臺灣博碩士論文加值系統

本實驗主要是利用合成的方式將天然的高分子與合成高分子相結合，以得到一具有親、疏水的兩性高分子接枝共聚物CSn-PLLA，並應用在藥物釋放及組織工程領域，以及探討材料對細胞生長的影響。
在合成方面，將軟骨細胞外間質的重成份之一Chondroitin sulfate (CS)上的OH官能基與L-lactide單體進行開環聚合反應，以得到接枝型的兩性共聚物。其中，CS在共聚物中的含量是以NMR來進行定量，在實驗的過程中，其在共聚物中的含量會隨進料比例、反應條件而改變，大致介於1.1%-15.4%之間。在材料性質方面，隨不同比例的材料進行分析(IR、DSC、XRD、酵素水解性質)，其材料的特性會隨CS在共聚物中的含量而有所改變，因此在實驗中也加以探討。
在藥物釋放應用方面，材料的特性對應用在藥物釋放有相當影響，因此在本研究主要是探討在實驗中所合成出來的兩性接枝型共聚物其在水溶液中的聚集行為，並量測材料的臨界聚集濃度(Critical aggregatiob concentration)大致介於4.3×10-3-9.1×10-3之間、粒子在水溶液中的平衡常數(1.41×106-3.65×105)、粒子在水溶液中具有良好的穩定性以及觀察粒子的形態(AFM、FE-SEM)，且將材料進行細胞毒性測試（MTT assay），觀察材料對細胞生長的影響，也利用共焦顯微鏡(Confocal microscopy)觀察到粒子能對細胞進行非定點式的穿透。同時，在本實驗中，也分別對親、疏水性藥物進行包覆及釋放，觀察材料對不同性質的藥物包覆的影響。
在組織工程應用方面，由於CS是軟骨細胞外間質的重要成份之一，將其導入合成材料中，使材料做為細胞培養用支架時能具有更類似細胞外間質的特性，以供軟骨細胞生長。在實驗中，將合成的兩性高分子CSn-PLLA與PLLA混摻，並以鹽析及溶劑揮發的方式做成具內部連通的多孔性支架，孔隙度可達85 %以上，並由XRD分析可知材料的混摻相當均勻，以及具有足夠的機械強度及親水性可供軟骨細胞貼覆生長。在細胞實驗方面，將軟骨細胞直接種在多孔性基材上，觀察細胞在基材上生長良好，在進行細胞長期培養，觀察軟骨細胞在基材上的生長狀況並已正常表現，包括以切片染色的方式以可看出細胞已生長出正常軟骨的軟骨腔(lacuna)結構、以及由軟骨細胞分泌細胞外間質的定量分析可知軟骨細胞已分泌出大量的新生細胞外間質(包括Collagen、GAGs)，並由RT-PCR的方式觀察細胞基因表現之情形也與天然軟骨的表現相符合(GAPDH、Type II collagen、aggrecan)，已表現出大量的aggrecan以及正常軟骨分泌的第二型膠原蛋白，因此藉由細胞生長之結果來評估所合成的材料對軟骨細胞具有促進其再生成組織的能力。

In this investigation, new biodegradable brush-like amphiphilic copolymers were synthesized by ring opening polymerization. Poly(L-lactide) (PLLA) was grafted onto chondroitin sulfate (CS), which is one of the physiologically significant specific glycosaminoglycans (GAGs), using a tin octanoate [Sn(Oct)2] catalyst in DMSO. The hydroxyl groups of the chondroitin sulfate were used as initiating groups. These functional groups enable specific mucoadhesion or receptor recognition. The degree of substitution (DS), the degree of polymerization (DP) and the chondroitin sulfate content (from 1.1% to 15.4%) were analyzed by 1H-NMR. The characteristics of these grafted copolymers, including the structure, the thermal properties and biodegradability etc., were examined with respect to CS content. Meanwhile, the amphiphilic core (PLLA) - corona (CS) nanoparticles, with size smaller than 200 nm, was examined by dynamic light scattering (DLS). Zeta potential analysis exhibited the value in the range -18.3 to -49.4 mV. The morphologies of the nanoparticles were observed by field-emission scanning electron microscopy (FE-SEM). The nanoparticles with lower cytotoxicity were examined by MTT assay. Furthermore, the in vitro BSA release kinetics of those CSn-PLLA nanoparticles was also determined in this study.
Novel polymeric amphiphilic copolymers were synthesized using chondroitin sulfate (CS) as a hydrophilic segment and poly(L-lactide) (PLLA) as a hydrophobic segment. Micelles of those copolymers were formed in an aqueous phase and were characterized by 1H NMR spectra, fluorescence techniques, dynamic light scattering (DLS), atomic force microscopy (AFM) and confocal microscopy. Their critical aggregation concentrations (CAC) are in the range of 0.0043 to 0.0091 mg/mL at 25oC. The partition equilibrium constants, Kv, of the pyrene probe in the aqueous solution were from 3.65×105 to 1.41×106 at 25 oC. The mean diameters of the micelles were below 200 nm, and their sizes were narrowly distributed. The AFM images revealed that the self-aggregates were spherical. Additionally, the CSn-PLLA micelles can efficiently transport within the cells via endocytosis as observed from confocal microscopy.
In cartilage tissue engineering, the graft copolymer was blended with poly(L-lactide) (PLLA) to form biomimic porous scaffolds. Natural CS was introduced into the polyester matrix to promote the proliferation of cells. Three-dimensional sponge-like scaffolds were fabricated by a combination of salt leaching and solvent casting methods. The morphology of the scaffolds was observed with scanning electron microscopy (SEM) with average pore size between 50~250 μm and its porosity was high (>85%). Compression analysis indicated that the mechanical properties of the scaffold were adequate to support the proliferation of cells. The hydrophilicity increased with increasing the copolymer content in the blend, as determined by measuring the contact angle. H&E, Masson and Safranin-O staining showed that cells formed a chondro-tissue gradually. Histological results revealed that abundant cartilaginous matrices surrounded spherical chondrocytes in the center of the explants. Chondrocytes cultured in this ECM-like scaffold maintained a round morphology phenotype, characterized by a significant quantity of extracellular matrix of sulfated glycosaminoglycans and collagens. Additionally, phenotypic gene expression (RT-PCR) indicated that chondrocytes expressed transcripts that encoded type II collagen and aggrecan, and generated sulfated glycosaminoglycans.

