Decellularized porcine annulus fibrosus to prepare a natural scaffold for intervertebral disc tissue engineering

椎間盤為人體內承受重量與吸收衝擊的重要器官，隨著人口老化成為目前世界的趨勢，椎間盤疾病越來越常見，尤其椎間盤退化引起的下背疼痛問題，更困擾著百分之八十的成年人。目前治療椎間盤疾病的方法偏向保守治療，藉由椎間盤切除術、脊柱融合術或物理治療、藥物治療，目的為減少患者的疼痛，而非修復、再生受損的椎間盤。而纖維環的修復是椎間盤治療的首要重點，若缺少纖維環的環繞，凝膠型態的髓核組織無法穩定存在於椎間盤，因此優先修復纖維環，才能使椎間盤修復治療繼續進行，達到治療之目的。 因此本研究希望以豬的纖維環去細胞化應用在椎間盤治療。因異種移植目前存在許多問題，本研究希望透過去細胞過程有效降低纖維環內細胞與免疫物質的數量，僅保留其胞外基質結構作為天然支架進行移植。 本研究為延續實驗室先前之研究，因此希望能夠比較各種不同影響因子，達到更好去細胞效果以及完成更多實驗驗證。因此首先比較不同冷凍解凍溫度、不同化學試劑與不同去細胞處理時間，選擇最有效的去細胞過程，其過程又可分為物理凍融、化學洗滌與酶洗滌三部分。去細胞過程完成同時測量其GAGs、Collagens、DNAs與α-Gals含量，及進行組織切片染色，評估去細胞過程的效果。另外進行機械壓縮測量其機械性質，並將纖維環與大鼠纖維環細胞共同培養測試其生物相容性。最後進行大鼠的動物實驗。 實驗結果顯示，經由去細胞處理能夠去除85%的DNA含量與88%的α-Gal抗原含量，並保存98%與94%的膠原蛋白與GAGs含量，且藉由H&E染色得知細胞可經由去細胞過程清洗去除。並由MTS細胞相容性實驗顯示，此去細胞化纖維環具有良好的細胞相容性，另外在壓縮試驗證明此去細胞處理纖維環與天然纖維環之機械性質無明顯差異。動物實驗中則顯示此支架能夠促使胞外基質分泌。以上結果證明此去細胞纖維環於椎間盤復原與再生應用上具有其發展潛力。Intervertebral disc (IVD) plays an important role to sustain weight and absorb shocks in human body. Low back pain associated with the disc degeneration causes lots of inconvenience and economic burden. Currently, the treatments of disc degeneration tend to be traditional and conservative. Treatments, such as physical therapy, discectomy, spinal fusion and drug treatment, had focused on relieving mechanical pain instead of repairing and regenerating the disc. Tissue engineering has attracted much attention and aim to be a effective method that could regenerate the IVD. Repairing the annulus fibrosus (AF) is the most significant matter in disc regeneration treatments. The gelatinous nucleus pulposus (NP) can’t exist steadily in the disc if the lack of AF. Therefore, taking priority to repair the AF enables the disc regeneration treatment easier. In this study, we took the decellularized techniques which combine physical, chemical and enzyme methods. We decellularized porcine AFs and created AF scaffolds for IVD tissue engineering. Since the xenotransplantation generated many immune problems, we not only try to reduce the cells and immune substances but retain the extracellular matrix (ECM). In this study, we evaluated different freeze-thaw temperatures, various chemical detergents and different decellularized time, followed by optimizing the best decellularizing method. Using the optimized decellularizing process, we found that the DNA content 85% and α-Gal content decreased 85% and 75%. Furthermore, we could maintain 94% of GAGs contents and 98% of collagen contents. The MTS assay revealed that the decellularized AFs had no cytoxicity for cell culture. And there were no significant mechanical differences between fresh and decellularized annulus fibrosus. These results suggested that the decellularized porcine AFs have great development for IVD repair and regeneration.摘要…………………………………………………………………………………………………………Ⅰ Abstract……………………………………………………………………………………………Ⅱ 目錄…………………………………………………………………………Ⅳ 圖目錄………………………………………………………………………Ⅶ 表目錄………………………………………………………………………Ⅸ 第一章 緒論…………………………………………………………………1 1.1 脊椎…………………………………………………………………1 1.2 椎間盤………………………………………………………………1 1.3 椎間盤退化…………………………………………………………3 1.4 椎間盤退化的傳統治療……………………………………………6 1.5 椎間盤退化的新興治療……………………………………………6 1.6 纖維環的修復………………………………………………………7 1.7 組織工程……………………………………………………………7 1.7.1 支架(Scaffold) ………………………………………………8 1.7.2 細胞(Cell) ……………………………………………………10 1.7.3 訊息因子(Signal) ……………………………………………10 1.8 去細胞化(Decellularization) …………………………………11 1.9 常見的移植方式……………………………………………………14 1.10 豬隻的異種移植……………………………………………………14 1.11 豬隻異種移植產生之排斥反應……………………………………15 1.12 α-Galactosyl抗原超急性排斥反應……………………………15 第二章 研究動機與目的……………………………………………………17 第三章 材料實驗與方法……………………………………………………19 3.1 實驗藥品……………………………………………………………19 3.2 實驗儀器……………………………………………………………21 3.3 豬隻纖維環(Annulus Fibrosus)之取得…………………………22 3.4 去細胞化(Decellularization) …………………………………22 3.4.1 以冷凍解凍方法去細胞…………………………………………22 3.4.2 以不同化學藥劑(Chemical agent)去細胞……………………23 3.5 GAG含量測定………………………………………………………25 3.6 膠原蛋白含量測定…………………………………………………27 3.7 DNA含量測定………………………………………………………28 3.8 細胞活性測試………………………………………………………29 3.8.1 纖維環細胞初代培養……………………………………………29 3.8.2 MTS測試…………………………………………………………31 3.9 掃描式電子顯微鏡(SEM)…………………………………………32 3.10 壓縮強度測試………………………………………………………33 3.11 組織石蠟切片………………………………………………………34 3.12 蘇木紫-伊紅染色(H&E Staining)………………………………35 3.13 過碘酸氧化反應染色(PAS staining)……………………………36 3.14 Masson’s Trichrome染色(MT staining)………………………37 3.15 Alpha galactosyl (α-GAL) 含量測定…………………………38 3.16 動物試驗(Animal Experiment)…………………………………40 第四章 結果與討論…………………………………………………………41 4.1 冷凍解凍溫度對纖維環組織的影響………………………………41 4.2 化學試劑對纖維環組織的影響……………………………………44 4.3 去細胞時間對纖維環組織的影響…………………………………46 4.4 去細胞處理前後纖維環之外觀與微結構…………………………49 4.5 去細胞處理前後纖維環之切片染色分析…………………………51 4.6 去細胞處理前後纖維環之GAGs含量………………………………53 4.7 去細胞處理前後纖維環之膠原蛋白含量…………………………54 4.8 去細胞處理前後纖維環之DNAs含量………………………………55 4.9 去細胞處理前後纖維環之α-Gals含量…………………………56 4.10 去細胞處理前後纖維環之細胞相容性…………………………58 4.11 去細胞處理前後纖維環之機械性質……………………………60 4.12動物試驗…………………………………………………………62 第五章 結論………………………………………………………………66 參考文獻……………………………………………………………………6