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系統識別號 U0026-0502201822365600
論文名稱(中文) 原子力顯微鏡量測多孔性牙支架材料及細菌機械性質之技術建立
論文名稱(英文) Establishment of AFM measurement protocol for biomechanical properties of porous dental scaffold and bacteria
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系
系所名稱(英) Department of Materials Science and Engineering
學年度 106
學期 1
出版年 106
研究生(中文) 林虹君
研究生(英文) Hung-Chun Lin
學號 N56044371
學位類別 碩士
語文別 中文
論文頁數 95頁
口試委員 指導教授-劉浩志
口試委員-郭瑞昭
口試委員-李旺龍
共同指導教授-姜永敏
中文關鍵字 生物力學  膠體探針  局部剛度  初始黏附力 
英文關鍵字 AFM  scaffold  local stiffness  initial adhesion  colloidal probe 
學科別分類
中文摘要 生物力學性質日漸倍受關注,近年來發現無論細胞自身或是細胞外環境的力學性質皆會對細胞的表現產生影響,細胞內的相關蛋白會因外界環境的力學性質而改變並調控細胞本身的型態,包含了細胞分化、運動方向、活性甚至細胞凋亡。已有許多團隊致力於探討細胞及其生長環境的機械性質,大多數研究結果皆以定性的方式呈現,且尚未有一量測的標準,本研究目的在於利用原子力顯微鏡以及自製膠體球形探針量測定量的生物機械性質。
本團隊將Polycaprolactone (PCL) /Silk fibroin (SF) /Cellulose nanocrystals (CNCs) 依不同比例混合溶液透過靜電紡絲製造五種不同成分之人造支架材料 (PCL、PCL/SF、PCL/SF/1%CNC、PCL/SF/3%CNC、PCL/SF/5%CNC),此材料被應用於使牙胚幹細胞再生,其異質性會隨著CNCs比例的增加而上升,先前文獻指出細胞的表現與基底的機械性質有密切的關係,本研究分別使用了傳統的拉伸試驗以及原子力顯微鏡來量測基板材料微區內的材料剛度,我們將此材料微米範圍的機械性質稱為局部剛度,因此將得到材料巨觀以及微觀的力學性質。在拉伸試驗的結果我們發現支架材料剛度與CNCs添加的比例呈現正相關,然而在原子力顯微鏡局部剛度量測的結果顯示了支架材料的剛度隨著CNCs的比例增加而下降,透過原子力顯微鏡所測得最低局部剛度的材料 PSC5 (PCL/SF/5%CNC) 與牙胚幹細胞之剛度最為匹配,且經過生物實驗也發現此材料對於骨組織的再生效果最佳,因此我們認為利用原子力顯微鏡搭配膠體探針對生物基底材料的量測能真正模擬出細胞所感測到基底材料的機械性質,研究中也指出當基底材料的局部剛度與生物體本身越相近越有利於細胞之分化及生長。
另外我們再進一步將自製膠體球形探針應用於量測生物體本身之機械性質。生物膜是造成許多疾病的主因之一,先前已有許多研究量測生物膜之機械性質,細菌黏附於基板是形成生物膜的關鍵要素之一,因此本團隊將量測細菌對基板的初始黏附力,先前有團隊對初始黏附力作了定性的量測,然而我們將透過原子力顯微鏡對變異鏈球菌初始黏附力作定量的量測,並探討在不同酸鹼值的口腔環境下細菌的機械性質將有何變化。量測的結果顯示出,細菌在弱酸性的環境下有最大的初始黏附力,推測是由於在酸性環境中,變異鏈球菌將排出更多的氫離子使環境pH值持續降低,且水合氫離子容易與磷酸鹽結合形成磷酸鹽陽離子,這些陽離子可以形成鈣酸螯合物 (Chelation),使周圍晶格中的礦物離子脫離,從而導致大量的脫鹽,牙釉質在酸性環境下將進行脫礦反應使牙釉質表面粗糙度提高,此結果造成變異鏈球菌對基板的黏附力增加,因此我們得知將口腔保持在中性及弱鹼性的環境將有利於預防生物膜的產生而避免齲齒的發生。
綜合以上觀點,本研究在於利用自製之球形探針量測纖維材料以及單細胞細菌之機械性質,期望未來能夠提供檢測人造纖維材料以及單細胞細菌機械性質的標準。
英文摘要 In this study, colloidal probe was successfully being used in measuring the local stiffness of scaffolds and the initial adhesion of single bacteria. In the first case, the scaffolds fabricated by electrospinning with different component containing PCL/SF/CNC (polycaprolactone / silk fibroin/ cellulose nanocrystals) were used for growing dental follicle stem cells (DFSCs). The macroscopic and microscopic stiffness of the scaffolds were measured though conventional tensile test and colloidal probe force spectroscopy using atomic force microscopy (AFM). The macroscopic stiffness increased with the CNC wt% increase, and the local stiffness decreased with the CNC wt% increase. The lowest local stiffness was 57 kPa which was consistent with DFSCs. With biological test, the result showed that the biological efficacy of scaffolds correlates with local stiffness. In the second case, the initial adhesion of bacteria to attach on the enamel is the important step in the process of biofilm colonization, and the modulus is a characteristic of bacteria activity. We use colloidal probe stick with single bacteria by AFM to get the quantitative results of the initial adhesion and the modulus of S.mutans. With using this method, for knowing how the pH value affect initial adhesion and modulus, the pH of PBS buffer we used was adjusted to be 5, 7 and 9. The result showed that the initial adhesion was strongest under pH 9, and the modulus did not be influenced by different pH value.