目 錄
摘要-----------------------------------------------------------------------------------------------I
一.緒論
1-1 研究背景與目的-----------------------------------------------------------------------1
1-2 藥物釋放應用 -----------------------------------------------------------------------3
1-3 組織工程應用 -----------------------------------------------------------------------5

二.文獻回顧
2-1 生醫用高分子材料 ----------------------------------------------------------------8
2-1-1生物可分解與非生物可分解材料-----------------------------------------8
2-1-2 天然高分子(Natural polymers) --------------------------------------9
2-1-2-1 透明質酸（Hyaluronic acid）----------------------------------------9
2-1-2-2 膠原蛋白（Collagen）---------------------------------------------------11
2-1-2-3 褐藻酸鹽（Alignate）---------------------------------------------------13
2-1-2-4 幾丁質（Chitin）與幾丁聚醣（Chitosan）------------------------14
2-1-2-5 明膠 ------------------------------------------------------------15
2-1-2-6 軟骨素（Chondroitin Sulfate） -------------------------------------15
2-1-3 人工合成材料（Synthetic polymers） ----------------------------------17
2-1-3-1 聚酯類高分子 （Polyester） ---------------------------------------18
2-1-3-2 聚醯胺 （Polyamide）--------------------------------------------------22
2-1-3-3 聚酸酐 （Polyanhydrides）-------------------------------------------23
2-1-3-4 聚醚類(Polyethers) --------------------------------------------------24
2-1-3-5聚丁基氰丙烯酸酯------------------------------------------------------24
2-2影響降解因素--------------------------------------------------------------------------25
2-2-1 高分子形態 ---------------------------------------------------------------- 26
2-2-2 分子量及分子量分佈--------------------------------------------------------27

Part Ⅰ. Drug Delivery
2-3 藥物傳輸系統 ----------------------------------------------------------------- 35
2-4高分子藥物載體之製備 ----------------------------------------------------------39
2-5藥物載體經細胞的吞噬機制 ------------------------------------------------42
2-6奈米粒子於癌症之應用 -------------------------------------------------------44
2-7奈米粒子（conventionalnanoparticles）----------------------------------------45
2-8具隱蔽性之奈米粒子（stealth nanoparticles）-------------------------------46
2-9具癌細胞辦識性之奈米粒子-----------------------------------------------------47

三.研究動機與目的 -------------------------------------------------------------48
四.實驗藥品、設備及步驟
4-1 實驗藥品 -----------------------------------------------------------------------51
4-2 實驗方法與步驟 ----------------------------------------------------------------52
4-2-1. CSn-PLLA之合成與鑑定 -------------------------------------------------52
4-2-2. PLLA之合成 --------------------------------------------------------------53
4-3 結構鑑定與分析 ----------------------------------------------------------------54
4-3-1 核磁共振（Nuclear magnetic resonance, 1H-NMR） ----------54
4-3-2 紅外線光譜儀（FT-IR）------------------------------------------------------54
4-3-3 掃瞄式熱差分析儀(DSC) ---------------------------------------------54
4-3-4 X光繞射分析 ------------------------------------------------------------------54
4-3-5 GPC分子量分析---------------------------------------------------------------54
4-3-6 粒子的製備（Particle preparation） -------------------------------------54
4-3-7 粒徑（size）測試及表面電位（zeta potential）------------------------55
4-3-8 臨界聚集濃度（critical aggregation concentration, CAC）-------55
4-3-9 原子力顯微鏡(Atomic force microscopy, AFM) --------------55
4-3-10 場發射式電子顯微鏡（Field emission scanning electron microscopy, FE-SEM） --------------------------------------------------------55
4-4 細胞毒性測試（Toxicity testing） ----------------------------------------------56
4-5 細胞型態觀察（cells morphology） ------------------------------------------56
4-6 體外藥物釋放模擬（In-vitro drug release）---------------------------------57
4-6-1 Bovine serum albumin(BSA) 包覆 -----------------------------------57
4-6-2 BSA釋放--------------------------------------------------------------------------57
4-6-3 Flutamide 包覆 ------------------------------------------------------------58
4-6-4 Flutamide 釋放 ------------------------------------------------------------58
4-7 Endocytosis of Micelles-------------------------------------------------------------58
4-8 材料水解測試 -----------------------------------------------------------------------59
4-8-1 含水量變化及重量損失 -------------------------------------------------59
4-8-2 分子量變化----------------------------------------------------------------------59
4-8-3 表面型態 ----------------------------------------------------------------------59

五.結果與討論
5-1 CSn-PLLA之合成與結構鑑定 ------------------------------------------------60
5-2 熱性質分析 -------------------------------------------------------------------------67
5-3 XRD分析 -------------------------------------------------------------------------68
5-4 奈米粒子之製備（Preparation of nanoparticles） ------------------------69
5-5 臨界聚集濃度（Critical aggregation concentration, CAC） -----------72
5-6 粒徑（Size）及表面電位(Zeta potential) -------------------------------------79
5-7 奈米微胞的微胞化行為 ---------------------------------------------------------82
5-8 奈米粒子表面型態（Surface morphology of nanoparticle）-----------84
5-9 奈米粒子之穩定性測試 ---------------------------------------------------------85
5-10 細胞毒性測試（Toxicity testing） --------------------------------------------87
5-11 材料水解特性 --------------------------------------------------------------------89
5-11-1 含水量（Water uptake）及重量損失(Weight loss) ----------------89
5-11-2 材料水解表面型態 （Surface morphology）-----------------------93
5-12 細胞穿透實驗（Confocal microscopy）-------------------------------------95
5-13 體外藥物釋放模擬----------------------------------------------------------------96
5-13-1 親水性藥物 (BSA) 包覆 ---------------------------------------------97
5-13-2 疏水性藥物(Flutamide)包覆 ------------------------------------100

六、結論------------------------------------------------------------------------------------103


