利用含脂肪幹細胞的聚二氧乙基噻吩：聚苯乙烯磺酸/甲基丙烯酸明膠水凝膠進行電刺激以促進傷口癒合

Abstract

研究目的：傷口癒合是一個複雜的過程，但大多數敷料缺乏電活性，無法對生理電信號或外部電刺激（EStim）作出反應。本研究旨在開發一種具有電活性且可光固化的脂肪幹細胞（ADSCs）負載水凝膠，通過將聚（3,4-乙烯二氧噻吩）聚苯乙烯磺酸鹽（PEDOT:PSS）的導電性與甲基丙烯酸明膠（GelMA）的相容性相結合，PEDOT:PSS/GelMA水凝膠可以作為類似於細胞外基質的再生模板並充當導電中間體，而脂肪幹細胞具有自我更新和分化的能力，用以增強組織工程策略。我們預計這種電活性水凝膠可以將局部電刺激傳遞到傷口部位，調節細胞功能以改善傷口癒合。 研究材料與方法：PEDOT:PSS/GelMA水凝膠是使用含7% GelMA和0.1% PEDOT:PSS的配方合成的。在合成之後，水凝膠進行了全面的表徵和生物相容性測試。此外，我們從老鼠的雙側腹股溝脂肪組織中分離出ADSCs，並將其加入PEDOT:PSS/GelMA預聚合物溶液中，通過紫外線輻射誘導凝膠化。為了驗證電刺激對含ADSC的PEDOT:PSS/GelMA水凝膠促進傷口癒合的效果，在大鼠背部製造全厚度皮下傷口。對這些傷口進行了各種處理：含ADSC的GelMA水凝膠（有或沒有電刺激）和含ADSC的PEDOT:PSS/GelMA水凝膠（有電刺激）。為了記錄癒合進程，在第4天、第7天和第14天拍攝了傷口的照片。在第14天，進行了組織學評估和角質化的免疫組織染色，以進一步評估癒合過程。 研究結果：PEDOT:PSS/GelMA水凝膠表現出良好的親水性、生物降解性和優異的電活性。此外，它還顯示出良好的生物相容性，對L929細胞的黏附和增殖沒有細胞毒性的作用。 在使用全厚度傷口缺損模型的體內評估中，含ADSC的PEDOT:PSS/GelMA水凝膠與電刺激一起加速了傷口閉合。它增加了上皮化、角質化和膠原沉積。結果顯示，在電刺激下使用含ADSC的PEDOT:PSS/GelMA水凝膠處理的傷口有更多且更有組織的膠原纖維形成。新形成的膠原基質與周圍細胞外基質的總體比例顯著高於其他組別。該組觀察到的傷口癒合顯著改善可能歸因於通過導電水凝膠對組織的直接電刺激應用，這顯著促進了成纖維細胞的遷移和基質合成。 結論：PEDOT:PSS/GelMA水凝膠表現出良好的生物降解性、良好的導電性和理想的生物相容性。通過將電刺激與含脂肪幹細胞的水凝膠敷料結合，這種可生物降解且具有電活性的支架提供了一種有效的協同治療策略，以加速傷口癒合過程。

Wound healing is a complex process, but most dressings lack electroactivity and cannot respond to physiological electrical signals or external electrical stimulation (EStim). This study aims to develop an electroactive, photocurable hydrogel loaded with adipose-derived stem cells (ADSCs). By combining the conductivity of Poly(3,4-ethylenedioxythiophene) Polystyrene Sulfonate (PEDOT:PSS) with the compatibility of Gelatin Methacrylate (GelMA), the PEDOT:PSS/GelMA hydrogel can serve as a regenerative template similar to the extracellular matrix and act as an electroconductive intermediate. ADSCs, capable of self-renewal and differentiation, are used to enhance the tissue engineering strategy. This electroactive hydrogel is expected to deliver localized EStim to wound sites, modulating cell behavior to improve wound healing. Materials and methods: The PEDOT:PSS/GelMA hydrogel was synthesized using a protocol involving 7% GelMA and 0.1% PEDOT:PSS. Following synthesis, the hydrogel underwent comprehensive characterization and biocompatibility testing. Additionally, ADSCs were isolated from bilateral inguinal adipose tissues and incorporated into the PEDOT:PSS/GelMA prepolymer solution, with UV radiation used to induce gelation. To validate the effect of EStim on the ADSC-laden PEDOT:PSS/GelMA hydrogel for promoting wound healing, full-thickness subcutaneous wounds were created on the dorsal surface of rats. Various treatments were applied to these wounds: ADSC-laden GelMA hydrogel with or without EStim, and ADSC-laden PEDOT:PSS/GelMA hydrogel with EStim. To document the healing progression, digital photographs of the wounds were taken on days 4, 7, and 14. On day 14, histological evaluations and immunohistochemical staining for keratinization were performed to further assess the healing process. This multi-faceted approach ensured a thorough examination of the hydrogel's effectiveness in promoting wound repair. Results: The PEDOT:PSS/GelMA hydrogel demonstrated great hydrophilicity, biodegradability, and excellent electroactivity. Additionally, it showed great biocompatibility, with no cytotoxic effects on L929 cell adhesion and proliferation. In the in vivo evaluation using a full-thickness wound defect model, the ADSC-laden PEDOT:PSS/GelMA hydrogel with EStim accelerated wound closure. It increased epithelialization, keratinization and collagen deposition. The results indicated that wounds treated with ADSC-laden PEDOT:PSS/GelMA hydrogel with EStim exhibited a greater abundance and more organized collagen fiber formation. The overall ratio of newly formed collagen matrix to the surrounding extracellular matrix[U1] was significantly greater than in the other groups. The notable enhancement in wound healing observed in this group may be attributed to the direct application of EStim to the tissue through the conductive hydrogel, which markedly facilitated fibroblast migration and matrix synthesis. Conclusion: The PEDOT:PSS/GelMA hydrogel exhibited good biodegradability, good conductivity and desirable biocompatibility. By coupling EStim with the ADSC-laden PEDOT:PSS/GelMA hydrogel, this electroactive scaffold provided an effective synergistic therapeutic strategy for accelerating the wound healing process.