β-三鈣磷酸鹽廣泛應用於醫療科學以及材料等領域，因其組成類似於骨骼中礦物結構，能夠促進骨骼的癒合進而受到諸多的關注。然而β-三鈣磷酸鹽在使用上缺乏抑制細菌的效果，在手術上恐有感染的風險，為了克服此一問題，本實驗中藉由添加銀與鋅等元素於β-三鈣磷酸鹽中，以提升材料的抑菌性質。
在此一研究中，採用一步合成的噴霧熱烈解法(Spray pyrolysis , SP)
來進行銀與鋅摻雜β-三鈣磷酸鹽的實驗。而在SP製備法中相較於溶膠-凝膠法以及濕化學合成法具有低汙染與製程較連續等優勢。本研究中分別透過X光繞射儀、場發射掃描式電子顯微鏡、能量色散譜以及氮氣吸/脫附分析儀來測定β-三鈣磷酸鹽、銀摻雜β-三鈣磷酸鹽、鋅摻雜β-三鈣磷酸鹽以及銀與鋅摻雜β-三鈣磷酸鹽等樣品之晶體結構、形貌、粒徑大小、化學組成與比表面積。此外藉由菌落數的計算比較銀摻雜β-三鈣磷酸鹽、鋅摻雜β-三鈣磷酸鹽以及銀與鋅摻雜β-三鈣磷酸鹽等樣品對於大腸桿菌的有效抑制細菌效果，而結果顯示2.87 mol% 銀與2.87 mol%鋅摻雜β-三鈣磷酸鹽比5.75 mol%銀摻雜β-三鈣磷酸鹽以及5.75 mol%鋅摻雜β-三鈣磷酸鹽還具有較強的抑菌效果。而相關的抑菌機制將於本文中近一步探討。

Beta-Tricalcium phosphate (β-TCP) particles are popular in the applications of medical science and material due to the significantly simulates the mineralogical structure of natural bones. However, β-TCP lacks of antibacterial activity. In order to overcome this problem doping β-TCP with Ag and Zn are proposed.
In this present study, one-step synthesized process by spray pyrolysis (SP) was carried out to prepare Ag and Zn doped β-TCP. The SP process have advantages low contamination and continuous production over the sol-gel and wet chemical precipitation synthesis. The crystallographic structure, morphology, particle size, chemical composition, and specific surface area of un-doped, Ag-doped β-TCP, Zn-doped β-TCP and AgZn-doped β-TCP, were characterized by X-ray diffraction, scanning electron microscopy, energy dispersive spectroscopy and Brunauer-Emmet-Teller nitrogen adsorption/desorption method, respectively. In addition, the efficacy antibacterial activity of Ag-doped β-TCP, Zn-doped β-TCP and AgZn-doped β-TCP against Escherichia coli are obtained by colony count method. The results indicated that 5.75 mol% AgZn-doped β-TCP (contain 2.87 mol% Ag and 2.87 mol% Zn) were better than those 5.75 mol% Ag-doped β-TCP and 5.75 mol% Zn-doped β-TCP. The antibacterial activity mechanism of Ag- and Zn-doped β-TCP are also discussed.

References

[1] L. L. Hench, Bioceramics From Concept To Clinic, (1991) 1487-1510.
[2] J. Lu, J. Descamps M Fau-Dejou, G. Dejou J Fau-Koubi, P. Koubi G Fau -Hardouin, J. Hardouin P Fau-Lemaitre, J.-P. Lemaitre J Fau-Proust, J.P. Proust, The biodegradation mechanism of calcium phosphate biomaterials in bone (1993) 21.
[3] N. Kondo, A. Ogose, K. Tokunaga, H. Umezu, K. Arai, N. Kudo, M. Hoshino, H. Inoue, H. Irie, K. Kuroda, H. Mera, N. Endo, Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation, Biomaterials, 27 (2006) 4419-4427.
[4] H. van de Belt, D. Neut, W. Schenk, J.R. van Horn, H.C. van der Mei, H.J. Busscher, Infection of orthopedic implants and the use of antibiotic loaded bone cements. A review, Acta orthopaedica Scandinavica, 72 (2001) 557-571.
[5] R.E. Simionescu, D.J. Kennedy, Prevention of Infection in Prosthetic Devices, The Bionic Human, Springer2006, pp. 159-185.
[6] W. Chen, S. Oh, A. Ong, N. Oh, Y. Liu, H. Courtney, M. Appleford, J. Ong, Antibacterial and osteogenic properties of silver‐containing hydroxyapatite coatings produced using a sol gel process, Journal of Biomedical Materials Research Part A, 82 (2007) 899-906.
