論文名稱:

熔融擠製成型聚乳酸-甘醇酸/膠原蛋白組合支架在骨組織工程之發

 

展與應用

 

The Development and Application of FDM-PLGA/Collagen

 

Composite Scaffold in Bone Engineering

研究生:

姚道禮  Dao-Li Yew

指導教授:

陳恆理 博士  Hen-Li Chen

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39417004

          學年度:

95

          語文別:

中文

          出版年:

96

關鍵字:

熔融擠製成型  Fused Deposition Modeling

 

聚乳酸-甘醇酸  Poly (lactide-co-glycolide)

 

膠原蛋白  Collagen

 

骨組織工程  Bone Tissue Engineering

全文說明:

(本論文 20100827 對校內公開)

        論文頁數:

78

摘要:

利用基因治療可持續給予促進組織再生的因子,在組織工程上扮演著重要的角色。活化基因基質(Gene-Activated MatrixGAM)由基質及內含所要表達因子基因序列的質體所組成。利用GAM療法給予骨生長因子已被證實可促進骨再生,惟傳統GAM使用膠原蛋白(collagen)為基質,機械性質不佳,而限制了其應用。聚乳酸-甘醇酸(Poly (lactide-co-glycolide), PLGA)具良好機械性,但為疏水性化合物,不利細胞生長貼附,而膠原蛋白支架雖然有利細胞生長,但機械性並不佳,有研究指出在溶劑鑄造/鹽析法製成PLGA支架孔洞內製造膠原蛋白的微支架(microsponge)可同時獲得較好的機械性和細胞的生長貼附。熔融擠製成型(Fused Deposition ModelingFDM)為製作支架深具潛力的一種新方式,可改善溶劑鑄造/鹽析技術孔洞分佈不均和連通性較差等缺點,並可有效增加支架機械性質。本篇研究目的為發展利用FDM-PLGA膠原蛋白組合支架,並應用此策略於加強膠原蛋白活化基因基質的機械強度。研究中首先在FDM-PLGA支架的孔洞內,製備膠原蛋白微支架,形成FDM-PLGA/Collagen支架,以電子顯微鏡觀察製成的FDM-PLGAFDM-PLGA/Collagen支架,並分別植入兔子股骨兩側骨缺損內,在四週及八週測量骨癒合。發現FDM-PLGA/Collagen支架骨再生作用較佳。進一步在FDM-PLGA支架孔洞內製造Collagen GAM,製成以FDM-PLGA加強機械性質之膠原蛋白活化基因基質(FDM-PLGA/Collagen GAM),發現FDM-PLGA/Collagen LacZ-GAM在兔骨缺損內可表達內含報告基因(reporter gene)。接著建構含人類BMP4的質體(pUMVC1-hBMP4)以供測試GAM用,在體外轉染C2C12細胞後可增加其鹼性磷酸酶活性的表達,證實所產生的BMP4具生物活性。最後再次以兔子股骨模型證實攜帶hBMP4基因的FDM-PLGA/Collagen GAM比攜帶LacZ基因者有更佳骨癒合能力。本研究發展了一種FDM-PLGA/Collagen創新組合支架,並測試其在骨組織工程上的應用,此外,也證實以FDM-PLGA支架來加強Collagen GAM機械性質為可行的策略。

 

