Synthesis, Characterization of Silver Doped Hydroxyapatite Nanoparticles for Biomedical ApplicationsSneha S Bandgar1,2, Tanaji V Kolekar3, Shailesh S Shirguppikar3, Mahesh A Shinde3, Rajendra V Shejawal1* and Sambhaji R Bamane2*
2Department of Chemistry, R.S.B.Mahavidyalaya, Aundh, Satara, 415002, M.S., India
3Rajarambapu Institute of Technology, Islampur, Sangli 415 414, M.S., India
Rajendra V Shejawal, Department of Chemistry, Lal Bahadur Shastri College, Satara, 415002, M.S., India, Sambhaji R Bamane, Department of Chemistry, R.S.B.Mahavidyalaya, Aundh, Satara, 415002, M.S., India,
Calcium based Bio ceramics are potentially attractive a wide range of medical applications. The effect of Silver substitution on the biocompatibility of hydroxyapatite (HA) under the physiochemical conditions has been investigated. Various samples of Silver doped hydroxyapatite (Ag-HA) with different concentration (0, 0.5, 1.0, 1.5, 2.0, 2.5 mol%) were successfully synthesized by solution combustion method and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), and thermal analysis techniques. XRD and TEM results reveal uniform and crystalline Ag-HA nanoparticles.
Solution combustion processes, Apatite composites, Antibacterial, Biomedical applications
During recent years, there have been efforts in developing Nanocrystaline Calcium orthophosphate to enhance their biological and mechanical properties for use in biomedical applications. Calcium orthophosphate based inorganic bio ceramic materials have a wide range of biomedical applications . Bioresorbable and Bioactive phases of calcium phosphate bioceramic materials are choice for bone-tissue engineering application because of their similar inorganic composition with the mineral phases of natural bone, excellent biocompatibility and osteoconductivity [2,3]. In 1920 it is reported the first successful medical application of calcium phosphate in humans . Recently most widely-used bioresorbable and bioactive ceramics include calcium orthophosphates (CaP). They are present in bones, teeth and the tendons of mammals, giving these organs hardness and stability. They are known as non-ion-substituted calcium orthophosphates with a Ca/P molar ratio between 0.5 and 2.0. The most widely used member of the family of CaPs is hydroxyapatite (HA) . Among different forms of calcium phosphate, hydroxyapatite (HA) is one of the most promising inorganic biomaterials. HA is the principal mineral constituents of natural bones and teeth .
Hydroxyapatite (HA) has been widely used as a biomaterial in orthopedics, bioengineering and dentistry, because of its good biocompatibility . Synthetic Hydroxyapatite (HA) is the most promising because of good cation exchange rate with metals, excellent biocompatibility and high affinity for the pathogenic microorganisms [8-10]. It is reported that around 70-80% of implants are made of biocompatible metals . With the introduction of a transient metal ion such as silver HA can be effective in controlling microorganisms Due to its ion-exchange capabilities .
Synthetic HA with metal doping for Biomedical applications has gained lot of attention because of the high flexibility and stability of apatite structure, a great number of cationic substitution are of potential application in the Biomedical field. There are many reports of the occupying of Ca sites by various divalent ( Mg2+, Sr2+, Cd2+, Ba2+) and Trivalent cations (Al3+, Fe3+) . However, now days the biggest current problem in the biomedical field is post-surgical infections arising from recent-implanted synthetic biomaterials, because these provide sites for potential bacterial adhesion . However, there are some limitations of these bioceramic materials involving the possible release of harmful metal ions through wear and corrosion processes when they are exposed to aggressive body environment [15-17].
The metal release of toxic ions might cause adverse effects to the surrounding cells [18-20]. Silver has been known as a disinfectant for many years and has a broad spectrum of antimicrobial activity while exhibiting low toxicity towards mammalian cells. Various studies reported that the silver ions doped in the HA coatings play an important role in preventing or minimizing bacterial adhesion . Furthermore, the reactivity of silver is high efficient when used in Nano sized particles due to their better contact with microorganisms . higher concentration of Ag more than 300 ppb in human blood can cause side-effects in the form of leukopenia, Kidney and liver damage . Therefore, optimization of Ag concentration in HA is critical to guarantee Ag/HA optimal antimicrobial ability without cytotoxicity .