[7] S. Samani, S. Hossainalipour, M. Tamizifar, H. Rezaie, In vitro antibacterial evaluation of sol–gel‐derived Zn‐, Ag‐, and (Zn+Ag)‐doped hydroxyapatite coatings against methicillin‐resistant Staphylococcus aureus, Journal of Biomedical Materials Research Part A, 101 (2013) 222-230.
[8] N. Iqbal, M.R.A. Kadir, N.A.N.N. Malek, N.H. Mahmood, M.R. Murali, T. Kamarul, Rapid microwave assisted synthesis and characterization of nanosized silver-doped hydroxyapatite with antibacterial properties, Materials letters, 89 (2012) 118-122.
[9] V.K. Sharma, R.A. Yngard, Y. Lin, Silver nanoparticles: green synthesis and their antimicrobial activities, Advances in colloid and interface science, 145 (2009) 83-96.
[10] X. Li, Y. Sogo, A. Ito, H. Mutsuzaki, N. Ochiai, T. Kobayashi, S. Nakamura, K. Yamashita, R.Z. LeGeros, The optimum zinc content in set calcium phosphate cement for promoting bone formation in vivo, Materials Science and Engineering: C, 29 (2009) 969-975.
[11] M. Otsuka, S. Marunaka, Y. Matsuda, A. Ito, P. Layrolle, H. Naito, N. Ichinose, Calcium level‐responsive in‐vitro zinc release from zinc containing tricalcium phosphate (ZnTCP), Journal of biomedical materials research, 52 (2000) 819-824.
[12] N. Matsumoto, K. Sato, K. Yoshida, K. Hashimoto, Y. Toda, Preparation and characterization of β-tricalcium phosphate co-doped with monovalent and divalent antibacterial metal ions, Acta biomaterialia, 5 (2009) 3157-3164.
[13] B. Liu, D.x. Lun, Current Application of β‐tricalcium Phosphate Composites in Orthopaedics, Orthopaedic surgery, 4 (2012) 139-144.
[14] Z. Nazemi, M.H. Nazarpak, M. Mehdikhani-Nahrkhalaji, H. Staji, M.M. Kalani, Synthesis, characterisation and antibacterial effects of sol–gel derived biphasic calcium phosphate nanopowders, Micro & Nano Letters, 9 (2014) 403-406.
[15] B. Singh, S. Kumar, N. Saha, B. Basu, R. Gupta, Phase stability of silver particles embedded calcium phosphate bioceramics, Bulletin of Materials Science, 38 (2015) 525-529.
[16] S.-J. Shih, I.-C. Chien, Preparation and characterization of nanostructured silver particles by one-step spray pyrolysis, Powder technology, 237 (2013) 436-441.
[17] B. Li, Z. Liu, J. Yang, Z. Yi, W. Xiao, X. Liu, X. Yang, W. Xu, X. Liao, Preparation of bioactive β-tricalcium phosphate microspheres as bone graft substitute materials, Materials Science and Engineering: C, 70 (2017) 1200-1205.
[18] S. Jaiswal, P. McHale, B. Duffy, Preparation and rapid analysis of antibacterial silver, copper and zinc doped sol–gel surfaces, Colloids and Surfaces B: Biointerfaces, 94 (2012) 170-176.
[19] A. Kinaci, V. Neuhaus, D.C. Ring, Trends in bone graft use in the United States, Orthopedics, 37 (2014) e783-e788.
[20] S. Bauer, P. Schmuki, K. von der Mark, J. Park, Engineering biocompatible implant surfaces: Part I: Materials and surfaces, Progress in Materials Science, 58 (2013) 261-326.
[21] A. Domanska, A. Boczkowska, Biodegradable polyurethanes from crystalline prepolymers, Polymer Degradation and Stability, 108 (2014) 175-181.
[22] Y.W. Frank K. Ko, Introduction to Nanofiber Materials, (2014) 147.
[23] R. Ghosh, R. Sarkar, Synthesis and characterization of sintered beta-tricalcium phosphate: A comparative study on the effect of preparation route, Materials Science and Engineering: C, 67 (2016) 345-352.
[24] H.J. Busscher, H.C. Van Der Mei, G. Subbiahdoss, P.C. Jutte, J.J. Van Den Dungen, S.A. Zaat, M.J. Schultz, D.W. Grainger, Biomaterial-associated infection: locating the finish line in the race for the surface, Science translational medicine, 4 (2012) 110-153.
[25] W. Suchanek, M. Yoshimura, Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants, Journal of Materials Research, 13 (1998) 94-117.
