Antibacterial borosilicate bioactive glasses for bone tissue engineering

Abstract

Event Abstract Back to Event Antibacterial borosilicate bioactive glasses for bone tissue engineering João Fernandes1*, Margarida Martins1*, Nuno N. Neves1*, Ricardo A. Pires1* and Rui L. Reis1* 1 University of Minho, 3Bs Research Group, Portugal Introduction: The bioactive glasses (BGs), including Bioglass® 45S5, have been widely investigated in bone tissue engineering (BTE) field due to their extraordinary feature of bonding with bone by the formation of a hydroxyapatite (HA) layer. However, the majority of BGs show slow degradation rates and present no effect against bacterial infections [1,3]. However the borosilicate bioactive glasses (BBGs) are known to possess a faster degradation rate, which may be used to achieve a more controlled release of specific component ions (e.g. Mg2+, Ca2+ and Sr2+) [2] enhancing the activity on HA formation and antibacterial properties. The aim of this study was to assess if borosilicate bioactive glasses (BBGs) are able to present antibacterial properties for BTE applications. Experimental Methods: BBGs compositions of general formula 0.05Na2O . xMgO . yCaO . (0.35-x-y)SrO . 0.20B2O3 . 0.40SiO2 (molar ratio, where x, y = 0.35 or 0.00, and x ≠ y) were synthesized by melt quenching (T≈1450ºC) and ground to obtain particles with a size < 63 µm. The capacity to form bone-like structures was assessed after immersion of the BBGs into simulated body fluid (SBF) for 7 days. Microbial susceptibility was measured using standardized inocula of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis in contact with the BBGs. Disk diffusion (DD) and broth dilution (BD) methods were adapted to microparticles, testing known amounts of BBGs. The cytotoxicity of the BBGs was assessed by direct contact with human osteosarcoma cell line during 7 days of incubation (37ºC and 5% CO2). Cell proliferation (DNA) and metabolic activity (MTS) profiles were followed along the culture time. Results and Discussion: The BBGs were successful obtained by melt quenching, ground and presented non-toxic effects up to 3 days. After the immersion of BBGs into SBF, Ca- (BBG-Ca) and Sr-containing glasses (BBG-Sr) revealed the presence of bone-like structures onto the surface at different stages of crystallization. However, after 7 days of immersion, the BBG-Ca glass displayed an increase in bone-like structures deposition onto the surface with a Ca/P ratio of ≈ 1.67, similar to HA (Figure 1). The DD data demonstrated that the Mg-containing (BBG-Mg) and the BBG-Sr glasses inhibited the growth of bacteria S. epidermidis and P. aeruginosa respectively. Clear halos appeared around the agar discs containing BBGs. The inhibition zone for each bacterial strain is shown in Figure 2. Moreover, the BD method data of BBG-Mg (Figure 3a) indicates that it prevents the growth of S. epidermidis, having a bacteriostatic effect. Surprisingly, the BBG-Sr (Figure 3c), presented and outstanding bactericidal activity against P. aeruginosa for all concentrations tested but 9 mg/mL. In fact, it was observed that BBG-Sr is able to eradicate P. aeruginosa at concentrations ≥ 18 mg/mL. This contrasts with 45S5 bioglass (Figure 4b and 4d) that only exhibits bactericidal activity at high concentrations and high dissolution times. Conclusion: Novel BBGs with different substituted ions (Ca2+, Sr2+ or Mg2+) were successfully synthesised by melt-quench not showing cytotoxic effects up to 3 days culture. Bioactivity assay provides convincing evidence that BBGs produce bone-like structures onto the surface (e.g.: BBG-Ca has a Ca/P ratio ≈ 1.67). BBG-Mg has bacteriostatic activity against S. epidermidis and BBG-Sr has bacteriostatic/bactericidal activity against P. aeruginosa, eradicating this bacterium at higher concentrations (≥ 18 mg/mL). Results also revealed a concentration dependence on the anti-bacterial properties. Portuguese Foundation for Science and Technology (Ph.D. grant BD/73162/2010); The European Union’s Seventh Framework Programme (FP7/2007–2013) under Grant No. REGPOT-CT2012-31633-POLARIS.; M. Martins is financed by the project “Tissue engineering of connective tissues” (Ref. RL3 – TECT - NORTE-01-0124-FEDER-000020) co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).