開發個人化具生物活性之功能性硬骨類組織

、線上人數：89

、訪客IP：3.15.34.233

姓名	張家齊(Jia-Ci, Jhang) 查詢紙本館藏	畢業系所	生物醫學工程研究所
論文名稱	開發個人化具生物活性之功能性硬骨類組織 (To Establish Bioactive Functional Bone-like Tissues for Personalized Use)
檔案	[Endnote RIS 格式] [Bibtex 格式] [相關文章] [文章引用] [完整記錄] [館藏目錄] 至系統瀏覽論文 ( 永不開放)
摘要(中)	骨缺損（Bone defect）所導致的創傷，在臨床上通常會以骨替代物的移植手術作為主要治療手段，常見的骨替代物選擇大致可分成三類；自體移植手術（Autografts）、異體異種移植手術（Allografts、Xenografts）、合成材料的替代物移植手術（Synthetic materials subsites），這些方案都各有部分缺點：自體移植手術受限於可使用的替代部位缺少，雖然具有良好生物活性，但並不能治療大範圍的骨缺損；異體及異種移植手術，可能來自於他人捐贈骨或是動物來源的豬骨、牛骨，經過高溫燒結加工處理後，再進行移植手術，但因此也缺少了生物活性；合成材料的優勢是可以被大量製造，大多來自於高分子或是陶瓷材料，能被使用的損傷部位有限。有鑑於目前臨床治療上有不足，科學家嘗試使用以組織工程（Tissue engineering）技術為核心，結合細胞治療（Cell therapy），期望打造出具有生物活性的類組織來滿足臨床需求；因此本研究建立體外三維（Three-dimensional）共同培養（Co-culture）系統，期望培養同時具有硬骨特徵且具有血管結構的類組織，選用具有能分化成成骨細胞（Osteoblasts）的人類間質幹細胞（Human mesenchymal stem cells）以及能夠形成血管（Blood vessels）的人臍靜脈內皮細胞（Human umbilical vein endothelial cells）；在二維（Two-dimensional）培養確認兩株細胞以1：1比例共培養具有最佳的生物性礦化表現，隨後進行了三維動態培養，有效促進細胞增生、存活率提升、尺寸團提升、分化之礦化物提升的效果。期許本技術能夠製作出符合個人化需求的生物活性硬骨移植功能性類器官，以改善目前骨替代物移植手術的不足之處。
摘要(英)	For current clinical practice, bone substitute transplantation is the main therapeutic choice for bone defect treatments. Common options of bone substitute can be roughly divided into four categories： autografts, allografts, xenografts, and synthetic bone substitutes. Each of these options has some benefits and disadvantages. Autologous transplantation is limited by the lack of alternative tissue source, although it is still the gold standard in clinical. Allografts and xenografts may come from other people′s donated bones or even other animal-derived bone tissues, which are sintered at high temperature for removing immune responses but lacks biological activity. The advantage of synthetic materials (such as polymer-based or ceramic-based materials) is that they can be manufactured in large quantities, however, these synthetic materials can be only applied on specific injury sites. In view of the current lack of optimal clinical treatment, scientists are trying to use tissue engineering technique combined with cell therapy, hoping to create bioactive tissue-like substitutes to meet clinical needs. The aim of this study is to cultivate human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) forming biomineralized and vascular structure in-vitro via three-dimensional dynamic co-culture system. From two-dimensional culture condition, we have confirmed the best biomineralization of the two cells co-cultured at a 1:1 ratio. Afterwards, we have confirmed the cell proliferation, differentiation and biomineralization were improved in three-dimensional dynamic co-culture system. This study is expected to be able to create bioactive and functional bone-like tissues that meet individual needs and improve the shortcomings of current bone substitute transplants.
