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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周涵怡 | zh_TW |
| dc.contributor.advisor | Han-Yi Chou | en |
| dc.contributor.author | 蔡㑊璇 | zh_TW |
| dc.contributor.author | Yi-Hsuan Tsai | en |
| dc.date.accessioned | 2026-08-28T16:37:10Z | - |
| dc.date.available | 2026-08-29 | - |
| dc.date.copyright | 2026-08-28 | - |
| dc.date.issued | 2026 | - |
| dc.date.submitted | 2026-08-13 17:46:15 | - |
| dc.identifier.citation | 1. Arvidson, K., Abdallah, B. M., Applegate, L. A., Baldini, N., Cenni, E., Gomez-Barrena, E., Granchi, D., Kassem, M., Konttinen, Y. T., Mustafa, K., Pioletti, D. P., Sillat, T., & Finne-Wistrand, A. (2011). Bone regeneration and stem cells. *Journal of Cellular and Molecular Medicine, 15*(4), 718–746. https://doi.org/10.1111/j.1582-4934.2010.01224.x
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/104599 | - |
| dc.description.abstract | 骨組織缺損常見於外傷、腫瘤切除、感染性壞死與先天性發育缺陷，當範圍超過臨界大小時，內源性修復機制無法橋接缺損而形成骨不癒合。現行骨移植材料分別受限於取材量與供區併發症、處理後骨誘導能力下降，或僅具骨傳導功能。間葉幹細胞之治療效益主要來自旁分泌作用，其分泌之細胞外囊泡(extracellular vesicles, EVs)可將 miRNA 與蛋白質遞送至受體細胞，已成為無細胞治療之核心；惟其體內半衰期極短，需藉載體達成局部持續釋放。
本研究以三維列印技術製備藻酸鹽／明膠複合水膠支架作為牙源幹細胞 EVs 之遞送載體。先比較 3% 與 5% 藻酸鹽配方之抗壓強度、降解速率與生物相容性以選定配方；繼以尺寸排阻層析法自牙髓幹細胞(DPSCs)與牙周韌帶幹細胞(PDLSCs)分離 EVs 並完成鑑定；最後以骨肉瘤細胞株 MG63 評估兩種來源 EVs 於不同劑量下之成骨反應。 結果顯示，3% 與 5% 配方之最大抗壓強度分別為 0.82 與 1.05 MPa，第 28 天降解率分別為 81% 與 32%，細胞存活率均高於 70%，符合 ISO 10993 標準；因降解時程與骨再生窗口相符，後續選用 3% 配方。紅外光譜佐證 EVs 已嵌入水膠網絡，釋放曲線呈兩相行為並持續七天。DPSC-EVs 與 PDLSC-EVs 之平均粒徑分別為 83.3 與 95.1 nm，均表現 CD9、CD81 與 TSG101。功能評估方面，第 14 天鹼性磷酸酶染色面積以 PDLSC-E9 組最高(約 31%)，高於 DPSC-E9 與 PDLSC-E8 組(約 15%)與對照組(約 5%)；第 21 天茜素紅吸光值亦以 PDLSC-E9 組最高(O.D. 540 nm 約 0.91)；PDLSC-EVs 組之 OCN 與 OPN 表現顯著高於對照組與 DPSC-EVs 組(p < 0.05)，且上述效果均具劑量依賴性。 綜合而言，三維列印天然水膠可有效搭載牙源幹細胞 EVs 並維持其活性與持續釋放；PDLSC-EVs 之成骨誘導效能優於 DPSC-EVs，顯示細胞來源對 EVs 功能具實質影響。惟本研究侷限於體外模式，體內效能仍待動物實驗驗證。 | zh_TW |
| dc.description.abstract | Bone defects arising from trauma, tumor resection, infection, or congenital disorders cannot heal spontaneously once they exceed the critical size. Current grafting materials are limited by donor availability, donor-site morbidity, or insufficient osteoinductivity. As the benefit of mesenchymal stem cells derives largely from paracrine signaling, their extracellular vesicles (EVs), which deliver miRNAs and proteins to recipient cells, have emerged as a promising cell-free alternative; their short half-life in vivo, however, necessitates a carrier for local sustained release.