論文目次 摘要 I
Extended abstract III
致謝 XI
目錄 XIII
圖目錄 XVI
表目錄 XIX
第一章 序論 1
1.1 前言 1
1.2 研究動機 3
1.3 研究目的 4
第二章 文獻回顧及理論基礎 5
2.1 人體口腔環境 5
2.1.1 口腔構造 5
2.2 原子力顯微鏡 8
2.2.1 原子力顯微鏡工作原理 8
2.2.2 接觸式 ( Contact mode ) 10
2.2.2非接觸模式 ( Non-contact mode ) 11
2.2.3輕敲式 ( Tapping mode ) 11
2.2.4剛度 ( Stiffness ) 與楊氏模量 ( Modulus ) 14
2.3原子力顯微鏡對生物樣品之應用 16
2.3.1 原子力顯微鏡對細菌之量測 16
2.3.2變異鏈球菌 ( Streptococcus mutans ) 19
2.3.3 pH值對細菌之影響 21
2.3.4多巴胺 ( Polydopamine ) 22
2.3.5 原子力顯微鏡對生物基底材料之量測 24
2.4奈米纖維支架 26
2.4.1 組織工程 26
2.4.2靜電紡絲原理 28
2.4.3 聚己內酯 ( Polycaprolactone ) 30
2.4.4絲素蛋白 ( Silk Fibroin ) 31
2.4.5纖維素奈米微晶 ( Cellulose nanocrystals ) 32
第三章 實驗方法與步驟 33
3.1 實驗設計原理 33
3.2 實驗流程 34
3.2.1 膠體探針製備 34
3.2.2 變異鏈球菌培養 36
3.2.3單細胞細菌力學性質量測 36
3.2.4奈米纖維支架製備 39
3.2.5 奈米纖維支架力學性值量測 40
3.3 實驗儀器與藥品 42
第四章 結果與討論 48
4.1 膠體探針量測高分子標準試片 48
4.1.1 高分子標準試片表面形貌分析 48
4.1.2 高分子標準試片機械性質分析 50
4.2 奈米纖維支架之機械性質分析 55
4.2.1 奈米纖維支架表面結構分析 56
4.2.2 奈米纖維支架拉伸試驗量測結果 63
4.2.4 纖維支架材料局部剛度量測參數之討論 70
4.2.5 拉伸試驗及局部剛度量測之綜合討論 73
4.3 膠體探針量測單細胞細菌之機械性質 75
4.3.1 單細胞細菌膠體生物探針 75
4.3.2 牙釉質基板表面形貌分析 77
4.3.3 單細胞細菌在不同酸檢環境之機械性質分析 78
第五章 結論與未來展望 88
5.1 結論 88
5.2 未來展望 89
第六章 參考文獻 90
參考文獻 B. H. Liu and L. C. Yu, "In-situ, time-lapse study of extracellular polymeric substance discharge in Streptococcus mutans biofilm," Colloids Surf B Biointerfaces, vol. 150, pp. 98-105, Feb 1 2017.