Part Ⅱ. Tissue Engineering
Part II 組織工程簡介 ----------------------------------------------------107
二.文獻回顧、相關原理及應用
2-1 高分子在組織工程扮演的角色 -------------------------------------------113
2-2 組織工程應用在軟骨組織 --------------------------------------------------115
2-3 軟骨組織生理學 ------------------------------------------------------------------119
2-3-1 軟骨組織的主要成份 -------------------------------------------------119
2-3-2 軟骨細胞 ----------------------------------------------------------------------119
2-3-3 軟骨細胞外間質-蛋白質多醣(proteoglycans) -------------------120
2-3-4 軟骨細胞外間質-膠原蛋白(collagen) ----------------------------121
2-3-5 關節透明軟骨組織生理學 --------------------------------------------121
2-3-6 關節軟骨的生物力學及機械功能 -----------------------------------123
2-3-7 軟骨細胞的生長 --------------------------------------------------------125
2-4 目前關節軟骨受損的療法及缺點 ------------------------------------126
2-4-1 Drilling、Microfracture和Abrasion-------------------------------------126
2-4-2 異體移植(allograft) ------------------------------------------------------127
2-4-3 自體移植(autograft or mosaicplasty) -------------------------------127
2-5 組織工程應用的支架設計與要求 -----------------------------------128
2-5-1 具有生物相容性（Biocompatibility） --------------------------------129
2-5-2 基材孔隙度及型態（Pore size and morphology）-----------------129
2-5-3 具生物可分解之特性（Biodegradability） -------------------------129
2-5-4 適當機械強度及加工性（Mechanical properties and processibility）-------------------------------------------------------------------129
2-6 支架的製備方法（Manufacture of polymer scaffolds）----------------130

三.研究動機與目的 ---------------------------------------------------------132

四. 實驗藥品、設備及實驗步驟
4-1 實驗藥品------------------------------------------------------------------------------134
4-2 實驗方法與步驟 ----------------------------------------------------------------135
4-2-1 多孔性支架之製備 --------------------------------------------------------135
4-2-2 親水性測試 ---------------------------------------------------------------135
4-2-3 孔隙度（Porosity）------------------------------------------------------------135
4-2-4 孔徑大小（Pore size）-------------------------------------------------------136
4-2-5 機械性質（Mechanical property）---------------------------------------136
4-2-5-1 材料性質 ------------------------------------------------------------136
4-2-5-2 支架機械性質 -----------------------------------------------------136
4-3 細胞培養溶劑配製----------------------------------------------------------------137
4-4 細胞培養儀器及器材 ---------------------------------------------------------137
4-5 細胞培養前準備 ----------------------------------------------------------------137
4-5-1 DMEM培養基配製 --------------------------------------------------------137
4-5-2 PBS配製 -----------------------------------------------------------------------138
4-5-3 細胞培養用支架前處理 -------------------------------------------------138
4-6 軟骨細胞分離-----------------------------------------------------------------------138
4-7 細胞培養（In vitro）----------------------------------------------------------------139
4-8 細胞毒性分析 (MTT分析)----------------------------------------------------139
4-9 DNA assay ------------------------------------------------------------------------140
4-10 醣胺素分析 -----------------------------------------------------------------------141
4-11 膠原蛋白分析（Collagen assay） -------------------------------------------142
4-12 掃瞄式電子顯微鏡（SEM）觀察 -------------------------------------------143
4-13 組織染色切片 -------------------------------------------------------------------144
4-14 細胞RNA分離(RNA Isolation)和反轉錄聚合脢鏈鎖反應(Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) )------------------------145
4-15 支架降解速率測試 ------------------------------------------------------------146

五.結果與討論
5-1 孔性支架製備（Preparation of porous scaffolds） -----------------------148
5-2 材料混摻之相容性 （Compatiability）-------------------------------------149
5-3 支架性質分析-----------------------------------------------------------------------152
5-3-1 親疏水性 (Hydrophilic property) ------------------------------152
5-3-2 支架機械性質分析 -----------------------------------------------------153
5-4 細胞培養------------------------------------------------------------------------------154
5-5 表面型態分析-----------------------------------------------------------------------155
5-6 細胞種植密度對細胞生長之影響-------------------------------------------159
5-7 細胞培養後支架機械性質分析 -------------------------------------------161
5-8 利用共焦顯微鏡觀察細胞在支架上分佈 -----------------------------162
5-9 軟骨組織化學定性分析---------------------------------------------------------164
5-9-1 蘇木紫-伊紅染色 （H＆E）----------------------------------------------164
5-9-2 膠原蛋白染色 (Masson) -------------------------------------------166
5-9-3 醣胺素GAGs染色 (Safranin-O)--------------------------------------167
5-9-4 免疫化學染色- Type II Collagen -----------------------------------168
5-10 軟骨組織細胞外間質定量分析 -------------------------------------------169
5-10-1 膠原蛋白(Collagen)分析------------------------------------------------169
5-10-2 醣胺素(GAGs)分析-------------------------------------------------------170
5-11 軟骨組織基因表現 (RT-PCR) ---------------------------------------------171
5-11-1 不同培養條件---------------------------------------------------------------172
5-11-2 不同培養時間---------------------------------------------------------------173

六、結論------------------------------------------------------------------------------------175

參考文獻 --------------------------------------------------------------------------------177

作者簡介----------------------------------------------------------------------------------187

個人著作 ---------------------------------------------------------------------------------188











