Influence of calcium and phosphorus release from bioactive glasses on viability and differentiation of dental pulp stem cells

Abstract

The release of ions that can significantly contribute toward cellular response is an important characteristic of bioactive glasses (BG). Here, ionic extracts of three different compositions of BG powders in 60 mol% SiO2, x mol% CaO (x = 28, 32 and 36), x mol% P2O5 (x = 12, 8 and 4) compositional system were utilized to study their effect on the viability, differentiation and mineralization of dental pulp stem cells (DPSCs) in vitro. ICP was applied to detect the exact ionic concentrations released from different composition of BG. DPSCs treated with conditioned media from the glass with 4 mol% P2O5 (BGCM1, media containing 44.01 ± 0.6 mg/L Si, 61.72 ± 0.1 mg/L Ca and 7.57 ± 0.01 mg/L P) were more metabolically active compared to conditioned media from the glass with 8 mol% P2O5 (BGCM2, media with 47.36 ± 0.7 mg/L Si, 57.4 ± 0.1 mg/L Ca and 14.54 ± 0.2 mg/L P), at all times tested, but in all cases the process was slower than the control. Cells exposed to media conditioned by the glass with 12 mol% P2O5 (BGCM3, 40.46 ± 0.5 mg/L Si, 61. 85 ± 0.3 mg/L Ca and 28.43 ± 0.3 mg/L P) responded differently, such that cells showed to be more metabolically active than control at day 3, but then similar to or lower than control at higher time points. Differentiation of DPSCs toward osteogenic lineage in the presence of BGCM was assessed by Alizarin red staining. Cells treated with high phosphate BGCM3 displayed a higher density of red mineralized nodules than cells treated with BGCM1 and BGCM2 after 21 days of culture in non-osteogenic medium. BGCM3 was therefore chosen for gene expression studies. Osteogenic differentiation of DPSCs in the presence and/or absence of BGCM3 or osteogenic supplements were studied by RT-PCR. Overall, the results demonstrated that, in the absence of osteogenic supplements, BGCM3 group showed a significantly higher mRNA expression levels for alkaline phosphatase at day 7, osteopontin and osteonectin at days 7 and 14, and a high level of collagen I at day 14, compared to negative control group (BM−). Overall, the results obtained from BGCM3 group are beneficial for the design and manufacture of scaffolds or particulates with tailored ion release for a range of bone repair applications

Long term effects of bioactive glass particulates on dental pulp stem cells in vitro

“…BG particulates were immersed in simulated [29] body fluid (SBF) in a shaking manner at a concentration of 600 µg/ml, based on our previous study [20] at time points of 0, 1, 3, 5 and 7 days. At the end of each time point the medium was removed and replaced with ethanol to terminate any further surface reactions followed by drying for analysis.…”

“…All experiments were conducted after patients had signed informed consent forms. The isolation procedure was applied as previously described [20]. Briefly, the pulp was separated from the crowns and roots, minced into small pieces, and digested in 3mg/mL collagenase type I (Sigma-Aldrich) and the cell suspensions were then collected.…”

“…The present results of ALP activity indicate that BG particulates could induce early differentiation. tion of stem cells without any additional chemical supplements [20,33,35]. This is mainly due to their degradation capability which liberates ions such as Ca, P, Si, Na, Sr etc [9,11,16,36] depending upon their initial chemical composition of the glass.…”

“…The in vivo environment has fluid flow, which can disperse the ions, but during static in vitro cell culture, high pH can damage cells. For this reason BG of various compositions prepared by sol-gel method are often preconditioned prior to cell culture [20]. Recent studies showed that increasing the phosphate content in the sol-gel glass led to formation of a phosphate glass network, which co-exists with the silicate network [21].…”

“…In our previous study, the effect of BG dissolution products (60 mol% SiO 2 , 28 mol% CaO and 12 mol% P 2 O 5 ) on osteogenic differentiation of dental pulp stem cells, was evaluated [20]. BG dissolution products affected differentiation of DPSCs according to higher mRNA expression levels for: alkaline phosphatase at day 7; osteopontin and osteonectin at days 7 and 14; and a high level of collagen I at day 14, compared to negative control group (basal medium).…”