Antibacterial Borosilicate Glasses for Bone Tissue Engineering

Bioactive glasses (BGs), including 45S5 glass, have been widely investigated in Bone Tissue Engineering (BTE) for more than three decades. They have been regarded as relevant materials for the repair and replacement of bone defects. However, the majority of BG particles show slow degradation rates and present no effect against bacterial infections^{1, 3}. The Borosilicate matrix is known to possess a faster degradation rate, increases the formation of hydroxyapatite (HA) and presents an enhanced release of specific cations (e.g.: Ca, Sr and Mg)². Such of those cations are known for their influence in the cell proliferation and differentiation, as well as, for their antimicrobial character. The aim of this study was to assess if borosilicate BGs are able to present anti-microbial properties for BTE applications.

EXPERIMENTAL METHODS

Borosilicate glass compositions of general formula 0.20B₂O₃ : 0.40SiO₂ : xMgO : yCaO : (0.35-x-y)SrO : 0.05Na₂O (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 calcium phosphate layer was tested after immersion of the BG samples 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 BG samples. Disk diffusion (DD) and broth dilution (BD) methods were adapted to BG samples, testing known amounts of glass particles.

The cytotoxicity of the glass particles 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 BG samples were successful obtained by melt quenching, ground and presented non-toxic effects up to 3 days.

After the immersion of BG samples into SBF, Ca- (BSCaO) and Sr-containing glasses (BSSrO) revealed the presence of bone-like apatite structures at their surface, although at different stages of crystallization.

However, after 7 days of immersion, the BSCaO glass displayed an increase in apatite-like deposition with a Ca/P ratio of ≈ 1.67, similar to HA.

The DD data demonstrate that the Mg-containing (BSMgO) and the BSSrO glasses inhibited the growth of bacteria S. epidermidis and P. aeruginosa respectively. Clear halos appeared around the glass-loaded agar discs.

Moreover, BD data showed a concentration dependent effect of BSSrO glasses against P. aeruginosa (e.g.: BSSrO_18mg exhibiting a reduction of ≈ 4 log₁₀CFU/ml). 

CONCLUSION

We were able to synthesise borosilicate-based glasses by melt quenching, and we observed no cytotoxic effects up to 3 days culture. Chemical characterization supported the high reactivity and their bioactivity (BSCaO sample with a Ca/P ≈ 1.67).

Bacterial tests with different concentrations (9, 18, 36 and 72 mg/ml of BG) indicate that BSMgO and BSSrO glasses have antibacterial activity against S. epidermidis and P. aeruginosa respectively. Moreover BSSrO glasses have specific bactericidal activity against P. aeruginosa for quantities equal or higher than 18 mg/ml. Results also revealed a concentration dependence on the anti-bacterial properties.