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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 | Kuan-Cheng Kuo | en |
dc.contributor.author | 郭寬程 | zh_TW |
dc.date.accessioned | 2021-06-17T03:11:50Z | - |
dc.date.available | 2020-08-27 | |
dc.date.copyright | 2020-08-27 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69272 | - |
dc.description.abstract | 以人體牙髓幹細胞(human dental pulp stem cells, hDPSCs)作為細胞來源,應用雙相型透明質酸膠體顆粒(biphasic hyaluronic acid granules, biHAG)作為支架,並載入洛伐他汀之聚乳酸聚甘醇酸奈米顆粒(Poly(lactic-co-glycolic acid) nanoparticle with lovastatin, PLGA-Lova)的組織工程研究模型,可藉由洛伐他汀(lovastatin)的長期緩釋效果達到促進鈣化組織生成和誘導血管新生的作用,具有達到牙本質牙髓組織再生目標的潛力。其中biHAG為交聯型透明質酸膠體顆粒(hyaluronic acid granules, HAG)和非交聯透明質酸(non-crosslinked HA, ncHA)的混合物,根據混合比例和膠體粒徑大小的不同,可表現出不同的可注射性、流體性質和膨脹比例等物理性質,然而其降解行為尚待進一步研究;此外biHAG搭載PLGA-Lova的最佳混合濃度也需要進一步的分析。因此本研究的目的是希望了解交聯程度、混合比例和膠體粒徑大小對材料降解行為的影響,並分析biHAG搭載PLGA-Lova的濃度對細胞行為的影響,研發理想的牙本質牙髓組織再生材料。 使用1,4-丁二醇二縮水甘油醚(1,4-butanediol diglycidyl ether, BDDE)做為交聯劑製備2% HAG,並經由過篩得到420 μm和250 μm兩種粒徑大小。接著以設定比例使用物理性方式混合HAG與ncHA製備biHAG,材料依所含HAG的比例分別命名為HAG100、HAG80和HAG60。然後將PLGA-Lova以濃度50 μg/ml和100 μg/ml與biHAG進行物理性混合,製備出搭載PLGA-Lova之雙相型透明酸膠體(Lova@biHAG)。並且進一步評估材料的交聯程度、降解行為與生物相容性。 研究結果顯示,HAG100、HAG80和HAG60三種材料的交聯修飾程度(degree of modification, MoD)介於19.14% ~ 20.08%,與ncHA的比例無關。而在體外降解行為的部分,粒徑大小420 μm的組別之一天降解率介於33.19% ~ 35.17%,而粒徑大小250 μm的組別則介於40.49% ~ 44.10%;增加ncHA的比例或是降低粒徑大小均會提升材料的體外一天降解率。活體降解行為方面,HAG100於6周的活體降解率僅有7.76%,16周時增加到12.80%;而biHAG於6周的活體降解率介於20.01% ~ 22.47%,於16周增加到25.10% ~ 30.66%。隨著時間增加,所有組別的組織生成量和材料降解率都有明顯提升,其中添加ncHA的biHAG明顯高於HAG100,而HAG60雖然略高於HAG80,但沒有顯著差異;此外小顆粒組別略高於大顆粒組別,但同樣沒有顯著差異。而在生物相容性分析的部分,將PLGA-Lova濃度為50 μg/ml和100 μg/ml的Lova@biHAG以萃取液模型培養細胞後,細胞致死率與控制組無顯著差異,顯示材料對細胞沒有毒性。另外在細胞生長行為的分析方面,可以看到細胞與Lova@biHAG共養後,隨著時間細胞數有增加的趨勢,且大多數觀察到的細胞具有正常的形態以及平滑的細胞表面,只有少於2 %的細胞呈現摺疊、破孔和缺陷的現象;此外五天後呈現圓球狀的細胞比例均有下降的趨勢,顯示材料具有良好的生物相容性。 總結來說,添加ncHA能夠提升材料在活體內的代謝速度和組織生成的誘導能力,然而在16周後降解率最高的材料也只有約三成,整體的分解時間較長,未來需要針對材料的降解速率進行改良。而在PLGA-Lova混合濃度的部分,可以看到當載入濃度為50 μg/ml和100 μg/ml時,材料對細胞均表現出良好的生物相容性和細胞生長行為,未來需要進一步評估促進細胞分化的能力,以及在活體內誘導組織生成的能力,來確定此藥物濃度的有效性。 | zh_TW |
dc.description.abstract | The study model of tissue engineering by using human pulp stem cells (hDPSCs) as cell sources, biphasic hyaluronic acid granules (biHAG) as scaffold and PLGA-lovastatin nanoparticles (PLGA-Lova) as stimulator via long-term and static lovastatin releasing, has highly potential to promote mineralization and angiogenesis in pulp and dentin regeneration. The mixtures of cross-linked hyaluronic acid granules (HAG) and non-crosslinked HA (ncHA), named biHAG, were demonstrated to have different injectabilities, rheologic properties and swelling ratios based on their granular sizes and mixing ratio of HAG and ncHA. But the information of their