圖 目 錄
Part I. Drug delivery
圖2-1 透明質酸(Hyaluronic acid)結構圖。--------------------------------------10
圖2-2 膠原蛋白在顯微鏡下呈現由三條螺旋狀聚胜肽鏈所組成的帶狀結構。---------------------------------------------------------------------------12
圖2-3. 褐藻酸鹽（Alignate）結構圖。-----------------------------------------------13
圖2-4. 幾丁質與幾丁聚醣結構圖。------------------------------------------------15
圖2-5. Chondroitin sulfate結構。-----------------------------------------------------16
圖2-6. 陽離子開環聚合反應機制圖-----------------------------------------------20
圖2-7. 以Sn(Oct)2為觸媒聚合PLLA的反應機構----------------------------21
圖2-8. 常使用的芳香族（CPP）與脂肪族（SA）聚酸酐單體。-------------24
圖2-9. Drug concentrations at site of therapeutic action after delivery as a conventional injection (thin line) and as a temporal controlled release system (bold line). ----------------------------------------------30
圖2-10. Drug delivery from an ideal distribution controlled release system. Bold line: drug concentrations at site of therapeutic action. Thin line: systemic levels at which side effects occur. --------------31
圖2-11. Examples of mechanisms of temporal controlled release.--------33
圖2-12. 藥物經皮膚吸收示意圖。--------------------------------------------------37
圖2-13. 奈米藥物載體以內吞作用（endocytosis）由細胞外進入細胞內之示意圖。-------------------------------------------------------------------42
圖4-1. 實驗所用到的化學藥品結構。---------------------------------------------51
圖4-2. CSn-PLLA之合成示意圖。---------------------------------------------------53
圖4-3. PLLA之合成示意圖。----------------------------------------------------------53
圖5-1. Reaction scheme for the synthesis of the grafted copolymers ----61
圖5-2. CS-PLLA的1H-NMR圖譜。---------------------------------------------------62
圖5-3. PLLA的1H-NMR圖譜。--------------------------------------------------------62
圖5-4. CS-PLLA的IR光譜圖。---------------------------------------------------------65
圖5-5. PLLA的IR光譜圖。--------------------------------------------------------------66
圖5-6. PLLA, CS及接枝共聚物的DSC熱分析圖。---------------------------68
圖5-7. CSn-PLLA之XRD光譜分析--------------------------------------------------69
圖5-8. 1H NMR spectroscopy of CS15.4-PLLA nanoparticles in (a)d6-DMSO and (b)D2O. --------------------------------------------------71
圖5-9. The schematic structure of nanoparticle of amphiphilic copolymer of chondroitin sulfate and poly(L-lactide) in an aqueous medium.--------------------------------------------------------------------------72
圖5-10.不同高分子濃度下pyrene在波長339 nm下吸收度變化情形。-76
圖5-11. 不同高分子濃度下pyrene吸收強度變化比值圖，其中兩段直線交會點即為臨界聚集濃度。----------------------------------------------78
圖5-12.不同含量CS對臨界聚集濃度的變化。---------------------------------78
圖5-13. CSn-PLLA在不同濃度下，螢光吸收強度變化（F-Fmin）/(Fmax-F)圖。------------------------------------------------------------------------------------79
圖5-14. CSn-PLLA奈米粒子的粒徑及粒徑分佈隨CS含量變化圖。----81
圖5-15. CS15.4-PLLA的粒徑分佈。---------------------------------------------------81
圖5-16. 表面電位隨CS含量之變化。----------------------------------------------82
圖5-17. 奈米微胞平均粒徑與粒徑分佈隨溶液組成之變化。------------83
圖5-18. CS15.4-PLLA奈米粒子的AFM影像。------------------------------------84
圖5-19. CS11.5-PLLA奈米粒子在FE-SEM下之表面型態。 ----------------85
圖5-20. 不同保存條件下之粒徑分佈。--------------------------------------------87
圖5-21. CSn-PLLA奈米粒子對Caco-2細胞毒性測試。-----------------------88
圖5-22. 毒性測試前後細胞型態。---------------------------------------------------88
圖5-23. CSn-PLLA共聚物在水解過程中的含水量變化圖。---------------91
圖5-24. PLLA共聚物在水解過程中的重量變化。-----------------------------91
圖5-25. CSn-PLLA共聚物在水解過程中的分子量變化。-------------------92
圖5-26. 不同水解時間的CSn-PLLA共聚物IR圖譜。-------------------------94
圖5-27. CS11.5-PLLA水解過程的表面型態變化。------------------------------94
圖5-28. 奈米粒子隨時間穿透細胞之共焦顯微鏡影像。-------------------96
圖5-29. BSA在紫外光下之吸收光譜隨濃度變化情形。--------------------98
圖5-30. BSA在不同PH值下之校正曲線。----------------------------------------99
圖5-31. 不同包覆方式之BSA藥物釋放曲線。---------------------------------99
圖5-32. Flutamide的UV吸收光譜。 ----------------------------------------------101
圖5-33. Flutamide的藥物釋放校正曲線。---------------------------------------101
圖5-34. 兩性接枝共聚物的藥物釋放曲線。 ---------------------------------102


Part II. Tissue engineering
圖2-14. 組織工程利用體外細胞培養的方式發展組織化器官替代物。-------------------------------------------------------------------------------109
圖2-15. 組織工程三要素。-----------------------------------------------------------110
圖2-16. 各種不同形式的高分子在組織工程上的應用。-----------------115
圖2-17. 關節軟骨。----------------------------------------------------------------------118
圖2-18. 軟骨組織的結構圖。-------------------------------------------------------123
圖2-19. Mosaicplasty手術，將非受外力部位的Osteochondral graft移植到受力缺損處。------------------------------------------------------------128
圖5-35. CSn-PLLA與PLLA混摻支架的表面。-------------------------------149
圖5-36. X-ray diffraction patterns of PLLA, CS11.5-PLLA, CS and PLLA/CS11.5-PLLA blend materials. ----------------------------151
圖5-37. SEM pictures: (a) A simple blend of CS and PLLA; (b) The CS/CSn-PLLA/PLLA composition exhibit a much better interfacial adhesion.--------------------------------------------------------151
圖5-38. 不同混摻比例的接觸角變化。-----------------------------------------152
圖5-39. 不同CS11.5-PLLA與PLLA混摻的細胞毒性分析。---------------155
圖5-40. 細胞在支架上生長前後比較（外觀）：左為培養前，右為培養四週後。--------------------------------------------------------------------------157
圖5-41. 軟骨細胞在支架PLLA/CS11.5-PLLA (20/80 wt/wt)（A）（B）及PLLA支架（C）上生長情形（2 days）。--------------------------------------------------------------------------------------157
圖5-42. 老鼠軟骨細胞在支架上培養四星期之表面結構。（A）、（B）為上表面;（C）、（D）為下表面;（E）、（F）為內部結構。--------------------------------------------------------------------------------------158
圖5-43. 不同種植密度之細胞生長變化圖。----------------------------------160
圖5-44. 不同種植密度之H＆E切片染色。 ----------------------------------161
圖5-45. Compression modulus of the scaffolds after different cells culture time intervals. -----------------------------------------------162
圖5-46. 利用共焦顯微鏡觀察細胞在支架上之分佈。--------------------163
圖5-47. 不同深度的共焦顯微鏡分析，由左至右，以及由上至下，為表面到深度200μm之細胞分佈圖。---------------------------------164
圖5-48. Histochemical examination of the cartilage-like tissue formed at various culture intervals on blend scaffold (PLLA/CS11.5-PLLA=20:80).-------------------------------------------165
圖5-49. Masson trichrome staining of the scaffolds cultured in vitro after 1, 2 and 4 weeks, respectively. -----------------------------------------166
圖5-50. Safranin-O staining of the scaffolds cultured in vitro after 1, 2, and 4 weeks, respectively. -----------------------------------------------167
圖5-51. . Immunohistological localization of cartilage-specific ECM molecules (positive staining for type II collagen) at various intervals during culture (PLLA/CS11.5-PLLA=20:80).----------168
圖5-52. Collagen content analysis of scaffold coacervate-chondrocyte constructs as a function of time. ---------------------------------------170
圖5-53. GAGs measurement for scaffolds of different composition at various culture intervals. -------------------------------------------------171
圖5-54. Expression of cartilage-specific genes was analyzed by RT-PCR. Total RNA was analyzed for type I collagen, type II collagen, aggrecan, with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as the control. ----------------------------------------------------------------------173
圖5-55. Expression of cartilage-specific genes was analyzed by RT-PCR with different cultural intervals. ---------------------------------------174








