關鍵字(中)	★ 組織工程 ★ 生物反應器 ★ 細胞治療 ★ 共培養 ★ 三維培養	關鍵字(英)	★ Tissue Engineering ★ Bioreactor ★ Cell therapy ★ Co-culture ★ 3D culture
論文目次	摘要… i Abstract iii 致謝…. v 目錄… vii 圖目錄 xi 表目錄 xiv 符號縮寫說明 xv 一、緒論 1 1-1骨損傷 2 1-2高齡化導致的骨密度下降 2 1-3傳統骨損傷治療方法 3 1-4三種骨替代物方案 3 1-5骨損傷治療的新方案 4 1-6研究目標 5 二、研究介紹與理論 7 2-1人體中的骨頭 7 2-1-1骨頭分類 7 2-1-2長骨結構 7 2-1-3長骨組織再生過程 8 2-2骨組織修復中的細胞作用 11 2-2-1成骨細胞（Osteoblast）與破骨細胞（Osteoclast） 11 2-3組織工程 12 2-3-1組織工程願景 12 2-3-2組織工程三元件 13 2-4細胞培養方式 14 2-4-1細胞培養 14 2-4-2二維細胞培養與三維細胞培養 15 2-4-3三維細胞培養方法 15 2-4-4共同細胞培養 16 2-5骨組織工程結合細胞療法之方式 18 2-5-1細胞治療中的體外培養與治療方式 18 2-5-2細胞療法操作及潛在治療效果 19 2-5-3細胞療法的法律規範與市場進程 21 2-6組織工程中的細胞培養優化 23 2-6-1間質幹細胞的三維培養（Mesenchymal stem cells, MSCs） 23 2-6-2血管內皮細胞（Endothelial cell of blood vessels）的三維培養與血管新生作用（Angiogenesis） 27 2-6-3間質幹細胞與血管內皮細胞的共同培養效應 29 2-6-4間質幹細胞（Mesenchymal stem cells, MSCs）與血管內皮細胞（Human umbilical vein endothelial cells, HUVECs）的共同培養系統優化 34 2-7海藻酸鈉支架（Alginate scaffold） 39 2-8生物反應器（Bioreactor）& 動態培養（Dynamic culture） 41 三、實驗架構 42 3-1實驗動機 42 3-2實驗參照 44 3-3實驗室成果與設計 46 四、材料與方法 50 4-1海藻酸鈉支架製備 51 4-2 hMSCs與HUVECs二維細胞培養 53 4-3 hMSCs與Co-culture培養之分化表現測試 54 4-4三維細胞培養植入操作與實驗測試組別 54 4-5自組裝生物反應器架設 57 4-6粒線體活性測試（Mitochondrion assay） 58 4-7細胞核染色（Nuclei staining）與細胞存活/死亡染色（Live/dead） 58 4-8 Xylenol-Orange染色 59 4-9培養基水質檢測 60 4-10共軛焦顯微鏡（Confocal microscopy） 61 4-11掃描式電子顯微鏡（Scanning Electron Microscope, SEM） 62 4-12傅立葉變換紅外光譜（Fourier Transform Infrared Spectroscopy, FT-IR） 62 4-13 X射線衍射儀（X-ray Diffraction, XRD） 63 4-14即時聚合酶連鎖反應（Real-time polymerase chain reaction, qPCR） 64 五、結果與討論 66 5-1細胞型態觀察 66 5-1-1人類骨隨間質幹細胞（hMSCs）與血管內皮細胞（HUVECs）的型態觀察 66 5-1-2人類骨隨間質幹細胞（hMSCs）與血管內皮細胞（HUVECs）的共同培養（Co-culture）型態觀察 66 5-2海藻酸鈉支架及細胞植入後測試 70 5-3掃描式電子顯微鏡（SEM）下觀察海藻酸鈉支架中的細胞團尺寸大小及分佈 71 5-4共軛焦顯微鏡下的死活細胞染色與礦化染色觀察 74 5-5生物性礦化物的蒐集檢測 78 5-5-1 XRD生物性礦化物檢測 78 5-5-2 FT-IR生物性礦化物檢測 79 5-5-3培養基的水質檢測 81 5-6即時聚合酶連鎖反應檢測（qPCR） 83 六、結論 84 七、文獻參考 86
參考文獻	[1] U.N.W.p.a. 2019, United Nations Department of Economic and Social Affairs; Population Division ;2020, 2019. [2] B. 醫療資訊團隊, 【骨折】如何急救和治療？一文睇清骨折種類、症狀和護理方法, 2020-04-20. https://www.bowtie.com.hk/blog/zh/骨折/. [3] 港島東醫院聯網社區服務, 長者常見疾病, 2020-6-16. https://www.healthyhkec.org/healthcare/eldercare/common-diseases/. [4] 葉峻榳, 一跌倒，骨折風險高！銀髮族骨質疏鬆別輕忽, 2016-09-06. https://www.top1health.com/Article/240/42962. [5] 康健知識庫, 什麼是骨質疏鬆症？. https://kb.commonhealth.com.tw/library/123.html#data-3-collapse. [6] 黃聖筑, 35歲開始流失！造成骨質疏鬆的4大原因是這些, 2018-07-06. https://heho.com.tw/archives/17267. [7] Y. Chang, B. Cho, S. Kim, J. Kim, Direct conversion of fibroblasts to osteoblasts as a novel strategy for bone regeneration in elderly individuals, Experimental & molecular medicine 51(5) (2019) 1-8. [8] J. Goldhahn, D. Little, P. Mitchell, N.L. Fazzalari, I.R. Reid, P. Aspenberg, D. Marsh, Evidence for anti-osteoporosis therapy in acute fracture situations—recommendations of a multidisciplinary workshop of the International