degradation behaviors were still lacking now and the optimal PLGA-Lova concentration in biHAG need to be further investigated. The purpose of this study was to investigate the effects of degree of cross-linking, granular size and mixing ratio of HAG and ncHA on the degradation behaviors of biHAG, and to evaluate the cellular behaviors to biHAG with different PLGA-Lova concentration, and then propose ideal materials for dentin and pulp regeneration. 2% HAG with granular size of 420 μm and 250 μm were synthesized by using 1,4-butanediol diglycidyl ether (BDDE) as crosslinking agent. biHAG were prepared by mixing ncHA with HAG, named HAG100, HAG80 and HAG60 according to the composition ratio of HAG. Then, biHAG with PLGA-Lova (Lova@biHAG) were obtained by adding 50 μg/ml and 100 μg/ml PLGA-Lova. The degree of modification, in vitro and in vivo degradation behaviors and biocompatibility of the materials were investigated. The data of the degree of modification for HAG100, HAG80 and HAG60 were between 19.14% ~ 20.08%, indicating ncHA had no effects on degree of cross-linking. The in vitro study showed the 1 day degradation rates of 420 μm HAGs were 33.19% ~ 35.17% and 250 μm HAGs were 40.49% ~ 44.10%. Increased the content of ncHA and decreased granular size would raise the 1 day degradation rate in vitro. The in vivo study demonstrated the degradation rate of HAG100 was 7.76% at 6-weeks and 12.80% at 16-weeks, while biHAG was 20.01% ~ 22.47% at 6-weeks and 25.10% ~ 30.66% at 16-weeks. In all groups, the degradation rate significantly increased from 6-weeks to 16-weeks. The biHAGs presented much faster degradation than HAG100, and the HAG 60, contained more ncHA, had higher degradation rate than HAG80 but without statistically significance, indicating ncHA may play roles for promoting in vivo degradation. 250 μm HAGs only degraded slightly faster than 420 μm HAGs without a significant difference. In addition, the results showed Lova@biHAG with two different PLGA-Lova concentrations were both biocompatible. No significant differences were found in cell viability and cell death among Lova@biHAGs and control groups using extract model. The hDPSC grew on Lova@biHAGs were less retained in rounded shape and increased in cell numbers with culture time. Most hDPSC presented normal morphology with smooth surface, only less than 2% cells were observed with folding, defects of small holes, or depression. In summary, addition of ncHA would promote degradation behavior and tissue formation in vivo. However, the degradation rate of biHAGs was less than 30% even after 16 weeks, which may be too long for dentin and pulp regeneration and need further investigation to improve. Although Lova@biHAGs with 50 μg/ml and 100 μg/ml PLGA-Lova, had good biocompatibility and no negative effects on cell behaviors, further evaluations would be also needed to clarify their effects on cell differentiation and tissue formation in vivo. