The Development of collagen-chitosan Dermal Filler or long-term stability

近年來由於生活品質的提高，人們逐漸開始注重自身的保養，由其是臉部皺紋。所以市面上出現了許多的除皺方法，不過最直接的治療方法莫過於皮下填充注射手術，該治療方法屬於不需要經過動刀即可達到除皺效果的微創手術，也目前普遍被選用來治療臉部上的皺紋。填充材料常以自體移植、異體移植、合成材料、生物合成等等來當作填充物質，雖說有部分填充材料已經通過美國藥物食品檢驗局。但對於一個理想的填充材料而言，目前所使用的填充材料都有相當程度上的缺點。研究之目的在於研發出一個理想的針劑填充材料，在目前使用的材料之中以膠原蛋白對人體的生物相容性情況最為良好，可缺點在於使用期限過短，所以本研究以豬皮萃取的第一型膠原蛋白為基材，加入具生物可分解性的高分子幾丁聚醣來進行研究，企圖營造出一個理想的填充材料。在本研究中，所製備的膠原蛋白幾丁聚醣膠體之特性將會透過流變儀、針筒注射、生物相容性、降解試驗等檢測後，可得知若以本材料為真皮填充材料則不會發生卡針的現象；且若施打進入病患體內，亦沒有材料位移的疑慮；至於降解試驗上，添加幾丁聚醣後的複合材料經本實驗證明確實可以延緩其降解結，因此若未來使用在真皮填充物及其應用上是相當具有潛力的。In recent decades, facial wrinkles is becoming the embarrassed question in developed countries. These minimally invasive procedures are becoming more popular with those who want to avoid having cosmetic surgery. Dermal filler is one of minimally invasive cosmetic procedures . Dermal filler is populated to cure the fine lines、rhytids and overlying folds.lthough some injectable agents was approved by the US Food and Drug Administration(FDA) but the ideal dermal filler has continued quest .n order to find the ideal dermal filler, we use the new material, which include porcine collagen and chitosan to achieve the goal which is long-lasting soft-tissue augmentation without side effects.n this study,the collagen-chitosan gel is investigated.The cross-linking agents is glutaradehyde. For the results,the gel viscosity is increased when collagen blended with chitosan and our material may be suitable for syringe injection.For in vitro,the cytotoxicity show that collagen-chitosan gel is biocompatible.In the test of degradation, results showed that collagen blended with chitosan may slow degradation when soaking in collagenase solution.The feature of collagen-chitosan gel is potential application to dermal filler.國立臺灣大學碩士學位論文..................................I文摘要.................................................IIbstract................................................III錄.....................................................IV目錄..................................................VII目錄...................................................IX一章 緒論..............................................1.1前言...................................................1.2 皮膚構造與生理功能....................................2.3皮膚衰老理論...........................................4.4 皺紋..................................................9.4.1皺紋的成因...........................................9.4.2 肌膚與皺紋的分類...................................10.4.3常見的除皺方法......................................11.4.4 皺紋與治療的方式...................................15.5 填充材料的介紹 .......................................16.5.1 理想的填充材料.....................................16.5.2 目前常見之顏面填充材料.............................17.6 研究動機.............................................21二章 基礎理論..........................................22.1 膠原蛋白(Collagen)...................................22.1.1膠原蛋白分子結構與合成 ..............................22.1.2第一型膠原蛋白之種類與特性..........................27.1.3膠原蛋白與老化......................................27.2幾丁聚醣的選用........................................29.3 交聯劑...............................................30三章 實驗步驟與方法....................................31.1實驗儀器..............................................31.2 實驗藥品.............................................32.3實驗方法與流程........................................34.4材料性質分析..........................................35.5高效能液相層析儀檢測(high performance liquid chromatography，HPLC)....................................36.6濃度檢測..............................................37.7 