Gene therapy plays an important role in tissue engineering due to its ability to sustain release of factors promoting tissue regeneration. Gene-activated matrix (GAM) consists of matrix and plasmid encoding gene sequence of interested factor. Use of GAM technology to deliver osteogenic factors is effective in bone regeneration. However, the interior mechanical properties of conventional collagen GAM limit its application. Poly (lactide-co-glycolide) (PLGA), though with good mechanical properties, is a hydrophobic material unfavorable for cell attachment. Collagen scaffold is a material with good cell response but with inferior mechanical property. Composites consist of porogen-leaching PLGA scaffold and collagen microsponge within its pores have been developed to simultaneously achieve better mechanical strength and cell response. Fused deposition modeling (FDM) is a new method for scaffold fabrication with great potential. FDM scaffolds not only can overcome drawbacks of porogen-leaching scaffolds such as uneven pore distribution and poor pore interconnectivity, but also greatly improve their mechanical properties. The purposes of this study was to develop a FDM-PLGA/Collagen composite scaffold and apply the composite strategy to reinforce the mechanical properties of collagen GAM. Collagen microsponge was fabricated in pores of prefabricated FDM-PLGA scaffold to form FDM-PLGA/Collagen scaffold. The structure of both FDM-PLGA scaffold and FDM-PLGA/Collagen scaffold were examined under scanning electron microscope. Later, both scaffolds were implanted into contralateral femoral osseous defects in rabbits. Bone regeneration was evaluated after 4 and 8 weeks. FDM-PLGA/Collagen scaffold was found to have a better osteogenic response. Furthermore, collagen GAM was made in pores of FDM-PLGA scaffold. The FDM-PLGA/Collagen LacZ GAM was able to express the encoded beta-galactosidase in vivo. A plasmid encoding gene of human BMP4 (pUMVC1-hBMP4) was constructed for GAM therapy. The bioactivity of the expressed BMP4 was proven by its ability to increase alkaline phosphatase activity of transfected C2C12 cells. FDM-PLGA/Collagen GAM containing pUMVC1-hBMP4 showed a better osteogenic effect than which containing pUMVC1-nt--gal in rabbit femoral osseous defect model. In summary, a novel scaffold composite with consisting of FDM-PLGA and collagen was developed and tested in bone tissue engineering application. In addition, the reinforcement of collagen GAM with FDM-PLGA scaffold aiming at improving mechanical property is proven to be a practical strategy.

        論文目次:

中文摘要:    1

 

英文摘要:    3

 

壹、緒論    5

 

一、    研究動機    5

 

二、    組織工程(Tissue Engineering    6

 

三、    活化基因基質(Gene Activated Matrix)    8

 

四、    生物支架(Scaffold)    9

 

五、    膠原蛋白(Collagen    10

 

六、    聚乳酸-甘醇酸Poly (lactide-co-glycolide), PLGA    11

 

七、    訊息因子    13

 

八、    BMP4 對骨組織的影響    14

 

九、    PLGA膠原蛋白複合式支架    15

 

十、    支架製備方法之改良    17

 

十一、    實驗目的    19

 

貳、實驗材料與方法    20

 

一、    實驗材料    20

 

二、    實驗方法    29

   
 

參、實驗結果    47

 

一、掃描式電子顯微鏡觀察FDM-PLGA/Collagen複合支架之結構    47

 

二、FDM-PLGA onlyFDM-PLGA/Colllagen 動物實驗    47

 

三、質體載體pUMVC1-hBMP4的建構    48

 

四、載體轉染後在哺乳類細胞中表達    49

 

五、載體轉染後鹼性磷酸酶活性測試    50

 

六、FDM-PLGA/Collagen GAM動物實驗    51

 

肆、討論    53

 

伍、結論    58

 

陸、附圖    60

 

圖一、PLGA/Collagen支架的SEM    60

 

圖二、動物手術流程圖    61

 

圖三、FDM-PLGA/ColFDM-PLGA的骨密度比較    62

 

圖四、pUMVC1-hBMP4基因重組流程圖    64

 

圖五、BMP基因片段    65

 

圖六、pUMVC1-hBMP4構建---限制酶分析    66

 

圖七、以pUMVC1-hBMP4轉染細胞後的表現(一)    67

 

圖八、以pUMVC1-hBMP4轉染細胞後的表現(二)    68

 

圖九、以pUMVC1-hBMP4轉染細胞後的表現(三)    69

 

圖十、以pUMVC1-LacZ轉染細胞後的表現    70

 

圖十一、FDM-PLGA/Collagen GAM在動物體內的基因表達    71

 

圖十二、FDM-PLGA/Collagen GAM的骨密度比較    72

 

柒、參考文獻    74