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研究生:劉人豪
研究生(外文):Zen-Hao Liu
論文名稱: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
論文名稱(外文):The Use of Decellularization Technique to Prepare Spinal Annulus Fibrosus Scaffold in Repair of Intervertebral Disc
指導教授:黃義侑黃義侑引用關係
指導教授(外文):Yi-You Huang
口試委員:鍾次文許馨云黃意真
口試委員(外文):Tze-Wen ChungHSIN-YUN HsuYi-Cheng Huang
口試日期:2012-07-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:50
中文關鍵詞:纖維環去細胞化支架
外文關鍵詞:annulus fibrosusdecellularizationscaffold
相關次數:
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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.

目錄
目錄................................................................................................................................ I
圖目錄........................................................................................................................... III
表目錄...........................................................................................................................IV
摘要...............................................................................................................................V
Abstract .........................................................................................................................VI
第一章 序論.................................................................................................................. 1
1.1 脊椎.................................................................................................................. 1
1.2 椎間盤.............................................................................................................. 1
1.3 椎間盤退化症.................................................................................................. 2
1.4 椎間盤退化症的治療...................................................................................... 3
1.4.1 椎間盤退化症保守療法............................................................................... 3
1.4.2 椎間盤退化症手術治療............................................................................... 3
1.5 常見的移植方式.............................................................................................. 4
1.6 組織工程.......................................................................................................... 5
1.6.1 棚架(scaffold) ............................................................................................... 6
1.6.2 細胞(cell) ...................................................................................................... 6
1.6.3 生長訊息(signal) .......................................................................................... 7
1.7 纖維環的修復.................................................................................................. 7
1.8 去細胞化.......................................................................................................... 8
第二章 研究動機與目的............................................................................................ 10
第三章 實驗材料與方法............................................................................................ 12
3.1 實驗藥品........................................................................................................ 12
3.2 實驗儀器........................................................................................................ 14
3.3 纖維環(Anulus Fibrosus)之取得................................................................... 15
3.4 去細胞化(decellulazation) ............................................................................. 15
3.4.1 以Freeze-Thaw 的方法去細胞 ................................................................. 15
3.4.2 以不同的decellularization agent 去細胞 .................................................. 16
3.5 脫水試驗........................................................................................................ 17
3.6 動物實驗........................................................................................................ 18
3.7 掃描式電子顯微鏡觀測................................................................................ 19
3.8 石蠟組織切片................................................................................................ 19
3.9 蘇木紫-伊紅染色(H&E) ............................................................................. 19
3.10 Alcian Blue Stain ......................................................................................... 20
3.11 Picrosirius Red Stain .................................................................................... 21
3.12 膠原蛋白的定量:Hydroxyproline Assay ................................................. 21
II
3.13 GAG assay ................................................................................................... 22
3.14 DNA assay ................................................................................................... 23
3.15 紫外光吸光值分析...................................................................................... 24
3.16 細胞活性測試.............................................................................................. 25
3.16.1 NIH/3T3 繼代培養 ........................................................................... 25
3.16.2 MTS 測試 ......................................................................................... 26
3.17 Tensile Test ................................................................................................... 26
第四章 結果與討論.................................................................................................... 28
4.1 去細胞化的結果............................................................................................ 28
4.2 脫水試驗........................................................................................................ 32
4.3 去細胞前後纖維環組織切片分析................................................................ 34
4.4 去細胞前後纖維環之Hydroxyproline assay ............................................... 37
4.5 去細胞前後纖維環之GAG assay ............................................................... 38
4.6 去細胞前後纖維環之DNA 含量檢測 ........................................................ 39
4.7 去細胞化前後的纖維環之生物相容性測試................................................ 40
4.8 去細胞前後纖維環之機械性質.................................................................... 41
4.9 動物試驗........................................................................................................ 43
第五章 結論.............................................................................................................. 47
第六章 參考文獻........................................................................................................ 48