[26] R.H. Fitzgerald, Microbiologic environment of the conventional operating room, Archives of surgery, 114 (1979) 772-775.
[27] K. Verkkala, A. Eklund, J. Ojajärvi, L. Tiittanen, J. Hoborn, P. Mäkelä, The conventionally ventilated operating theatre and air contamination control during cardiac surgery–bacteriological and particulate matter control garment options for low level contamination, European journal of cardio-thoracic surgery, 14 (1998) 206-210.
[28] D. Campoccia, L. Montanaro, P. Speziale, C.R. Arciola, Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use, Biomaterials, 31 (2010) 6363-6377.
[29] M.J. Calhoun J, Musculosketal Disorder, (2003) 680.
[30] http://www.microbiologyinfo.com/different-size-shape-and-arrangement-of bacterial-cells/.
[31] Different size, shape and arrangement of bacterial cells http://www.microbiologyinfo.com/different-size-shape-and-arrangement ofbacterial-cells/.
[32] C. Allen, J.F. Loo, S. Yu, S. Kong, T.-F. Chan, Monitoring bacterial growth using tunable resistive pulse sensing with a pore-based technique, Applied microbiology and biotechnology, 98 (2014) 855-862.
[33] H. Kubitschek, Cell volume increase in Escherichia coli after shifts to richer media, Journal of bacteriology, 172 (1990) 94-101.
[34] M.T. Madigan, J.M. Martinko, Microorganisms and microbiology, Brock biology of microorganisms. 11th ed. Upper Saddle River, New Jersey (NJ): Pearson Prentice Hall, (2006) 1-20.
[35] J.D. Wang, P.A. Levin, Metabolism, cell growth and the bacterial cell cycle, Nature Reviews Microbiology, 7 (2009) 822.
[36] S. Cooper, C.E. Helmstetter, Chromosome replication and the division cycle of Escherichia coli Br, Journal of molecular biology, 31 (1968) 519-540.
[37] P. Cornelis, Expressing genes in different Escherichia coli compartments, Current opinion in biotechnology, 11 (2000) 450-454.
[38] J. Díaz-Visurraga, C. Gutiérrez, C. Von Plessing, A. García, Metal nanostructures as antibacterial agents, Science and technology against microbial pathogens: Research, development and evaluation. Badajoz: Formatex, (2011) 210-218.
[39] R. Dastjerdi, M. Montazer, A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties, Colloids and Surfaces B: Biointerfaces, 79 (2010) 5-18.
[40] A. Simchi, E. Tamjid, F. Pishbin, A. Boccaccini, Recent progress in inorganic and composite coatings with bactericidal capability for orthopaedic applications, Nanomedicine: Nanotechnology, Biology and Medicine, 7 (2011) 22-39.
[41] M.J. Hajipour, K.M. Fromm, A.A. Ashkarran, D.J. de Aberasturi, I.R. de Larramendi, T. Rojo, V. Serpooshan, W.J. Parak, M. Mahmoudi, Antibacterial properties of nanoparticles, Trends in biotechnology, 30 (2012) 499-511.
[42] G.V. Vimbela, S.M. Ngo, C. Fraze, L. Yang, D.A. Stout, Antibacterial properties and toxicity from metallic nanomaterials, International journal of nanomedicine, 12 (2017) 3941.
[43] J. Hardes, H. Ahrens, C. Gebert, A. Streitbuerger, H. Buerger, M. Erren, A. Gunsel, C. Wedemeyer, G. Saxler, W. Winkelmann, Lack of toxicological side-effects in silver-coated megaprostheses in humans, Biomaterials, 28 (2007) 2869-2875.
[44] R.O. Darouiche, I.I. Raad, S.O. Heard, J.I. Thornby, O.C. Wenker, A. Gabrielli, J. Berg, N. Khardori, H. Hanna, R. Hachem, A comparison of two antimicrobial-impregnated central venous catheters, New England Journal of Medicine, 340 (1999) 1-8.
[45] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms, FEMS microbiology letters, 279 (2008) 71-76.
[46] Y. Liu, L. He, A. Mustapha, H. Li, Z. Hu, M. Lin, Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157: H7, Journal of applied microbiology, 107 (2009) 1193-1201.
[47] Y. Xie, Y. He, P.L. Irwin, T. Jin, X. Shi, Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni, Applied and environmental microbiology, 77 (2011) 2325-2331.
[48] F.H. Albee, Studies in bone growth: triple calcium phosphate as a stimulus to osteogenesis, Annals of surgery, 71 (1920) 32.
