臺灣博碩士論文加值系統

骨質疏鬆症是一種可能導致骨折的常見疾病，需要進一步研究以開發更有效的治療方法。骨骼是高度血管化的器官，血管系統和礦化基質的發育需要成骨和血管生成之間的協調。我們專注於開發 3D 生物列印策略來製造血管化骨類器官 （VBO）。骨支架是使用聚己內酯（PCL）製造的，在其上接種骨前驅細胞（OPC）。由甲基丙烯酸酐化明膠（GelMA） 製成的血管支架，透過將血管支架放置在兩個骨支架之間，使 VBO 血管化。本研究採用壓縮力刺激研究機械壓縮對骨形成的影響，主要著重在骨支架培養物的壓縮刺激。透過 CCK-8 分析測量細胞活力，揭示成骨細胞在機械刺激下在支架內存活和增殖。共聚焦顯微鏡結果顯示成骨轉錄因子的獨特分佈。西方點墨法顯示，機械壓縮會增加成骨細胞中 Runx2 的表達，但會降低鹼性磷酸酶 （ALP）和骨橋蛋白（OPN）的表達。為了評估 VBO 的生理功能，將它們植入到 S.D.大鼠外側髁上鑽出的直徑 3 毫米、深 3 毫米的孔中。顯微 CT 影像顯示，移植 VBO 後的骨癒合有所增加。骨螢光標記結果表明，植入VBO的大鼠具有明顯的成骨作用。 VBO 模仿天然骨髓微環境，是治療骨質疏鬆症藥物開發的理想平台，並可能成為移植的合適替代品。這些發現為骨組織中的力傳導、成骨和血管生成的耦合以及更安全、更有效的骨質疏鬆症和骨折癒合治療提供了見解。

Osteoporosis is a common disease that may lead to fractures, needing further research to develop more effective treatments. The skeleton is a highly vascularized organ, and the development of the vascular system and mineralized matrix requires coordination between osteogenesis and angiogenesis. We focus on developing 3D bioprinting strategies to manufacture vascularized bone organoids (VBOs). Bone scaffold is fabricated using polycaprolactone (PCL), on which seeding osteoprogenitor cells (OPCs). A vascular scaffold made of gelatin-methacrylate (GelMA) is seeded with human umbilical vein endothelial cells (HUVECs), and VBOs are vascularized by placing the vascular scaffold between two bone scaffolds. This study employs compressive force stimulation to investigate the effects of mechanical compression on bone formation, primarily focusing on single-axis compression stimulation of the bone scaffold cultures. Cell viability is measured via CCK-8 analysis, revealing that osteoblasts survive and proliferate within the scaffold under mechanical stimulation. Confocal microscopy results show results showed unique distribution of osteogenic transcription factor. Western blotting reveals that mechanical compression increases the expression of Runx2 in osteoblasts but decreases the expression of alkaline phosphatase (ALP) and osteopotin (OPN). To assess the physiological function of VBOs, they are implanted into 3 mm diameter, 3 mm deep holes drilled into the lateral condyles of S.D. rats. Micro-CT imaging shows increased bone healing in transplanted with VBOs. Bone fluorescent labeling results show that rats implanted with VBO have obvious bone formation effect. Imitating the natural bone marrow microenvironment, VBOs serve as an ideal platform for drug development for treating osteoporosis and may potentially be suitable alternatives for transplantation. These findings provide insights into mechanotransduction in bone tissue, the coupling of osteogenesis and angiogenesis, and safer and more effective treatments for osteoporosis and fracture healing.

正文目錄 III
圖目錄 V
縮寫表 VI
中文摘要 VII
Abstract VIII
第一章、緒論 9
第一節、骨質疏鬆症 9
第二節、骨組織與骨質疏鬆症 12
第三節、骨組織工程 15
第四節、骨機械生物學 18
第五節、研究動機與目的 20
第二章、實驗材料與方法 21
第一節、實驗設計 21
第二節、研究材料 25
壹、細胞來源 25
貳、實驗藥劑與材料 25
第三節、研究方法 30
壹、成骨前驅細胞Osteoprogenitor cells（OPCs）培養 30
貳、3D支架列印 30
參、3D細胞培養 31
肆、壓力刺激 32
伍、分析細胞在壓力刺激後的差異與表現 32
陸、動物實驗 （In vivo experiment） 37
第三章、實驗結果 43
第一節、分析骨支架單培養系統中F-ACTIN經壓縮後的影響與表現 43
第二節、分析骨支架單培養系統中RUNX2經壓縮後的影響與表現 43
第三節、分析骨支架單培養系統中ALP經壓縮後的影響與表現 44
第四節、分析骨支架單培養系統中OPN經壓縮後的影響與表現 45
第五節、使用MICRO-CT觀察支架對於骨生長的影響 45
第六節、利用連續螢光標記觀察骨小樑及礦化沉積速度 45
第七節、以骨染液觀察骨小樑及礦化沉積速度 46
第四章、討論 47
第五章、結果附圖 50
第六章、結論 60
第七章、參考文獻 61
圖目錄
附圖 1、機械夾爪與操作介面 21
附圖 2、模型設計 22
附圖 3、實驗組別 23
附圖 4、動物實驗模型與流程 24

圖 1、3D OSTEOBLASTS單培養經壓縮後的F-ACTIN表現 50
圖 2、3D OSTEOBLASTS單培養經壓縮後的RUNX2表現 51
圖 3、使用WESTERN BLOT分析RUNX2的相對表現量 52
圖 4、使用WESTERN BLOT分析ALP的相對表現量 53
圖 5、使用WESTERN BLOT分析再培養後ALP的相對表現量 54
圖 6、使用WESTERN BLOT分析OPN的相對表現量 55
圖 7、動物實驗手術後支架放入位置 56
圖 8、使用MICRO-CT觀察支架對於骨生長的影響 57
圖 9、利用連續螢光標記觀察骨小樑及礦化沉積速度 58
圖 10、以骨染色觀察骨小樑及礦化沉積速度 59

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