| 摘要: | 體積性肌肉缺損（Volumetric Muscle Loss, VML）常因大範圍組織破壞與神經再支配不足，導致骨骼肌結構與功能無法有效恢復。雖然近年組織工程技術可藉由細胞片或生物反應器促進肌管排列與分化，但移植後因缺乏及時且穩定的神經支配，仍容易發生肌纖維萎縮與功能退化。因此，如何在維持肌肉結構完整性的同時，促進神經分化與神經肌肉接點（neuromuscular junction, NMJ）的形成，仍為肌肉組織工程中亟待解決的關鍵問題。本研究提出一種結合「肌肉細胞片轉移」與「神經生長因子（nerve growth factor, NGF）持續釋放」之策略，利用纖維蛋白凝膠（fibrin gel, FG）作為可降解且具生物相容性的藥物載體，以建立具神經誘導潛力的神經肌肉共培養系統。首先，透過調控纖維蛋白原、凝血酶及離子條件，製備具有適當力學性質之 FG，並評估其成膠行為與壓縮模數。隨後，將 NGF 包埋於 FG 中進行釋放行為分析，並觀察其對 PC12 細胞神經分化的影響。在生物反應器培養條件下，將排列良好之 C2C12 肌肉細胞片，成功利用載藥型 FG 進行細胞片轉移。最後以神經細胞種植於載有肌管細胞片的水膠進行共培養，來評估NMJ形成的效果，結果顯示，NMJ 後突觸相關基因（如 CHRNE、RAPSN 及 CHRNA1）表現顯著上調，而神經結構相關基因亦呈現分化趨勢。免疫螢光染色進一步顯示，神經軸突與乙醯膽鹼受體簇集於肌管表面呈現空間共位，顯示本系統已啟動具 NMJ 特徵之神經肌肉整合過程。綜合以上結果，本研究證實載藥型纖維蛋白凝膠結合肌肉細胞片轉移，可在提供神經誘導微環境，以促進神經分化並啟動 NMJ 相關分子與結構特徵。此研究成果可作為未來應用於體積性肌肉缺損修復與神經肌肉組織工程之重要基礎。;Volumetric muscle loss (VML) often results in impaired muscle regeneration due to extensive tissue damage and inadequate reinnervationof the affected muscles. Although tissue-engineered muscle constructs can be generated using cell sheet and bioreactor-based approaches, the lack of effective neural integration remains a major limitation. In this study, we developed a nerve growth factor (NGF) loaded fibrin gel (FG) which can be used not only to transfer myotube sheets from the bioreactor but also to sustain the delivery of NGF, promoting neurogenesis.
To prepare FG with the appropriate mechanical properties, the concentrations of fibrinogen, thrombin, and NaCl were tuned, and the gelation behavior and compressive modulus were evaluated. Then, NGF was loaded into FG, and its release profile was monitored, and its effects on PC12 neurite outgrowth/neuronal differentiation were evaluated. When FG was used to harvest C2C12 myotubes from the bioreactor, the transferred myotubes on FG maintained good morphology and high viability. Finally, PC12 cells were co-cultured with the myotube sheets on the FG to evaluate the efficiency of NMJ formation. Gene expression analysis revealed upregulation of postsynaptic NMJ-related markers, including CHRNE, RAPSN, and CHRNA1, under coculture conditions. Immunofluorescence staining of βIII-tubulin and α-bungarotoxin further demonstrated spatial colocalization of neuronal projections and acetylcholine receptor clusters on myotubes.
These results indicate that NGF-loaded FG combined with muscle cell sheet transfer provides a neural inductive microenvironment that promotes neural differentiation and initiates NMJ-associated molecular and structural features. This platform may serve as a foundation for future neuromuscular tissue engineering applications. |
