幹細胞結合3D生物列印GelMA材料對STZ誘導糖尿病大鼠之傷口癒合影響

Abstract

若治療如糖尿病患者所擁有的慢性難癒合傷口，需要耗費極大的醫療資源，故必需針對其治療方式進行改善。為此我們製作了含有幹細胞的水凝膠敷料，並評估其對STZ誘導的糖尿病大鼠傷口癒合試驗的效用。在本實驗中，我們將可光交聯性的明膠甲基丙烯(GelMA)作為敷料原料，其具有可變化的幾何形狀及明膠濃度，透過3D列印技術印製，可將幹細胞培養於3D及2D的環境。實驗中所使用的幹細胞為自婦女胎盤中分離的胎盤蛻膜間質幹細胞(pcMSC)及自成人血液中純化的血液微小幹細胞(SB)。於3D環境中培養幹細胞的水凝膠敷料包括薄片型、魚骨頭型及無特定形狀、可塗佈式的GelMA。pcMSC於敷料印製完成後存活率高達80%以上，其中薄片型及魚骨頭型的GelMA敷料在培養4天後仍能維持80%左右的存活率；在2D環境培養下，pcMSC具良好的細胞型態，且貼附的細胞約占培養表面的30%。此外，我們使用大鼠及豬隻模型來評估這些幹細胞敷料對傷口癒合是否具可行性。最初我們以切除大鼠背部全層皮膚直徑8mm的圓形傷口來模擬難癒合傷口，但由於模擬失敗，此方法很快地被進行修改。在修改後的模型中，我們模擬難癒合傷口的方式改為切除大鼠背部全層皮膚2×3cm方形傷口，並縫合傷口邊緣以防因組織攣縮所造成的傷口早期閉合。我們的研究結果顯示此方式確實使傷口再上皮化速度遲緩，並且與對照組相比，在幹細胞治療組中觀察到較佳的再上皮化。此外，我們也發現在豬隻模型中使用pcMSC敷料的治療組，其傷口中的VEGF表達量較對照組高。這些結果表明在目前的實驗設置下，含有pcMSC及SB的GelMA敷料對促進傷口癒合是具可行性的，但效果並不明顯優於對照組。推測可能為有效作用的幹細胞量不足所致，故未來需再進一步改良，提高幹細胞敷料對難癒合傷口的治療效果。

There is an unmet need for the improvement of treatment for chornic, hard-to-heal wounds such as that in diabetic patients owing to that it consumes a wealth amount of medical resources. In this regard, we fabricated stem-cell-embedded hydrogel patch and evaluated its effect on wound healing in STZ-induced diabetes rats. In this study, photocrosslinkable gelatin methacryloyl (GelMA) hydrogel with a variety of geometrical forms and gelatin concentration fabricated with 3D printing technology and cultured with stem cells in 3D or 2D conditions was employed. The 3D constructs included a regular film, an injectable, fishbone-like thread, and an amprphous, spreadable form. The stem cells utilized in this study were pcMSC and SB cells, abbreviated for human placenta chorionic decidual-derived mesenchymal stem cell and small blood stem cell, respectively. The cell viability in the 3D constructs was up to 80%. In particular, the viability of the pcMSCs cultured in the regular film and fishbone-like thread constructs over 4 days was still around 80%. The morphology of the pcMSCs cultured on the 2D films was well-spreaded, with that around 30% of the culture surface was occupied by the spreaded cells. The feasibility of these cell-laden patchs for wound healing was evaluated using rat and pig models. Initially, the hard-to-heal wound was simulated by excising a round-shaped, full-thickness skin of 8mm in diameter on the back of rats. But the approach was soon revised owing to the failure in simulation of the hard-to-heal wound. In the revised model, the hard-to-heal wound was simulated by removal of a rectangular-shaped, full-thickness skin of size 2×3cm, followed by suturing at the wound corners to impede early wound closure by contracture of the underlying tissue. Our results show that the wound re-epithelialization was markedly delayed in this approach and better re-epithelializion was seen in the groups treated with stem cell-laden patches when compared with that treated with standard approach. We also found that a higher VEGF expression in the wound treated with the pcMSCs laden patch than that of regular treatment in swine model. Our data indicated that the pcMSC and SB laden GelMA patches were feasible for wound healing but not significantly superior than standard treatment in the current setup. Possible reasons include insufficient amount of effective stem cells. Further studies are required to improve the efficacy of the stem cell-laden dressing in the treatment of hard-to-heal wounds.