Nano sized HA have been synthesized by many routes, including co-precipitation , sol-gel synthesis , emulsion methods , microwave precipitation  and mechano chemical methods . Solution combustion synthesis (SCS) is invented in 1986, a large number of research efforts have been focused toward the preparation of important materials, mostly oxides, with improved properties . In solution combustion synthesis, aqueous reactive solutions are used, where the precursors are mixed on the molecular level. The advantage of this process has been demonstrated by the use of different fuels and oxidizers, varying the oxidizer/fuel ratio and ignition sources, as well as combination of various synthesis approaches .
In the present investigation, Nano crystalline HA were prepared by a facile Solution combustion method. This work aims to study the effect of Ag on the Bioactivity of Nanocrystalline Hydroxyapatite (HA) under the physiological Conditions. Antibacterial effect was evaluated quantitatively against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The cytotoxicity assessments have been utilized to evaluated the bio-compability of nanocrystalline HA and Ag-HA with different concentration. Cytotoxicity of the prepared material has been studied by utilizing L929 (mouse Fibroblast) cell line for 12 and 24 h. For this trypan blue dye exclusion (TBDE) and MTT assays were performed to identify the possible toxicity of nanoparticles. The ultra-trace Ag doped HA nanocrystals may provide new opportunities in non-cytotoxic implant with antibacterial ability in bone tissue Engineering.
Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), and di-Ammonium hydrogen orthophosphate (NH4)2HPO4 were purchased from Sigma-Aldrich and Silver nitrate (AgNO3) were purchased from Thomas Baker. All chemicals used here were of analytical grade and used without further purification.
Synthesis of Ag-doped hydroxyapatite
Ag-doped hydroxyapatite (Ag-HA) with different concentration (0, 0.5, 1.0, 1.5, 2.0, 2.5 mol%) were prepared by modifying solution combustion method. For this, polyvinyl alcohol (PVA) was used as a fuel. In brief, the stoichiometric amounts of the nitrate precursors Ca(NO3)2.4H2O, AgNO3 and phosphate precursor (NH4)2HPO4 were dissolved in double distilled water to form the solution of 0.1 M. The equimolar solution of PVA was prepared in double distilled water. The mixture of oxidants and fuel was placed onto a magnetic stirrer for 30 min. to get uniform mixing. Evaporation of water to form a gel of precursors was carried out at 100ºC and then the gel was heated at 300ºC to obtain a powder. The obtained powder of Ag-HA was then annealed at 950ºC for 6 h to remove carbon residues and then used for further analysis. The atomic ratio of (Ag+Ca)/P in the precursor were fixed at 1.67 in all of the cases. The dried mixture possesses the characteristics of combustion and can be ignited to start combustion reaction using muffle furnace. Various Ag-doped hydroxyapatite samples containing Ag content 0, 0.5, 1.0, 1.5, 2.0, 2.5 mol% were denoted as HA, Ag-HA-1, Ag-HA-2, Ag-HA-3, Ag-HA-4, Ag-HA-5, respectively.
Structural and morphological studies
The structural and morphological studies of the samples were studied using Thermo gravimetric analysis (TGA), X-ray Diffractometer (XRD), Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy and Transmission Electron Microscopy (TEM) and. Phase identification and structural analysis of Ag-HA were studied using X-ray diffraction (Philip-3710) with Cu-Kα radiation in the 2θ range from 10ºC to 80ºC. The all patterns were analysed by X-pert high score plus software and compared with the Joint Committee on Powder Diffraction Standards (JCPDS) (JCDPS card No. 01-074-0565 and 01-074-1743). The surface morphology and particles sizes of the Ag-HA were determined by using transmission electron microscope (Philips CM 200 model) with an operating voltage of 20-200 kV and a resolution of 2.4 Å. The compositional analysis was done by energy dispersive spectroscopy (EDS, JEOL JSM 6360). A Perkin-Elmer spectrometer (Model No-783 USA) was used to obtain FTIR spectra of Al-HA samples in the range of 450-4000 cm-1 using KBr pellets.
