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研究生:陳于正
研究生(外文):Yu-Zheng Chen
論文名稱:膠原蛋白/玻尿酸多孔隙支架之試作及其對成骨細胞生物相容性之評估
論文名稱(外文):Evaluation of biocompatibility of osteoblast seeded on the porous scaffolds which manufactured by collagen/hyaluronic acid from chicken combs
指導教授:劉登城
指導教授(外文):Deng-Cheng Liu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:畜產學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:100
中文關鍵詞:膠原蛋白玻尿酸骨髓基質細胞生物相容性支架
外文關鍵詞:collagenhyaluronic acidbone marrow stromal cellbiocaompatibilityscaffold
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當人體骨骼發生粉碎性骨折,或切除惡性骨腫瘤後即會造成大範圍的骨組織缺損,傳統的骨科醫療對於延遲癒合患部通常是採用自體骨移植(autografts)、異體骨移植(allografts)或人工植入物如鋼螺絲固定骨碎片的方法以促進傷口骨頭新生並與周圍骨組織重新融合成一個完整的結構。但各種方法都各有其缺點存在,如造成病人額外的傷口與痛楚、高昂的手術費用、應力遮蔽效應與感染傳染病等問題。倘若能仿照人體骨骼結構製造人工支架、植入具成骨能力的細胞、配合生長因子誘導細胞執行正確的生理活動進而製造人體骨骼以取代原本失去功能和缺損的部位,促使傷口癒合並恢復應有的功能,則自體骨移植與異體骨移植的缺點皆可同時避免,此即為骨組織工程的完美構想。
本試驗的目的在於以禽類分離出來的第一型膠原蛋白與玻尿酸,利用兩者特殊的生理性質,並以1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)化學交聯的方式製備多孔隙的膠原蛋白/玻尿酸支架,培養源自哺乳動物的骨母細胞與骨先質細胞。進而評估其跨物種的使用對其生物相容性的影響。
結果顯示:添加玻尿酸於膠原蛋白支架中可提高其含水率,但會降低支架結構強度。隨著支架交聯度與結構穩定度的增加,含水率有隨之下降的趨勢,而以雙式交聯支架的結構穩定性最好。雙式交聯支架中以95%酒精為二次交聯液溶劑的組別有最好的結構穩定性與含水率。自大鼠萃取而來的骨母細胞與雙式交聯支架於體外環境共同培養後發現,以交聯劑處理的膠原蛋白與膠原蛋白/玻尿酸等所有支架對骨母細胞的生物相容性皆無負面影響。且以Von kossa染色法得知,將骨髓基質細胞植入雙式交聯之膠原蛋白及膠原蛋白/玻尿酸支架於第七天時,皆能探知鈣結晶沉積於支架上。
綜觀以上結果,雙式交聯之膠原蛋白及膠原蛋白/玻尿酸支架皆可誘導骨髓基質細胞進行成骨作用,故本研究所開發之雙式交聯支架可作為未來硬骨組織工程的進一步研究材料。
Skeletal abnormalities, wound and tumor bone resection all cause bone loss. Traditional methods used to repair bone wound include autograft or allograft and artificial implants such as steel screw which usually makes additional trauma and high cost of patient. In addition to pain of patient, stress shielding effect will also weak the mechanical strength of the regenerative bone near the implants. Bone tissue engineering, a new developed therapy for healing bone wound.
Type I collagen and hyaluronic acid from rooster’s combs were combined together to make porous scaffolds and treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for intermolecular crosslinked will be studied in this research. Three dimensional scaffolds were seeded with rat bone marrow stromal cells(BMSC), or cultured with osteoblasts for 0, 3 and 7days. Biocompatibility of the scaffolds were evaluated with the morphology of osteoblast, 3-(4,5-dimethythiazol-2-yl) -5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS), and Von kossa stain.
The results indicated that hyaluronic acid decreased the mechanical stability of crosslinked collagen/hyaluronic acid scaffolds, it should be treated with second crosslinking to get more stable. The 95% alcohol is the best choice of solvent as a second crosslinking agent due to their stable structure in water and high water resorbance ability of all double crosslinked scaffolds. All double crosslinked scaffolds did not have any negative effect on osteoblast by MTS test. At the 7th day, all double crosslinked collagen or collagen/hyaluronic acid scaffolds seeded with BMSC significantly observed calcium deposit in the scaffold by Von kossa staining.
However , the results demonstrate that both of double crosslinked collagen scaffolds and double crosslinked collagen/hyaluronic acid scaffolds are suitable for bone cells development and can be used as a biomedical material for bone healing or repair in the future.
目次
頁次
壹、中文摘要---------------------------------------------------1
貳、前言-------------------------------------------------------3
參、文獻檢討---------------------------------------------------4
一、硬骨結構組成及功能-----------------------------------4
(一)硬骨成分、結構-------------------------------------4
(二)硬骨功能-------------------------------------------5
(三)硬骨的細胞種類-------------------------------------6
(四) 骨折修復與重建------------------------------------9
二、玻尿酸的生理功能及醫學上的應用-----------------------11
(一) 玻尿酸的結構特性與分佈---------------------------11
(二)玻尿酸於組織修復時所扮演的角色--------------------13
(三)玻尿酸於生物醫學上的應用--------------------------18
三、 組織工程--------------------------------------------22
(一)組織工程的定義------------------------------------22
(二)骨組織工程----------------------------------------23
(三)骨組織工程之細胞與訊號----------------------------26
(四)骨組織工程支架與材料選擇--------------------------29
(五)、膠原蛋白/玻尿酸支架製作、特性分析---------------33
肆、材料與方法------------------------------------------------36
一、藥品配置---------------------------------------------36
(一) 細胞培養液的配置---------------------------------36
(二)分化劑的配製--------------------------------------37
(三) Phosphate buffered saline, (PBS)溶液的配製-------37
二、大鼠細胞的取得與培養---------------------------------38
(一)初生大鼠骨母細胞的萃取----------------------------38
(二)大鼠骨髓基質細胞的萃取----------------------------39
三、膠原蛋白/玻尿酸支架製作、特性分析--------------------40
(一)一次交聯支架之製作--------------------------------40
(二)雙式交聯支架之製作--------------------------------40
(三)細胞植入方法--------------------------------------41
(四)支架含水率測定------------------------------------42
(五)顯微構造觀察(SEM) --------------------------------42
(六)孔隙度測試----------------------------------------43
四、支架生物相容性評估-----------------------------------43
(一)細胞活性測試--------------------------------------43
(二)Von kossa組織切片染色-----------------------------46
五、統計分析---------------------------------------------47
伍、結果與討論------------------------------------------------47
一、支架架構分析-----------------------------------------47
(一)支架外部結構及內部孔徑比較------------------------46
(二)各種支架含水率的比較------------------------------56
(三)支架間孔隙度的比較--------------------------------68
二、細胞於體外培養、分化---------------------------------71
(一)骨母細胞的萃取與培養------------------------------71
(二)骨髓基質細胞的萃取與培養--------------------------72
三、支架生物相容性測試-----------------------------------73
(一) 支架降解物對細胞活性的影響-----------------------73
(二)骨髓基質細胞植入不同支架後對其生化特徵的影響------75
陸、結論------------------------------------------------------88
柒、參考文獻--------------------------------------------------90
捌、英文摘要--------------------------------------------------99
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