表 目 錄
表2-1. Functions and applied regions of biodegradable polymers.----------8
表2-2. 常見的生物可分解性聚酯及其結構。----------------------------------22
表2-3. 影響生物分解性高分子降解因素。--------------------------------------26
表5-1. 1H-NMR比較。--------------------------------------------------------------------63
表5-2. 接枝共聚物的進料與回收結果。------------------------------------------64
表5-3. CS-PLLA的IR特徵吸收峰位置。-------------------------------------------66
表5-4. PLLA的IR特徵吸收峰位置。---------------------------------------------66
表5-5. 不同組成CS-PLLA之微胞特性。------------------------------------------79
表5-6. 支架的組成及特性。---------------------------------------------------------153

參 考 文 獻
[1] A.S. Sawheney, C.P. Pathak, and J.A. Hubbel, Macromolecules 26 (1993) 581-587.
[2] J.B. Park, R.S. Lakes “BIOMATERIALS An Introduction 2nd ed.” Plenum Press, New York (1992) P.2-3.
[3] J.D. Abraham, K. Joseph, D. Wiseman, “Handbook of biodegradable polymers” harwood academic publishers, 1997.
[4] T. Livingston, P. Ducheyne, J. Garino, Journal of Biomedical Materials Research. 62 (2002) 1-13.
[5] G.H. Altman, R.L. Horan, H.H. Lu, J. Moreau, I. Martin, J.C. Richmond, D.L. Kaplan, Biomaterials 23 (2002) 4131-4141.
[6] L.A. Solchaga, J. Gao, J.E. Dennis, A. Awadallah, M. Lundberg, A.I. Caplan, V.M. Goldberg, Tissue Engineering 8 (2002) 333-347.
[7] K. Kakehi, M. Kinoshita, S.I. Yasueda, Journal of Chromatography B 797 (2003) 347-355.
[8] K. Mabuchi, T. Obara, K. Ikegami, T. Yamaguchi, T. Kanayama, Clinical Biomechanics 14 (1999) 352-356.
[9] E. Bell, Journal of Biomechanical Engineering. 113 (1991) 113.
[10] J.M. Pachence, Journal of Biomedical Materials Research. 33 (1996) 35-40.
[11] K. Kühn, Matrix Biology 14 (1994) 439-445.
[12] L. Shapior, S. Cohen, Biomaterials 18 (1997) 583.
[13] W.C. Chen, S.S. Cheng, Water Science and Technology 34 (1996) 51-58.
[14] S.V. Madihally, H.W.T. Mattew, Biomaterials 20 (1999) 1133-1142.
[15] Y. Okamoto, R. Yano, K. Miyatake, I. Tomohiro, Y. Shigemasa, S. Minami, Carbohydrate Polymers 53 (2003) 337-342.
[16] R.A.A. Muzzarelli, N. Frega, M. Miliani, C. Muzzarelli, M. Cartolari, Carbohydrate Polymers 43 (2000) 263-268.
[17] D.K.F. Meijer, G. Molema, F. Moolenaar, D. De Zeeuw, P.J. Swart, Journal of controlled release 39 (1996) 163-172.
[18] H.Y. Park, I.H. Song, J.H. Kim, W.S. Kim, International Journal of Pharmaceutics 175 (1998) 231-236.
[19] J. Wang, Y. Tabata, D. Bi, K. Morimoto, Journal of controlled release 73 (2001) 223-231.
[20] O. Toledano, S. Magdassi, Journal of Colloid and Interface science 200 (1998) 235-240.
[21] L.F. Wang, S.S. Shen, S.C. Lu, Carbohydrate Polymers 52 (2003) 389-396.
[22] K.R. Kirker, Y. Luo, J.H. Nielson, J. Shelby, G.D. Prestwich, Biomaterials 23 (2002) 3661-3671.
[23] R.U. Margolis, R.K. Margolis, Cell & Tissue Research 290 (1997) 343-348.
[24] J.P. Bali, H. Cousse, E.S. Neuzil, Arthritis Rheum 31 (2001) 58-68.
[25] R. Zhang, P.X. Ma, Processing of polymer scaffolds: phase separation. In Methods of Tissue engineering, Academic Press, San Diego (2001) 715.
[26] J.M. Pachence, J. Kohn, Biodegradable Polymers. In Principle of Tissue engineering, 2nd edition. Academic Press, San Diego (2000) 263.
[27] S. Li, Journal of Biomedical Materials Research 48 (1999) 342.
[28] J.E. Bergsma, F.R. Rozema, R.R.M. Bos, G. Boering, W.C. Bruijn, A.J. Pennings, Biomaterials 16 (1995) 267-274.
[29] V. Hasirci, K. Lewandrowski, J.D. Gresser, D.L. Wise, D.J. Trantolo, Journal of Biotechnology 86 (2001) 135-150.
[30] J.C. Middleton, A.J. Tipton, Biomaterials 21 (2000) 2335-2346.
[31] Y.J. Du, G.J. Lemstra, A,J, Nijenhuis, A.M. Hubb, Macromolecules 28 (1995) 2124-2132.
[32] P.X. Ma, R. Langer, In Polymers in Medicine and Pharmacy. Materials Research Society (1995) 99.
[33] A.M. Reed, D.K. Gilding, Polymer 22 (1981) 494-498.
[34] Y. You, S.W. Lee, J.H. Youk, B.M. Min, S.J. Lee, W.H. Park, Polymer Degradation and stability 90 (2005) 441-448.
[35] C.G. Pitt, M.M. Gratzl, G.L. Kimmel, J. Surles, A. Schindler, Biomaterials 2 (1981) 215-220.
[36] S. Namekawa, S. Suda, H. Uyama, S. Kobayashi, International Journal of Biological Macromolecules 25 (1999) 145-151.
