臺灣博碩士論文加值系統

矽酸鈣材料（Calcium silicate-based material，CS）已成功且廣泛的運用在牙科臨床上。過去研究顯示，由於CO2雷射的光熱機制，讓CO2雷射具有良好的抗菌能力。本研究的目的除了分析CO2雷射的照射對於 CS 材料特性的影響外，也同時對於人類牙周韌帶細胞（human periodontal ligament cells，hPDLs）的生物性質進行評估。本研究將hPDLs培養於CS上，並使用牙科CO2雷射且裝製 0.25 cm2 光點面積雷射光纖頭並且刺激細胞，後續再分析細胞的生長以及牙本 質分化的研究。結果顯示，CO2雷射的照射可以增加CS中鈣離子和矽離子釋放至培養液的濃度，並影響細胞的生理行為。此外，CO2雷射照射也會促進 hPDLs細胞培養於CS上的牙本質分化以及骨礦化結節的形成，並且促進牙本質生成蛋白和黏附蛋白的生成表現。三氧礦化物 (Mineral trioxide aggregate，MTA)是一種生物性材料，近年來被廣泛的應用在臨床根管治療上，這篇研究我們也將分析經由CO2雷射照射後對於MTA材料特性及細胞能力的影響進行評估，實驗的結果是與CO2雷射照射CS後的表現是相同的，並且其固化時間在經由照射CO2雷射後為118分鐘與未照射CO2雷射相比是有顯著降低，在最大徑向拉伸強度和X-光繞射的結果是與未照射CO2雷射相似的。目前的研究結果提供了新的並且重要的數據在CO2雷射在照射CS 及MTA的影響，現階段的結果可得知CO2雷射照射CS和MTA是可以增加矽離子的，並且是低於造成細胞凋亡的臨界濃度，同時可以促進hPDLs的分化行為，這些結果在未來是有機會被應用於牙本質生成和牙周組織的再生醫學治療 。

Calcium silicate-based material (CS) has been successfully used in dental clinical applications. Studies show that the anti-bacterial effects of CO2 laser irradiation are highly efficient when bacteria are embedded in biofilm, due to a photo-thermal mechanism. The purpose
of this study was to confirm the effects of CO2 laser irradiation on CS, with regard to both material characterization and human periodontal ligament cell (hPDLs) viability. CS was irradiated with a dental CO2 laser using directly mounted fiber optics in wound healing mode with a spot area of 0.25 cm2, and then stored in an incubator at 100% relative humidity and 37 °C for 1 d to set. The hPDLs cultured on CS was analyzed, along with their proliferation and cementogenic/odontogenic differentiation behaviors. The results indicate that the CO2 laser irradiation increased the amount of Ca and Si ions released from the CS, and regulated cell behavior. CO2 laser-irradiated CS promoted cementogenic/odontogenic differentiation of hPDLs, with the increased formation of mineralized nodules on the substrate’s surface. It also up-regulated the protein expression of multiple markers of cementogenic and the expression of cementum attachment protein.
Mineral trioxide aggregate (MTA) was a biomaterial with several clinical applications in endodontics. We also were to confirm the effects of CO2 laser irradiation on MTA with regard to both material characterization and cell viability. The results were the same the effect of CO2 laser irradiation on CS, and the setting time after irradiation by the CO2 laser was significantly reduced to 118 minutes rather than the usual 143 minutes. The maximum diametral tensile strength and x-ray diffraction patterns were similar to those obtained without CO2 laser irradiation. The current study provides new and important data about the effects of CO2 laser irradiation on CS and MTA. Taking cell functions into account, the Si concentration released from CS and MTA with laser irradiated may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue.

