臺灣博碩士論文加值系統

本研究將聚乳酸—聚甘醇酸(Polylactic-co-glycolic acid , PLGA) 或PLGA-OH、Dioxane與水的三相系統充分混和後，再利用冷凍乾燥法製備3維多孔支架，並添加氫氧基磷灰石(Hydroxyapatite , HAP) 及奈米二氧化矽(Silica)來增加對材料的生物相容性與機械性質。研究中探討不同的Dioxane:Water的比例、冷浸溫度、冷浸時間以及不同成分的PLGA及濃度，對多孔型支架的型態、孔隙率、孔洞大小、機械性質、X-ray繞射分析以及降解試驗的影響。結果顯示，改變不同的反應條件，就會得到不同的孔洞大小，再依照我們所需要的結構去挑選適當的製備條件。當PLGA(w/v)及PLGA-OH(w/v)濃度為10%、 Dioxane:Water比例9:1，冷浸時間30分鐘及冷浸溫度為4℃時，孔洞會較為均一及穩固，所得到的孔洞大小分別約為107.2 μm與108.7 μm孔隙率高達82.4%與83.7%。添加HAP(w/v)及奈米Silica(v/v)的比例分別為4%及1.5%時，並不會對孔洞結構有任何的影響，使用DSC 測試，得知PLGA及PLGA-OH在製程中並無改變性質。由XRD可看出隨著添加物的增加，HAP的繞射強度會減弱。由壓縮測試可得知添加HAP並不會增加支架的強度，但加入奈米Silica後，卻可以有效的提升強度，壓縮模數18 MPa及最大壓縮應力約在32 MPa。在降解試驗中，添加HAP及奈米Silica可延緩降解速率至16個星期。本研究將UMR-106成骨細胞植覆於支架上，由Cell counts和SEM觀察，可得知支架隨著天數增加其細胞數量也隨之增加，並且可觀察出添加HAP會提升材料的生物相容性。

This study is to fabricate there dimensional porous scaffolds through freeze-drying method by completely mixing of either polylactic-co-glycolic acid (PLGA) or PLGA-OH, dioxane and water. It’s biocompatibility and mechanical properties could be enhanced by adding hydroxyapatite (HAP) and nano-SiO2. The different ratio of dioxane and water, dipping temperature, and the concentration of PLGA on the effect of morphology, porosity, pore size, and degradation test of scaffolds were investigated in this study. The SEM results showed that the pore size changed with different reaction conditions. The pore size of scaffolds which was dipped at 4°C for 30 minutes was uniform when the ratio of dioxane and water was 9：1. The pore size and porosity were 107.2 μm and 82.4% in 10 % PLGA(w/v), and those were 108.7 μm and 83.7% in 10 % PLGA-OH(w/v). The addition of HAP and nano-SiO2 would not change the structure of pore and synthesis procedure of the scaffolds. The XRD peak of HAP decreased when the amount of nano-SiO2 increased. The compression test results revealed that the strength of scaffolds increased with nano-SiO2, and the compression modulus was 18MPa. The degradation of scaffolds with HAP and nano size SiO2 addition could sustain up to 16 weeks.

