以豬的纖維環去細胞化製備天然支架應用於椎間盤組織工程

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

孫芙雯(Fu-Wen Sun)

指導教授 ： 黃義侑

國立臺灣大學/工學院/醫學工程學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.11064

摘要

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。

關鍵字

椎間盤 ； 纖維環 ； 去細胞化 ； 組織工程 ； 支架 ； 胞外基質

並列摘要

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.

並列關鍵字

intervertebral discs ； annulus fibrosus ； decellularized ； tissue engineering ； scaffold ； extracellular atrix

參考文獻

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