材料性質分析.........................................38.7.1膨潤試驗(Swelling ratio,SR).........................38.7.2材料熱性質分析(Thermal analysis)....................38.7.3降解試驗(Degradation)...............................39.7.4流變性質分析(Rheology) ..............................41.7.5注射試驗分析(Injectability test)....................41.8 生物相容性...........................................42.8.1 WST-1細胞活性測試(Water Soluble Tetrazolium Salt-1 Assay)...................................................42.8.2 LDH 乳酸脫氫酶檢定(Lactate Dehydrogenase Assay, LDH).....................................................43四章 結果與討論........................................45.1 HPLC檢測.............................................45.2濃度檢測..............................................48.3 材料性質分析.........................................50.3.1膨潤試驗............................................50.3.2流變性質分析........................................52.3.3材料熱性質分析......................................54.3.4降解試驗............................................56.3.5注射試驗分析........................................64.4生物相容性............................................65.4.1 WST-1細胞活性測試..................................65.4.2 LDH 乳酸脫氫酶檢定.................................66五章 結論.............................................67考文獻.................................................68目錄 1. 1皮膚結構示意圖(Diagram of Skin Structure)..........3 1. 2 紫外光線與人體皮膚穿透深度關係圖.................6 1. 3紫外光導致老化機制示意圖...........................6 1. 4老化導致於皮膚結構變化之組織切片圖.................7 1. 5真皮填充物植入點與皺紋的關係圖....................14 1. 6(A) 真皮層結構變化導致細紋的產生與治療方式(B) 深層皺紋治療的方式.............................................142. 1膠原蛋白三股螺旋圖.................................232. 2生物體內膠原蛋白合成圖.............................242. 3膠原蛋白結構圖 ..............................272. 4幾丁質與幾丁聚醣...................................292. 5戊二醛交聯膠原蛋白示意圖...........................303. 1實驗流程圖.........................................353. 2 BCA試劑示意圖....................................373. 4注射器設計圖(左圖).................................413. 5 注射器實體圖(右圖)................................413. 6WST-1作用機制......................................423. 7 LDH作用機制.......................................434. 1膠原蛋白與幾丁聚醣的全波長圖譜.....................464. 2藉由HPLC分析幾丁聚醣之圖譜.........................464. 3藉由HPLC分析膠原蛋白之圖譜.........................474. 4藉由HPLC分析膠原蛋白基丁聚醣材料之圖譜.............474. 5膠原蛋白之校正曲線.................................494. 6各實驗組中膠原蛋白的含量...........................494. 7各材料於磷酸緩衝溶液中之膨潤試驗...................504. 8 各材料於磷酸緩衝溶液中之膨潤試驗..................514. 9將材料浸泡於不同溶劑之中...........................514. 10剪切應力對剪切應變的關係圖........................534. 11黏度對剪切應變的關係圖............................534. 12純材料之熱重損失與裂解溫度圖......................554. 13複合材料之熱重損失與裂解溫度圖....................554. 14膠原蛋白標準曲線圖................................564. 15不同濃度材料於磷酸緩衝溶液降解圖..................574. 16材料於磷酸緩衝溶液降解圖..........................574. 17膠原蛋白標準曲線圖................................584. 18不同濃度材料於培養基之降解圖......................594. 19材料於培養基之中降解圖............................594. 20不同濃度材料於膠原蛋白分解酶溶液中之降解圖........624. 21材料於膠原蛋白分解酶溶液中之降解圖................624. 22材料於膠原蛋白分解酶溶液中之降解圖................634. 23不同比例材料的注射性質............................644. 24 WST-1測試結果....................................654. 25 LDH測試結果......................................66目錄1. 1 2000-2005年接受軟組織填充物治療人數................11. 2 皮膚病變與其成因...................................71. 3老化於皮膚相關病變..................................81. 4膚色分類與其特性...................................101. 5以雷射治療皮膚老化之限制...........................111. 6 化學脫皮法治療皺紋................................131. 7目前常用填充材料整理表.............................2