This study developed a three-dimensionally printed alginate/gelatin hydrogel scaffold as a carrier for dental stem cell-derived EVs. Formulations containing 3% and 5% alginate were compared for compressive strength, degradation, and biocompatibility; EVs were isolated from human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) by size-exclusion chromatography, and their osteogenic effects were evaluated in MG63 cells at two doses. Maximum compressive strengths were 0.82 and 1.05 MPa and 28-day degradation rates were 81% and 32% for the 3% and 5% formulations, respectively, with L929 viability above 70% for both, meeting ISO 10993. The 3% formulation was selected because its degradation profile better matched the window of bone regeneration. Infrared spectroscopy confirmed EV incorporation, and release was biphasic and sustained through day 7. DPSC-EVs and PDLSC-EVs showed mean diameters of 83.3 and 95.1 nm and expressed CD9, CD81, and TSG101. Alkaline phosphatase staining at day 14 was highest in the PDLSC-E9 group (approximately 31%), exceeding DPSC-E9 and PDLSC-E8 (approximately 15%) and the control (approximately 5%); alizarin red staining at day 21 was likewise highest in PDLSC-E9 (O.D. 540 nm approximately 0.91), and PDLSC-EV treatment significantly upregulated OCN and OPN (p < 0.05), all in a dose-dependent manner. In summary, this customizable hydrogel system carried dental stem cell-derived EVs while preserving bioactivity and sustaining release. PDLSC-derived EVs showed superior osteoinductive efficacy, indicating that the cellular source materially influences EV function. As the evaluation was limited to in vitro models, in vivo efficacy awaits verification. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2026-08-28T16:37:10Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2026-08-28T16:37:10Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii 英文摘要 iv 圖 次 x 表 次 xi 第一章 文獻回顧 1 1.1 骨組織再生的生理基礎與臨床需求 1 1.2 現行骨缺損治療策略及其限制 2 1.3 幹細胞治療於骨再生之應用與其瓶頸 4 1.3.1 間葉幹細胞於骨再生之應用 4 1.3.2 幹細胞的旁分泌作用 4 1.3.3 細胞治療的臨床轉譯瓶頸 5 1.4 細胞外囊泡 5 1.5 細胞外囊泡之分離純化與鑑定 8 1.6 細胞外囊泡於再生醫學與臨床之應用 10 1.7 牙髓幹細胞(Dental Pulp Stem Cells, DPSCs) 12 1.8 牙周韌帶幹細胞(Periodontal Ligament Stem Cells, PDLSCs) 12 1.9 水膠於骨再生材料之應用現況 16 1.9.1 水膠的基本特性與分類 16 1.9.2 藻酸鹽(Alginate) 16 1.9.3 明膠(Gelatin) 17 1.9.4 藻酸鹽／明膠複合水膠 17 1.10 三維列印技術於骨組織工程中的發展 18 1.11 生物支架中生物活性因子之控制釋放 19 1.12 研究動機、目的與假設 22 1.12.1 研究動機 22 1.12.2 研究目的 22 1.12.3 研究假設 23 第二章 研究材料與方法 24 2.1 支架材料配製 24 2.2 水膠支架列印 24 2.3 支架材料表面官能基分析(傅立葉紅外光譜FTIR) 25 2.4 支架材料抗壓強度試驗 25 2.5 支架材料降解性能分析 25 2.6 支架材料生物毒性試驗 26 2.7 牙髓幹細胞萃取及細胞培養 26 2.8 牙周韌帶幹細胞細胞培養 27 2.9 細胞外囊泡萃取 27 2.10 細胞外囊泡濃度及尺寸鑑定 28 2.11 細胞外囊泡型態觀察 29 2.12 細胞外囊泡專一性標記鑑定 29 2.13 水膠支架之細胞外囊泡釋放速率 31 2.14 細胞貼附型態觀察 31 2.15 骨細胞分化試驗 31 2.16 