Contents

Abstract I
Chinese Abstract III
Acknowledgement IV
Contents V
List Figures VII

Chapter 1 Introduction 1
1.1. Laser 1
1.2. Bone cement 2
1.3. Si effect 3
1.4. Silicate-based materials 4
1.4.1. Calcium silicate-base (CS) 5
1.4.2. Bioglass 6
1.4.3. Mineral trioxide aggregate (MTA) 7
Chapter 2 Materials and Methods 9
2.1 Preparation of specimens 9
2.2 Setting time, PH variation, and strength 10
2.3 Phase composition and morphology 10
2.4 In vitro soaking 11
2.5 Ion concentration 11
2.6 Antibacterial properties 11
2.7 hPDLs isolation and culture 12
2.8 Collagen secretion 13
2.9 Cell adhesion and proliferation 13
2.10 Fluorescent staining 14
2.11 Osteogenesis assay 14
2.12 Western blot 15
2.13 Alizarin red stain 16
2.14 Statistical analysis 16

Chapter 3 Results 17
3.1 Apatite precipitate 17
3.2 Ion concentrations 17
3.3 Antibacterial properties 17
3.4 Cell adhesion and collagen secretion 18
3.5 Cell proliferation 18
3.6 Osteogenic and cementogenic differentiation 19
3.7 Physicochemical properties 19
3.8 Antibacterial properties 21
3.9 Ion concentrations 21
3.10 Cell viability 22
3.11 Odontogenic differentiation in hDPCs 22

Chapter 4 Discussion 24
Chapter 5 Conclusion 27
Reference 28

List of Figures

Figure 1 SEM micrographs of CS surfaces before and after immersion in SBF for different time points. 33

Figure 2 (A) Ca and (B) Si ion concentrations of SBF after immersion for different times. 34

Figure 3 (A) The growth inhibition zones and (B) the anti-bacterial effects of CS with and without CO2 laser irradiation after culture in Staphylococcus aureus. 35

Figure 4 (A) Cell adhesion and (B) collagen I secretion from hPDLs cultured on CS 36

Figure 5 Immunofluorescence images of nuclei (blue), and F-actin (red) in the hDPCs were cultured on CS 37

Figure 6 Cell proliferation assay for hDPCs cultured on CS 38

Figure 7 (A) ALP activity and (B) OC amount of hPDLs were cultured on CS for different time-points. 39

Figure 8 (A) Immunodetection and (B) quantification of CAP and CEMP1-1 protein expression in hPDLs were cultured on CS 40

Figure 9 (A) Alizarin Red S staining and (B) quantification of calcium mineral deposits by hPDLs cultured on CS 41

Figure 10 The effects of CO2 laser treatment on (A) setting time and (B) diametral tensile strength (DTS) of MTA cements. (C) XRD patterns of MTA with and without CO2 laser irradiation after hydration at 37oC for one day. (D) SEM micrographs of MTA surfaces before and after immersion in SBF for one day 42

Figure 11 (A) The growth inhibition zones and (B) the antibacterial effects of MTA with and without CO2 laser irradiation after culture in S. aureus. (C) Ca and (D) Si ion concentrations of DMEM after immersion for different times. 43

Figure 12 (A) Cell proliferation assay for hDPCs cultured on MTA (B) Immunofluorescence images of nuclei (blue) and F-actin (red) in the hDPCs were cultured on MTA 44

Figure 13 (A) Immunodetection of DSPP and DMP-1 protein expression in hDPCs cultured on MTA (B) ALP activity and (C) OC amount of hDPCs cultured on MTA for different times. (D) Alizarin red S staining and quantification of calcium mineral deposits by hDPCs cultured on MTA 45

