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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃義侑(Yi-You Huang) | |
| dc.contributor.author | Lien-Chen Wu | en |
| dc.contributor.author | 吳連禎 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:25:14Z | - |
| dc.date.available | 2022-07-20 | |
| dc.date.copyright | 2017-07-20 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-06-14 | |
| dc.identifier.citation | 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.
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Sandrin, Alpha1,3-galactosyltransferase knockout pigs are available for xenotransplantation: are glycosyltransferases still relevant? Immunol Cell Biol, 2005. 83(6): p. 687-93. 38. Puga Yung, G.L., et al., Complete absence of the alphaGal xenoantigen and isoglobotrihexosylceramide in alpha1,3galactosyltransferase knock-out pigs. Xenotransplantation, 2012. 19(3): p. 196-206. 39. Goncalves, A.C., et al., Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens. J Heart Valve Dis, 2005. 14(2): p. 212-7. 40. Choi, S.Y., et al., Elimination of alpha-gal xenoreactive epitope: alpha-galactosidase treatment of porcine heart valves. J Heart Valve Dis, 2012. 21(3): p. 387-97. 41. Zheng, M.H., et al., Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation. J Biomed Mater Res B Appl Biomater, 2005. 73(1): p. 61-7. 42. 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Gilbert, Immune response to biologic scaffold materials. Semin Immunol, 2008. 20(2): p. 109-16. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59484 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:25:14Z (GMT). No. of bitstreams: 1 ntu-106-D98548019-1.pdf: 2657062 bytes, checksum: ba408e231c1d8826806624c146abe05c (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 中文摘要……………………………………………………………………Ⅰ
Abstract……………………………………………………………………Ⅱ 目錄…………………………………………………………………………Ⅳ 圖目錄………………………………………………………………………Ⅵ 表目錄………………………………………………………………………Ⅶ Chapter 1 Background…………………………………………………………1 Chapter 2 Research motives and purposes………………………………… 4 Chapter 3 Materials and methods……………………………………………6 3.1 Tissue harvest……………………………………………………………6 3.2 Part 1………………………………………………………………………6 3.2.1 Decellularization methods…………………………………………7 3.2.2 Histology……………………………………………………………7 3.2.3 Scanning electron microscopy (SEM) ……………………………8 3.2.4 Determination of proteoglycan, collagen, and DNA content……8 3.2.5 Cytotoxicity of decellularized AF in transwell insert model……9 3.2.6 Mechanical test…………………………………………………10 3.2.7 Statistical analysis………………………………………………11 3.3 Part 2……………………………………………………………………12 3.3.1 Comparison of decellularization methods………………………13 3.3.2 Optimal Decellularization Method………………………………15 3.3.3 Biochemical assays………………………………………………16 3.3.4 Histology…………………………………………………………17 3.3.5 Scanning electron microscopy (SEM) …………………………18 3.3.6 Compression (indentation) test…………………………………18 3.3.7 α-Gal ELISA test sample processing ……………………………18 3.3.8 Quantification of α-Gal content…………………………………19 3.3.9 Cytotoxicity of decellularized AF in a transwell insert model…19 3.3.10 In vivo immunocompatibility study……………………………20 3.3.11 Statistical analysis………………………………………………21 Chapter 4 Result…………………………………………………………22 4.1 Part 1………………………………………………………………22 4.1.1 Histological analysis ……………………………………………22 4.1.2 Scanning electron microscopy (SEM) analysis…………………24 4.1.3 Biochemical assays………………………………………………25 4.1.4 Cytotoxicity assay………………………………………………26 4.1.5 Mechanical test…………………………………………………27 4.2 Part 2…………………………………………………………………27 4.2.1 Comparison of decellularization methods by biochemical assays…27 4.2.2 Histologic analysis ………………………………………………30 4.2.3 Scanning electron microscopy (SEM) analysis………………32 4.2.4 Biomechanical test………………………………………………33 4.2.5 Cytotoxicity assay………………………………………………34 4.2.6 In vivo immuno-compatibility study……………………………35 4.2.7 Quantification of α-Gal content…………………………………38 Chapter 5 Discussion……………………………………………………39 Chapter 6 Conclusion……………………………………………………49 Chapter 7 Reference……………………………………………………50 圖目錄 Figure 1 Parallel bone-AF-bone specimens………………………………………11 Figure 2 Histology of annulus fibrosus with H&E stain………23 Figure 3 Fresh and decellularized annulus fibrosus stained with alcian blue…23 Figure 4 Fresh and decellularized annulus fibrosus stained with sirius red……24 Figure 5 SEM images of annulus fibrosus…………………………………………24 Figure 6 Cytotoxicity studies of the control and decellularized AF………………26 Figure 7 Histology of annulus fibrosus with H&E stain…………………………30 Figure 8 Fresh and ODM AF stained with MT staining ………………31 Figure 9 Fresh and ODM AF stained with PAS staining ……………31 Figure 10 SEM images of annulus fibrosus………………………………………32 Figure 11 Biomechanical testing of ODM AF……………………………………33 Figure 12 Cytotoxicity studies of the control and ODM AF 34 Figure 13 In vivo immuno-compatibility studies at 7 days…36 Figure 14 In vivo immuno-compatibility studies at 14 days ……………………37 Figure 15 Quantification of α-Gal content ………………………………………38 表目錄 Table 1 GAGs, hydroxyproline, DNA contents of the fresh and decellularized annulus fibrosus……………………………………25 Table 2 Biomechanical properties of fresh and decellularized annulus fibrosus…………………………………………………………………27 Table 3 Results of Biochemical Assays of AF in Decellularization ……29 | |
| dc.language.iso | en | |
| dc.subject | 去細胞 | zh_TW |
| dc.subject | 組織工程 | zh_TW |
| dc.subject | 纖維環 | zh_TW |
| dc.subject | α-Gal表面抗原 | zh_TW |
| dc.subject | 椎間盤 | zh_TW |
| dc.subject | 脊椎 | zh_TW |
| dc.subject | tissue engineering | en |
| dc.subject | annulus fibrosus | en |
| dc.subject | intervertebral disc | en |
| dc.subject | decellularization | en |
| dc.subject | spine | en |
| dc.subject | α-Gal epitope | en |
| dc.title | 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 | zh_TW |
| dc.title | Fabrication and Properties of Decellularized Porcine Annulus Fibrosus for Tissue Engineering in Spine Surgery | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 曾永輝,孫瑞昇 | |
| dc.contributor.oralexamcommittee | 林?輝,陳志華 | |
| dc.subject.keyword | α-Gal表面抗原,纖維環,椎間盤,去細胞,脊椎,組織工程, | zh_TW |
| dc.subject.keyword | α-Gal epitope,annulus fibrosus,intervertebral disc,decellularization,spine,tissue engineering, | en |
| dc.relation.page | 53 | |
| dc.identifier.doi | 10.6342/NTU201700952 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-06-15 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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