2.1

Bioactive glass synthesis and characterization

ZnBGs (compositions are shown in Fig. 2 A) were synthesized by the melt-quench route . Glass frits were grounded and sieved to powders ≤38 μm. The particle size and amorphous nature was confirmed by master particle size analyzer and X-ray diffraction (XRD). ZnBGs powders were characterized as described previously . Briefly, 75 mg of BGs powder (≤38 μm) was added to 50 ml Tris buffer and incubated on an orbital shaker (60 rpm at 37 °C). Post shaking, the pH values were recorded. The solution was filtered, diluted by a factor of ten in distilled H ₂ O and acidified with 69% nitric acid (final concentration 2%) prior to determining the elemental concentrations of calcium (Ca), phosphorus (P), silica (Si), and zinc (Zn) using an inductively coupled plasma-optical emission spectrometer (ICP-OES). The residual powders were dried overnight and analyzed to evaluate the extent of apatite formation using XRD, and Fourier transform infrared spectroscopy (FTIR).

Effects of free Zn ion on hDPSCs. (A): MTT activity of hDPSCs treated with Zn (0–20 ppm). Data is presented as fold change upon comparison with Zn 0 ppm group at day 1. (B): ALP activity of hDPSCs following Zn (0–20 ppm) treatment. The ALP activity was measured in each group and normalized to cell number. (C): Crystal violet staining of hDPSCs after 10 days exposure to Zn (0–20 ppm). Scale bar = 100 μm. All the data are expressed as mean ± SD from three independent experiments. * indicates significance value of p < 0.05 when compared to Zn 0 ppm group.

Characterization of ZnBGs. (A): ZnBG compositions, the network connectivity for all the ZnBGs are fixed at 2.1. (B): XRD patterns of all unreacted BGs confirm amorphous nature. (C)–(F): XRD pattern of ZnBGs after Tris buffer dissolution from 0 to 168 h, two peaks at 26 and 32 2θ ^o indicate apatite formation. (G)–(J): ICP-OES analysis of ZnBGs after immersion in Tris buffer for 168 h. All ionic (P, Ca, Si, and Zn) data in ppm was plotted as a function of time.

2.2

Cell culture

Human dental pulp stem cells (hDPSCs, Lonza) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 10% fetal calf serum (FCS) and antibiotics (10 U/L penicillin and 100 mg/L streptomycin) in a humidified atmosphere with 10% CO ₂ at 37 °C.

Free ionic Zn (0.5, 1, 5, 10 and 20 ppm) was added as ZnCl ₂ . ZnBG conditioned medium (ZnBG-CM) was prepared by immersing 75 mg of glass powders in 50 ml DMEM for set periods of time. Before being added to the cells, ZnBG-CM was filter-sterilized, serum and antibiotics were added and equilibrated to 37 °C, 10% CO ₂ in an incubator.

Cells between passages 3–5 were used in all experiments. For cell proliferation, MTT and ALP activity assays, 1000 cells/well were seeded in 96-well plates and cultured for 1, 4, 7 and 10 days. 1 × 10 ⁵ cells/dish and 2 × 10 ⁴ cells/well were placed in 10 cm dishes and 6-well plate for Western blot and qPCR studies, respectively. 5 × 10 ³ cells were seeded onto sterilized coverslips and placed in 12-well plates for immunocytochemistry. 24-wells plate with 5 × 10 ³ cells/well was used for the mineralization study.

2.3

MTT activity assay

The MTT activity assay reflects mitochondrial activity—an indication of viable cells. After rinsing twice with PBS, 50 μl of MTT solution (5 mg/ml MTT in PBS) was added to cell culture and incubated for 4 h at 37 °C. After this, medium was removed and the formazan was solubilized in 100 μl dimethyl sulfoxide (DMSO). The absorbance of the solubilized formazan supernatant was measured at 560 nm.

2.4

Quantitative assay of ALP activity

Alkaline phosphatase (ALP) activity was measured by adding 100 μl of ALP reaction solution (20 mg 4-nitropheyl-phosphate disodium salt hexahydrate tablet dissolved in 8 ml Tris buffer solution (pH = 9.5) containing 15 μl of 2 M MgCl ₂ ) to cell lysate, incubated in the dark at 37 °C for 1 h. Afterwards, the reaction was stopped by adding 50 μl of 1 M NaOH. The absorbance of the supernatant was measured at 405 nm and ALP activity was calculated according to a standard curve (Fig. S1).