[2] Wikipedia. Human tooth, from
(https://en.wikipedia.org/wiki/Human_tooth)
[3] 張. 中. 名譽理事長. 牙周病病因及其預防,取自:
(http://minitwo.mdjh.tn.edu.tw/health/wp-content/uploads/2014/06/%E7%89%99%E5%91%A8%E7%97%85%E7%97%85%E5%9B%A0%E5%8F%8A%E5%85%B6%E9%A0%90%E9%98%B2.pdf)
[4] 王. 醫師. 認識牙周病,取自: (http://epaper.ntuh.gov.tw/health/201106/PDF/%E8%AA%8D%E8%AD%98%E7%89%99%E5%91%A8%E7%97%85.pdf)
[5] C. F. Q. G. Binnig, and C. Gerber, "Atomic Force Microscope," Physical Review Letters, vol. 56, pp. 930-933, 1986.
[6] 黃英碩, "掃描探針顯微術的原理及應用," 科儀新知, vol. 144, pp. 7-17, 2005.
[7] J. Ďurkovič, Kardošová, M. and Lagaňa, R., "Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves " Bio-protocol, vol. 4, 2014.
[8] L. Kailas, E. C. Ratcliffe, E. J. Hayhurst, M. G. Walker, S. J. Foster, and J. K. Hobbs, "Immobilizing live bacteria for AFM imaging of cellular processes," Ultramicroscopy, vol. 109, pp. 775-80, Jun 2009.
[9] Y. T. Laila Abu-Lail, Paola A. Pinzón-Arango, Amy Howell, Terri A. Camesano, "Using Atomic Force Microscopy to Measure Anti-Adhesion Effects on Uropathogenic Bacteria, Observed in Urine after Cranberry Juice Consumption," Journal of Biomaterials and Nanobiotechnology, vol. 3, pp. 533-540, 2012.
[10] K. H. Metwalli, S. A. Khan, B. P. Krom, and M. A. Jabra-Rizk, "Streptococcus mutans, Candida albicans, and the human mouth: a sticky situation," PLoS Pathog, vol. 9, p. e1003616, 2013.
[11] J. K. Clarke, "On the Bacterial Factor in the Ætiology of Dental Caries," British journal of experimental pathology, vol. 5, p. 141, 1924.
[12] Gram-positive and Gram-negative bacteria, from
(https://www.dreamstime.com/stock-illustration-gram-positive-gram-negative-bacteria-difference-bacterial-image45337024)
[13] 中國科普博覽. 微生物的生命活動,取自: (http://www.kepu.net.cn/gb/lives/microbe/microbe_life/200310170061.html)
[14] J. Welin-Neilands and G. Svensater, "Acid tolerance of biofilm cells of Streptococcus mutans," Appl Environ Microbiol, vol. 73, pp. 5633-8, Sep 2007.
[15] H. Lee, S. M. Dellatore, W. M. Miller, and P. B. Messersmith, "Mussel-inspired surface chemistry for multifunctional coatings," Science, vol. 318, pp. 426-30, Oct 19 2007.
[16] R. Louise Meyer, X. Zhou, L. Tang, A. Arpanaei, P. Kingshott, and F. Besenbacher, "Immobilisation of living bacteria for AFM imaging under physiological conditions," Ultramicroscopy, vol. 110, pp. 1349-57, Oct 2010.
[17] H. Lee, N. F. Scherer, and P. B. Messersmith, "Single-molecule mechanics of mussel adhesion," Proc Natl Acad Sci U S A, vol. 103, pp. 12999-3003, Aug 29 2006.
[18] H. Lee, B. P. Lee, and P. B. Messersmith, "A reversible wet/dry adhesive inspired by mussels and geckos," Nature, vol. 448, pp. 338-41, Jul 19 2007.
[19] D. E. Discher, P. Janmey, and Y. L. Wang, "Tissue cells feel and respond to the stiffness of their substrate," Science, vol. 310, pp. 1139-43, Nov 18 2005.
[20] Wikipedia. Tissue engineering, from
(https://en.wikipedia.org/wiki/Tissue_engineering)
[21] 醫. 王. 助理教授. 高雄醫學大學e快報 第128期  生命科學院 專題,取自: (http://enews2.kmu.edu.tw/index.php/Enews128_%E6%B7%BA%E8%AB%87%E9%AA%A8%E7%B5%84%E7%B9%94%E5%86%8D%E7%94%9F)
[22] F. R. S. Sir Geoffrey Taylor, "Disintegration of water drops in an electric field," Proceedings of the Royal Society A, vol. 280, pp. 383-397, 1964.
[23] H. J. Sung, C. Meredith, C. Johnson, and Z. S. Galis, "The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis," Biomaterials, vol. 25, pp. 5735-42, Nov 2004.
[24] M. C. Serrano, R. Pagani, M. Vallet-Regi, J. Pena, A. Ramila, I. Izquierdo, et al., "In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts," Biomaterials, vol. 25, pp. 5603-11, Nov 2004.