[1] Kandel R, Roberts S, Urban JP. Tissue engineering and the intervertebral disc: the
challenges. European spine journal : official publication of the European Spine
Society, the European Spinal Deformity Society, and the European Section of the
Cervical Spine Research Society. 2008;17 Suppl 4:480-91.
[2] Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, et al.
The human lumbar intervertebral disc: evidence for changes in the biosynthesis and
denaturation of the extracellular matrix with growth, maturation, ageing, and
degeneration. The Journal of clinical investigation. 1996;98:996-1003.
[3] Raghavan SS, Woon CY, Kraus A, Megerle K, Choi MS, Pridgen BC, et al. Human
Flexor Tendon Tissue Engineering: Decellularization of Human Flexor Tendons
Reduces Immunogenicity In Vivo. Tissue engineering Part A. 2011.
[4] Alini M, Li W, Markovic P, Aebi M, Spiro RC, Roughley PJ. The potential and
limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an
intervertebral disc-like matrix. Spine. 2003;28:446-54; discussion 53.
[5] Chang G, Kim HJ, Kaplan D, Vunjak-Novakovic G, Kandel R. Porous silk scaffolds
can be used for tissue engineering annulus fibrosus. European Spine Journal.
2007;16:1848-57.
[6] Masuda K, Imai Y, Okuma M, Muehleman C, Nakagawa K, Akeda K, et al.
Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc
height and structural changes in the rabbit anular puncture model. Spine.
2006;31:742-54.
[7] Costi JJ, Freeman BJ, Elliott DM. Intervertebral disc properties: challenges for
biodevices. Expert review of medical devices. 2011;8:357-76.
[8] Bron JL, Helder MN, Meisel HJ, Van Royen BJ, Smit TH. Repair, regenerative and
supportive therapies of the annulus fibrosus: achievements and challenges.
European spine journal : official publication of the European Spine Society, the
European Spinal Deformity Society, and the European Section of the Cervical Spine
Research Society. 2009;18:301-13.
[9] Masuda K, Lotz JC. New challenges for intervertebral disc treatment using
regenerative medicine. Tissue engineering Part B, Reviews. 2010;16:147-58.
[10] Korecki CL, Costi JJ, Iatridis JC. Needle puncture injury affects intervertebral disc
mechanics and biology in an organ culture model. Spine. 2008;33:235-41.
[11] Elliott DM, Yerramalli CS, Beckstein JC, Boxberger JI, Johannessen W, Vresilovic EJ.
The effect of relative needle diameter in puncture and sham injection animal models
of degeneration. Spine. 2008;33:588-96.
49
[12] Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs.
Biomaterials. 2006;27:3675-83.
[13] Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ
decellularization processes. Biomaterials. 2011;32:3233-43.
[14] Suto K, Urabe K, Naruse K, Uchida K, Matsuura T, Mikuni-Takagaki Y, et al.
Repeated freeze-thaw cycles reduce the survival rate of osteocytes in bone-tendon
constructs without affecting the mechanical properties of tendons. Cell and tissue
banking. 2010.
[15] Jung HJ, Vangipuram G, Fisher MB, Yang G, Hsu S, Bianchi J, et al. The effects of
multiple freeze-thaw cycles on the biomechanical properties of the human
bone-patellar tendon-bone allograft. Journal of orthopaedic research : official
publication of the Orthopaedic Research Society. 2011;29:1193-8.
[16] Huang H, Zhang J, Sun K, Zhang X, Tian S. Effects of repetitive multiple
freeze-thaw cycles on the biomechanical properties of human flexor digitorum
superficialis and flexor pollicis longus tendons. Clin Biomech (Bristol, Avon).
2011;26:419-23.
[17] Vavken P, Joshi S, Murray MM. TRITON-X is most effective among three
decellularization agents for ACL tissue engineering. Journal of orthopaedic research :
official publication of the Orthopaedic Research Society. 2009;27:1612-8.
[18] Deeken CR, White AK, Bachman SL, Ramshaw BJ, Cleveland DS, Loy TS, et al.
Method of preparing a decellularized porcine tendon using tributyl phosphate.
Journal of Biomedical Materials Research Part B: Applied Biomaterials.
2011;96B:199-206.
[19] Osti OL, Vernon-Roberts B, Fraser RD. 1990 Volvo Award in experimental studies.
Anulus tears and intervertebral disc degeneration. An experimental study using an
animal model. Spine. 1990;15:762-7.
[20] Fraser RD, Osti OL, Vernon-Roberts B. Intervertebral disc degeneration.
European spine journal : official publication of the European Spine Society, the
European Spinal Deformity Society, and the European Section of the Cervical Spine
Research Society. 1993;1:205-13.
[21] Chiang CJ, Cheng CK, Sun JS, Liao CJ, Wang YH, Tsuang YH. The effect of a new
anular repair after discectomy in intervertebral disc degeneration: an experimental
study using a porcine spine model. Spine. 2011;36:761-9.
[22] Kheir E, Stapleton T, Shaw D, Jin Z, Fisher J, Ingham E. Development and
characterization of an acellular porcine cartilage bone matrix for use in tissue
engineering. Journal of biomedical materials research Part A. 2011;99:283-94.
[23] Enobakhare BO, Bader DL, Lee DA. Quantification of Sulfated
Glycosaminoglycans in Chondrocyte/Alginate Cultures, by Use of
50
1,9-Dimethylmethylene Blue. Analytical Biochemistry. 1996;243:189-91.
[24] Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of
sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica et
biophysica acta. 1986;883:173-7.
[25] Giannini S, Buda R, Di Caprio F, Agati P, Bigi A, De Pasquale V, et al. Effects of
freezing on the biomechanical and structural properties of human posterior tibial
tendons. International orthopaedics. 2008;32:145-51.
[26] Huber M, Trattnig S, Lintner F. Anatomy, biochemistry, and physiology of
articular cartilage. Investigative radiology. 2000;35:573-80.
[27] Pickard J, Ingham E, Egan J, Fisher J. Investigation into the effect of proteoglycan
molecules on the tribological properties of cartilage joint tissues. Proceedings of the
Institution of Mechanical Engineers Part H, Journal of engineering in medicine.
1998;212:177-82.
[28] Brown BN, Valentin JE, Stewart-Akers AM, McCabe GP, Badylak SF. Macrophage
phenotype and remodeling outcomes in response to biologic scaffolds with and
without a cellular component. Biomaterials. 2009;30:1482-91.
[29] Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. Circulating
mitochondrial DAMPs cause inflammatory responses to injury. Nature.
2010;464:104-7.
[30] Cartmell JS, Dunn MG. Effect of chemical treatments on tendon cellularity and
mechanical properties. Journal of biomedical materials research. 2000;49:134-40.
[31] Galili U. The alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) in
xenotransplantation. Biochimie. 2001;83:557-63.

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