氧氣在細胞調節、細胞增殖及能量產生上都扮演著重要的角色，若缺乏氧氣使細胞處於缺氧狀態，就容易因細胞代謝遭受破壞、ATP耗盡等現象，刺激細胞在短時間內死亡，導致組織壞死，產氧材料在臨床上是被廣泛需要的，像是骨折因常伴隨著血管結構的破壞，使得局部組織缺氧，進而導致骨折癒合不良或不癒合的情形。氧氣在骨折癒合中扮演重要的角色．其涉及了成骨細胞的分化、骨細胞增殖及骨礦化等等。為了改善細胞缺氧情形，並將其運用致臨床上，本論文開發一產氧載體，以聚乳酸-乙醇酸(poly latic-co-glycotic acid, PLGA)包覆過氧化鈣(CaO2)及二氧化錳(MnO2)。當PLGA降解時，水會滲透進入載體中和CaO2反應產生過氧化氫(H2O2)，並進一步被MnO2催化產生氧氣。實驗結果顯示，我們能夠以微流道系統製備出PLGA產氧微粒載體。將載體的各項參數最佳化後，該產氧微粒可使低氧環境下磷酸緩衝溶液中的溶氧量顯著上升，且最多可持續四天。在細胞實驗方面，我們也證實此PLGA產氧微粒載體不具有明顯細胞毒性。缺氧誘導因子的免疫螢光染色結果顯示，本論文開發之PLGA產氧微粒載體可提供足夠氧氣，使低氧(1% O2)培養環境中的細胞不致有明顯的缺氧情形發生。未來我們亦擬將此氧氣載體做更廣泛的應用，以了解本論文開發之氧氣載體在臨床上的潛能。

As a crucial molecule to life, oxygen is required in several cellular processes, such as aerobic respiration, enzyme activation, cell differentiation, collagen synthesis, that are involved in fracture healing. Therefore, a major hurdle in promoting bone regeneration is to provide sufficient oxygen to support the survival and function of the newly formed tissue. In this work, a composite oxygen-generating system consisting calcium peroxide (CaO2)/manganese dioxide (MnO2)-encapsulated poly lactic-co-glycolic acid (PLGA) microparticles (MPs) was developed for improving local oxygenation and preventing hypoxia-induced cell death. Once exposed to water, solid CaO2 particles embedded in the PLGA MPs can generate hydrogen peroxide (H2O2), which can be further converted into oxygen under the catalysis of MnO2. According to our results, the developed oxygen-generating MPs could produce oxygen continuously for four days without dramatic change in pH or accumulation of H2O2. The results of in vitro studies demonstrated that the oxygen-producing MPs could effectively relieve cellular hypoxia, suggesting their great potential for enhancing local oxygenation of the hypoxic tissues.

摘要 I
Abstract II
誌謝辭 III
目錄 IV
圖目錄 VI
第一章　緒論 1
1.1呼吸作用 1
1.2 產氧材料 2
1.3 聚乳酸-聚乙醇酸(Poly latic-co-glycotic acid, PLGA) 5
1.4 改善細胞缺氧之應用 6
1.5 骨骼缺損微環境 7
1.6 研究動機與實驗目的 9
第二章　材料與方法 10
2.1 包覆CaO2及MnO2之PLGA載體的製備 10
2.2 包覆CaO2及MnO2之PLGA載體的特性分析 11
2.2.1 產氧載體之大小與型態 11
2.2.2 產氧載體之氧氣產生情形 11
2.2.3 PLGA載體產生之酸鹼微環境 11
2.2.4 PLGA載體之H2O2濃度測試 12
2.3 細胞毒性測試 13
2.3.1 H2O2對細胞的影響 13
2.3.2 乳酸脫氫脢(Lactic Dehydrogenase, LDH)分析 13
2.3.3細胞存活率分析 14
2.3.4 細胞存活觀察 14
2.4 細胞回氧測試 14
第三章　結果與討論 15
3.1 PLGA濃度對產氧微粒載體所造成的影響 15
3.1.1 PLGA微粒載體之氧氣產生情形 15
3.1.2 PLGA微粒載體產氧後之酸鹼微環境 16
3.1.3 PLGA微粒載體產氧後之H2O2濃度 17
3.1.4 H2O2濃度之細胞毒性 18
3.1.5 PLGA產氧微粒載體之細胞毒性 19
3.2 CaO2濃度對產氧微粒載體所造成的影響 21
3.2.1 PLGA微粒載體之氧氣產生情形 21
3.2.2 PLGA微粒載體產氧後之酸鹼微環境 22
3.2.3 PLGA微粒載體產氧後之H2O2濃度 23
3.2.4 PLGA產氧微粒載體之細胞毒性 23
3.3 MnO2濃度不同對氧氣載體之影響 25
3.3.1 PLGA微粒載體之氧氣產生情形 25
3.3.2 PLGA微粒載體產氧後之酸鹼微環境 26
3.3.3 PLGA微粒載體產氧後之H2O2濃度 27
3.3.4 PLGA產氧微粒載體之細胞毒性 27
3.4 PLGA產氧微粒載體的大小與型態 30
3.5 PLGA產氧微粒載體劑量對細胞的影響 31
3.6 PLGA載體改善細胞缺氧之情形 32
第四章　結論 33
參考文獻 34
 