Society for Fracture Repair, Elsevier, 2010, pp. 267-271. [9] V. Campana, G. Milano, E. Pagano, M. Barba, C. Cicione, G. Salonna, W. Lattanzi, G. Logroscino, Bone substitutes in orthopaedic surgery: from basic science to clinical practice, Journal of Materials Science: Materials in Medicine 25(10) (2014) 2445-2461. [10] S. Paderni, S. Terzi, L. Amendola, Major bone defect treatment with an osteoconductive bone substitute, Musculoskeletal Surgery 93(2) (2009) 89-96. [11] W. Wang, K.W. Yeung, Bone grafts and biomaterials substitutes for bone defect repair: A review, Bioactive materials 2(4) (2017) 224-247. [12] B. Bank, Bone Bank Allografts. https://www.bonebank.com/ [13] M. Health, Spinal bone graft, (2020-07-25). https://medlineplus.gov/ency/presentations/100136_3.htm. [14] 愛科學見真理學常識, 羥基磷灰石為代表的骨替代材料逐漸成為臨床應用的寵兒, 2021-09. http://www.ifuun.com/a20179215419959/. [15] 百科知識, 羥基磷灰石https://www.easyatm.com.tw/wiki/羥基磷灰石. [16] A. Khademhosseini, R. Langer, A decade of progress in tissue engineering, Nature protocols 11(10) (2016) 1775-1781. [17] J. Walker, Skeletal system 1: the anatomy and physiology of bones, (2020-2-27). [18] Wikipedia, Long bone. https://en.wikipedia.org/wiki/Long_bone [19] Bone structure. https://www.sciencedirect.com/topics/materials-science/bone-structure [20] R.A. Carano, E.H. Filvaroff, Angiogenesis and bone repair, Drug discovery today 8(21) (2003) 980-989. [21] V. Devescovi, E. Leonardi, G. Ciapetti, E. Cenni, Growth factors in bone repair, La Chirurgia degli organi di movimento 92(3) (2008) 161-168. [22] Y. Kubo, C.J. Wruck, A. Fragoulis, W. Drescher, H.C. Pape, P. Lichte, H. Fischer, M. Tohidnezhad, F. Hildebrand, T. Pufe, Role of Nrf2 in fracture healing: clinical aspects of oxidative stress, Calcified tissue international 105(4) (2019) 341-352. [23] D.A. Towler, The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair, Current osteoporosis reports 6(2) (2008) 67-71. [24] V. Devescovi, E. Leonardi, G. Ciapetti, E. Cenni, Growth factors in bone repair, Chir Organi Mov 92(3) (2008) 161-8. [25] R.C. Riddle, T.L. Clemens, Bone cell bioenergetics and skeletal energy homeostasis, Physiological Reviews (2017). [26] X. Cao, RANKL-RANK signaling regulates osteoblast differentiation and bone formation, Bone research 6(1) (2018) 1-2. [27] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423(6937) (2003) 337-342. [28] T. Britannica, Editors of Encyclopaedia, Argon. Encyclopedia Britannica (2020). [29] F.J. O′brien, Biomaterials & scaffolds for tissue engineering, Materials today 14(3) (2011) 88-95. [30] M.S. Chapekar, Tissue engineering: challenges and opportunities, Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 53(6) (2000) 617-620. [31] C. Imashiro, M. Hirano, T. Morikura, Y. Fukuma, K. Ohnuma, Y. Kurashina, S. Miyata, K. Takemura, Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration, Scientific reports 10(1) (2020) 1-11. [32] C. Chen, X. Bai, Y. Ding, I.