- 學位論文
己二酸二醯肼交聯氧化透明質酸水膠結合明膠微粒應用於軟骨組織工程之研究
A study of adipic acid dihydrazide crosslinked oxi-hyaluronic acid hydrogel combined with gelatin microparticle as scaffold for articular cartilage tissue engineering
摘要
隨者人口老化關節炎患者不斷增加，軟骨修復治療為現今再生醫學的重點發展項目之一。由於軟骨組織中缺乏神經及血管，自我修復能力低，受到傷害若沒妥善治療往往會持續惡化，最後導致退化性關節炎產生。然而目前臨床上已有許多針對軟骨受損的治療方法，但仍還是有許多限制存在。近年來組織工程發展迅速，結合細胞、支架和信號三元素，使軟骨修復技術邁向新領域。在過去研究中，已有相當多的軟骨修復性材料被開發，但材料本身仍有諸多限制存在，至今還未找到一個最佳的條件，能提供軟骨細胞一個最佳的仿生環境。 本實驗將以組織工程概念，開發新型生物性支架，使軟骨細胞經過體外培養過程依然能維持它的細胞型態，解決過去材料上的限制。實驗中，利用氧化開環法製備氧化玻尿酸水膠與二次交聯法製備高交聯明膠微粒，並以傅立葉轉換紅外線光譜儀、掃描式電子顯微鏡與生物相容性測試等來分析材料特性，且更進一步將軟骨細胞與明膠微粒包埋於氧化玻尿酸水膠，形成水膠複合物，經培養後，以掃描式電子顯微鏡、組織學染色、醣胺聚醣定量和基因表現等體外測試評估軟骨細胞的生長狀況。
並列摘要
In recent years, the cartilage injury repair has been a popular topic in regenerative medicine. The cartilage has limited self-repair capacity due to lack of nerves and blood vessels in the articular cartilage. So far, some treatment surgeries have been developed in clinic; however, some limitations still exist such as fibrocarilage formation. Until the tissue engineering is well developed, cartilage repair will reach to a higher level. To date, the most suitable bioscaffold for chondrocytes in the cartilage repair is still not finding. In this study, we have developed the hyaluronic acid-based hydrogel and incorporated it with gelatin particles to evaluate the possibility as the bioscaffold for cartilage tissue engineering. We expected the bioscaffold not only mimic ECM functions, but also have the ability to promote chondrocytes proliferation and maintain the phenotype. In this study, the oxidized hyaluronic acid with aldehyde functional groups prepared by sodium periodate was crosslinked by adipic acid dihydrazide (ADH) to form hydrogel. The gelatin was modified by glutaraldehyde and proanthocyanidins crosslink agents to form gelatin particles. Then we incorporated hydrogel and particles as the bioscaffold. To investigate the characterization of bioscaffold, the IR, SEM, EDS, degradation time and biocompatibility test were evaluated. The gene expression for chondrocytes encapsulated in hydrogel was also evaluated by real-time PCR. The results shown the microparticles had uniform pore size about 80-150 μm and the crosslinking index remained above 90%.The hydrogel combined with microparticles through WST-1, LDH and Live/Dead analysis are non-cytotoxicity; also has good results in SEM analysis, histological evaluation, GAGs content, gene expression and tissue culture assay. Importantly, the construct also promoted chondrocytes synthesis of COL2A1, AGN, and SOX-9 mRNA. In this study, we prepared hyaluronic acid-based hydrogel combined with gelatin particles as chondrocytes bioscaffold. The oxidized hyaluronic acid /ADH crosslinked hydrogel can be prepared in a liquid form at room temperature and easily mixed with gelatin particles and chondrocytes. These preliminary data indicate that the hydrogel and gelatin particles have good biocompatibility, and contribute chondrocytes maintain gene expression. Based on the results, the oxi-HA hydrogel combined with gelatin particles may have potential uses in cartilage tissue engineering applications.