[37] K.Park, W.S.W. Shalaby, H. Park, Biodegradable hydrogels for drug delivery (1993) P.13~34.
[38] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Chemica Reviews 99 (1999) 3181-3198.
[39] P. Beck, J. Kreuter, I. Fichtner, Journal of Microencapsulation, 10 (1993) 101.
[40] J.O. Hollinger, Biomedical Applications of Synthetic Biodegradable Polymers (1995) P. 224.
[41] R. Langer, Nature 392 (1998) 5-10.
[42] J.R. Brouwers, Pharmaceutical World Science 18 (1996) 153-162.
[43] W. Dang, O.M. Colvin, H. Berm, W.M. Saltzman, Cancer Research 54 (1994) 1729-1735.
[44] K.A. Walter, R. Tamargo, A. Oliver, P.C. Burger, H. Brem, Neurosurgery 37 (1995) 1129-1145.
[45] N. Katayama, R. Tanaka, Y. Ohno, C. Ueda, T. Houjou, K. Takada, International Journal of Pharmaceutical 115 (1995) 87-93.
[46] W. Kalala, R. Kinget, G. Van Den Mooter, C. Samyn, International Journal of Pharmaceutical 139 (1996) 187-195.
[47] P. Omathanu, P. Ramesh, Chemical Biology 5 (2001) 447-451.
[48] D.W. Rafferty, J.L. Koenig, Journal of controlled release 83 (2002) 29-39.
[49] S.W. Sanders, Journal of controlled release 39 (1996) 389-397.
[50] H. Brem, S. Piantadosi, P.C. Burger, M. Walker, R. Selker, N.A. Vick, K. Black, M. Sisti, The lancet 345 (1995) 1008-1012.
[51] S.P. Rigby, C.F. Van der Walle, J.H. Raistrick, Journal of controlled release 96 (2004) 97-100.
[52] P. Sriamornsak, J. Nunthanid, International Journal of Pharmaceutics 160 (1998) 207-212.
[53] I. Krogel, R. Bodmeier, Journal of controlled release 61 (1999) 43-50.
[54] G.A. Peyman, D. Yang, B. Khoobehi, H.H. Rahimy, S.Y. Chin, Ophthalmic Literature 50 (1999) 28.
[55] A.K. Dash, G.C. Cudworth, Journal of Pharmaceutical and Toxicological Methods 40 (1998) 1-12.
[56] F. Marianna, Pharmaceutical Science & Technology Today 3 (2000) 417-425.
[57] J. Elisseeff, A. Kristi, S. Derek, M. Winnette, R. Mark, Y. Michael, L. Robert, Plastic & Reconstructive Surgery 104 (1999) 1014-1022.
[58] D.L. Elbert, J.A. Hubbell, Biomacromolecules 2 (2001) 430-441.
[59] L.Q. Wang, K. Tu, Y. Li, J. Zhang, L. Jiang, Z. Zhang, Reactive & Functional Polymers 53 (2002) 19-27.
[60] J.V.S. Gobburu, W.J. Jusko, Advanced Drug Delivery Reviews 46 (2001) 45-47.
[61] A.J. Hubbell, Molecular Medicine Today 1 (1995) 134-139.
[62] J.A. Hubbell, Current Opinion in solid state & Material Science 3 (1998) 246-251.
[63] G.G.P. van der Wijdeven, Journal of controlled release 85 (2002) 145-162.
[64] A. Hatefi, B. Amsden, Journal of controlled release 80 (2002) 9-28.
[65] P.J. Gillbar, B. Pharm, Journal of Pain and Symptom Management 17 (1999) 197-207.
[66] M.C. Jones, J.C. Leroux, European Journal of Pharmaceutics and Biopharmaceutics 48 (1999) 101.
[67] K. Okuyama, I.W. Lenggoro, Chemical Engineering Science 58 (2003) 537-547.
[68] F. Iskandar, L. Gradon, K. Okuyama, Journal of Colloid and Interface Science 265 (2003) 296-303.
[69] C. Schmidt, R. Bodmeier, Journal of controlled release 57 (1999) 115-125.
[70] H. Murakami, M. Kobayashi, H. Takeuchi, Y. Kawashima, International Journal of Pharmaceutics 187 (1999) 143-152.
[71] Y. Efendiev, M.R. Zachariah, Chemical Engineering Science 56 (2001) 5763-5769.
[72] A.M. Hillery, A.W. Lioyd, J. Swarbrick “Drug Delivery and Targeting” London and New York (2001) p.17-20.
[73] N.B. Graham, M. Zulfiqar, Polymer 30 (1989) 2130.
[74] R. Hejazi, M. Amiji, Journal of controlled release 89 (2003) 151-165.
[75] V.R. Sinha, Rachna Kumria, International journal of pharmaceutics 224 (2001) 19-38.
[76] J.K. Thomas, K. Kalyanasundaram, Journal of American Chemical Society 99 (1977) 2039-2044.
[77] I. Astafieva, X.F. Zhong, A. Eisenberg, Macromolecules 26 (1993) 7339-7352.
[78] H.R. Kricheldorf, C. Boettcher, K. Uwe. Tonnes, Polymer 33 (1992) 2817-2824.
[79] K. Kalyanasundaram, J.K. Thomas, Journal of American Chemical Society 99 (1997) 2039-2044.
[80] K.P. Ananthapadamanabhan, E.D. Goddard, N.J. Turro, P.L. Kuo, Langmuir 1 (1985) 352-355.
[81] C. Allen, Y. Yu, D. Maysinger, A. Eisenberg, Bioconjugate Chemistry 9 (1998) 564-572.
[82] C. Zhao, M.A. Winnik, G. Riess, M.D. Croucher, Langmuir 6 (1990) 514-516.
[83] K. Kalyanasundoram, J.K. Thomas, Journal of American Chemical Society 99 (1988) 2039-2044.