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:11:50Z (GMT). No. of bitstreams: 1 U0001-1808202015472700.pdf: 8908805 bytes, checksum: 49d504a39e54f3973d95c35276a37ac3 (MD5) Previous issue date: 2020 | 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 xiii 縮寫表 xv 第一章 前言 1 第二章 文獻回顧 3 2.1 組織工程於牙本質牙髓組織再生的運用 3 2.1.1 傳統根管治療的限制與發展 3 2.1.2 組織工程技術於牙本質牙髓組織再生的發展 4 2.2 幹細胞 5 2.2.1 人體牙髓幹細胞的純化與繼代培養 6 2.2.2 特定標記幹細胞的篩選與在牙本質牙髓組織再生的運用 7 2.3 支架 9 2.3.1 支架於牙本質牙髓組織再生的運用 9 2.3.2 透明質酸的特性與在醫學上的運用 9 2.3.3 交聯型透明質酸在牙本質牙髓組織再生研究上的運用 10 2.3.4 雙相型透明質酸膠體 11 2.3.5 交聯型透明質酸之交聯程度檢測 12 2.3.6 交聯型透明質酸之體外降解行為檢測 13 2.4 生長因子 14 2.4.1 生長因子於牙本質牙髓組織再生的運用 14 2.4.2 Statin類藥物 14 2.4.3 Statin類藥物於牙本質牙髓組織再生研究的運用 15 2.4.4 PLGA-lovastatin於牙本質牙髓組織再生研究的運用 16 2.4.5 搭載PLGA-lovastatin之雙相型透明質酸膠體 17 第三章 動機與目的 18 第四章 材料與方法 19 4.1 儀器裝置 19 4.2 藥品材料 20 4.3 材料製備 21 4.3.1 製備交聯型透明質酸膠體顆粒(HAG)材料 21 4.3.2 製備雙相型透明質酸膠體顆粒(biHAG)材料 21 4.3.3 製備洛伐他汀之聚乳酸聚甘醇酸奈米顆粒(PLGA-Lova)材料 21 4.3.4 製備雙相型透明質酸膠體(biHAG)及搭載洛伐他汀之雙相型透明質酸膠體(Lova@biHAG)材料22 4.4 交聯程度分析 23 4.5 體外降解行為分析 24 4.5.1 呈色反應之最佳實驗條件設定 24 4.5.2 完全水解之實驗模型 25 4.5.3 體外一天降解率 26 4.5.4 統計分析 26 4.6 活體降解行為之分析 26 4.6.1 小鼠背部皮下注射模型 26 4.6.2 組織學觀察與半定量分析 27 4.6.3 統計分析 27 4.7 Lova@biHAG的生物相容性分析 27 4.7.1 細胞選擇與培養 28 4.7.2 材料萃取液製備 28 4.7.3 細胞存活率 29 4.7.4 細胞致死率 29 4.7.5 統計分析 30 4.8 細胞與Lova@HAG共養之細胞生長行為 30 4.8.1 樣本製備 30 4.8.2 細胞生長行為之分析 31 第五章 結果 32 5.1 交聯程度分析 32 5.2 體外降解行為分析 32 5.2.1 呈色反應之最佳實驗條件設定 32 5.2.2 完全水解之實驗模型 33 5.2.3 一天體外降解率 33 5.3 活體降解行為分析 34 5.3.1 組織學發現 34 5.3.2 活體降解率之半定量分析 35 5.4 生物相容性分析 35 5.4.1 細胞存活率分析 35 5.4.2 細胞致死率分析 35 5.4.3 細胞生長行為之觀察 36 第六章 討論 38 6.1 交聯程度之探討 38 6.2 體外降解行為之探討 39 6.2.1 呈色反應之最佳實驗條件設定 39 6.2.2 完全水解之實驗模型 41 6.2.3 一天體外降解率 42 6.3 活體降解行為之探討 43 6.3.1 實驗模型設計之探討 43 6.3.2 組織學發現之探討 44 6.3.3 體外與活體降解行為之比較 45 6.4 PLGA-Lova濃度對生物行為的影響 46 第七章 結論 48 參考文獻 50 附錄 54 | |
dc.language.iso | zh-TW | |
dc.title | 搭載洛伐他汀之雙相型透明質酸膠體之降解行為及生物相容性分析 | zh_TW |
dc.title | Degradation Behavior and Biocompatibility of Biphasic Hyaluronic Acid Gel with Lovastatin | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林峯輝(Feng-Huei Lin),李伯訓(Bor-Shiunn Lee) | |
dc.subject.keyword | 支架,透明質酸膠體,交聯程度,體外降解,動物模型,皮下注射,洛伐他汀,生物相容性, | zh_TW |
dc.subject.keyword | scaffolds,hyaluronic acid granules,degree of cross-linking,in-vitro degradation,animal model,subcutaneous injection,lovastatin,biocompatibility, | en |
dc.relation.page | 93 | |
dc.identifier.doi | 10.6342/NTU202003989 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-19 | |
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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