[1]Eldeniz AU, Hadimli HH, Ataoglu H, Orstavik D. Antibacterial Effect of Selected Root-End Filling Materials 2006;32:345–9.
[2]Ricucci D, Siqueira JF. Biofilms and Apical Periodontitis: Study of Prevalenceand Association with Clinical and Histopathologic Findings. J Endod 2010;36:1277–88.
[3]Ricucci D, Siqueira JF Jr. Apical Actinomycosis as a Continuum of Intraradicular and Extraradicular Infection: Case Report and Critical Review on Its Involvement with Treatment Failure. J Endod 2008;34:1124–9.
[4]Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J 2006;39:249–81.
[5]Huang TH, Chen CL, Hung CJ, Kao CT. Comparison of antibacterial activities of root-end filling materials by an agar diffusion assayand Alamar blue assay. J Dent Sci 2012;7:336–41.
[6]Kim JH, Kim Y, Shin SJ, Park JW, Jung IY. Tooth Discoloration of Immature Permanent Incisor Associated with Triple Antibiotic Therapy: A Case Report. J Endod 2010;36:1086–91.
[7]Tosun E, Tasar F, Strauss R, Kıvanc DG, Ungor C. Comparative evaluation of antimicrobial effects of Er:YAG, diode, and CO2 lasers on titanium discs: an experimental study. J Oral Maxillofac Surg 2012;70:1064–9.
[8]Alves F, Mima EG, Dovigo LN, Bagnato VS, Jorge JH, de Souza Costa CA, et al. The influence of photodynamic therapy parameters on the inactivation of Candida spp: in vitro and in vivo studies. Laser Phys Lett 2014;24:045601.
[9]Baum OI, Zheltov GI, Omelchenko AI, Romanov GS, Romanov OG, Sobol EN. Thermomechanical effect of pulse-periodic laser radiation on cartilaginous and eye tissues. Laser Phys Lett 2013;23:085602.
[10]Sobol E, Zakharkina OL, Baskov A, Shekhter A, Borschenko I, Guller A, et al. Laser engineering of spine discs 2009;19:825–35.
[11]Huang TH, Liu SL, Chen CL, Shie MY, Kao CT. Low-Level Laser Effects on Simulated Orthodontic Tension Side Periodontal Ligament Cells. Photomed Laser Surg 2013;31:72–7.
[12]Sobol E, Shekhter A, Guller A, Baum O, Baskov A. Laser-induced regeneration of cartilage. J Biomed Opt 2011;16:080902.
[13]Sobol EN, Milner TE, Shekhter AB, Baum OI, Guller AE, Ignatieva NY, et al. Laser reshaping and regeneration of cartilage. Laser Phys Lett 2007;4:488–502.
[14]Cohen J, Featherstone J, Le CQ, Steinberg D, Feuerstein O. Effects of CO 2laser irradiation on tooth enamel coated with biofilm. Lasers Surg Med 2014;46:216–23.
[15]Dederich DN, Pickard MA, Vaughn AS. Comparative bactericidai exposures for selected oral bacteria using carbon dioxide laser radiation - Dederich - 2005 - Lasers in Surgery and Medicine - Wiley Online Library. Lasers Surg Med 1990;10:591–4.
[16]Huang TH, Lu YC, Kao CT. Low-level diode laser therapy reduces lipopolysaccharide (LPS)-induced bone cell inflammation. Lasers Med Sci 2012;27:621–7.
[17]Kuo CL, Kao CT, Fang HY, Huang TH, Chen YW, Shie MY. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages. Laser Phys Lett 2015;12:035601.
[18]Huang TH, Chen CC, Liu SL, Lu YC, Kao CT. A low-level diode laser therapy reduces the lipopolysaccharide (LPS)-induced periodontal ligament cell inflammation. Laser Phys Lett 2014;11:075602.
[19]Hsieh WH, Chen YJ, Hung CJ, Huang TH, Kao CT, Shie MY. Osteogenesis differentiation of human periodontal ligament cells by CO 2laser-treatment stimulating macrophages via BMP2 signalling pathway. Laser Phys Lett 2014;24:1–8.
[20]Wilson PD, Amstutz HC, Czerniecki A, Salvati EA, MENDES DG. Total Hip Replacement with Fixation by Acrylic Cement. J Bone Joint Surg Am 1972;54:207–21.