[84] G.S. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakurai, K. Kataoka, Langmuir 9 (1993) 945-949.
[85] K.N. Prasad, T.T. Luong, A.T. Florence, J. Paris, C. Vautin, M. Seiller, F. Puisieux, Journal of Colloid Interface Science 90 (1982) 303-309.
[86] G.S. Kwon, K. Kataoka, Advanced drug delivery reviews 16 (1995) 295-309.
[87] A. Rosler, Vandermeulen, W.M. Guido, Advanced Drug Delivery Reviews 53 (2001) 95-108.
[88] M. Wilhelm, C.L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J.L. Mura, G. Riess, M.D. Croucher, Macromolecules 24 (1991) 1033-1040.
[89] S.C. Lee, Y. Chang, J.S. Yoon, C. Kim, I.C. Kwon, Y.H. Kim, S.Y. Jeong, Macromolecules 32 (1999) 1847-1852.
[90] Y. Nagasaki, T. Okada, C. Scholz, M. Iijima, M. Kato, K. Kataoka, Macromolecules 31 (1998) 1473-1479.
[91] D.M. Small, In The Bile Acids, Chemistry, Physiology, and Metabolism; Nair, P.P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; p249.
[92] M. Almgren, F. Grieser, J.K. Thoms, J. Am. Chem. Soc. 101 (1979) 279-291.
[93] S.C. Lee, Y. Chang, J.S. Yoon, C. Kim, I.C. Kwon, Y.H. Kim, S.Y. Jeong, Macromolecules 32 (1999) 1847-1852.
[94] L. Zhang, A. Eisenberg, Journal of American Chemical Society 118 (1996) 3168-3181.
[95] L. Zhang, H. Shen, A. Eisenberg, Macromolecules 30 (1997) 1001-1011.
[96] A.A. Salyers, American Journal of Clinical Nutrition 32 (1979) 158-163.
[97] A.A. Salyers, M. Brien, Journal of Bacteriol 143 (1980) 772-780.
[98] A. Schindler, Y.M. Hibionda, C.G. Pitt, Journal of Polymer Science 20 (1982) 319.
[99] Y. Li, J. Nothnagel, T. Kissel, Polymer 38 (1997) 6197-6206.
[100] R.A. Miller, J.M. Brady, D.E. Cutright, Journal of Biomedical Materials Research 11 (1977) 711.
[101] Y. Li, C. Volland, T. Kissel, Polymer 39 (1998) 3087-3097.
[102] A. Eisenberg, C. Allen, D. Maysinger, Colloids and Surface B: Biointerfaces 16 (1999) 3-27.
[103] J.S. Rodrigues, N.S. Santos-Magalhaes, L.C.B.B. Coelho, P. Couvreur, G. Ponchel, R. Gref, Journal of controlled release 92 (2003) 103-112.
[104] K.A. Janes, P. Calvo, M.J. Alonso, Advanced Drug Delivery Reviews 47 (2001) 83-97.
[105] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Journal of Applied Polymer Science 63 (1997) 125-132.
[106] R. Langer, J.P. Vacanti, Science 260 (1993) 920.
[107] P.X. Ma, Tissue engineering, In encyclopedia of polymer science and technology, 3rd edition, John Wiley & Sons, N.J. (2004)
[108] J.A. Buckwalter, H.J. Mankin, AAOS Inst Course Lect 47 (1998) 487-504.
[109] L. Peterson, M. Brittberg, I. Kiviranta, E.L. Akerlund, A. Lindahl, American Journal of Sports Medicine30 (2002) 2-12.
[110] J. Bloch, E.G. Fine, N. Bouche, A.D. Zurn, P. Aebischer, Experiental Neurology 172 (2001) 425-432.
[111] G.N. King, N. King, A.T. Cruchley, J.M. Wozney, F.J. Hughes, Journal of Dental Research 76 (1997) 1460-1470.
[112] R. Zhang, P.X. Ma, Journal of Biomedical Materials Research 52 (2000) 430-438.
[113] D. Palmes, H.U. Spiegel, Biomaterials 25 (2004) 1601-1611.
[114] M.W. Dacis, J.P. Vacant, Biomaterials 17 (1996) 365-372.
[115] J.J. Marler, J. Upton, R. Langer, J.P. Vacanti, Advanced Drug Delivery Review 33 (1998) 165-182.
[116] L.E. Meredith, Materials Today 7 (2004) 48-55.
[117] M. Nomi, A. Atala, P.D. Coppi, S. Soker, Molecular Aspects of Medicine 23 (2002) 463-483.
[118] R.M. Nerum, International Congress Series 1262 (2004) 122-125.
[119] T. Ahsan, R.L. Sah, Osteoarthritis and Cartilage 7 (1999) 29-40.
[120] J.K. Francis Suh, Howard W.T. Matthew, Biomaterials 21 (2000) 2589-2598.
[121] D.W. Hutmacher, Biomaterials 21 (2000) 2529-2543.
[122] E. Bell, Journal of Biomechanical Engineering 113 (1991) 113.
[123] K.Y. Lee, D.J. Mooney, Chemical Reviews 101 (2001) 1869-1879.
[124] C.M. Klapperich, C.R. Bertozzi, Biomaterials 25 (2000) 5631-5641.
[125] J. Raghunath, H.J. Salacinski, K.M. Sales, P.E. Butler, A.M. Seifalian, Current Opinion in Biotechnology 16 (2005) 503-509.
[126] R. Cortesini, Transplant Immunology 15 (2005) 81-89.
[127] T.A. Holland, A.G. Mikos, Journal of controlled release 86 (2003) 1-14.
[128] V. Luginbuehl, L. Meinel, H.P. Merkle, B. Gander, European Journal of pharmaceutics and Biopharmaceutics 158 (2004) 197-208.
