Elsevier

Applied Surface Science

Volume 257, Issue 9, 15 February 2011, Pages 4510-4518
Applied Surface Science

Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering

https://doi.org/10.1016/j.apsusc.2010.12.113Get rights and content

Abstract

Monophase silver-doped hydroxyapatite (AgxCa10−x(PO4)6(OH)2; 0.002  x  0.04) nanoparticles were prepared using a neutralization method and investigated with respect to potential medical applications. This method consists of dissolving Ag2O in solution of H3PO4, and the slow addition to suspension of Ca(OH)2 was applied for the purpose of homogenous distribution of silver ions. Characterization studies from XRD, TEM and FTIR spectra showed that obtained crystals are monophase hydroxyapatites and that particles of all samples are of nano size, with average length of 70 nm and about 15–25 nm in diameter. Antimicrobial studies have demonstrated that all silver-doped hydroxyapatite samples exhibit excellent antimicrobial activity in vitro against the following pathogens: Staphylococcus aureus, Escherichia coli and Candida albicans. The hydroxyapatite sample with the highest content of silver has shown the highest antimicrobial activity; killed all cells of E. coli and brought to more than 99% reduction in viable counts of S. aureus and C. albicans. The atomic force microscopic studies illustrate that silver-doped hydroxyapatite sample causes considerable morphological changes of microorganism cells which might be the cause of cells’ death. Hemolysis ratios of the silver-doped hydroxyapatite samples were below 3%, indicating good blood compatibility and that are promising as biomaterials.

Research highlights

▶ The neutralization method has been used for synthesis of silver-doped hydroxyapatite samples. ▶ Particles of silver-doped hydroxyapatite samples are of nano size and homogenous in composition. ▶ The silver-doped hydroxyapatite samples showed excellent antimicrobial activity. ▶ AFM showed that silver-doped sample causes morphological changes of microorganism cells. ▶ Silver-doped samples are nonhemolytic and seen promising for medical applications.

Introduction

Inorganic biomaterials based on calcium orthophosphate have their wide range of applications in medicine [1], [2], [3]. Among them, synthetic hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is the most promising because of its biocompatibility, bioactivity, and osteoconductivity. Hydroxyapatite has been used to fill a wide range of bony defects in orthopedic and maxillofacial surgeries and dentistry [4], [5], [6], [7]. It has also been widely used as a coating for metallic prostheses to improve their biological properties [8], [9], [10]. Post surgical infections associated with the presence of implant materials are present at up to 5% of patients; the problem usually requires their removal [11], [12]. Microorganism adhesions on implant surfaces represent an initial crucial step in infection and lead to the formation of biofilm, in which microorganisms are more resistant to antimicrobial agents [13]. To solve the problem of contamination of hydroxyapatite, it has been proposed to use antimicrobial agents such as antibiotics, fluorine and biocides metal ions [14], [15], [16]. The main concern with antibiotics is that the development of resistant microorganisms and adsorbed antibiotics are quickly washed out by body fluids and cannot prevent post-surgical infections when long-term. Metal ions (Ag+, Cu2+ and Zn2+) are widely used in medicine as antimicrobial agents. Silver ions in particular show oligodynamic effect with a minimal development of microorganism's resistance [17]. Low concentrations are not toxic to mammals, but high concentrations can cause cytotoxicity and argyria [18], [19], [20]. Several in vitro studies reported that the silver ions in the HAP coatings play an important role in preventing or minimizing initial bacterial adhesion [21], [22], [23], [24], [25], [26].

Several studies describe the synthesis of silver-doped hydroxyapatite with the use of nitrate and/or ammonium precursors. Kim et al. [27] synthesized silver ion substituted hydroxyapatite and nitrate-apatite by using a wet-chemical method. Chung et al. [28] suggested the use of a sol–gel procedure; however, these materials, besides the hydroxyapatite and nitrate-apatite phases, include an Ag2O crystalline phase depending on the substitution degree of silver. Rameshbabu et al. [29] and Oh et al. [30] have used ammonium phosphates as precursors in synthesis of silver doped hydroxyapatite. Namely, the use of the ammonium and nitrate precursors may cause the incorporation of NH4+ and NO3 ions into the hydroxyapatite and then affect its structure [31], [32]. The main concern with this impurity is its elimination. Ammonia with silver ions in the basic conditions builds a very stable complex ion [Ag(NH3)2]+ that can affect the amount of silver to be incorporated into hydroxyapatite [33].

The aim of this work was to synthesize the monophasic silver-doped hydroxyapatite nanopowders with high crystallinity and to examine their antimicrobial and hemolytic activities. This method involves dissolving Ag2O in a solution of H3PO4, and slow addition to suspension of Ca(OH)2 was applied for the homogenous distribution of silver ions. Slow reactant addition was found to yield pure, well-crystallized HAP, due to low supersaturation and avoidance of local inhomogeneity [34], [35]. The main reason for using the neutralization method to produce silver-doped hydroxyapatite materials in this study is the possibility of preparing pure products from relatively inexpensive chemicals, and the suitability for an industrial production. The main disadvantage of neutralization method is that it is not reproducible. The antimicrobial activity of silver-doped samples against different pathogens: Staphylococcus aureus (S. aureus, ATCC 25923), Escherichia coli (E. coli, ATCC 25922), and Candida albicans (C. albicans, ATCC 24433) was evaluated in vitro.

Section snippets

Preparation of the materials

Pure HAP and doped HAP with various concentrations of silver ions were prepared by neutralization method as described in our previous work [36]. Calcium carbonate (CaCO3), silver oxide (Ag2O) and phosphoric acid (H3PO4) (all analytical grade, Merck) were used as the starting chemicals. Calcium oxide (CaO) was obtained by calcinations of CaCO3 for 24 h at 1000 °C. Experiments were performed with inert atmosphere (N2). Double distilled water was used throughout all the experiments. The Ca(OH)2

Results and discussion

The results of the quantitative elemental analysis of pure HAP and AgHAP samples are listed in Table 1. The silver contents in doped samples are somewhat greater than the amount of starting material. This may be attributed to nonstoichiometric products. The (Ca + Ag)/P ratios for all samples were smaller than 1.67. The stoichiometric product of hydroxyapatite has a Ca/P ratio of 1.67, but powders obtained by wet methods are usually nonstoichiometric [41].

The XRD patterns of all the samples are

Conclusion

In this study, the neutralization method has been successfully used for synthesis of monophase silver-doped hydroxyapatite nanoparticles, which have antimicrobial properties. The analysis of XRD, FTIR and TEM showed that particles of silver-doped hydroxyapatite samples are of nano size and homogenous in composition. Antimicrobial study shows that silver-doped hydroxyapatite samples with very low silver content have excellent activity against E. coli, S. aureus, and C. albicans. AgHAP3 sample

Acknowledgements

This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia. Special thanks to Dr. Danijela Randjelovic (IHTM - Institute of Microelectronic Technologies and Single Crystals, University of Belgrade) for performing AFM characterization of the samples, AFM image analysis and helpful discussions.

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