-S. Lee, Electrical stimulation as a novel tool for regulating cell behavior in tissue engineering, Biomaterials research 23(1) (2019) 1-12. [33] ARSHADCHAUDRY, CELL CULTURE, THE SCIENCE CREATIVE QUARTERLY (2004-08). [34] M. Kapalczynska, T. Kolenda, W. Przybyla, M. Zajaczkowska, A. Teresiak, V. Filas, M. Ibbs, R. Blizniak, L. Luczewski, K. Lamperska, 2D and 3D cell cultures - a comparison of different types of cancer cell cultures, Arch Med Sci 14(4) (2018) 910-919. [35] J.C. Fontoura, C. Viezzer, F.G. Dos Santos, R.A. Ligabue, R. Weinlich, R.D. Puga, D. Antonow, P. Severino, C. Bonorino, Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance, Materials Science and Engineering: C 107 (2020) 110264. [36] J.W. Haycock, 3D cell culture: a review of current approaches and techniques, 3D cell culture (2011) 1-15. [37] Y. Miki, K. Ono, S. Hata, T. Suzuki, H. Kumamoto, H. Sasano, The advantages of co-culture over mono cell culture in simulating in vivo environment, The Journal of steroid biochemistry and molecular biology 131(3-5) (2012) 68-75. [38] L. Goers, P. Freemont, K.M. Polizzi, Co-culture systems and technologies: taking synthetic biology to the next level, Journal of The Royal Society Interface 11(96) (2014) 20140065. [39] D. Chen, S. Liu, H. Ma, X. Liang, H. Ma, X. Yan, B. Yang, J. Wei, X. Liu, Paracrine factors from adipose-mesenchymal stem cells enhance metastatic capacity through Wnt signaling pathway in a colon cancer cell co-culture model, Cancer cell international 15(1) (2015) 1-13. [40] H. Li, J. Chang, Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect, Acta biomaterialia 9(6) (2013) 6981-6991. [41] H. Yoshida, M. Nakamura, S. Makita, K. Hiramori, Paracrine effect of human vascular endothelial cells on human vascular smooth muscle cell proliferation: transmembrane co-culture method, Heart and vessels 11(5) (1996) 229-233. [42] C.J. Kirkpatrick, S. Fuchs, R.E. Unger, Co-culture systems for vascularization—learning from nature, Advanced drug delivery reviews 63(4-5) (2011) 291-299. [43] 維基百科, 旁分泌. https://zh.wikipedia.org/wiki/旁分泌. [44] M.F. Pittenger, D.E. Discher, B.M. Péault, D.G. Phinney, J.M. Hare, A.I. Caplan, Mesenchymal stem cell perspective: cell biology to clinical progress, NPJ Regenerative medicine 4(1) (2019) 1-15. [45] A.W. James, Review of signaling pathways governing MSC osteogenic and adipogenic differentiation, Scientifica 2013 (2013). [46] A. Augello, C. De Bari, The regulation of differentiation in mesenchymal stem cells, (2010). [47] M. Kassem, B.M. Abdallah, H. Saeed, Osteoblastic cells: differentiation and trans-differentiation, Archives of biochemistry and biophysics 473(2) (2008) 183-187. [48] R. Cancedda, G. Bianchi, A. Derubeis, R. Quarto, Cell therapy for bone disease: a review of current status, Stem