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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李苑玲(Yuan-Ling Lee) | |
dc.contributor.author | Ching-Ching Liu | en |
dc.contributor.author | 柳青青 | zh_TW |
dc.date.accessioned | 2021-06-17T08:10:21Z | - |
dc.date.available | 2024-08-27 | |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73792 | - |
dc.description.abstract | 當牙髓發炎亦或感染時,清創根管後若能保留甚至再生活髓組織,維持牙齒感知外界刺激的功能,並使齒質持續生長,是現今牙髓疾病治療的目標。Tideglusib是FDA認證可於臨床治療阿茲罕默症的藥物,本身為肝醣合成酶激酶(glycogen synthase kinase-3, GSK-3)抑制劑,會參與牙齒生長發育過程並促進牙本質再生(dentinogenesis)。本研究目的是以人類牙髓幹細胞(human dental pulp stem cell, hDPSC)作為細胞來源,使用Tideglusib調控細胞的生長效應,同時以雙相型透明質酸膠體顆粒(biphasic hyaluronic acid granules, biHAG)作為支架,研發出可注射性的組織再生材料,誘導牙本質牙髓複合體(dentin-pulp complex)的再生。
支架材料是使用BDDE(1,4-butanediol diglycidyl ether)作為交聯劑製備出透明質酸膠體顆粒(hyaluronic acid granules, HAG),改變交聯反應環境和降低粒徑大小以改良製備流程,並與非交聯型透明質酸(non-crosslinked hyaluronic acid, ncHA)混合製備出biHAG,分別以HAG比例命名為HAG100、HAG80和HAG60,以流變性質及膨脹比例評估製程因素對物理性質的影響。同時使用hDPSC與材料共同培養,觀察材料生物相容性和細胞生長行為。也利用反轉錄聚合酶連鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)檢測不同Tideglusib濃度對細胞分化之效應。動物實驗則將各組材料與hDPSC混合後進行免疫抑制鼠皮下注射,六周後犧牲,觀察材料在活體組織的代謝分解行為和組織反應。 物理性質分析發現,當交聯反應環境自烘箱改為水浴槽,材料的流體性質和均質性增加,但不影響交聯程度。而粒徑大小方面,結果顯示250 μm膠體材料的固態性質、流動性和可注射性較420 μm者高,其中250 μm HAG80、HAG60可通過27G的針頭進行注射,並且HAG80的結構維持性較HAG60穩定,因此選用HAG80進行後續研究。 細胞實驗方面,結果顯示Tideglusib濃度小於300 nM時,細胞存活率都超過八成。而搭載Tideglusib的Tide@HAG具備良好的生物相容性,並且使用萃取液模型檢測出透明質酸膠體顆粒並不會影響藥物釋放,亦不具備緩釋效果,約一天內會釋放所承載的藥物量。而與hDPSC共養的研究中,粒徑大小不會影響細胞的生長行為,所有組別的細胞形態正常無破孔,無論粒徑大小,添加100 nM Tideglusib有較好的細胞生長行為。檢測細胞分化發現,添加100 nM Tideglusib可提升DSPP及VEGF的基因表現,因此選用100 nM Tideglusib作為活體實驗的添加濃度。 而動物實驗結果顯示,所有組別的材料生物相容性良好,不會造成發炎反應。在不加藥的420 μm組別中,HAG100材料完整,沒有明顯纖維組織生成,HAG80和HAG60的材料間都有纖維組織和血管形成。粒徑大小方面,420 μm和250 μm材料其組織生長及血管增生情狀差異不大。而無論粒徑大小,添加100 nM Tideglusib組別相較於未加藥者,都有較多的組織生長量及血管形成,其中250 μm的組別在組織生成有顯著差異。 總結來說,此研發材料在改變交聯反應環境為水浴槽後,可提升流動性跟均質性;而降低粒徑大小可提升可注射性,其中以HAG80的結構維持性較佳。Tide@HAG生物相容性佳,搭載100 nM Tideglusib對細胞生長以及分化有提升的趨勢。活體組織反應觀察到,添加ncHA和Tideglusib都會促進纖維組織和血管生成,其中又以250 μm的組別較具備牙本質牙髓複合體再生的潛力。 | zh_TW |
dc.description.abstract | Maintain the pulp vitality or regenerate the dentin-pulp complex to keep the tooth perception to external stimuli and root growth is the goal of modern endodontic treatment. Tideglusib, a FDA-approved drug for Alzheimer's disease treatment, is a glycogen synthase kinase-3 (GSK-3) inhibitor that participates in tooth growth and promotes dentinogenesis. The purpose of this study was to develop an injectable, Tideglusib contained biphasic hyaluronic acid granules (biHAG) with human dental pulp stem cells (hDPSC) as cell sources for dentin-pulp complex regeneration.
