Balancing antibacterial activity and toxicity in silver-loaded hydroxyapatite: the impact of the silver nanoparticle incorporation method

Abstract

Hydroxyapatite (Hap) has limited intrinsic antimicrobial properties, which can be significantly enhanced by incorporating silver nanoparticles (AgNPs). The antibacterial properties of silver, particularly in the form of silver ions (Ag⁺) and silver nanoparticles (AgNPs), are well-documented and extensively utilized in both biomedical and industrial applications. However, high concentrations of Ag⁺ or poorly dispersed silver nanoparticles can exert cytotoxic effects on mammalian cells, making it essential to optimize silver content and distribution to achieve an effective balance between antimicrobial efficacy and biocompatibility. In our study, we sought to identify the optimal conditions for achieving this balance in silver-loaded hydroxyapatite (Ag–Hap). To this end, we prepared a series of Ag–Hap samples by employing different silver incorporation strategies and varying the silver content. Hence, nanopowders of pure hydroxyapatite (Hap), silver-substituted hydroxyapatite (Ca_10−xAg_x(PO₄)₆(OH)₂), and silver/hydroxyapatite (Ag_x/Hap) composites were synthesized via a simple precipitation method. Two distinct Ag_x/Hap composites were prepared: one by precipitating Hap nanoparticles in a nano-silver slurry (Method 1), and the other by precipitating AgNPs into a Hap suspension (Method 2). The synthesized materials were characterized using X-ray powder diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FTIR) to assess their structure, morphology, elemental composition, and purity. Antibacterial activity and cytotoxicity were evaluated across all formulations using two bacterial strains and C2C12 murine muscle precursor cells. Results revealed that embedding silver nanoparticles into the hydroxyapatite matrix enhanced antibacterial efficacy more effectively than ionic substitution of silver for calcium within the Hap lattice. The formulation that demonstrated the most favorable combination of bactericidal activity and cell viability was the Ag_x/Hap composite synthesized via reverse precipitation (Method 2), particularly within the silver content range of x = 0.2–0.3. This method likely promoted a uniform dispersion of AgNPs within the Hap matrix, thereby enabling sustained antibacterial action while minimizing cytotoxicity. This optimized formulation offers a promising strategy for developing bioactive coatings and scaffolds with enhanced antimicrobial properties, biocompatibility, and structural stability, making it well-suited for use in orthopedic, dental, and other biomedical implant applications.