Antibacterial activity assay
The antibacterial ability of Nanocrystaline HA, Ag-HA-1, Ag-HA-2, Ag-HA-3, Ag-HA-4, Ag-HA-5 was carried out at 0.2, 0.4, 0.6, 0.8, 1.0 ppm by bacteriological plate counting methods using Gram negative Escherichia coli (E. coli, ATCC 8739) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 6538). The bacterial strains were purchased from National Chemical laboratory, Pune. The bacteria were cultured in liquid nutrient broth medium at 35-37ºC for 12 h and adjusted to a concentration of about 107 CFU/mL. All used laboratory supplies below were sterilized in an autoclave at 121ºC for 20 min. Briefly, 0.5 g of each sterile Sample was dispersed in centrifugal tube containing Buffered sodium chloride peptone (BECP) (9 mL) and bacteria suspension (1 mL) and incubated at 30-35ºC for 4, 8, 12, 16, 32, 48 and 72 h. For subsequent bacteria counting, 100 μL of suspension was extracted from centrifugal tube and inoculated into solid nutrient agar medium followed by 24 h incubation at 35ºC. The bacterial colonies were counted. The antibacterial rates (R %) were calculated based on the formula which is already Reported elsewhere [32,33].
In present work The structural, composition and cytotoxic properties of the pure HA and Ag-HA nanoparticles with different stoichiometric ratios prepared by a modified solution combustion technique have been studied in great detail. The physicochemical properties of the pure HA have been affected with the existence of Silver. The pure HA and Ag-HA nanoparticles having pure phase and almost identical particle sizes. EDS analysis confirmed the presence of pure HA and Ag-HA with stoichiometric ratio and this material could be applicable for the formation of new bone in vivo. HA nanoparticles doped with Ag possess effective antibacterial activity and in vitro noncytotoxicity. The antibacterial rate increases from 65% to 99% while the cell viability decreases from 97% to 75% when the Ag-doping concentration varies from 0.05 to 190 ppm. The optimal properties with excellent cytocompatibility and antibacterial ability can be achieved when the Ag-doping concentration is between 0.30 ppm and 2.3 ppm. The antibacterial HA with Ag is non-toxicity to living cells and tissues even if the particles are internalized by cells. Hence, it is revealed that Taken together, Ag-HA nanocrystals are potentially applicable as bone substitution materials in tissue engineering. Ag-HA is an excellent candidate in the biomedical field.
 A. Bigi, E. Boanini, K. Rubini, J. Solid. State. Chem., 2004, 177, 3092-3098.
 L.L. Hench, J. Bioceramics, Am. Ceram. Soc., 1998, 81, 1705-1728.
 K.D. Groot, C.P.A.T. Klein, J.G.C. Wolke, J.M.A. Blieck-Hogervorst, In: T. Yamamuro, L.L. Hench, J. Wilson, CRC Press, Boca Raton, FL, 1990, P-3.
 R.Z. LeGeros, Osaka University, 2002.
 M. Supovan, Ceramics International., 41, 2015, 9203-9231
 H.J. Qiu, J. Yang, P. Kodali, J. Koh, G.A. Ameer, Biomaterials., 2006, 27, 5845-5854.
 Y. Tanaka, Y. Hirata, R. Yoshinaka, J. Ceram. Process. Res., 2003, 4 (4), 197-201.
 I. Smiciklas, A. Onjia, J. Markovic, S. Raicevic, Mater. Sci. Forum, 2005, 494, 405-410.
 R.Z. LeGeros, Chem. Rev., 2008, 108, 4742-4753.
 E.D.Berry, G.R. Siragusa, Appl. Environ. Microbial., 1997, 63, 4069-4074.
 M. Niinomi, M. Nakai, J. Hieda, Acta Biomaterialia, 2012, 8, 3888-3903.
 K.S. Oh, K.J. Kim, Y.K. Jeong, Y.H. Choa, Key. Eng. Mater., 2003, 240-242, 583-586.
 M. Wakamura, K. Kandori, T. ishikawa, Colloids Surface: A., 2000, 164, 297.
 E.M. Hetrick, M.H. Schoenfisch, Chem. Soc. Rev., 2006, 35, 780-789.
