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姓名	林楷崴(Kai-Wei Lin)  查詢紙本館藏	畢業系所	生物醫學工程研究所
論文名稱	利用體外循環培養系統模擬腦源性神經分泌促使 硬骨類組織成熟化 (To Establish In-vitro Circulating System Mimicking Brain-Derived Neurosecretion for Accelerating Bone-like Tissue Maturation)
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摘要(中)	隨著高齡社會來臨，年長者因鈣質流失、骨質疏鬆等問題加劇，骨損傷復原的速度明顯較一般人慢且不佳。傳統上的骨移植治療方式大多是自體、異體骨移植，或是骨粉等生物性材料，但卻存在免疫排斥或骨材料硬度篩選的問題，而現行的細胞療法多採用單細胞（single cell）的方式來治療患者，不但價格昂貴且效果受限，因此想藉由細胞治療以及組織工程的方式來加速傷者骨修復速度。根據衛福部公告，細胞治療已經在多家醫院實行，藉由選取特定細胞加以培養再植入患部，能夠達成減少手術次數、降低感染風險等優勢。臨床醫學案例統計上，發現因車禍意外造成腦損傷患者，其骨頭修復速度較一般患者快速，這與腦神經分泌物突破血腦屏障（blood-brain barrier, BBB）有密切關聯，因此我們使用人類腦神經幹細胞株（human neural progenitor cells/ReNcell™ CX cell line）與初代人類骨髓間質幹細胞（primary human mesenchymal stem cell, hMSCs）在生物反應器中來模擬上述環境，除了添加骨分化試劑外，利用培養神經幹細胞時所分泌出之生長因子，透過系統洄流滋養人類骨髓間質幹細胞，以刺激成骨生長速度加速硬骨類組織成熟化。為了維持神經細胞的生長，本次實驗使用了明膠（gelatin）及人類腦血管內皮細胞株 （human brain endothelial capillary cells, hCMEC/D3 cell line）來做為神經細胞的生物支架，配合海藻膠（alginate）生物支架來促進人類骨髓間質幹細胞形成類硬骨團塊（bone-like tissues）。透過實驗室自組裝的生物反應器，藉由生物反應器的回流裝置輸送生長因子與滋養物質，能夠更貼近真實的人類體循環，最後達到類硬骨團塊成熟化（maturation）之目的。希望透過建立此系統，來突破目前骨科細胞治療市場的困境，造福更多的患者。
摘要(英)	With the growing share of aging population, the elderly has crucial problems such as calcium loss and osteoporosis. Thereupon, these aggravated problems lead to bone reconstruction more slowly than the average person. The traditional treatments of bone defects are mostly autologous/allogeneic bone transplantation, but there are problems of immune rejection. Or the clinical doctor would like to use biomaterial substitution, however, how to choose the appropriate material source is very confusing. Moreover, the current cell therapy is expensive and mostly restricted to single cells. According to the announcement of the Ministry of Health and Welfare in Taiwan, the cell therapy has been implemented in many hospitals. The statistics of clinical medical cases showed, it is found that the speed of bone repair in patients with traumatic brain injury (TBI) caused by car accidents is faster than that of ordinary patients. From previous reference we found that is closely related to the breakthrough of blood-brain barrier (BBB) by brain-derived neural secretions. In this study, human neural progenitor cell line (ReNcell™ CX) and primary human mesenchymal stem cells (hMSCs) were applied in the designed bioreactor system to biomimic the micro-environment. The neural-related growth factors secreted by ReNcell™ CX cell line and stimulated hMSCs toward osteogenesis and accelerated bone-like tissue maturation during the dynamic cultivation. In order to maintain the growth of ReNcell™ CX cells, gelatin scaffolds were used as biological scaffolds and human brain endothelial capillary cells (hCMEC/D3) were co-cultured with ReNcell™ CX cells in bioreactor system. At the same time, alginate scaffolds were applied hMSCs to form bone-like tissues. Through the self-designed bioreactor system, growth factors and nourishing substances are delivered through the refluxing device. It is closer to the real human physiological circulation, and finally achieve the purpose of in-vitro bone-like tissue maturation. We sincerely hope the strategy can break through the current predicament of the orthopedic cell therapy market and benefit more patients.