Cells 21(5) (2003) 610-619. [49] P. Rosset, F. Deschaseaux, P. Layrolle, Cell therapy for bone repair, Orthopaedics & Traumatology: Surgery & Research 100(1) (2014) S107-S112. [50] M. Herrmann, S. Verrier, M. Alini, Strategies to stimulate mobilization and homing of endogenous stem and progenitor cells for bone tissue repair, Frontiers in bioengineering and biotechnology 3 (2015) 79. [51] M.S. Trust, Stem cells and MS - AHSCT, (2021-07). [52] N.K. Garg, S. Gaur, S. Sharma, Percutaneous autogenous bone marrow grafting in 20 cases of ununited fracture, Acta Orthopaedica Scandinavica 64(6) (1993) 671-672. [53] J.F. Connolly, R. Guse, J. Tiedeman, R. Dehne, Autologous marrow injection as a substitute for operative grafting of tibial nonunions, Clinical orthopaedics and related research (266) (1991) 259-270. [54] P. Hernigou, A. Poignard, F. Beaujean, H. Rouard, Percutaneous autologous bone-marrow grafting for nonunions: influence of the number and concentration of progenitor cells, JBJS 87(7) (2005) 1430-1437. [55] M. Mathieu, S. Rigutto, A. Ingels, D. Spruyt, N. Stricwant, I. Kharroubi, V. Albarani, M. Jayankura, J. Rasschaert, E. Bastianelli, Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures, Bone 53(2) (2013) 391-398. [56] 衛生福利部, 衛福部9月正式開放細胞治療嘉惠病人推動醫療生技發展, 2018-09-04. https://www.mohw.gov.tw/cp-16-43698-1.html. [57] 衛福部, 細胞治療技術資訊專區, 2010. https://www.mohw.gov.tw/cp-4628-55268-1.html. [58] 杜蕙容, 義大醫院攜手三顧《特管法》自體軟骨細胞移植獲准, 2019. http://pro.metatech.com.tw/news/c75b4e92-3248-49a8-ad6e-c6a1652685e6. [59] 衛福部, 細胞治療技術資訊專區. https://celltherapy.mohw.gov.tw/tech_search.htm. [60] Y. Petrenko, E. Syková, Š. Kubinová, The therapeutic potential of three-dimensional multipotent mesenchymal stromal cell spheroids, Stem cell research & therapy 8(1) (2017) 1-9. [61] T.J. Bartosh, J.H. Ylöstalo, A. Mohammadipoor, N. Bazhanov, K. Coble, K. Claypool, R.H. Lee, H. Choi, D.J. Prockop, Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties, Proceedings of the National Academy of Sciences 107(31) (2010) 13724-13729. [62] I.S. Park, J.-W. Rhie, S.-H. Kim, A novel three-dimensional adipose-derived stem cell cluster for vascular regeneration in ischemic tissue, Cytotherapy 16(4) (2014) 508-522. [63] I.A. Potapova, P.R. Brink, I.S. Cohen, S.V. Doronin, Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells, Journal of Biological Chemistry 283(19) (2008) 13100-13107. [64] S.H. Bhang, S. Lee, J.-Y. Shin, T.-J. Lee, B.-S. Kim, Transplantation of cord blood mesenchymal stem cells as spheroids enhances vascularization, Tissue Engineering Part A 18(19-20) (2012) 2138-2147. [65] L. Barile, G. Vassalli, Exosomes: Therapy delivery tools and biomarkers of diseases, Pharmacology & therapeutics 174 (2017) 63-78. [66] T.B. Steinbichler, J. Dudás, S. Skvortsov, U. Ganswindt, H. Riechelmann, I.