Silver- and/or titanium-doped calcium phosphate highly porous bioceramic with antibacterial activity

Introduction

Regeneration and reconstruction of bone defects is significant challenge in modern medicine and often requires surgery where biomaterials are used. About 10% of these procedures are at risk of bacterial infections [1]. Due to the possible systemic toxicity and growing emergence of antibiotic-resistance caused by systemic use of antibiotics methods are being sought to ensure local delivery of antibacterial agents at the site of implantation. The use of alternative antibacterial agents such as bacteriophages, bacteriophins and inorganic agents (Ag, Cu, Zn, Ti compounds, etc.) with far lower susceptibility to induce resistance than common antibiotics has resurge recently [2]. Broadening of the spectrum of calcium phosphate (CaP) materials by modification with metal ions to impart advanced biological and physicochemical properties has been observed. Attaining antibacterial properties is one of the aims and the most common dopants studied in this regard are Ag(I), Cu(II), Zn(II), Co(II) etc. [3]. Combining Ag(I) with Ti(IV) is a potential way to promote bone-implant integration, with reduced bacterial infection and enhanced bioactivity [4], [5].
The aim of this study was to impart antibacterial properties to highly porous CaP bioceramics, which could be used in orthopaedic surgery, by doping them with Ag(I) and Ti(IV) ions, and to evaluate the synergy and cumulative effects of dopants.

Experimental Methods

Method of producing highly porous Ag- and/or Ti-doped CaP bioceramic scaffolds compromised following steps:(i) preparation of porous bioceramic scaffolds (the substrates), through PU foam replica method, (ii) aqueous precipitation of Ag- and/or Ti-doped CaP (hydroxyapatite (HAp) and apatitic-tricalcium phosphate), (iii) surface modification of the substrates by depositing the Ag- and/or Ti-doped CaP layer by vacuum-assisted impregnation.
Prepared bioceramic scaffolds were analyzed for microstructure and composition using common analytical techniques - SEM, XRD, FT-IR, EDX etc.
Minimum inhibitory concentration (MIC) of the Ag- and/or Ti-doped CaP against gram-positive S. aureus (ATCC 25923) was determined by microdilution method. The antibacterial properties of Ag- and/or Ti-doped CaP bioceramic scaffolds were determined by the broth dilution method, which involves inoculating a diluted bacterial suspension into TSB agar medium and incubating at 37 oC. A suspension of S. aureus bacterial reference culture ATCC 25923 (106 CFU/mL) was used.
In vitro bioactivity of Ag- and/or Ti-doped CaP bioceramic scaffolds in semi-dynamic conditions in the SBF was evaluated. The dynamics of the decrease of Ca ions concentration in the SBF after exposure to the bioceramic scaffolds was performed using an automated titration station and Ca-ion-selective electrode. The formation of a biomimetic apatite layer on the sacaffolds surface was assessed using SEM.

Results and Discussion

The obtained bioceramic scaffolds had an open porous structure and highly interconnected pores (Fig. 1). A porosity of 92 ± 2%, pore sizes in a range from 50 to 450 µm. According to FT-IR and XRD analyses the bioceramic scaffolds were composed of biocompatible phases. No impurities form the raw materials were detected at least at the detection limits of the instruments. No significant differences in positions and intensities of the characteristic XRD diffraction peaks and FT-IR absorbance bands were observed among the compounds of various compositions.
EDX results confirmed the presence of Ag and Ti in the synthesis products. The content of Ag in the samples was higher than planned, while Ti was close to the planned. However, it was observed that with increasing calcinations temperature, the Ag content decreases. Thus, for sintering the coating 900 oC was chosen to maintain Ag concentration as close as possible to the non-calcined CaP.
The MIC values for all Ag- and / or Ti-doped CaPs were 10 mg / mL, except for Ag-doped and Ag- and Ti-co-doped, which showed MICs of 5 and 2.5 mg / mL, respectively. This also coincides with the results of antibacterial evaluation of Ag- and / or Ti-doped CaP bioceramic substrates. Namely, the respective bioceramic substrates showed the highest antibacterial activity.
All scaffolds, regardless of composition, showed in vitro bioactivity.

Conclusion

The combination of polymeric foam replica and vacuum-assisted impregnation technologies is suitable for preparation of the Ag- and/or Ti-doped CaP bioceramic scaffolds with highly interconnected porous architecture. The physical properties, as well as a phase and chemical purity are sufficient to provide a suitable initial three-dimensional support for cells to grow into it and make a new bone and allows the scaffolds produced to be used for osteoconductive bone implants, tissue engineering implants. Antibacterial activity of the scaffolds depends on the phase composition which affects concentration of antibacterial ions released into the surrounding environment.