The 2% cross-linked hyaluronic acid granules (HAG) with granular size of 420 μm and 250 μm were synthesized by using BDDE (1,4-butanediol diglycidyl ether) as a crosslinking agent under oven or water bath environments. Then, HAG was mixing with non-crosslinked hyaluronic acid (ncHA) to produce biHAG, named HAG100, HAG80 and HAG60 according to the composition ratio of HAG. Rheology and swelling ratios of different biHAG were assessed . The biocompatibility and the induction of cell differentiation of biHAG and Tideglusib contained biHAG (Tide@HAG) were also evaluated. In addition, the tissue response to the biHAG and Tide@HAG with hDPSC mixtures were investigated using subcutaneous injection model on immunosuppressive mice, sacrificed at the sixth weeks. The results showed biHAG cross-linked at water bath presented the similar swelling ratio, but more homogeneity and fluid properties than that at oven. biHAG with 250 μm not only had better injectability and fluidity but also structure maintenance property than those of 420 μm. 250 μm HAG80 and HAG60 could be injected through a 27G needle, and HAG80 could maintain its shape than HAG60 after injection. Therefore, HAG80 was selected for further in vitro and in vivo studies. No cytotoxicity of Tideglusib with more than 80% cell viability was found when its concentration less than 300 nM. Tide@HAG presented good biocompatibility using extract model. In addition, almost all Tideglusib would release from HAG within 24 hours, indicating HAG did not have a sustained release effect. In all biHAG and Tide@HAG, hDPSC attached and growth on the materials were observed and granular sizes did not affect the cell growth behaviors, in which, HAG80 with 100 nM Tideglusib had best cell growth behavior and more expression of DSPP and VEGF in vitro. Therefore, 100 nM Tideglusib was used for in vivo experiment. The histological findings of all biHAG and Tide@HAG confirmed they were all biocompatible and no inflammatory response were found. In the 420 μm groups without drug, HAG100 showed more materials retained with few fibrous tissue formation and both HAG80 and HAG60 presented more fibrous tissue and blood vessels formation. No obvious differences on fibrous tissue growth and angiogenesis were found between HAG80 with 420 μm and 250 μm. The addition of 100 nM Tideglusib did promote more tissue growth and angiogenesis than other groups, and the 250 μm groups had significant better tissue formation compare to 420 μm groups. In summary, the biHAG cross-linked at water bath had more fluidity and homogeneity than those at oven condition and biHAG with 250 μm showed better injectability, in which HAG80 presented better properties for injection and structure maintenance. Tide@HAG had good biocompatibility and 100 nM Tideglusib could enhance cell growth and cell differentiation in vitro. In addition, the additon of ncHA and 100nM Tideglusib in biHAG would promote fibrous tissue and vessels formation in vivo. 250 μm biHAG with 100 nM Tideglusib presented the better fibrous tissue formation and angiogenesis, showing the highly potential in dentin-pulp complex regeneration. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:10:21Z (GMT). No. of bitstreams: 1 ntu-108-R05422017-1.pdf: 5809255 bytes, checksum: 4175f539a6ef6d4089c1f346cc934832 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii 英文摘要 iv 目錄 vii 圖目錄 xi 表目錄 xiv 縮寫表 xvi 第一章 前言 1 第二章 文獻回顧 3 2.1 現代根管治療的發展與限制 3 2.2 組織工程的再生技術 5 2.3 幹細胞 6 2.4支架 8 2.4.1 透明質酸的特性 8 2.4.2雙相型透明質酸膠體顆粒在牙科的應用 9 2.5生長因子 10 2.5.1 生長因子的選擇 10 2.5.2 Wnt訊息傳遞路徑在牙齒發育扮演的角色 10 2.5.3 GSK-3抑制劑的選用 11 第三章 動機與目的 14 第四章 材料與方法 15 4.1 儀器裝置 15 4.2 藥品材料 16 4.3 材料製備 16 4.3.1 製備交聯型透明質酸膠體顆粒(HAG)材料 16 4.3.2 製備雙相型透明質酸膠體顆粒(biHAG)材料 17 4.4 物理性質分析 17 4.4.1 流變性質分析 17 4.4.2 膨脹比例分析 18 4.5 Tideglusib濃度對細胞活性的作用 18 4.5.1 細胞選擇與培養 18 4.5.2 細胞活性之分析 19 4.5.3 細胞存活率 19 4.5.4 統計分析 20 4.6 Tide@HAG的生物相容性 20 4.6.1 製備搭載Tideglusib之雙相型透明質酸膠體(Tide@biHAG)材料 20 4.6.2 細胞選擇與培養 21 4.6.3 材料萃取液製備 21 4.6.4 細胞存活率 21 4.6.5 細胞致死率 22 4.6.6 統計分析 22 4.7 細胞與Tide@HAG共養之細胞生長行為 22 4.7.1 細胞生長行為之分析 23 4.8 不同濃度Tideglusib對細胞分化的影響 23 4.8.1 細胞選擇與培養 23 4.8.2 RNA萃取及反轉錄聚合酶鏈鎖反應 24 4.8.3 統計分析 25 4.9 小鼠背部皮下注射材料之組織反應 25 4.9.1 注射材料製備 25 4.9.2 小鼠背部皮下注射模型 25 4.9.3 檢體觀察及組織學切片分析 26 4.9.3 統計分析 26 第五章 結果 27 5.1 雙相型透明質酸膠體顆粒之物理性質分析 27 5.1.1 流變性質(rheology property)分析 27 5.1.2 膨脹比例(swelling ratio)分析 28 5.2 搭載Tideglusib之雙相型透明質酸膠體顆粒對細胞之影響 28 5.2.1 Tideglusib的細胞毒性分析 28 5.2.2 Tide@HAG的生物相容性分析 29 5.2.3 細胞生長行為之觀察分析 29 5.2.4 不同濃度Tideglusib對細胞分化之影響分析 30 5.3 小鼠背部皮下注射之活體實驗結果 30 5.3.1 檢體外觀及體積大小 30 5.3.2 組織切片觀察及半定量分析 31 第六章 討論 33 6.1 透明質酸膠體的物理性質探討 33 6.1.1 溫度反應環境對膠體影響之探討 33 6.1.2 粒徑大小對膠體影響之探討 34 6.2 Tide@HAG對於細胞影響之探討 36 6.2.1 Tideglusib的細胞毒性之探討 36 6.2.2 Tide@HAG的生物相容性探討 36 6.2.3 細胞和Tide@HAG共養的生長行為探討 37 6.2.4 不同濃度Tideglusib對細胞分化行為之探討 38 6.3 小鼠背部注射biHAG和Tide@HAG對活體組織影響之探討 39 6.3.1 實驗模型設計之探討 39 6.3.2 檢體外觀及尺寸之探討 40 6.3.3 組織學觀察之探討 41 第七章 結論 44 參考文獻 46 附錄 51 | |
dc.language.iso | zh-TW | |
dc.title | 研發搭載Tideglusib之雙相型透明質酸膠體在牙本質牙髓組織再生的應用 | zh_TW |
dc.title | Development of Biphasic Hyaluronic Acid Gel with Tideglusib in Dentin-Pulp Tissue Regeneration | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林?輝(Feng-Huei Lin),李伯訓(Bor-Shiunn Lee) | |
dc.subject.keyword | 透明質酸膠體,交聯反應環境,粒徑大小,物理性質,Tideglusib,生物相容性,細胞分化,動物模型,纖維組織生成,血管新生, | zh_TW |
dc.subject.keyword | hyaluronic acid granules,cross-linking reaction environment,granular size,physical properties,Tideglusib,biocompatibility,cell differentiation,subcutaneous model,fibrous tissue formation,angiogenesis, | en |
dc.relation.page | 91 | |
dc.identifier.doi | 10.6342/NTU201903468 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
顯示於系所單位: | 臨床牙醫學研究所 |
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