 U. Türkan, O. Öztürk, A.E. Eroğlu, Orthopedic Implant Material., 2006, 200, 5020-5027.
 N. Espallargas, C. Torres, A.I. Muñoz, WEAR., 2014, 332-333.
 S.J. Lee J.J. Lai, J Materials Processing Technol., 2003, 140, 206-210.
 C.C. Shih, C.M. Shih, Y.Y. Su, L.H.J. Su, M.S. Chang, S.J. Lin, 2004, 46, 427-441.
 T.F. Zhang, Q.Y. Deng, B. Liu, B.J. Wu, F.J. Jing, Y.X. Leng, CoCrMo and Ti6Al4V Substrate.,2003.
 B. Alemón, M. Flores, W. Ramírez, J. C. Huegel, E. Broitman, 2015, 81, 159-168.
 V. Stanic, D. Janackovic, S. Dimitrijevic, S.B. Tanaskovic, M. Mitrica, M.S. Pavlovic, A. Krstic, D. Jovanovic, S. Raicevic, Appl. Surf. Sci., 2011, 257, 4510-4518.
 J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramirez, M.J. Yacaman, Nanotechnology., 2005, 2346-2353.
 C. Shi, J. Gao, M. Wang, J. Fu, D. Wang, Y. Zhu, Mater. Sci. Eng. C., 2015, 55, 497-505.
 R. Murugan, S. Ramakrishna, Acta. Biomater., 2006, 2, 201-206.
 M.H. Fathi, A. Hanifi, V. Mortazavi, J. Mater. Process. Technol., 2008, 202, 536-542.
 G.C. Koumoulidis, A.P. Katsoulidis, A.K. Ladavos, P.J. Pomonis, C.C. Trapalis, A.T. Sdoukos, T.C. Vaimakis, J. Colloid Interface. Sci., 2003, 259, 254-260.
 Z. Zou, K. Lin, L. Chen, J. Chang, Ultrason. Sonochem., 2012, 19, 1174-1179.
 S. Lala, S. Brahmachari, P.K. Das, D. Das, T. Kar, S.K. Pradhan, Mater. Sci. Eng., 2014, 42, 647-656.
 A.G. Merzhanov, Bentham Sci., 2012, 112.
 S.T. Aruna, Bentham Publishers., 2010, 206-221.
 C. Shi, J. Gao, M. Wang, J. Fu, D. Wang, Y. Zhu, Mat. Sci. Eng., 2015, C55, 497-505.
 N.D. Thorat, S.V Otari, R.M. Patil, V.M. Khot, A.I. Prasad, R.S. Ningthoujam, Colloids. Surf. B. Biointerfaces., 2013, 111 264-269.
 F.A.C. Andradea, L.C.O. Vercikb, F.J. Monteiroc, E.C. Silva Rigoa, Ceramics Int., 2016, 42, 2271-2280.
 M.J. Phillips, J.A. Darr, Z.B. Luklinska, I. Rehman, Mater. Sci. Mater. Med., 2003, 14, 875-882.
 S.V. Dorozhkin, Prog. Cryst. Growth Charact. Mater., 2002, 44, 45-61.
 S. Kang, M. Herzberg, D.F. Rodrigues, M. Elimelech, Langmuir, 2008, 24, 6409-6413.
 Z.M. Xiu, Q.B. Zhang, H.L. Puppala, V.L. Colvin, P.J.J. Alvarez, Nano Lett., 2012, 12, 4271-4275.
 W.K. Jung, H.C. Koo, K.W. Kim, S. Shin, S.H. Kim, Y.H. Park, Appl. Environ. Microbiol., 2008, 74, 2171-2178.
 Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, J. Biomed. Mater. Res., 2000, 52, 662-668.
 R.J. Chung, M.F. Hsieh, C.W. Huang, L.H. Perng, H.W. Wen, T.S. Chin, J. Biomed. Mater. Res., 2006, 76B 169-178.
 T.V. Kolekar, N.D. Thorat, H.M. Yadav, V.T. Magalad, M.A. Shinde, S.S. Bandgar, J.H. Kim, G.L. Agawane, Ceram. Int., 2016, 42, 5304-5311.
 A.E. Nel, L. Mädler, D. Velegol, T. Xia, E.M.V. Hoek, P. Somasundaran, Nat. Mater., 2009, 8, 543-557.