以去細胞技術製備脊椎纖維環支架應用於脊椎椎間盤修復

Abstract

摘要 超過百分之八十的成年人有下背疼痛的經驗，造成下背痛的原因很多， 有越來越多證據顯示下背痛與椎間盤退化有相關性，此外纖維環的破裂、椎間盤 突出也是幾個常見的原因。治療椎間盤的退化又分為保守療法及手術治療。當採 用椎間盤切除術時，手術過程會對纖維環造成破壞，而目前對修復椎間盤並無一 個良好的方法。本研究的目的是希望開發出以豬的纖維環去細胞化應用在脊椎纖 維環的修復。雖然自體移植和異體移植是比較不容易造成免疫反應的移植方式， 但自體移植來源有限且數量往往不敷使用，而異體移植數量依然有限、且會有疾 病傳染及免疫排斥的問題，因此採用異種移植。 異種移植易造成嚴重的免疫，因此希望透過去細胞化有效降低纖維環內細胞 的數量，而只保留其組織架構。去細胞化的過程先採用物理凍融的方法，接著採 用化學方法，以0.1%的SDS(一種常用來溶解細胞及核膜的洗滌劑)。並以脫水試 驗去判斷是否符合臨床應用的需求。組織切片染色有H&E、Picrosirius Red、Alcian Blue 三種。同時以生化評估方法去了解DNA 含量、GAG 含量、Collagen 含量。 並以拉伸測試去測試機械性質。將纖維環和3T3 共培養，最後再以MTS 測試去 了解生物相容性。 六次freeze-thaw 去細胞化的結果優於三次，因此採用六次freeze-thaw 作為 製備過程。DNA 含量下降86%，GAG 含量下降15.9%，Collagen 處理前後並無 明顯差異。MTS 的結果去細胞化的纖維環具有良好相容性。機械強度在剛性與 楊氏係數也無明顯差異。雖然埋入老鼠體內仍會產生免疫反應的問題，但綜合以 上結果，本實驗製備一個去細胞化的纖維環，將此支架應用於修復椎間盤纖維環 上具有發展潛力。

Abstract Low back pain is the common civil disease in the recent decades, and more than 80% of adult people are persecuted by low back pain. There are many factors that cause low back pain, and the degeneration of intervertebral disc may be the major relational factor. Besides, rupture of annulus fibrosus and herniated intervertebral disc (HIVD) may also the common aetiologies of low back pain. To treat the degeneration of intervertebral disc, there are mainly two therapeutics including conservative therapy and surgical therapy. The process of discectomy may cause the damage of spinal annulus fibrosus, and the repair of the annulus fibrosus is always a big challenge of the surgeons. The objective purpose of this study is to prepare a implant of annulus fibrosus made by porcine lumber disc. Because of the shortage donated source of allograft implant, development of xenograft implant may be the tendency of annulus fibrosus repair associated tissue engineering. Because xenograft always cause serious immunological rejection or infection, decellularization of annulus fiber may be practiced by physical freeze-thaw and chemical method to minimize cell content in implant tissue to prevent immunological reaction. In this study, porcine annulus fibrosus after freeze-thaw treatment were treated with 0.1% SDS solution to make cell lysed and washed out the cell lysate. Subsequently, histological section with H&E stain, Picrosirius Red stain, and Alcian Blue stain were used to observe the morphology, and biochemical assays were performed to identify the phenotype of treated annulus fiber. The results demonstrated that the porcine annulus fibrosus treated with six-time freeze-thaw performed better condition than three-time freeze-thaw groups. The DNA content of decellularized VII tissue was 86% less than fresh tissues, and the GAG content of decellularized tissue was 15.9% less than fresh tissues. Collagen content were no significant differences with fresh tissues. MTS assay showed that decellularized annulus fibrosus were non-toxic to normal cells. It demonstrated the biocompatibility of the decellularized annulus fibrosus. Stiffness and Young’s modulus by mechanical property test also showed that there were no significant differences between treated and fresh annulus fibrosus. Although there was still immunological response when the decellularized annulus fibrosus implanted subcutaneous in rat for a long period of time, the decellularized method applied for preparing the xenograft implant of annulus fibrosus repair in this study may be potential in clinical repair of intervertebral disc.