1. Semenza, G.L., Life with oxygen. Science, 2007. 318(5847): p. 62-64.
2. Lunt, S.Y. and M.G. Vander Heiden, Aerobic Glycolysis: Meeting the Metabolic Requirements of Cell Proliferation, in Annual Review of Cell and Developmental Biology, Vol 27, R. Schekman, L. Goldstein, and R. Lehmann, Editors. 2011. p. 441-464.
3. Park, S. and K.M. Park, Hyperbaric oxygen-generating hydrogels. Biomaterials, 2018. 182: p. 234-244.
4. Lemasters, J.J., et al., The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochimica Et Biophysica Acta-Bioenergetics, 1998. 1366(1-2): p. 177-196.
5. Kaelin, W.G. and P.J. Ratcliffe, Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Molecular Cell, 2008. 30(4): p. 393-402.
6. Farris, A.L., A.N. Rindone, and W.L. Grayson, Oxygen delivering biomaterials for tissue engineering. Journal of Materials Chemistry B, 2016. 4(20): p. 3422-3432.
7. Weaver, L.K., K.J. Valentine, and R.O. Hopkins, Carbon monoxide poisoning - Risk factors for cognitive sequelae and the role of hyperbaric oxygen. American Journal of Respiratory and Critical Care Medicine, 2007. 176(5): p. 491-497.
8. Krafft, M.P., Fluorocarbons and fluorinated amphiphiles in drug delivery and biomedical research. Advanced Drug Delivery Reviews, 2001. 47(2-3): p. 209-228.
9. Camci-Unal, G., et al., Oxygen-releasing biomaterials for tissue engineering. Polymer International, 2013. 62(6): p. 843-848.
10. Oh, S.H., et al., Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials, 2009. 30(5): p. 757-762.
11. Pedraza, E., et al., Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(11): p. 4245-4250.
12. Wang, J.P., et al., Oxygen-Generating Nanofiber Cell Scaffolds with Antimicrobial Properties. Acs Applied Materials & Interfaces, 2011. 3(1): p. 67-73.
13. Jain, R.A., The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials, 2000. 21(23): p. 2475-2490.
14. Makadia, H.K. and S.J. Siegel, Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers, 2011. 3(3): p. 1377-1397.
15. Kashi, T.S.J., et al., Improved drug loading and antibacterial activity of minocycline-loaded PLGA nanoparticles prepared by solid/oil/water ion pairing method. International Journal of Nanomedicine, 2012. 7: p. 221-234.
16. Anderson, J.M. and M.S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres. Advanced Drug Delivery Reviews, 1997. 28(1): p. 5-24.
17. Siegel, S.J., et al., Effect of drug type on the degradation rate of PLGA matrices. European Journal of Pharmaceutics and Biopharmaceutics, 2006. 64(3): p. 287-293.
18. Wischke, C. and S.P. Schwendeman, Principles of encapsulating hydrophobic drugs in PLA/PLGA microparticles. International Journal of Pharmaceutics, 2008. 364(2): p. 298-327.
19. Zhang, H.F., et al., Preservation of Blood Vessels with an Oxygen Generating Composite. Advanced Healthcare Materials, 2018. 7(21).
20. Lu, C.Y., et al., The role of oxygen during fracture healing. Bone, 2013. 52(1): p. 220-229.
21. Burge, R., et al., Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025. Journal of Bone and Mineral Research, 2007. 22(3): p. 465-475.
22. Hu, K. and B.R. Olsen, Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. Journal of Clinical Investigation, 2016. 126(2): p. 509-526.
23. Gomez-Barrena, E., et al., Bone fracture healing: Cell therapy in delayed unions and nonunions. Bone, 2015. 70: p. 93-101.
24. Loi, F., et al., Inflammation, fracture and bone repair. Bone, 2016. 86: p. 119-130.
25. Claes, L., S. Recknagel, and A. Ignatius, Fracture healing under healthy and inflammatory conditions. Nature Reviews Rheumatology, 2012. 8: p. 133.
26. Marsell, R. and T.A. Einhorn, The biology of fracture healing. Injury-International Journal of the Care of the Injured, 2011. 42(6): p. 551-555.
27. Kanczler, J.M. and R.O.C. Oreffo, Osteogenesis and angiogenesis: The potential for engineering bone. European Cells & Materials, 2008. 15: p. 100-114.
28. Ray, P.D., B.W. Huang, and Y. Tsuji, Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cellular Signalling, 2012. 24(5): p. 981-990.
29. Circu, M.L. and T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biology and Medicine, 2010. 48(6): p. 749-762.
30. Wei, H., et al., Apoptosis of Mesenchymal Stem Cells Induced by Hydrogen Peroxide Concerns Both Endoplasmic Reticulum Stress and Mitochondrial Death Pathway Through Regulation of Calspases, p38 and JNK. Journal of Cellular Biochemistry, 2010. 111(4): p. 967-978.
31. Min, S.K., et al., Endoplasmic reticulum stress is involved in hydrogen peroxide induced apoptosis in immortalized and malignant human oral keratinocytes. Journal of Oral Pathology & Medicine, 2008. 37(8): p. 490-498.
32. Prasad, P., et al., Multifunctional Albumin-MnO2 Nanoparticles Modulate Solid Tumor Microenvironment by Attenuating Hypoxia, Acidosis, Vascular Endothelial Growth Factor and Enhance Radiation Response. Acs Nano, 2014. 8(4): p. 3202-3212.