利用人類脂肪幹細胞衍生細胞外基質與奈米纖維製備複合支架應用於促進傷口癒合

Abstract

細胞外間質對於組織工程的應用一直是生物醫學研究人員很感興趣的一環。細胞外間質生物支架能透過使用自體細胞來創造而成，使用自體細胞所生產的生物支架能有效減少使用異種和同種異體時所產生的免疫或排斥反應等等，更可改善自體組織供應不足的問題。 因為在之前的研究當中，我們已經發現在不同的方向性(有方向性與無方向性)的聚乳酸共聚物電紡絲纖維不織布上，細胞的型態會因為方向性的不同，而有型態與生長的情形的不同。我們發現，在有方向性的電紡絲上面生長的細胞，型態相對起來是比較拉長的，而且生長的情形也相對沒有方向性的電紡絲來得好。 因此在這個研究當中，我主要生產具有方向性的聚乳酸共聚物電紡絲纖維不織布作為細胞培養的基板。透過培養大量的人類脂肪幹細胞在聚乳酸共聚物纖維不織布上，可以在培養7天後取得細胞─細胞外間質─不織布纖維混成物。之後，利用冷凍溶解週期的方式把材料上的細胞物質去除，我們留下聚乳酸共聚物不織布模板，以增加混成物的可操作性與機械強度。 最後，人類臍帶血內皮細胞會被重新培養在我們所生成的細胞外間質─不織布纖維混成物支架上來測試其生物相容性。我們也會透過使用老鼠的傷口癒合實驗來驗證我們所生產出的細胞外間質生物支架是否具有促進血管新生和傷口修復的能力。而透過是否含有細胞外間質的實驗組別，我能夠直接比較細胞外間質對於傷口修復的效果如何。

Nanofibers have been considered as a matured technique that have been studied in various biomedical applications. In addition, biomedical researchers always have great interest in extracellular matrix (ECM) scaffolds derived from cultured cells for tissue engineering applications. ECM materials can be prepared from cultured cells to generate cell derived ECM (cECM) scaffolds. It can avoid the undesired host responses which are induced by either allogenic or xenogenic materials and circumvents which limit the supply of autologous tissues. In the previous study, it was found out that on the different orientation of PLGA nanofiber matrices, which are aligned ones and random ones, the morphology of cells were different. It could also be observed that the morphology of cells were more prolonged in the aligned mats than the other one. In this study, we fabricated the nanofibers and evaluated the performance of human adipose-derived stem cells (hASCs) cultured on the electrospun fibers by quantifying the cECM amounts and over cell proliferation behavior as well as the materials characterization of the nanofibers. To produce the PLGA-cECM Mesh, the hASCs were seeded on the PLGA fibers for 7 days followed by decellularization process to remove hASCs. To investigate the potential of PLGA-cECM meshes toward wound healing, fibroblasts (NCTC clone 929 of strain L, L929s) were seeded onto the PLGA-cECM meshes and cell viability and growth were evaluated. Finally, the ability of PLGA-cECM meshes accelerating wound healing procedure and possible induced angiogenesis were assessed via an in vivo mice wound healing model. The efficacy and potential of using this mesh was demonstrated with the promising results.