2.5

Crystal violet staining

Cells were fixed with 10% neutral buffered formalin for 15 min, rinsed twice with PBS and stained in 0.5% (w/v) crystal violet for 15 min at room temperature. Before visualizing, the unbound stains were removed by extensive washing in PBS.

2.6

Western blot analysis

Cell pellets were re-suspended in RIPA buffer (containing 1% Triton X-100 and 1% cocktail proteinase inhibitors) and the protein content measured using DC™ Protein Assay. Intracellular expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1) and vascular endothelial growth factor (VEGF) was evaluated by Western blotting using indicated antibodies (Tables S1) on a PVDF membrane. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Cell culture supernatant was also collected and analyzed for DSPP and DMP-1 secretion, Coomassie blue was used to visualize sample separation. The band densities were quantified by ImageJ software.

2.7

Immunocytochemistry

4% paraformaldehyde in PBS was used to fix the cells. Subsequently, cells were permeabilized with 0.1% Triton X-100 for 10 min, and incubated with 10% goat serum for 30 min to block nonspecific binding. Following 1 h incubation in primary antibody (Table S1) at room temperature, cells were washed and incubated with secondary antibody for 1 h and counter stained with DAPI. Finally, the cells were mounted on slides and viewed under confocal microscopy (LSM710, Zeiss). The images were then analyzed with ImageJ software.

2.8

Quantitative real-time polymerase chain reaction-qPCR

Message RNA (mRNA) was isolated from cells with Dynabeads ^® (Thermo Fisher Scientific) and reverse-transcribed into cDNA using Transcriptor cDNA Synthesis Kit (Roche). The forward and reverse primers used for PCR were designed by Roche universal probe library according to the complementary DNA sequences available in GenBank (Table S2). qPCR amplifications were performed on LightCycler 480 qPCR system (Roche) using LightCycler ^® 480 SYBR Green I Master. For each PCR reaction, 2 μl of 10 fold-diluted cDNA was used for amplification of the target genes. The thermal cycling conditions were as follows: 95 °C for 5 min and 55 cycles of 95 °C for 10 s, 60 °C for 6 s, 72 °C for 6 s, 76 °C for 1 s. Relative expression levels of runt-related transcription factor 2 (RUNX2), osteonectin (ON), osteocalcin (OCN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), and bone morphogenetic protein 2 (BMP-2) were determined with reference to GAPDH .

2.9

Alizarin Red S staining

After 3–5 weeks culture, cells were fixed in 4% glutaraldehyde for 30 min and stained with 2% Alizarin Red S (Sigma–Aldrich) for 1 h at room temperature. After three washes in dH ₂ O to remove unbound stain, samples were air-dried and photographed. For quantification, the bound stain was extracted with 10% cetylpyridinum chloride, and the absorbance of solution was measured at 560 nm.

2.10

Scanning electron microscope (SEM)

Cells were fixed in 2.5% glutaraldehyde for 1 h and dehydrated in graded ethanol (50%, 70%, 80%, 90%, 95%, and 100%), followed by critical-point drying with hexamethyldisilazane. The samples were coated with carbonate and viewed using SEM with an energy-dispersive X-ray (EDX) detector operated from 10–20 kV to scan and analyze mineralized nodule formation and composition.

2.11

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5. All data were collected from three independent experiments and presented as mean ± standard deviation (SD). Differences were considered significant at p < 0.05, determined using one-way ANOVA with Bonferroni’s post hoc multiple comparison test.