中文摘要………………...………………………………………...……...I
目錄……………………...…………………………………...………….II
表目錄…………………...……………………………………...………VI
圖目錄…………………...…………………………………........……VIII
第一章 緒論………………………………..……………………………1
1-1人工骨……...…………………………………..………….…………1
1-2組織工程….……………………….…………………………………3
1-3生物可分解之高分子材料………………………………..…………7
1-4現代支架製備方法……………....………………………..…………9
1-5骨骼組織簡介……………....………………………..……………..12
第二章 文獻回顧……………………………………..………………15
2-1 國內外發展趨勢……………………………………..………….…15
2-2 研究動機與目的……………………………..………….…………18
第三章 利用冷凍乾燥法製備多孔形支架及物性分析……………...21
3-1材料與方法...……………………..…………………….………..…21
3-1-1藥品…....……………………………………………….…....……21
3-1-2實驗儀器……………...………………………………....…..……21
3-1-3實驗流程……...……………………………………………..……22
3-1-4以冷凍乾燥法製備PLGA、PLGA-OH及多孔形支架.....…..…23
3-1-5以冷凍乾燥法製備PLGA-OH/HAP 多孔形的複合支架………26
3-1-6以冷凍乾燥法製備PLGA-OH/HAP/Silica多孔形的複合架….26
3-1-7 SEM觀察切面形態……………..…………..………………....…27
3-1-8 DSC測試…………………………………………………………27
3-1-9孔洞大小的評估………….…………………………………..…..27
3-1-10孔隙率的評估…..……….….………………………….………..28
3-1-11 X光繞射分析(XRD)……………………………………………28
3-1-12機械性質測試….…………….……………..…………….…..…29
3-1-13降解試驗……………………………………...…………………29
3-1-14 UMR-106 細胞珠培養藥品配置……….……………………...30
3-1-15 UMR-106 細胞株植入支架…………..……………………….32
3-1-16細胞培養……………………….………………………………..22
3-1-17 Cell counts……..…….……………………………………….33
3-1-18 SEM觀察細胞生長情形………………………………………33
3-2結果與討論………….……………………………………..…….…34
3-2-1以冷凍乾燥法製備多孔形支架之探討……………………….…34
3-2-2 PLGA之SEM形態觀察….....................................................…...37
3-2-2-1有機溶劑Dioxane:Water的比例……..……..…….…..….….…37
3-2-2-2 PLGA濃度………………………………………….....….....…40
3-2-2-3冷浸溫度…..…………………………………………....…....…42
3-2-3 PLGA-OH之SEM形態觀察…………………..…..................…45
3-2-3-1有機溶劑Dioxane:Water的比例……..……………………...…45
3-2-3-2 PLGA-OH濃度…..………………………………………......…47
3-2-3-3冷浸時間…..…………………………………………....…....…49
3-2-4 PLGA-OH/HAP之SEM形態觀察………………………………53
3-2-5 PLGA-OH/HAP/Silica之SEM形態觀察……………………….56
3-2-6多孔隙支架DSC測試....…..……………………………...……...59
3-2-7 X光繞射分析…………….….………………….………………..61
3-2-8 PLGA-OH/HAP/Silica機械性質測試-壓縮測試………………..62
3-2-9降解試驗…………………………...……………………………..65
3-2-10 Cell counts…………………………………………..…………..66
第四章 結論…………………………………………………..……..…68
參考文獻……………………………………………………………..…69

1.王盈錦, 生物醫學材料, 合計圖書出版社, 民國91年
2.詹惠雯, 探討脈衝式電磁場促進類骨母細胞增生之機轉, 私立中原大學醫學工程學系, 民國92年
3.蔡明慈, 不同刺激時間單脈衝電磁場對造骨細胞骨髓細胞共同培養形成之類蝕骨細胞凋亡的影響, 私立中原大學醫學工程學系, 民國91年
4.黃仁波, 以冷凍式快速原型法製作組織工程之架, 國立中央大學機械工程研究所, 民國94年
5.葉郁仁, 應用於骨組織工程多孔性褐藻酸鹽/氫氧基磷灰石複合支架之研究, 嘉南藥理科技大學生物科技研究所,民國91年
6.吳侑峻, 利用聚乳酸-聚乙醇酸共聚物當作支架, 國立成功大學生物科技研究所, 民國92年
7.湯正明, 可降解軟骨細胞之架之製備與評估, 國立中興大學化工所, 民國88年
8.闕山璋, 生醫材料於組織器官修補與再生應用之展望, 材料會訊,7(1), 3-7,民國89年
9.杜逸虹, 物理化學, 三民書局出版社, 民國76年
10.蘇曉風, 複合材料學報, 第16卷 第2期, 民國88年
11.陳皇鈞, 材料科學與工程(上冊), 曉園出版社, 民國74年
12.Akao M., Aoki H., Kato K., “Mechanical properties of sintered hydroxyapatite for prosthetic application” J Mater Sci., 16, 809-812, 1981
13.Atkins P., “The Elements of Physical Chemistry 2nd ED”, Oxford University Press, 1996
14.Chu C. R, Coutts R. D., Yoshiokz M., Harwood F. L., Monosov A. Z. , Amiel D., “Articular cartilage repair using allogeneic perichondrocyte seeded biodegradable porous poly lactic acid (PLA): A tissue engineering study” J. Biomed. Mater. Res., 29, 1147-1154, 1995
15.Eaglstein W. H., Falanga V., “Tissue engineering and the development of Apligraf a human skin equivalent, ” Adv Wound Care 11, 1-8, 1998
16.Freed L. E., Marquis J. C., Nohria A., Emmanua J. I., Mikos A. G., Langer R., “Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers” J. Biomed. Mater. Res., 27, 11-23, 1993