[49] N. Eliaz, N. Metoki, Calcium phosphate bioceramics: a review of their history, structure, properties, coating technologies and biomedical applications, Materials, 10 (2017) 334.
[50] E. Verron, I. Khairoun, J. Guicheux, J.-M. Bouler, Calcium phosphate biomaterials as bone drug delivery systems: a review, Drug discovery today, 15 (2010) 547-552.
[51] S.V. Dorozhkin, Calcium orthophosphates as bioceramics: state of the art, Journal of functional biomaterials, 1 (2010) 22-107.
[52] H.E. Roscoe, C. Schorlemmer, A. Harden, H. Colman, A Treatise on Chemistry: Volume 1: the Non-metallic Elements, Macmillan1905.
[53] W.F. Muhlenberg, In Transactionsof the Medical Society of the State of Pennsylvania, 14 (1832) 90.
[54] M. Lassaigne, Solubility of carbonate of lime in water containing carbonic acid, Philosophical Magazine Series 3, 30 (1847) 297-298.
[55] X. Yang, Z. Wang, Synthesis of biphasic ceramics of hydroxyapatite and β-tricalcium phosphate with controlled phase content and porosity, Journal of Materials Chemistry, 8 (1998) 2233-2237.
[56] D. Yang, T.R. Tadjiev, J.W. Kim, C.K. You, S. Choi, K. Park, K. Ryoo, S.Y. Kim, Comparative study of the degradation behavior of mechanically mixed and chemically precipitated biphasic calcium phosphates, Key Engineering Materials, Trans Tech Publ, 2006, pp. 227-230.
[57] I. Manjubala, T. Sastry, R.S. Kumar, Bone in-growth induced by biphasic calcium phosphate ceramic in femoral defect of dogs, Journal of biomaterials applications, 19 (2005) 341-360.
[58] F.H. Lin, C.J. Liao, K.S. Chen, J.S. Sun, C.Y. Lin, Preparation of βTCP/HAP biphasic ceramics with natural bone structure by heating bovine cancellous bone with the addition of (NH4) 2HPO4, 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, 51 (2000) 157-163.
[59] S.R. Levitt, P.H. Crayton, E.A. Monroe, R.A. Condrate, Forming method for apatite prostheses, Journal of Biomedical Materials Research, 3 (1969) 683-684.
[60] E. Monroe, W. Votava, D. Bass, J.M. Mullen, New calcium phosphate ceramic material for bone and tooth implants, Journal of dental research, 50 (1971) 860-861.
[61] T. Driskell, C. Hassler, V. Tennery, L. McCoy, W. Clarke, Calcium phosphate resorbable ceramics-potential alternative to bone grafting, Journal of dental research, sage publications (1973), 123.
[62] W. Hubbard, Physiological calcium phosphate as orthopedic implant material, Diss. Abstr. Int, 35 (1974) 1683B.
[63] E. Nery, K. Lynch, W. Hirthe, K. Mueller, Bioceramic implants in surgically produced infrabony defects, Journal of periodontology, 46 (1975) 328-347.
[64] B. Zhao, G. yan Xing, Synthesis of beta-Tricalcium phosphate Powder through Aqueous Precipitation Method, Biomedical Engineering and Informatics, 2009. BMEI'09. 2nd International Conference on, IEEE, (2009), pp. 1-6.
[65] W.I. Abdel-Fattah, F.M. Reicha, T.A. Elkhooly, Nano-beta-tricalcium phosphates synthesis and biodegradation: 1. Effect of microwave and SO42− ions on β-TCP synthesis and its characterization, Biomedical Materials, 3 (2008) 034121.
[66] S. Loher, W.J. Stark, M. Maciejewski, A. Baiker, S.E. Pratsinis, D. Reichardt, F. Maspero, F. Krumeich, D. Günther, Fluoro-apatite and calcium phosphate nanoparticles by flame synthesis, Chemistry of materials, 17 (2005) 36-42.
[67] R.L. Price, L.G. Gutwein, L. Kaledin, F. Tepper, T.J. Webster, Osteoblast function on nanophase alumina materials: Influence of chemistry, phase, and topography, Journal of Biomedical Materials Research Part A, 67 (2003) 1284-1293.
[68] N.R. Washburn, K.M. Yamada, C.G. Simon Jr, S.B. Kennedy, E.J. Amis, High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation, Biomaterials, 25 (2004) 1215-1224.
[69] Y. Wan, Y. Wang, Z. Liu, X. Qu, B. Han, J. Bei, S. Wang, Adhesion and proliferation of OCT-1 osteoblast-like cells on micro-and nano-scale topography structured poly (L-lactide), Biomaterials, 26 (2005) 4453-4459.
