。為了達成此效果，我們將數種細胞株單獨或合併植入人造真皮中，後再植入雞胚尿絨毛膜培養，觀察其對加速血管新生的影響並驗證其生物相容性。材料及方法：本實驗使用的人工真皮是以Integra為骨架加入培養液後，第一組為對照組，僅加入培養液；第二組加入人類纖維母細胞；第三組加入含綠色螢光基因（pCX-EGFP）的小鼠胚幹細胞株；第四組加入一半小鼠胚幹細胞株和一半纖維母細胞；第五組加入攜帶外源性胰島素（insulin）基因之纖維母細胞；第六組加入攜帶外源性酸性纖維母細胞生長因子（acidic fibroblast growth factor; aFGF）基因之纖維母細胞。培養兩周後，再將上述六組分別植入孵育七天的雞胚尿絨毛膜，於培養箱內培養三天，之後將部份標本進行組織切片及免疫染色；第三及第四組則取剩餘標本抽取全部核醣核酸（total RNA），以反轉錄聚合酶連鎖反應（RT-PCR）來檢驗外源性纖維母細胞的酸性纖維細胞生長因子和胰島素的表現。在評估血管新生方面，則採用血管密度指數（vascular density index, VDI）作為指標。結果：病理組織切片及免疫染色顯示植入之小鼠胚幹細胞株及纖維母細胞皆成功附於Integra中；種入雞胚尿絨毛膜三天後，觀察細胞浸潤情形，並無急性發炎反應產生。在雞胚尿絨毛膜血管密度指數方面，外源性aFGF纖維母細胞組最高，外源性insulin纖維母細胞組次之，接下來依次為一半小鼠胚幹細胞株和一半纖維母細胞組，小鼠胚幹細胞株組和纖維母細胞組；對照組的血管密度指數最低。在抽取全部RNA，以RT-PCR檢驗aFGF和insulin的表現方面，證實兩種外源性纖維母細胞可分別分泌出aFGF和insulin。結論：本實驗發現外源性aFGF纖維母細胞，外源性insulin纖維母細胞以及攜有一半小鼠胚幹細胞株一半纖維母細胞組別有較佳的促進血管新生能力，同時這些組織工程合成的人供真皮在雞胚尿絨毛膜上有良好的生物相容性。

並列摘要

Background: In cases of extensive deep dermal and full-thickness defects, one is often faced with the difficulty of limited available donor sites. Tissue-engineered skin substitutes provide an alternative to skin autografts. One of the problems in developing the skin substitutes is how to establish early vascular networks in wound beds to facilitate earlier skin grafting.Aim and Objectives: In this pre-clinical study, we investigated the effect upon angiogenesis of pre-seeding the skin substitutes with different kinds of human fibroblasts, mouse embryonic stem cells, and co-cultures of human fibroblasts and mouse embryonic stem cells (ESCs) and checked the bio-compatibility of the tissue-engineered skin substitutes by histologic examination.Materials and Methods: We pre-seeded Integra with human fibroblasts, human fibroblasts with exogenous acidic fibroblast growth factor (aFGF) gene, human fibroblasts with exogenous insulin gene, mouse ESCs, and co-cultures of human fibroblasts and mouse ESCs, and then grouped them into six subgroups, including control group, by different combinations. Using chorioallanotonic membrane (CAM) assay, biocompatibility was checked by histologic examination, and vascular density index (VDI) was obtained. Total RNA was extracted from the cultured skin substitutes and we confirmed the expression of aFGF and insulin by reverse transcription-polymerase chain reaction (RT-PCR). Results: In CAM, no acute inflammatory process was found on histologic examination, and fibroblasts with exogenous aFGF gene, fibroblasts with exogenous insulin gene, and co-cultures showed an increase of VDI. RT-PCR confirmed the expression of aFGF and insulin from fibroblasts. Conclusions: Fibroblasts with exogenous aFGF gene, fibroblasts with exogenous insulin gene, and co-cultures of mouse ESCs and fibroblasts have positive effect to angiogenesis. Using the CAM assay, we provide a model with good biocompatibility, and the result is consistent with previous studies. (J Taiwan Soc of Plast Surg 2012;21:212~221)

並列關鍵字

skin substitutes ； human fibroblasts ； mouse embryonic stem cells ； CAM assay ； angiogenesis ； tissue engineering