17.Flahiff C. M., Blackwell A. S., Hollis J. M., Feldman D. S., “Analysis of a biodegradable composite for bone healing” J Biomed Mater Res., 32, 419-424, 1996
18.Gepstein R., Weiss R. E., Halle T., “Bridging large defects in bone by demineralized bone matrix in the form of a powder”. A radiographic, histological, and radioisotope-uptake study in rats," J Bone Joint Surg Am 69, 984-992 1987
19.Gerhart T. N., Renshaw A. A., Miller R. L., Noecker R. J., Hayes W. C., “ In vivo histologic and biomechanical characterization of a biodegradable particulate composite bone cement” J. Biomed Mater Res., 23, 1-16, 1989
20.Gugala Z., Gogdewski S., “In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold” J. Biomed. Mater. Res. 49, 183-191, 2000
21.Guobao W., Ma P. X., “Structure and properties of nano-hydroxyapatite /polymer composite scaffolds for bone tissue engineering” Biomaterials 25, 4749-4757, 2004
22.Higashi S., Yamamuro T., Nakamura T., Ikada Y., Hyon S. H., Jamshidi K., “Polymer-hydroxyapatite composites for biodegradable bone fillers” Biomaterials., 7, 183-187, 1986
23.Hua F. J., Park T. G., Lee D. S., “A facile preparation of highly interconnected macroporous poly(D,L-lactic acid-co-glycolic acid) (PLGA) scaffolds by liquid–liquid phase separation of a PLGA–dioxane–water ternary system”, Polymer 44, 1911–1920, 2003
24.Jarcho M., “Calcium phosphate ceramics as hard tissue prosthetics”, Clin. Orthop., 157, 259-278, 1981
25.Keskin D. S., Tezcaner A., Korkusuz P., Korkusuz F., “Collagen–chondroitin sulfate-based PLLA–SAIB-coated rhBMP-2 delivery system for bone repair” Biomaterials, 26, 4023-4034, 2005
26.Lo H., Kadiyala S., Guggino S. E., Leong K. W., “Poly(L-lactic acid) foams with cell seeding and controlled-release capacity” J Biomed Mater Res 30, 475-484 1996
27.Wu L., Ding J., “In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide)scaffolds for tissue engineering” Biomaterials 25, 5821-5830, 2004
28.Mikos A. G., Bao Y., Cima L. G., Ingber D. E., Vacant J. P., Langer R. “Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation” J Biomed Mater Res 27, 183-189, 1993
29.Miyamoto S., Takaoka K., “Bone induction and bone repair by composites of bone morphogenetic protein and biodegradable synthetic polymers” Ann. Chir. Gynaecol., 82, 69-75, 1993
30.Mathiowitz E., Amato G., Langer R., “Polyanhydride microspheres : Morphology and characterization of systems made by solvent removal” Polymer., 31, 547-555, 1990
31.Mikos G., Bao Y., Cima L. G., Ingber D. E., Vacanti J. P., Lange R., “Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation ” J Biomed Mater Res 27, 183-189, 1993
32.Mikos G., Sarakinos G., Leite S. M., Vacanti J. P. , and Langer R., “Laminated three-dimensional biodegradable foams for use in tissue engineering ” Biomaterials 145, 323-330, 1993
33.Mikos G., Lyman M. D., Freed R. Langer L. E., “Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture” Biomaterials 15, 55-58, 1994
34.Mooney J., Baldwin D. F., Suh N. P., Vacanti J. P., Langer R., “Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents” Biomaterials 17, 1417-1422, 1996
35.Metsger D. S., Driskell T. D., Paulsrud J. R., “Tricalcium phosphate ceramic, a resorbable bone implant : review and current status” J. Am. Dent. Assoc., 105, 1035-1038, 1982
