In the present study, bioactive glasses (BGs) in 60SiO2-(31-x) CaO–4P2O5–5Li2O-xAg2O systems (x = 0, 1, 5, and 10 mol %) with optimum fixed amount of Li2O (5 mol %) were successfully synthesized through sol-gel technique and the effects of Ag2O addition on in-vitro investigations, biological properties, and bactericidal efficiency were evaluated. Structural and morphological evaluations by X-ray diffraction (XRD), Fourier transform infrared (FTIR), transmission electron microscopy (TEM) as well as scanning electron microscopy (SEM) revealed that BG with chemical composition of 60SiO2–30CaO–4P2O5–5Li2O–1Ag2O (mol%) had a higher rate of hydroxyapatite (HA) formation than other synthesized specimens. While, BGs containing 5 and 10 mol% of Ag had a lower bioactivity because of the reduced dissolution rate of Si and Ca. The results obtained from the 3-(4,5dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and evaluation of alkaline phosphatase (ALP) activity exhibited that proliferation and differentiation of MC3T3-E1 osteoblast-like cells and osteoblastic activity were intensified by the presence of Ag in the BGs҆ composition. Meanwhile, BGs containing 1 mol% of Ag not only had no toxic effect on the cells, but also caused a significant enhancement in proliferation and differentiation of the cells within 7 days (**p < 0.01), as well as the highest ALP activity in this specimen (*p < 0.05). Additionally, all the synthesized specimens had significant antibacterial efficiency against E-coli bacteria. Our results showed that addition of 1 mol% of Ag to BG containing Li led to a significant antibacterial efficiency compared to the control (*p < 0.05). Afterwards, DAPI/actin and live/dead cell staining methods were applied, which well confirmed results of MTT assay. Finally, BG with chemical composition (LA01) of 60SiO2–30CaO–4P2O5–5Li2O–1Ag2O was introduced as an optimal novel co-doped BG in biomedical applications due to causing higher differentiation and proliferation of MC3T3-E1 cells and more increase in ALP activity and bactericidal efficiency.
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