[129] D. Pfander, T. Cramert, G. Weseloh, O. Pullig, D. Schuppan, M.Bauer, B. Swoboda, Osteoarthritis and Cartilage 7 (1999) 548-559.
[130] B. Pellaumail, A. Wartrin, D. Loeuille, P. Netter, G. Berger, P. Laugier, A. Saied, Osteoarthritis and Cartilage 10 (2002) 535-541.
[131] J.K. Suh, Z. Li, S.L.Y. Woo, Journal of Biomechanics 28 (1995) 357-364.
[132] G. Chen, T. Ushida, T. Tateishi, Materials Science and Engineering C 17 (2001) 63-69.
[133] S.P. Baldwin, W.M. Saltzman, Trends in Polymer Science 4 (1996) 177-182.
[134] C.J. Hunter, A.G. Imler, P. Malaviya, R.M. Nerem, M.E. Levenston, Biomaterials 23 (2002) 1249-1259.
[135] A.J. Nixon, Clinical Techniques in Equine Practice 1 (2002) 257-269.
[136] J.K. Suh, S. Scherping, T. Marui, R. Steadman, S.L.Y. Woo, Operative Techniques in Sport Medicine 3 (1995) 78-86.
[137] J.A. Buckwalter, Operative Techniques in Orthopaedics 7 (1997) 263-269.
[138] L. Lippiello, Osteoarthritis and Cartilage 11 (2003) 335-342.
[139] C.D. Hoemann, J. Sun, A. Legare, M.D. Mckee, M.D. Buschmann, Osteoarthritis and Cartilage 13 (2005) 318-329.
[140] T. Kelly, S. Samuel, Aesthetic Surgery Journal 20 (2000) 471-475.
[141] S. Tanaka, C. Hamanishi, H. Kikuchi, K. Fukuda, Seminars in Arthritis and Rheumatism 27 (1998) 392-399.
[142] T.G. Benedek, Osteoarthritis and Cartilage 14 (2006) 203-209.
[143] I.V. Yannas, Journal of Cellular Biochemistry 56 (1994) 188-191.
[144] S.M. Mueller, S. Shortkroff, T.O. Schneider, H.A. Breinan, I.V. Yannas, M. Spector, Biomaterials 20 (1999) 701-709.
[145] P.S. Donzelli, R.L. Spilker, G.A. Ateshian, V.C. Mow, Journal of Biomechanics 32 (1999) 1037-1047.
[146] A. Hayes, S.E. Clift, A.W. Miles, Medical Engineering & Physics 19 (1997) 242-252.
[147] J.C. McCarthy, J.A. Lee, Clinical Orthopaedics and Related Research 405 (2002) 122-128.
[148] M.L. Cameron, K.K. Briggs, J.R. Steadman, Am. Journal of Sports Medicine 31 (2003) 83-86.
[149] A.E. Gross, Journal of Arthroplasty 18 (2003) 14-17.
[150] G. Bentley, D. Norman, F. Haddad, Operative Techniques in Sport Medicine 8 (2000) 163-167.
[151] G. Kordas, J.S. Szabo, L. Hangody, Arthroscopy: The journal of Arthroscopic and Related Surgery 22 (2006) 414-421.
[152] T. Makino, H. Fujioka, M. Terukina, S. Yoshiya, N. Matsui, M. Kurosaka, Arthroscopy: The journal of Arthrosonic and Related Surgery 20 (2004) 837-840.
[153] L. Hangody, G. Kish, Z. Karpati, R. Eberhart, Operative Techniques in Orthopaedics 7 (1997) 312-322.
[154] P. Feczko, L. Hangody, J. Varga, L. Bartha, Arthroscopy: The Journal of Arthroscopic and Related Surgery 19 (2003) 755-761.
[155] P.M. van der Kraan, P. Buma, T. van Kuppevelt, W.B. van Den Berg, Osteoarthritis and Cartilage 10 (2002) 631-637.
[156] C.H. Chang, T.F. Kuo, C.C. Lin, C.H. Chou, K.H. Chen, F.H. Lin, H.C. Liu, Biomaterials 27 (2006) 1876-1888.
[157] P.X. Ma, R. Langer, In Tissue Engineering Methods and Protocols. Humana Press, NJ, (1999) 47.
[158] L. Lu, S.J. Peter, M.D. Lyman, H.L. Lai, S.M. Leite, J.A. Tamada, J.P. Vacanti, R. Langer, A.G. Mikos, Biomaterials 21 (2000) 1595-1605.
[159] P.X. Ma, R. Zhang, G. Xiao, R. Franceschi, Journal of Biomedical Materials Research 54 (2001) 284-293.
[160] S.N. Park, J.C. Park, H.O. Kim, M.J. Song, H. Suh, Biomaterials 23 (2002) 1205-1212.
[161] M.H. Ho, P.Y. Kuo, H.J. Hsieh, T.Y. Hsien, L.T. Hou, J.Y. Lai, D.M. Wang, Biomaterials 25 (2004) 129-138.
[162] Q. Hou, D.W. G.J. Feijen, Biomaterials 24 (2003) 1937-1947.
[163] S.H. Oh, S.G. Kang, E.S. Kim, S.H. Cho, J.H. Lee, Biomaterials 24 (2003) 4011-4021.
[164] P.X. Ma, B. Schloo, D. Mooney, R. Langer, Journal of Biomedical Materials Research 29 (1995) 1587-1595.
[165] C.A. Vacanti, R. Langer, B. Schloo, J.P. Vacanti, Plastic & Reconstructive Surgery 88 (1991) 753-759.
[166] P. Dubois, M. Krishnan, R. Narayan, Polymer 40 (1999) 3091-3100.
[167] T. Kawakatsu, K. Kawasaki, M. Furusaka, H. Okabayashi, T. Kanaya, Journal of Chemical Physics 102 (1995) 2247-2253.
[168] T. Groth, G. Altankov, Cell-surface interactions and the tissue compatibility of biomedical materials. (DC: ISO Press, Wasgington, 1998) P.12.
[169] Q. Cai, J. Yang, J. Bei, S. Wang, Biomaterials 23 (2002) 4483-4492.