[21]Petty W. The effect of methylmethacrylate on chemotaxis of polymorphonuclear leukocytes. J Bone Joint Surg Am 1978;60:492–8.
[22]Skripitz R, Aspenberg P. Attachment of PMMA cement to bone: force measurements in rats. Biomaterials 1999;20:351–6.
[23]Barralet JE, Grover L, Gaunt T, Wright AJ, Gibson IR. Preparation of macroporous calcium phosphate cement tissue engineering scaffold. Biomaterials 2002;23:3063–72.
[24]Chow LC. Calcium phosphate materials: reactor response. Adv Dent Res 1988;2:181–4–discussion185–6.
[25]Effah Kaufmann EA, Ducheyne P, Shapiro IM. Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis. Tissue Eng 2000;6:19–28.
[26]Christodoulou I, Buttery LDK, Tai G, Hench LL, Polak JM. Characterization of human fetal osteoblasts by microarray analysis following stimulation with 58S bioactive gel-glass ionic dissolution products. J Biomed Mater Res 2006;77:431–46.
[27]Valerio P, Pereira MM, Goes AM, Leite MF. The effect of ionic products from bioactive glass dissolution on osteoblast proliferation and collagen production. Biomaterials 2004;25:2941–8.
[28]Tsigkou O, Jones JR, Polak JM, Stevens MM. Differentiation of fetal osteoblasts and formation of mineralized bone nodules by 45S5 Bioglass conditioned medium in the absence of osteogenic supplements. Biomaterials 2009;30:3542–50.
[29]Gough JE, Jones JR, Hench LL. Nodule formation and mineralisation of human primary osteoblasts cultured on a porous bioactive glass scaffold. Biomaterials 2004;25:2039–46.
[30]Ding SJ, Shie MY, Takashi H, Naoki K, Chen G, Chang HC. Osteogenic differentiation and immune response of human bone-marrow-derived mesenchymal stem cells on injectable calcium-silicate-based bone grafts. Tissue Eng Part A 2010;16:2343–54.
[31]Chen C-C, Ho C-C, David Chen C-H, Wang W-C, Ding S-J. In vitro bioactivity and biocompatibility of dicalcium silicate cements for endodontic use. J Endod 2009;35:1554–7.
[32]Chen CC, Ho CC, Chen C, Ding SJ. Physicochemical Properties of Calcium Silicate Cements for Endodontic Treatment. J Endod 2009;35:1288–91.
[33]Xu S, Lin K, Wang Z, Chang J, Wang L, Lu J, et al. Reconstruction of calvarial defect of rabbits using porous calcium silicate bioactive ceramics. Biomaterials 2008;29:2588–96.
[34]Carlisle EM. Silicon: an essential element for the chick. Science 1972;178:619–21.
[35]Reffitt DM, Ogston N, Jugdaohsingh R, Cheung HFJ, Evans BAJ, Thompson RPH, et al. Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 2003;32:127–35.
[36]Xynos ID, Edgar AJ, Buttery LD, Hench LL, Polak JM. Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis. Biochem Biophys Res Commun 2000;276:461–5.
[37]Ding M, Shi X, Dong Z, Chen F, Lu Y, Castranova V, et al. Freshly fractured crystalline silica induces activator protein-1 activation through ERKs and p38 MAPK. J Biol Chem 1999;274:30611–6.
[38]Daniel LN, Mao Y, Williams AO, Saffiotti U. Direct interaction between crystalline silica and DNA - a proposed model for silica carcinogenesis. Scand J Work Environ Health 1995;21 Suppl 2:22–6.
[39]Sun J, Wei L, Liu X, Li J, Li B, Wang G, et al. Influences of ionic dissolution products of dicalcium silicate coating on osteoblastic proliferation, differentiation and gene expression. Acta Biomater 2009;5:1284–93.
