豬椎間盤纖維環的去細胞製備與組織性質於脊椎外科手術之應用

背景:多年來，針對椎間盤受損之新治療對策包含椎間盤的修補、取代及再生，然而似乎成效不彰，這可能歸因於對受損之椎間盤纖維環的治療著墨不足。本研究則著力於開發異種纖維環組織去細胞的生醫製程，並定量去細胞纖維環組織上α-Gal表面抗原的含量，以期開發並能臨床運用於脊椎手術之產品。方法：將豬的纖維環組織分組採取不同的凍融溫度、離子洗劑、溶製時間進行去細胞化，來決定去細胞的最佳製程。接著使用生化和機械測試確定去細胞化材料的完整性。最後定量並比較α-Gal表面抗原在去細胞化前後之差異。結果與討論：第1部分：由H＆E染色顯示去細胞化成功透過去細胞製程達成。纖維環組織的醣胺聚醣,膠原蛋白含量和機械特性等皆能被維持而無顯著變化，且無明顯的細胞毒性。第2部分：研究中採單變因測試決定了液態氮凍融、離子洗劑（0.1％SDS）、及溶製24小時等為豬纖維環組織去細胞化的最佳製程。綜合來看，這個去細胞最佳化製程除了保留更多的醣胺聚醣，同時也去除了與第1部分實驗相仿量的DNA。生物免疫相容性則透過在去細胞化材料能成功在動物體內重塑及α-Gal表面抗原的明顯減少來證實。結論：豬纖維環組織能成功去細胞，並保存重要的生物組成與機械特性。這些結果表明脫細胞豬纖維環材料將可能臨床應用在脊椎外科手術上。

關鍵字

α-Gal表面抗原；纖維環；椎間盤；去細胞；脊椎；組織工程

並列摘要

Background: Over the last few years, new treatments for a damaged intervertebral disc (IVD) have included strategies to repair, replace or regenerate the degenerative disc. However, these techniques are likely to have limited success, due to insufficiently effective means to address the damaged annulus fibrosus (AF). This study aimed to develop a bioprocess method for decellularization of the xenogeneic AF tissue and to quantify potentially antigenic α-Gal epitopes in the decellularized AF tissue, with a view to developing a scaffold as a potential candidate for clinical application in spinal surgery. Methods: Porcine AF tissue was decellularized using different freeze-thaw temperatures, chemical detergents, and incubation times in order to determine the optimal method for cell removal. The integrity of the decellularized material was determined using biochemical and mechanical tests. The α-Gal epitope was quantified before and after decellularization. Results & Discussion: Part1: H&E staining showed that decellularization was achieved through the decellularization protocols. GAG, collagen and mechanical properties of the AF scaffold were maintained with no significant cytotoxicity. Part 2 : The study defined the optimal conditions for decellularization of porcine AF tissues with dry freeze-thaw in liquid nitrogen, an ionic detergent (0.1% SDS), and a 24-hour incubation period when tested as single variables. Combined, these optimal decellularization conditions preserved more GAG while removing the same amount of DNA as the conditions used in Part 1 study. Under these conditions, immunocompatibility was evidenced by successful in vivo remodeling and reduction of the α-Gal epitope in the decellularized material. Conclusions: Porcine AF tissue were effectively decellularized with preservation of biologic composition and mechanical properties. These results demonstrate that decellularized AF scaffolds are potential candidates for clinical applications in spinal surgery.

並列關鍵字

α-Gal epitope ； annulus fibrosus ； intervertebral disc ； decellularization ； spine ； tissue engineering

參考文獻

1. Chan, S.C. and B. Gantenbein-Ritter, Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction? Swiss Med Wkly, 2012. 142: p. w13598.

2. Bron, J.L., et al., Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges. Eur Spine J, 2009. 18(3): p. 301-13.

3. Liu Yang, R.A.K., Godfrey Chang, J. Paul Santerre, Polar surface chemistry of nanofibrous polyurethane scaffold affects annulus fibrosus cell attachment and early matrix accumulation. Journal of Biomedical Materials Research Part A, 2008.

4. Mercuri, J.J., S.S. Gill, and D.T. Simionescu, Novel tissue-derived biomimetic scaffold for regenerating the human nucleus pulposus. Journal of Biomedical Materials Research Part A, 2011. 96A(2): p. 422-435.

5. Schek, R.M., A.J. Michalek, and J.C. Iatridis, Genipin-Crosslinked Fibrin Hydrogels as a Potential Adhesive to Augment Intervertebral Disc Annulus Repair. European Cells & Materials, 2011. 21: p. 373-383.

國際替代計量

豬椎間盤纖維環的去細胞製備與組織性質於脊椎外科手術之應用

全文下載

主題瀏覽