關鍵字(中)	★ 生物反應器 ★ 硬骨 ★ 成熟化 ★ 神經幹細胞 ★ 間質幹細胞 ★ 神經分泌物	關鍵字(英)	★ Bioreactor ★ hard bone ★ maturation ★ neural stem cell ★ mesenchymal stem cell ★ neurosecretion
論文目次	摘 要 i ABSTRACT ii 誌謝 iv 目錄 v 圖目錄 x 表目錄 xii 符號縮寫說明 xiii 第一章 緒論 1 1-1骨骼 2 1-1-1硬骨的功能 2 1-1-2硬骨的組成 2 1-1-3人類骨髓間質幹細胞 4 1-1-3 破骨細胞 5 1-1-4人類骨髓間質幹細胞之分化 6 1-2骨損傷 8 1-2-1硬骨修復機制 8 1-2-2現行硬骨移植方式 9 1-2-3 骨損傷之統計及其相關市場 10 1-3腦損傷 11 1-3-1腦部微結構 11 1-3-2血腦障壁 11 1-4 腦損傷與硬骨關係 13 1-4-1腦損傷的影響對硬骨的影響 13 1-4-2異位骨化相關機制 13 1-4-3 腦創傷動物模型對骨修復影響 14 第二章 研究動機與實驗目的 16 2-1細胞治療 16 2-1-1細胞治療原理 16 2-1-2現行細胞治療法規 18 2-1-3 臨床的細胞治療 19 2-2細胞培養 20 2-2-1二維細胞培養 20 2-2-2三維細胞培養 20 2-3海藻膠支架 21 2-3-1海藻酸鹽 21 2-3-2 海藻酸鹽之交聯與細胞培養 22 2-4明膠支架 23 2-4-1 明膠 23 2-4-2明膠支架交聯 24 2-4-3谷氨醯胺轉氨酶 25 2-5生物反應器 26 2-5-1組織工程 26 2-5-2傳統生物反應器 27 2-5-3自製生物反應器 28 2-6 串聯系統之生物反應器 29 2-7 實驗設計與架構 30 第三章 材料和方法 32 3-1 細胞培養 32 3-1-1人類骨髓間質幹細胞 32 3-1-2人類腦神經幹細胞 33 3-1-3人類腦血管內皮細胞 34 3-2二維共培養測試 35 3-2-1培養液比例測試 35 3-3骨分化 35 3-3-1骨分化試劑添加 35 3-4 海藻膠支架 36 3-4-1海藻酸支架製備 36 3-4-2海藻酸支架細植入率 37 3-4-3海藻酸支架細胞存活率 37 3-5明膠支架 38 3-5-1谷氨醯胺轉氨酶製備 38 3-5-2明膠支架製備 38 3-6生物反應器 40 3-6-1生物反應器管線配置 40 3-6-2細胞支架載入反應器 40 3-7 支架內細胞分析 40 3-7-1 支架內細胞死活染色 40 3-7-2 酒精固定 41 3-7-3超臨界二氧化碳乾燥 41 3-7-4離子鍍層濺射 42 3-7-5掃描式電子顯微鏡 42 3-8 骨礦化之鑑定 42 3-8-1礦化物結晶分析 42 3-8-2成分分析 42 3-9 硬骨相關基因表達 43 第四章 結果與討論 45 4-1培養液比例測試 45 4-2細胞支架製備 46 4-2-1海藻酸支架 46 4-2-2明膠支架 46 4-3細胞支架植入率 47 4-4細胞支架結構 48 4-5細胞於反應器培養一周後型態觀察 49 4-6 支架內細胞死活染色 50 4-7細胞礦化物分析 52 4-8沉澱物分析 53 4-8-1 X光繞射儀分析 53 4-8-2 傅立葉轉換紅外光譜分析 54 4-9 即時聚合酶連鎖反應 55 4-10 培養液檢測 58 第五章 結論 60 第六章 參考文獻 61
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指導教授	陳靖昀 周中興(Ching-Yun Chen Chung-Hsing Chou)	審核日期	2022-9-27
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