纖維蛋白膠混合明膠/透明質酸/軟骨素共聚物作為關節軟骨組織工程支架之研究

Fibrin glue mixed with gelatin/hyaluronic acid/ chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering

周正鴻(Cheng-Hung Chou)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.10400

摘要

關節軟骨是一為透明且具彈性的結締組織，其覆蓋於骨頭末端以提供運動時骨頭之間潤滑及吸收壓力的保護作用，但是其一旦受到損傷，並無法自體修復。因此本研究將以組織工程方法利用明膠、透明質酸、軟骨素與纖維蛋白膠於體外培養出軟骨組織，以達到修復受損軟骨的目的。
在支架材料的製備上，選取由豬皮煉製的明膠（Gelatin），並加入透明質酸(HA)和軟骨素(C6S)，透過1-ethyl-3-(3-dimethylaminopropyl) carbodiimide（EDC）的雙式交聯反應形成共聚物（GHC6S），並藉由冷凍乾燥法移除水分，在液態氮中研磨成微顆粒；纖維蛋白原由豬血漿經冷凍沈澱後取得，另外豬血漿中的第二凝血因子加入10% CaCl2活化後與纖維蛋白原反應聚合成纖維蛋白膠。經過一週及二週的軟骨細胞培養測試，進行Hematoxylin & Eosin、Alcian blue之組織化學染色、S-100蛋白和第二型膠原蛋白免疫組織化學染色，藉以分析細胞生長分佈與基因表現的情形；配合real time-PCR分析TIMP-1, MMP-2, MT1-MMP, aggrecan, decorin, IL-b1, TGF-b1, and FADD以及一、二、十型膠原蛋白的表現與否，檢測軟骨細胞基因表現的能力及狀態。並且進行總GAGs和硫化GAGs的定量分析。
實驗結果顯示GHC6S顆粒的大小約在50-300μm左右。GHC6S顆粒所製備而成的纖維蛋白膠支架能夠有效促進細胞外基質分泌，並抑制細胞外基質的降解，適合於軟骨組織工程。

關鍵字

纖維蛋白 ； 明膠 ； 透明質酸 ； 軟骨素 ； 關節軟骨 ； 組織工程

並列摘要

Articular cartilage provides functions of lubrication to shear stress and protection from compressive force, but it has poor ability to repair itself after suffering damage. The advanced method of tissue engineering is developed and used to maintain cell functions for tissue regeneration. Autologous fibrin glue has been demonstrated as a potential scaffold with very good biocompatibility for neocartilage formation. However, fibrin glue has been reported not to provide enough mechanical strength, but with many growth factors to interfere the tissue growth. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) tricopolymer sponge has been prepared as scaffold for cartilage tissue engineering and showed very good results, but problems of cell seeding and cell distribution troubled the researchers. In this study, GHC6S particles would be added into the fibrin glue to provide better mechanical strength, better cell distribution, and easier cell seeding, which would be expected to improve cartilage regeneration in vitro. Porcine cryo-precipitated fibrinogen and thrombin prepared from prothrombin activated by 10% CaCl2 solution were used in two groups. One is the fibrin glue group in which porcine chondrocytes were mixed with thrombin–fibrinogen solution, which was then converted into fibrin glue. The other is GHC6S-fibrin glue in which GHC6S particles were added into the thrombin-fibrinogen solution with porcine chondrocytes. After culturing for 1-2 weeks, the chondrocytes cultured in GHC6S-fibrin glue showed a round shape with distinct lacuna structure and showed positive in S-100 protein immunohistochemical stain. The related gene expressions of tissue inhibitor of metalloproteinases-1, matrix metalloproteinase- 2, MT1-MMP, aggrecan, decorin, type I, II, X collagen, interleukin-1 b, transforming growth factor-b 1 (TGF-b1), and Fas-associating death domain were checked by real-time PCR. Total GAGs and sulfated GAGs were also quantified by p-dimethylaminobenzaldehyde reaction and 1,9- dimethymethylene blue (DMMB) assay, respectively. The results indicated that the chondrocytes cultured in GHC6S-fibrin glue would effectively promote extracellular matrix (ECM) secretion and inhibit ECM degradation. The evidence could support that GHC6S-fibrin glue would be a promising scaffold for articular cartilage tissue engineering.

並列關鍵字

fibrin ； gelatin ； hyaluronic acid ； chondroitin-6-sulfate ； articular cartilage ； tissue engineering

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