[25] 台灣wiki. 絲素蛋白,取自: (http://www.twwiki.com/wiki/%E7%B5%B2%E7%B4%A0%E8%9B%8B%E7%99%BD)
[26] 王益真, "簡介奈米微晶纖維素," 林業研究專訊, vol. 19, pp. 16-18, 2012.
[27] R. N. Palchesko, L. Zhang, Y. Sun, and A. W. Feinberg, "Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve," PLoS One, vol. 7, p. e51499, 2012.
[28] J. Solon, I. Levental, K. Sengupta, P. C. Georges, and P. A. Janmey, "Fibroblast adaptation and stiffness matching to soft elastic substrates," Biophys J, vol. 93, pp. 4453-61, Dec 15 2007.
[29] M. Y. Chiang, Y. Yangben, N. J. Lin, J. L. Zhong, and L. Yang, "Relationships among cell morphology, intrinsic cell stiffness and cell-substrate interactions," Biomaterials, vol. 34, pp. 9754-62, Dec 2013.
[30] R. M. Domingues, M. E. Gomes, and R. L. Reis, "The potential of cellulose nanocrystals in tissue engineering strategies," Biomacromolecules, vol. 15, pp. 2327-46, Jul 14 2014.
[31] H. E. S. F. S. A. O. S. R. W.Rudieb, "Cellulose nanocrystals as a reinforcing material for electrospun poly(methyl methacrylate) fibers: Formation, properties and nanomechanical characterization," Carbohydrate Polymers, vol. 87, pp. 2488-2495, 2012.
[32] Q. X. Xu He, Canhui Lu, Yaru Wang, Xiaofang Zhang, Jiangqi Zhao, Wei Zhang, Ximu Zhang, and Yulin Deng, "Uniaxially Aligned Electrospun All-Cellulose Nanocomposite Nanofibers Reinforced with Cellulose Nanocrystals: Scaffold for Tissue Engineering," Biomacromolecules, vol. 15, pp. 618-627, 2014.
[33] Q. L. Loh and C. Choong, "Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size," Tissue Eng Part B Rev, vol. 19, pp. 485-502, Dec 2013.
[34] R. C. Chengjun Zhou, Rhonna Wu, and Qinglin Wu, "Electrospun Polyethylene Oxide/Cellulose Nanocrystal Composite Nanofibrous Mats with Homogeneous and Heterogeneous Microstructures," Biomacromolecules, vol. 12, pp. 2617–2625, 2011.
[35] R. G. Breuls, T. U. Jiya, and T. H. Smit, "Scaffold stiffness influences cell behavior: opportunities for skeletal tissue engineering," Open Orthop J, vol. 2, pp. 103-9, May 29 2008.
[36] A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, "Matrix elasticity directs stem cell lineage specification," Cell, vol. 126, pp. 677-89, Aug 25 2006.
[37] A. Engler, L. Bacakova, C. Newman, A. Hategan, M. Griffin, and D. Discher, "Substrate compliance versus ligand density in cell on gel responses," Biophys J, vol. 86, pp. 617-28, Jan 2004.
[38] S. J. Hollister, "Porous scaffold design for tissue engineering," Nat Mater, vol. 4, pp. 518-24, Jul 2005.
[39] S. P. Davide Tranchida, andMaria Soliman, "Nanoscale Mechanical Characterization of Polymers by AFM Nanoindentations:  Critical Approach to the Elastic Characterization," Macromolecules, vol. 39, pp. 4547–4556, 2006.
[40] S. R. C. a. E. Kalfon-Cohen, "Dynamic nanoindentation by instrumented nanoindentation and force microscopy: a comparative review," Beilstein J. Nanotechnol, pp. 815-833, 2013.
[41] V. J. S. V. Mark R. , William F. Guthrie, Greg F. Meyer, "Nanoindentation of polymers: an overview," ACS Polymer Preprints, vol. 167, pp. 15-44, 2001.
[42] G. Moeller, "AFM nanoindentation of viscoelastic materials with large end-radius probes," Polymer Science, vol. 47, pp. 1573-1587, 2009.
[43] S. Adriana N.Frone, Jean-François Chailan, Denis M.Panaitescu, "Morphology and thermal properties of PLA–cellulose nanofibers composites," Carbohydrate Polymers, vol. 91, pp. 377-384, 2013.
[44] Y. H. Maria S. Peresin, Justin O. Zoppe, Joel J. Pawlak and Orlando J. Rojas, "Nanofiber Composites of Polyvinyl Alcohol and Cellulose Nanocrystals: Manufacture and Characterization," Biomacromolecules, vol. 11, pp. 674–681, 2010.