[40]Christodoulou I, Buttery LDK, Saravanapavan P, Tai G, Hench LL, Polak JM. Dose- and time-dependent effect of bioactive gel-glass ionic-dissolution products on human fetal osteoblast-specific gene expression. J Biomed Mater Res 2005;74B:529–37.
[41]Zhao W, Chang J, Wang J, Zhai W, Wang Z. In vitro bioactivity of novel tricalcium silicate ceramics. J Mater Sci : Mater Med 2007;18:917–23.
[42]Jung G-Y, Park Y-J, Han J-S. Effects of HA released calcium ion on osteoblast differentiation. J Mater Sci : Mater Med 2010;21:1649–54.
[43]McCullen SD, Zhan J, Onorato ML, Bernacki SH, Loboa EG. Effect of varied ionic calcium on human adipose-derived stem cell mineralization. Tissue Eng Part A 2010;16:1971–81.
[44]Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, et al. Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc Natl Acad Sci USa 2004;101:5140–5.
[45]Godwin SL, Soltoff SP. Extracellular calcium and platelet-derived growth factor promote receptor-mediated chemotaxis in osteoblasts through different signaling pathways. J Biol Chem 1997;272:11307–12.
[46]Eklou-Kalonji E, Denis I, Lieberherr M, Pointillart A. Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro. Cell Tissue Res 1998;292:163–71.
[47]Shie MY, Ding SJ, Chang HC. The role of silicon in osteoblast-like cell proliferation and apoptosis. Acta Biomater 2011;7:2604–14.
[48]Shie MY, Ding SJ. Integrin binding and MAPK signal pathways in primary cell responses to surface chemistry of calcium silicate cements. Biomaterials 2013;34:6589–606.
[49]Wu BC, Youn SC, Kao CT, Huang SC, Hung CJ, Chou MY, et al. The effects of calcium silicate cement/fibroblast growth factor-2 composite on osteogenesis accelerator in human dental pulp cells. J Dent Sci 2014:1–9.
[50]Liu CH, Huang TH, Hung CJ, Lai WY, Kao CT, Shie MY. The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro. Int Endod J 2014;47:843–53.
[51]Wu BC, Kao CT, Huang TH, Hung CJ, Shie MY, Chung HY. Effect of Verapamil, a Calcium Channel Blocker, on the Odontogenic Activity of Human Dental Pulp Cells Cultured with Silicate-based Materials. J Endod 2014;40:1105–11.
[52]Torabinejad M, Watson T, Ford T. Sealing Ability of a Mineral Trioxide Aggregate When Used As a Root End Filling Material. J Endod 1993;19:591–5.
[53]Kao CT, Shie MY, Huang TH, Ding SJ. Properties of an Accelerated Mineral Trioxide Aggregate–like Root-end Filling Material. J Endod 2009;35:239–42.
[54]Silva EJ, Rosa TP, Herrera DR, Jacinto RC, Gomes BP, Zaia AA, et al. Evaluation of Cytotoxicity and Physicochemical Propertiesof Calcium Silicate-based Endodontic Sealer MTA Fillapex. J Endod 2013;39:274–7.
[55]Moritz N, Vedel E, Ylanen H, Jokinen M, Hupa M, Yli-Urpo A. Characterisation of bioactive glass coatings on titanium substrates produced using a CO2 laser. J Mater Sci : Mater Med 2004;15:787–94.
[56]Eid AA, Niu L, Primus CM, Opperman LA, Pashley DH, Watanabe I, et al. In vitro osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement 2013;39:1161–6.
[57]Hung CJ, Kao CT, Shie MY, Huang TH. Comparison of host inflammatory responses between calcium-silicate base material and intermediate restorative material. J Dent Sci 2014;9:158–64.
[58]Wei W, Qi Y, Nikonov SY, Niu L, Messer RLW, Mao J, et al. Effects of an experimental calcium aluminosilicate cement on the viability of murine odontoblast-like cells. J Endod. 2012;38:936–42.
[59]Huang SC, Wu BC, Kao CT, Huang TH, Hung CJ, Shie MY. Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro. Int Endod J 2014;48:236–45.
