Synthesis of antimicrobial monophase silver-doped hydroxyapatite nanopowders for bone tissue engineering
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.
References (58)
- et al.
Calcium phosphates as substitution of bone tissues
Prog. Solid State Chem.
(2004) - et al.
Hydroxyapatite cement in craniofacial reconstruction
Otolaryngol. Head Neck Surg.
(2005) - et al.
Synthesis of functionally graded bioactive glass-apatite multistructures on Ti substrates by pulsed laser deposition
Appl. Surf. Sci.
(2007) - et al.
The effect of hydroxyapatite coating on bony ingrowth into grooved titanium implants
Biomaterials
(1999) - et al.
The significance of infection related to orthopedic devices and issues of antibiotic resistance
Biomaterials
(2006) - et al.
Staphylococci and implant surfaces: a review
Injury
(2006) Antibacterial coating systems
Injury
(2006)- et al.
Bacterial adhesion studies on titanium, titanium nitride and modified hydroxyapatite thin films
Mater. Sci. Eng. C
(2007) - et al.
Bacterial resistance to silver in wound care
J. Hosp. Infect.
(2005) - et al.
Study of cytotoxicity mechanisms of silver nitrate in human dermal fibroblasts
Toxicol. Lett.
(1998)