The osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses

Abstract

Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real-time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium-substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration.

Keywords: bioactive glass; mesenchymal stromal cells; osteogenic differentiation; real-time PCR; strontium.

Figure 1

(A) Differential volume (%) distribution of particle sizes of Sr0, Sr50 and Sr100 bioactive glass powders. (B) Differential number (%) distribution of particle sizes of Sr0, Sr50 and Sr100 bioactive glass powders. Only one line can be observed in (B) due to the overlap of the data for the three samples. (C–E) SEM images of Sr0, Sr50 and Sr100 bioactive glass powders, showing great variation in particle shape and size within each sample

Figure 2

X‐ray diffraction spectra of Sr0, Sr50 and Sr100 bioactive glasses

Figure 3

(A) Experimentally measured density, and density calculated using Doweidar's model of glass density, of Sr0, Sr50 and Sr100 bioactive glasses. (B) Oxygen density of bioactive glass plotted in relation to the level of strontium substitution in its composition. (C) Bioactive glass solubility plotted in relation to the level of strontium substitution in its composition

Figure 4

Energy‐dispersive spectra patterns for (A) Sr0, (B) Sr50 and (C) Sr100 bioactive glass samples, before and after the solubility studies were performed: Pre, bioactive glass surface before the solubility study was performed; Post, bioactive glass surface after the solubility study was performed and the modified surface layer was removed; Detached, sample of the modified and detached bioactive glass surface layer after the solubility study was performed

Figure 5

Fluorescence emission levels obtained from cell culture media used in the study of the cytotoxic effect of increasing amounts (i.e. 0–320 mg in 3 ml of medium) of Sr0, Sr50 and Sr100 bioactive glass powders. After an incubation period of 72 h in the presence of bioactive glass powders, a resazurin dye‐based assay was used to measure the metabolic activity of a monolayer culture of MSCs. Statistically significant differences with the control samples (p < 0.05) were observed when using amounts of glass powder ≥ 40 mg for Sr0 and Sr50, and when using amounts ≥ 20 mg for Sr100

Figure 6

qRT–PCR analyses of the expression levels for selected genes associated with the osteoblastic differentiation process of BM‐MSCs cultured in standard cell culture medium (Std) and osteogenic cell culture medium (Ost) at 1, 3 and 6 days of exposure to 20 mg Sr0, Sr50 and Sr100 bioactive glass powders: (A) Bmp2; (B) Runx2; (C) Alpl; (D) Col1a1; (E) Spp1; and (F) Bglap. All fold changes were normalized to the values of the expression of each gene in the control samples (i.e. BM‐MSCs cultured in standard cell culture medium and not exposed to any amount of bioactive glass powder)