[60]Chou MY, Kao CT, Hung CJ, Huang TH, Huang SC, Shie MY, et al. Role of the P38 Pathway in Calcium Silicate Cement–induced Cell Viability and Angiogenesis-related Proteins of Human Dental Pulp Cell In Vitro. J Endod 2014;40:818–24.
[61]Lai WY, Kao CT, Hung CJ, Huang TH, Shie MY. An evaluation of the inflammatory response of lipopolysaccharide-treated primary dental pulp cells with regard to calcium silicate-based cements. Int J Oral Sci 2014;6:94–8.
[62]Liu CH, Hung CJ, Huang TH, Lin CC, Kao CT, Shie MY. Odontogenic differentiation of human dental pulp cells by calcium silicate materials stimulating via FGFR/ERK signaling pathway. Mater Sci Eng C 2014;43:359–66.
[63]Wu C, Chang J, Fan W. Bioactive mesoporous calcium–silicate nanoparticles with excellent mineralization ability, osteostimulation, drug-delivery and antibacterial properties for filling apex roots of teeth. J Mater Chem 2012;22:16801.
[64]Kao CT, Huang TH, Chen YJ, Hung CJ, Lin CC, Shie MY. Using calcium silicate to regulate the physicochemical and biological properties when using β-tricalcium phosphate as bone cement. Mater Sci Eng C 2014;43:126–34.
[65]Islam I, Chng HK, Yap AUJ. X-ray diffraction analysis of mineral trioxide aggregate and Portland cement. Int Endod J 2006;39:220–5.
[66]Hung CJ, Kao CT, Shie MY, Huang TH. Comparison of host inflammatory responses between calcium-silicate base material and IRM. J Dent Sci 2014;9:158–64.
[67]Wei W, Qi Y, Nironov SY, Niu L, Messer RL, Mao J, et al. Effects of an Experimental Calcium Aluminosilicate Cementon the Viability of Murine Odontoblast-like Cells. J Endod 2012;38:936–42.
[68]Su CC, Kao CT, Hung CJ, Chen YJ, Huang TH, Shie MY. Materials Science and Engineering C. Mater Sci Eng C 2014;37:156–63.
[69]Ding SJ, Kao CT, Shie MY, Hung JC, Huang TH. The Physical and Cytological Properties of White MTA Mixed with Na2HPO4 as an Accelerant. J Endod 2008;34:748–51.
[70]Bottino MC, Kamocki K, Yassen GH, Platt JA, Vail MM, Ehrlich Y, et al. Bioactive Nanofibrous Scaffolds for Regenerative Endodontics. Journal of Dent Res 2013;92:963–9.
[71]Su YF, Lin CC, Huang TH, Chou MY, Yang JJ, Shie MY. Materials Science and Engineering C. Mater Sci Eng C 2014;42:672–80.
[72]Hung CJ, Kao CT, Chen YJ, Shie MY, Huang TH. Antiosteoclastogenic Activity of Silicate-based Materials Antagonizing Receptor Activator for Nuclear Factor KappaB Ligand–induced Osteoclast Differentiation of Murine Marcophages. J Endod 2013;39:1557–61.
[73]Hung CJ, Hsin HI, Lin CC, Huang TH, Wu BC, Kao CT, et al. The Role of Integrin av in Proliferation and Differentiation ofHuman Dental Pulp Cell Response to Calcium Silicate Cement. J Endod 2014;40:1802–9.
[74]Huang TH, Shie MY, Kao CT, Ding SJ. The Effect of Setting Accelerator on Properties of Mineral Trioxide Aggregate. J Endod 2008;34:590–3.
[75]Matsui N, Nozaki K, Ishihara K, Yamashita K, Nagai A. Concentration-dependent effects of fibronectin adsorbed on hydroxyapatite surfaces on osteoblast adhesion. Mater Sci Eng C 2015;48:378–83.
[76]Zhang X, Han P, Jaiprakash A, Wu C, Xiao Y. A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells. J Mater Chem B 2014;2:1415.
[77]Shie MY, Chang HC, Ding SJ. Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment. Int Endod J 2012;45:337–45.
[78]Alvarez-Pérez MA, Narayanan S, Zeichner-David M, Rodríguez Carmona B, Arzate H. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23). Bone 2006;38:409–19.