Strontium and Zinc Co-Doped Mesoporous Bioactive Glass Nanoparticles for Potential Use in Bone Tissue Engineering Applications

Abstract

Mesoporous bioactive glass nanoparticles (MBGNs) have attracted significant attention as multifunctional nanocarriers for various applications in both hard and soft tissue engineering. In this study, multifunctional strontium (Sr)- and zinc (Zn)-containing MBGNs were successfully synthesized via the microemulsion-assisted sol-gel method combined with a cationic surfactant (cetyltrimethylammonium bromide, CTAB). Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs exhibited spherical shapes in the nanoscale range of 100 ± 20 nm with a mesoporous structure. Sr and Zn were co-substituted in MBGNs (60SiO₂-40CaO) to induce osteogenic potential and antibacterial properties without altering their size, morphology, negative surface charge, amorphous nature, mesoporous structure, and pore size. The synthesized MBGNs facilitated bioactivity by promoting the formation of an apatite-like layer on the surface of the particles after immersion in Simulated Body Fluid (SBF). The effect of the particles on the metabolic activity of human mesenchymal stem cells was concentration-dependent. The hMSCs exposed to Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs at 200 μg/mL enhanced calcium deposition and osteogenic differentiation without osteogenic supplements. Moreover, the cellular uptake and internalization of Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs in hMSCs were observed. These novel particles, which exhibited multiple functionalities, including promoting bone regeneration, delivering therapeutic ions intracellularly, and inhibiting the growth of Staphylococcus aureus and Escherichia coli, are potential nanocarriers for bone regeneration applications.

Keywords: antibacterial activity; bioactive glass nanoparticle; bioactivity; intracellular delivery; mesoporous.

Figure 1

Sr− and Zn−doped MBGN synthesis by the microemulsion-assisted sol–gel process.

Figure 2

Bright-field SEM images of (a) MBGNs, (b) Sr−MBGNs, (c) Zn−MBGNs, and (d) Sr−Zn−MBGNs operating at EHT = 1 kV and 100 kX. Scale bar = 200 nm.

Figure 3

(a) FTIR and (b) XRD spectra of MBGNs, Sr−MBGNs, Zn−MBGNs, and Sr−Zn−MBGNs.

Figure 4

N₂ physisorption isotherms of (a) MBGNs, (b) Sr−MBGNs, (c) Zn−MBGNs, and (d) Sr−Zn−MBGNs.

Figure 5

SEM images and EDS-SEM of in vitro apatite formation on (a) MBGNs, (b) Sr−MBGNs, (c) Zn−MBGNs, and (d) Sr−Zn−MBGNs after immersion in SBF for 21 days.

Figure 6

(a) FTIR and (b) XRD spectra of apatite formation on MBGNs, Sr−MBGNs, Zn−MBGNs, and Sr−Zn−MBGNs after immersion in SBF for 21 days.

Figure 7

The viability of hMSCs exposed to particles was compared to the positive control (cells cultured in media without nanoparticles). Six technical replicates (n = 6) were conducted within each experiment, repeated in triplicate (N = 3). The data are expressed as mean ± standard deviation (SD), and statistical analysis was performed using ANOVA along with the appropriate post hoc comparison test (Tukey’s test), with significance denoted by * for p < 0.05.

Figure 8

(a) Alizarin red S staining of hMSCs treated with MBGNs, Sr−MBGNs, Zn−MBGNs, and Sr−Zn−MBGNs at particle concentration of 200 μg/mL following 3 weeks using inverted light microscopy. Original magnification is ×10 and scale bar is 50 µm in length. (b) Semi-quantification of calcium deposits of hMSCs (* p < 0.05 versus the basal control group).

Figure 9

q-PCR analysis was used to assess the expression of osteogenic marker genes in hMSCs subjected to treatment with Sr−MBGNs, Zn−MBGNs, and Sr−Zn−MBGNs, all in the absence of osteogenic supplements, for 3 weeks. The quantification was performed by normalizing gene expression to GAPDH. The data presented are the results of two separate experiments, with values indicated as the mean ± SD (n = 3). Significant upregulation of osteogenic marker genes was observed in cells exposed to doped MBGNs compared to traditional MBGNs. (*) denotes a statistically significant difference between cells treated with doped MBGNs and traditional MBGNs under basal conditions at the corresponding time intervals (* p < 0.05).

Figure 10

(a) The viability of hMSCs exposed to FITC-conjugated MBGNs was compared to the positive control (cells cultured in media without nanoparticles). Six technical replicates (n = 6) were conducted within each experiment, repeated in triplicate (N = 3). The data are expressed as mean ± standard deviation (SD). (b) Fluorescent images indicated internationalization of the FITC-conjugated MBGNs (green) at a concentration of 200 mg/mL by the hMSCs. Nuclei were counterstained with DAPI (blue). Scale bar is 50 µm in length.

Figure 11

In vitro antibacterial activity (halo zone diameter) of MBGN, Sr−MBGNs, Zn−MBGNs, and Sr−Zn−MBGNs against S. aureus and E. coli after 18 h of incubation. (a) The different normalized widths of the antimicrobial “halo”. (b) Antimicrobial diffusion “halo” results: diameters of clear zones (n = 3). * indicating p < 0.05 compared to MBGNs group. Disk size was 6 mm diameter × 1 mm thickness.