[70] J.S. Cho, Y.C. Kang, Nano-sized hydroxyapatite powders prepared by flame spray pyrolysis, Journal of Alloys and Compounds, 464 (2008) 282-287.
[71] J.S. Cho, Y.N. Ko, H.Y. Koo, Y.C. Kang, Synthesis of nano-sized biphasic calcium phosphate ceramics with spherical shape by flame spray pyrolysis, Journal of Materials Science: Materials in Medicine, 21 (2010) 1143-1149.
[72] G. Solero, Synthesis of Nanoparticles through Flame Spray Pyrolysis: Experimental Apparatus and Preliminary Results, Nanoscience and Nanotechnology, 7 (2017) 21-25.
[73] S.-J. Shih, Y.-J. Chou, I.-C. Chien, One-step synthesis of bioactive glass by spray pyrolysis, Journal of Nanoparticle Research, 14 (2012) 1299.
[74] K. Kim, J. Park, C. Kim, H. Park, H. Chang, S. Choi, Synthesis of SrTiO3: Pr, Al by ultrasonic spray pyrolysis, Ceramics international, 28 (2002) 29-36.
[75] A. Ewald, D. Hösel, S. Patel, L.M. Grover, J.E. Barralet, U. Gbureck, Silver-doped calcium phosphate cements with antimicrobial activity, Acta biomaterialia, 7 (2011) 4064-4070.
[76] O. Gokcekaya, K. Ueda, K. Ogasawara, H. Kanetaka, T. Narushima, In vitro evaluation of Ag-containing calcium phosphates: Effectiveness of Ag-incorporated β-tricalcium phosphate, Materials Science and Engineering: C, 75 (2017) 926-933.
[77] G.L. Messing, S.C. Zhang, G.V. Jayanthi, Ceramic powder synthesis by spray pyrolysis, Journal of the American Ceramic Society, 76 (1993) 2707-2726.
[78] S.J. Shih, W.L. Tzeng, R. Jatnika, C.J. Shih, K.B. Borisenko, Control of Ag nanoparticle distribution influencing bioactive and antibacterial properties of Ag‐doped mesoporous bioactive glass particles prepared by spray pyrolysis, Journal of Biomedical Materials Research Part B: Applied Biomaterials, 103 (2015) 899-907.
[79] V. Orlovskii, V. Komlev, S. Barinov, Hydroxyapatite and hydroxyapatite-based ceramics, Inorganic Materials, 38 (2002) 973-984.
[80] B. Clarke, Normal bone anatomy and physiology, Clinical journal of the American Society of Nephrology, 3 (2008) S131-S139.
[81] S. Weiner, W. Traub, Bone structure: from angstroms to microns, The FASEB journal, 6 (1992) 879-885.
[82] P. Bianco, P.G. Robey, Stem cells in tissue engineering, Nature, 414 (2001) 118.
[83] R.S. Breed, W. Dotterrer, The number of colonies allowable on satisfactory agar plates, Journal of Bacteriology, 1 (1916) 321.
[84] E. Thian, T. Konishi, Y. Kawanobe, P. Lim, C. Choong, B. Ho, M. Aizawa, Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties, Journal of Materials Science: Materials in Medicine, 24 (2013) 437-445.
[85] T. Hamouda, A. Myc, B. Donovan, A.Y. Shih, J.D. Reuter, J.R. Baker, A novel surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against bacteria, enveloped viruses and fungi, Microbiological research, 156 (2001) 1-7.
[86] Q.L. Feng, J. Wu, G. Chen, F. Cui, T. Kim, J. Kim, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, Journal of biomedical materials research, 52 (2000) 662-668.
[87] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, Journal of colloid and interface science, 275 (2004) 177-182.
[88] N.A. Amro, L.P. Kotra, K. Wadu-Mesthrige, A. Bulychev, S. Mobashery, G.-y. Liu, High-resolution atomic force microscopy studies of the Escherichia coli outer membrane: structural basis for permeability, Langmuir, 16 (2000) 2789-2796.
[89] T. Sun, H. Hao, W. Hao, S. Yi, X. Li, J. Li, Preparation and antibacterial properties of titanium-doped ZnO from different zinc salts, Nanoscale research letters, 9 (2014) 98.
[90] S. Atmaca, G. Kadri, R. Cicek, The effect of zinc on microbial growth, Turkish Journal of Medical Sciences, 28 (1998) 595-598.
[91] A. Top, S. Ülkü, Silver, zinc, and copper exchange in Na-clinoptilolite and resulting effect on antibacterial activity, Applied Clay Science, 27 (2004) 13-19.