骨細胞礦化試驗 32 2.17 即時定量核酸鏈結反應 32 2.18 統計分析 33 第三章 結果與分析 34 3.1 複合水膠支架製備及性能測試 34 3.1.1 水膠支架製備及顯微結構 34 3.1.2 支架表面官能基鑑定分析 34 3.1.3 支架抗壓強度測試 34 3.1.4 支架降解性能分析 35 3.1.5 支架生物相容性試驗 35 3.2 細胞外囊泡特徵鑑定 35 3.2.1 奈米粒徑追蹤分析 35 3.2.2 穿透式電子顯微鏡形貌觀察 36 3.2.3 細胞外囊泡專一性膜蛋白表現 36 3.2.4 細胞外囊泡搭載於水凝膠支架之釋放速率檢測 36 3.3 細胞外囊泡結合水膠支架之骨再生活性試驗 36 3.3.1 人類骨肉瘤MG63細胞貼附觀察 36 3.3.2 人類骨肉瘤MG63細胞成骨分化試驗 37 3.3.3 人類骨肉瘤MG63細胞成骨礦化試驗 37 3.3.4 成骨相關基因表達鑑定 37 第四章 討論 39 4.1 支架配方之選擇與物理化學性質 39 4.2 EVs 嵌合之光譜學證據及其解釋範圍 40 4.3 EVs 之釋放行為與定量方法之解釋範圍 40 4.4 細胞貼附形態與成骨表現之關係 41 4.4.1 藻酸鹽支架之細胞黏附特性 41 4.4.2 細胞貼附受限與成骨標誌上升並存之意義 42 4.5 PDLSC-EVs 成骨效能優於 DPSC-EVs 之可能機制 42 4.5.1 組織生態區位與分泌表型 42 4.5.2 miRNA 貨物之差異 43 4.5.3 膜蛋白標誌表現之差異 43 4.5.4 細胞層次與囊泡層次效能之區辨 44 4.6 EVs 之劑量依賴性反應 44 4.7 評估模式之考量 44 4.8 與現有 EVs 遞送系統之比較 45 4.9 研究限制 46 4.10 未來研究方向 46 第五章 結論 48 第六章 參考文獻 49 附錄 57 圖 次 圖一、細胞外囊泡萃取流程圖 57 圖二、藻酸鹽+明膠複合水膠支架列印 58 圖三、掃描式電子顯微鏡下支架形貌 59 圖四、支架表面官能基鑑定分析-傅立葉紅外光譜法 60 圖五、支架抗壓強度測試-應力-應變曲線圖 61 圖六、支架降解曲線圖 62 圖七、支架生物毒性試驗-MTT assay 63 圖八、細胞外囊泡奈米粒徑追蹤分析 64 圖九、穿透式電子顯微鏡下細胞外囊泡形貌 65 圖十、細胞外囊泡專一性膜蛋白CD9、CD81、TSG101表現量 66 圖十一、細胞外囊泡搭載於水凝膠支架之釋放速率曲線 67 圖十二、人類骨肉瘤細胞MG63貼附於支架表面之螢光染色圖 68 圖十三、人類骨肉瘤細胞MG63成骨分化鹼性磷酸酶染色 69 圖十四、人類骨肉瘤細胞MG63成骨礦化茜素紅染色 71 圖十五、成骨相關OCN、OPN基因表現 72 表 次 表一、成骨相關基因 OCN、OPN 引子序列設計 73 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 骨再生 | - |
| dc.subject | 細胞外囊泡 | - |
| dc.subject | 三維列印水膠支架 | - |
| dc.subject | 牙髓幹細胞 | - |
| dc.subject | 牙周韌帶幹細胞 | - |
| dc.subject | bone regeneration | - |
| dc.subject | 3D printing hydrogel scaffold | - |
| dc.subject | extracellular vesicles | - |
| dc.subject | dental pulp stem cells | - |
| dc.subject | periodontal ligament stem cells | - |
| dc.title | 三維列印水膠搭載控制釋放牙源幹細胞細胞外囊泡對骨再生之效能評估 | zh_TW |
| dc.title | Evaluation of Bone Regeneration Efficacy Using 3D-Printed Hydrogel Loaded with Controlled-Release Dental Stem Cell-Derived Extracellular Vesicles | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 114-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林俊彬;陳文斌 | zh_TW |
| dc.contributor.oralexamcommittee | Chun-Pin Lin;Wen-Pin Chen | en |
| dc.subject.keyword | 骨再生; 細胞外囊泡; 三維列印水膠支架; 牙髓幹細胞; 牙周韌帶幹細胞 | zh_TW |
| dc.subject.keyword | bone regeneration; 3D printing hydrogel scaffold; extracellular vesicles; dental pulp stem cells; periodontal ligament stem cells | en |
| dc.relation.page | 64 | - |
| dc.identifier.doi | 10.6342/NTU202604256 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2026-08-14 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 口腔生物科學研究所 | - |
| dc.date.embargo-lift | N/A | - |
| 顯示於系所單位： | 口腔生物科學研究所 | |
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| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-114-2.pdf 未授權公開取用 | 2.96 MB | Adobe PDF |
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