Introduction
Calcium phosphates (CaP) (i.e. hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP)) are widely used due to their excellent biocompatibility (Kalita et al., 2007). Porous bioceramic is the material of choice for non-load bearing bone implants. Besides filling bone defects, porous bioceramic can be used as bone tissue engineering scaffolds and as drug delivery devices. Ideally, porous bioceramic has interconnected macroporous network with pore size ≥100 µm to allow the bone tissue ingrowth. The aim was to develop Ag- and/or Ti-doped CaP (Ag- and/or Ti-CaP) bioceramic scaffolds. Due of their promising antibacterial properties, Ag(I) with Ti(IV) doping was used to reduce risk of bacterial infection.
Experimental
Porous scaffolds (substrates) were prepared by polymer (polyurethane) replica method and sintering at 1300 ^oC. Ag- and/or Ti-CaP (HAp or β-TCP) powders were synthesized via aqueous precipitation. Ag- and/or Ti-CaP coatings were applied on the substrates surface by vacuum-assisted impregnation followed by sintering at 900 ^oC. To adjust the thickness of the coatings, the minimum inhibitory concentration of the Ag- and/or Ti-CaP powders against S.aureus was determined. Based on MIC values (≤10 mg/mL), it was chosen to apply 10 mg of the powders per sample. Physicochemical properties, e.g. phase composition and microstructure using XRD and SEM, respectively, were determined. In vitro bioactivity was assessed under semi-dynamic conditions in simulated body fluid (SBF) at 37 ^oC under mild orbital shaking (70 rpm). Antibacterial activity was determined as reduction in the CFU of S.aureus using bactericidal assay. Cytotoxicity was assessed according to ISO 10933 test guidelines using the Balb/c 3T3 and L929 cell lines. Effect on the cell proliferation was assessed using the MG-63 osteoblast cell line.
Results
The obtained scaffolds have porosity up to 92±2%, pores size from 50 to 450 µm, strut thickness from 30 to 100 µm and coating thickness ~5 µm. After 7 days of immersion in SBF apatite agglomerates with their characteristic globular and cauliflower-like shape (Li et al., 2015) started to form on the scaffolds surface. Increasing immersion time up to 14 and 28 days the scaffolds was completely covered with apatite layer. Statistical differences between control measurements indicated that the Ti-HAp and Ag- and/or Ti-β-TCP coated substrates have the highest antibacterial activity. Compared to the control (duodecyl sulfate (100 µg/ml)), the cell viability in the Balb/c 3T3 cell line ranged from 99.94 to 117.32%, and in the L929 cell line - from 96.67 to 119.06%. The Ag- and Ti-β-TCP coated scaffolds had the most significant effect on the cell proliferation.
Acknowledgements
This work has been supported by the ERDF within the Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment” (No.1.1.1.2/VIAA/2/18/339). The authors acknowledge financial support from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No. 857287.