[45] F. S. J. H. Sugandha Chahal, Anuj Kumar, Mashitah M. Yusoff and Mohammad Syaiful Bahari Abdull Rasad, "Electrospun hydroxyethyl cellulose nanofibers functionalized with calcium phosphate coating for bone tissue engineering," The Royal Society of Chemistry, vol. 5, pp. 29797-29504, 2015.
[46] W. M. Dunne, Jr., "Bacterial adhesion: seen any good biofilms lately?," Clin Microbiol Rev, vol. 15, pp. 155-66, Apr 2002.
[47] D. Cunliffe, C. A. Smart, C. Alexander, and E. N. Vulfson, "Bacterial adhesion at synthetic surfaces," Appl Environ Microbiol, vol. 65, pp. 4995-5002, Nov 1999.
[48] L. Weiss, "Cell Movement and Cell Contac the Measurement of Cell Adhesion," Experimental Cell Research, pp. 141-153, 1961.
[49] S. Baliga, S. Muglikar, and R. Kale, "Salivary pH: A diagnostic biomarker," J Indian Soc Periodontol, vol. 17, pp. 461-5, Jul 2013.
[50] R. Matsui and D. Cvitkovitch, "Acid tolerance mechanisms utilized by Streptococcus mutans," Future Microbiol, vol. 5, pp. 403-17, Mar 2010.
[51] J. A. Lemos, J. Abranches, and R. A. Burne, "Responses of cariogenic streptococci to environmental stresses," Curr Issues Mol Biol, vol. 7, pp. 95-107, Jan 2005.
[52] J. Zhang, Y. Du, Z. Wei, B. Tai, H. Jiang, and M. Du, "The prevalence and risk indicators of tooth wear in 12- and 15-year-old adolescents in Central China," BMC Oral Health, vol. 15, p. 120, Oct 9 2015.
[53] A. Wiegand and T. Attin, "Occupational dental erosion from exposure to acids: a review," Occup Med (Lond), vol. 57, pp. 169-76, May 2007.
[54] A. Lussi and T. Jaeggi, "Erosion--diagnosis and risk factors," Clin Oral Investig, vol. 12 Suppl 1, pp. S5-13, Mar 2008.
[55] I. Torres-Gallegos, V. Zavala-Alonso, N. Patino-Marin, G. A. Martinez-Castanon, K. Anusavice, and J. P. Loyola-Rodriguez, "Enamel roughness and depth profile after phosphoric acid etching of healthy and fluorotic enamel," Aust Dent J, vol. 57, pp. 151-6, Jun 2012.
[56] J. Song, J. F. Duval, M. A. Stuart, H. Hillborg, U. Gunst, H. F. Arlinghaus, et al., "Surface ionization state and nanoscale chemical composition of UV-irradiated poly(dimethylsiloxane) probed by chemical force microscopy, force titration, and electrokinetic measurements," Langmuir, vol. 23, pp. 5430-8, May 8 2007.
[57] C. D. Ma, C. Wang, C. Acevedo-Velez, S. H. Gellman, and N. L. Abbott, "Modulation of hydrophobic interactions by proximally immobilized ions," Nature, vol. 517, pp. 347-50, Jan 15 2015.
[58] P. Yu, C. Wang, J. Zhou, L. Jiang, J. Xue, and W. Li, "Influence of Surface Properties on Adhesion Forces and Attachment of Streptococcus mutans to Zirconia In Vitro," Biomed Res Int, vol. 2016, p. 8901253, 2016.
[59] A. Al-Ahmad, M. Wiedmann-Al-Ahmad, J. Faust, M. Bachle, M. Follo, M. Wolkewitz, et al., "Biofilm formation and composition on different implant materials in vivo," J Biomed Mater Res B Appl Biomater, vol. 95, pp. 101-9, Oct 2010.
[60] C. do Nascimento, C. da Rocha Aguiar, M. S. Pita, V. Pedrazzi, R. F. de Albuquerque, Jr., and R. F. Ribeiro, "Oral biofilm formation on the titanium and zirconia substrates," Microsc Res Tech, vol. 76, pp. 126-32, Feb 2013.
[61] T. D. Morgan and M. Wilson, "The effects of surface roughness and type of denture acrylic on biofilm formation by Streptococcus oralis in a constant depth film fermentor," J Appl Microbiol, vol. 91, pp. 47-53, Jul 2001.
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