-I. Skvortsova, Therapy resistance mediated by exosomes, Molecular cancer 18(1) (2019) 1-11. [67] D.G. Phinney, M.F. Pittenger, Concise review: MSC-derived exosomes for cell-free therapy, Stem cells 35(4) (2017) 851-858. [68] M. Kim, H.-W. Yun, B.H. Choi, B.-H. Min, Three-dimensional spheroid culture increases exosome secretion from mesenchymal stem cells, Tissue engineering and regenerative medicine 15(4) (2018) 427-436. [69] Y. Li, Q.-M. Pi, P.-C. Wang, L.-J. Liu, Z.-G. Han, Y. Shao, Y. Zhai, Z.-Y. Zuo, Z.-Y. Gong, X. Yang, Functional human 3D microvascular networks on a chip to study the procoagulant effects of ambient fine particulate matter, RSC Advances 7(88) (2017) 56108-56116. [70] S.J. Grainger, A.J. Putnam, Assessing the permeability of engineered capillary networks in a 3D culture, PLoS One 6(7) (2011) e22086. [71] M. Grellier, N. Ferreira‐Tojais, C. Bourget, R. Bareille, F. Guillemot, J. Amédée, Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells, Journal of cellular biochemistry 106(3) (2009) 390-398. [72] H. Li, R. Daculsi, M. Grellier, R. Bareille, C. Bourget, M. Remy, J. Amedee, The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network, PloS one 6(2) (2011) e16767. [73] H. Peng, A. Usas, A. Olshanski, A.M. Ho, B. Gearhart, G.M. Cooper, J. Huard, VEGF improves, whereas sFlt1 inhibits, BMP2‐induced bone formation and bone healing through modulation of angiogenesis, Journal of Bone and Mineral Research 20(11) (2005) 2017-2027. [74] S.J. Bidarra, C.C. Barrias, M.A. Barbosa, R. Soares, J. Amédée, P.L. Granja, Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells, Stem cell research 7(3) (2011) 186-197. [75] D.N. Heo, M. Hospodiuk, I.T. Ozbolat, Synergistic interplay between human MSCs and HUVECs in 3D spheroids laden in collagen/fibrin hydrogels for bone tissue engineering, Acta biomaterialia 95 (2019) 348-356. [76] M. Bruchet, A. Melman, Fabrication of patterned calcium cross-linked alginate hydrogel films and coatings through reductive cation exchange, Carbohydrate polymers 131 (2015) 57-64. [77] D.R. Sahoo, T. Biswal, Alginate and its application to tissue engineering, SN Applied Sciences 3(1) (2021) 1-19. [78] K. Dong, X. Wang, Y. Shen, Y. Wang, B. Li, C. Cai, L. Shen, Y. Guo, Maintaining Inducibility of Dermal Follicle Cells on Silk Fibroin/Sodium Alginate Scaffold for Enhanced Hair Follicle Regeneration, Biology 10(4) (2021) 269. [79] J. Kim, K. Kennedy, G. Vunjak-Novakovic, Bioreactors in regenerative medicine, Principles of Regenerative Medicine, Elsevier2019, pp. 787-803. [80] D.A. Gaspar, V. Gomide, F.J. Monteiro, The role of perfusion bioreactors in bone tissue engineering, Biomatter 2(4) (2012) 167-175. [81] Y. Park, S.T. Ji, U. Yong, S. Das, W.B. Jang, G. Ahn, S.