36.Nielsen F. F., Karring T., Gogolewski S., “ Biodegradable guide for bone regeneration : polyureathane membranes tested in rabbit radius defects” Acta Orthop Scand., 63, 66-69, 1992
37.Nam Y. S., Yoon J. J., Park T. G., “A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive” J Biomed Mater Res 53, 1-7, 2000
38.Nakafuku C., Takehisa S. Y., “Glass transition and mechanical properties of PLLA and PDLLA-PGA copolymer blends” J. Appl. Polym. Sci., 93, 2164-2173, 2004
39.Solheim E., Pinholt E. M., G. Bang., Sudmann E., “Regeneration of calvarial defects by a composite of bioerodiable polyorthoester and demineralized bone in rats” J Neurosurg., 76, 275-279,1992
40.Schugens C., Maquet V., Grandfils C., Jerome R., Teyssie P., “Polylactide macroporous biodegradable implants for cell transplantation. I. Preparation of polylactide foams by solid-liquid phase separation” (to appear)
41.Sopyan I., Mel M., Rameshc S., Khald K.A., “Porous hydroxyapatite for artificial bone applications”, Science and Technology of Advanced Materials, 8, 116-123, 2007
42.Shin K. C., Kim B. S., Kim J. H., Park T. G., Nam J. D., Lee D. S., “A facile preparation of highly interconnected macroporous PLGA scaffolds by liquid-liquid phase separation II”, Polymer, 46, 3801-3808, 2005
43.Tiedeman J. J., Garvin K. L., Kile T. A., Connolly J. F., “The role of a composite, demineralized bone matrix and bone marrow in the treatment of osseous defects” Orthopedics , 18, 1153-1158 1995
44.Thomson R. C., Yaszemski M. J., Powers J. M., Mikos A.G.. “Fabrication of biodegradable polymer scaffolds to engineer trabecular bone” J. Biomater. Sci., Polym Edn., 7, 23-38, 1995
45.Thomson R. C., Yaszemski M. J., Powers J. M. Mikos A.G., Murphy R. M., Bernstein H., Peppas N. A., “Biomaterials for Drug and Cell Delivery” Pittsburgh, Materials Research Society, 33-40, 1994
46.Thomson R. C., Wake M. G., Yaszemski M. J., Mikos A. G., “Biodegradable Polymer scaffolds to regenerate organs” In: Peppas NA, Langer RS, eds. Advances in Polymer Science., 122, 245-274, 1995
47.Vacanti C. A., Kim W., Upton J., “ Tissue engineered growth of bone and cartilage” Transplant Proc., 25, 1019-1021, 1993
48.Vert M., Manduit J., Li S., “Biodegradation of PLA/GA polymers : Increasing complexity”, Biomaterials, 15, 1209-1213, 1994
49.Verheyen C. C ., Klein C. P., Blieck-Hogervorst J. M. , Wolkevan J. G., Blitterswijn C. A., “Evaluation of hydroxyapatite/poly(l-lactide) composites: physico-chemical properties”, J Mater Sci:Mater Med., 4, 58-65, 1993
50.Wang H. T. , Palmer H., Linhardt R. J., Flanagan D. R., Schmitt E., “Degradation of poly(ester) microspheres” Biomaterials 11, 679-685, 1990
51.Wang Y. C., Lin M. C., Wang D. M., Hsieh H. J., “Fabrication of a novel porous PGA-chitosan hybrid matrix for tissue engineering” Biomaterials, 24, 1047-1057, 2003
52.Whang K., Thomas C. H., Healy K. E., “A novel method to fabricate bioabsorbable scaffolds”, Polymer 30, 837~742, 1995
53.Wilson K. S., Zhang K., Antonucci J. M., “Systematic variation of interfacial phase reactivity in dental nanocomposites”, Biomaterials, 26, 5095-5103, 2005
54.Zhang R., Ma P. X., “Poly(-hydroxy acids)/hydroxyapatite porous composites for bone – tissue engineering. I. Preparation and morphology”, J. Biomed Mater Res., 44, 446-455, 1999