-M. Kwon, J. Jang, 3D bioprinted tissue-specific spheroidal multicellular microarchitectures for advanced cell therapy, Biofabrication 13(4) (2021) 045017. [82] N. Chaicharoenaudomrung, P. Kunhorm, W. Promjantuek, N. Heebkaew, N. Rujanapun, P. Noisa, Fabrication of 3D calcium‐alginate scaffolds for human glioblastoma modeling and anticancer drug response evaluation, Journal of cellular physiology 234(11) (2019) 20085-20097. [83] C.-Y. Chen, C.-J. Ke, K.-C. Yen, H.-C. Hsieh, J.-S. Sun, F.-H. Lin, 3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy, Theranostics 5(6) (2015) 643. [84] 昶安生技有限公司, 粒線體功能與活性分析 Mitochondrion functional test. https://www.exbio.com.tw/blog/粒線體功能與活性分析-mitochondrion-functional-test-70.html. [85] 泰鼎生物科技有限公司, 細線體活性分析mitochrondria-assay. http://biopioneer.com.tw/?news=【-粒線體活性分析】mitochrondria-assay-系列商品. [86] L.C. Crowley, B.J. Marfell, N.J. Waterhouse, Analyzing cell death by nuclear staining with Hoechst 33342, Cold Spring Harbor Protocols 2016(9) (2016) pdb. prot087205. [87] 太鼎生物科技有限公司, Calcein-am活細胞螢光染劑. http://biopioneer.com.tw/?news=【calcein-am-活細胞螢光染劑】calcein-am-cell-viability-assay-kit-貨號1755-250. [88] 太鼎生物科技有限公司, PI-Propidium iodide死細胞核酸染劑. http://biopioneer.com.tw/?p=6269. [89] A. Stuart, D. Smith, Use of the fluorochromes xylenol orange, calcein green, and tetracycline to document bone deposition and remodeling in healing fractures in chickens, Avian diseases (1992) 447-449. [90] Y.H. Wang, Y. Liu, P. Maye, D.W. Rowe, Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes, Biotechnology progress 22(6) (2006) 1697-1701. [91] 葉柏安, 共軛焦顯微鏡的使用原理, 2009-03-16 https://highscope.ch.ntu.edu.tw/wordpress/?p=917. [92] 維基百科, 共軛焦顯微鏡. https://zh.wikipedia.org/zh-tw/共聚焦显微镜. [93] 精志科技, 掃描電子顯微鏡原理. https://www.amtech.com.tw/custom_85383.html. [94] 維基百科, 掃描電子顯微鏡. https://zh.wikipedia.org/zh-tw/扫描电子显微镜. [95] 楊仲準, C.-C. Yang, X 光繞射分析技術與應用 X-ray Diffraction Analysis: Techniques and Applications, 2011-06. https://www.tiri.narl.org.tw/Files/Doc/Publication/InstTdy/182/01820640.pdf. [96] 維基百科, 布拉格定律. https://zh.wikipedia.org/zh-tw/布拉格定律. [97] 台美檢驗, Real-time PCR食品檢測. https://www.superlab.com.tw/real_time_pcr/. [98] 維基百科, 即時聚合酶連鎖反應. https://zh.wikipedia.org/zh-tw/即時聚合酶鏈式反應. [99] C. Feng, L. Xiao, J. Yu, D. Li, T. Tang, W. Liao, Z. Wang, A. Lu, Simvastatin promotes osteogenic differentiation of mesenchymal stem cells in rat model of osteoporosis through BMP-2/Smads signaling pathway, Eur Rev Med Pharmacol Sci 24(1) (2020) 434-43. [100] C.-Y. Chen, T.-S. Chiang, L.-L. Chiou, H.-S. Lee, F.-H. Lin, 3D cell clusters combined with a bioreactor system to enhance the drug metabolism activities of C3A hepatoma cell lines, Journal of Materials Chemistry B 4(43) (2016) 7000-7008. [101] RRUFF, Hydroxylapatite R060180. https://rruff.info/hydroxylapatite/R060180
指導教授	陳靖昀孫瑞昇(Ching-Yun Chen Jui-Sheng Sun)	審核日期	2022-9-27
推文	facebook plurk twitter funp google live udn HD myshare reddit netvibes friend youpush delicious baidu
網路書籤	Google bookmarks del.icio.us hemidemi myshare

博碩士論文 109827021 詳細資訊