2.1

Bioactive glass synthesis and characterization

ZnBGs (compositions are shown in Fig. 2 A) were synthesized by the melt-quench route . Glass frits were grounded and sieved to powders ≤38 μm. The particle size and amorphous nature was confirmed by master particle size analyzer and X-ray diffraction (XRD). ZnBGs powders were characterized as described previously . Briefly, 75 mg of BGs powder (≤38 μm) was added to 50 ml Tris buffer and incubated on an orbital shaker (60 rpm at 37 °C). Post shaking, the pH values were recorded. The solution was filtered, diluted by a factor of ten in distilled H ₂ O and acidified with 69% nitric acid (final concentration 2%) prior to determining the elemental concentrations of calcium (Ca), phosphorus (P), silica (Si), and zinc (Zn) using an inductively coupled plasma-optical emission spectrometer (ICP-OES). The residual powders were dried overnight and analyzed to evaluate the extent of apatite formation using XRD, and Fourier transform infrared spectroscopy (FTIR).

Effects of free Zn ion on hDPSCs. (A): MTT activity of hDPSCs treated with Zn (0–20 ppm). Data is presented as fold change upon comparison with Zn 0 ppm group at day 1. (B): ALP activity of hDPSCs following Zn (0–20 ppm) treatment. The ALP activity was measured in each group and normalized to cell number. (C): Crystal violet staining of hDPSCs after 10 days exposure to Zn (0–20 ppm). Scale bar = 100 μm. All the data are expressed as mean ± SD from three independent experiments. * indicates significance value of p < 0.05 when compared to Zn 0 ppm group.

Characterization of ZnBGs. (A): ZnBG compositions, the network connectivity for all the ZnBGs are fixed at 2.1. (B): XRD patterns of all unreacted BGs confirm amorphous nature. (C)–(F): XRD pattern of ZnBGs after Tris buffer dissolution from 0 to 168 h, two peaks at 26 and 32 2θ ^o indicate apatite formation. (G)–(J): ICP-OES analysis of ZnBGs after immersion in Tris buffer for 168 h. All ionic (P, Ca, Si, and Zn) data in ppm was plotted as a function of time.

2.2

Cell culture

Human dental pulp stem cells (hDPSCs, Lonza) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 10% fetal calf serum (FCS) and antibiotics (10 U/L penicillin and 100 mg/L streptomycin) in a humidified atmosphere with 10% CO ₂ at 37 °C.

Free ionic Zn (0.5, 1, 5, 10 and 20 ppm) was added as ZnCl ₂ . ZnBG conditioned medium (ZnBG-CM) was prepared by immersing 75 mg of glass powders in 50 ml DMEM for set periods of time. Before being added to the cells, ZnBG-CM was filter-sterilized, serum and antibiotics were added and equilibrated to 37 °C, 10% CO ₂ in an incubator.

Cells between passages 3–5 were used in all experiments. For cell proliferation, MTT and ALP activity assays, 1000 cells/well were seeded in 96-well plates and cultured for 1, 4, 7 and 10 days. 1 × 10 ⁵ cells/dish and 2 × 10 ⁴ cells/well were placed in 10 cm dishes and 6-well plate for Western blot and qPCR studies, respectively. 5 × 10 ³ cells were seeded onto sterilized coverslips and placed in 12-well plates for immunocytochemistry. 24-wells plate with 5 × 10 ³ cells/well was used for the mineralization study.

2.3

MTT activity assay

The MTT activity assay reflects mitochondrial activity—an indication of viable cells. After rinsing twice with PBS, 50 μl of MTT solution (5 mg/ml MTT in PBS) was added to cell culture and incubated for 4 h at 37 °C. After this, medium was removed and the formazan was solubilized in 100 μl dimethyl sulfoxide (DMSO). The absorbance of the solubilized formazan supernatant was measured at 560 nm.

2.4

Quantitative assay of ALP activity

Alkaline phosphatase (ALP) activity was measured by adding 100 μl of ALP reaction solution (20 mg 4-nitropheyl-phosphate disodium salt hexahydrate tablet dissolved in 8 ml Tris buffer solution (pH = 9.5) containing 15 μl of 2 M MgCl ₂ ) to cell lysate, incubated in the dark at 37 °C for 1 h. Afterwards, the reaction was stopped by adding 50 μl of 1 M NaOH. The absorbance of the supernatant was measured at 405 nm and ALP activity was calculated according to a standard curve (Fig. S1).

2.5

Crystal violet staining

Cells were fixed with 10% neutral buffered formalin for 15 min, rinsed twice with PBS and stained in 0.5% (w/v) crystal violet for 15 min at room temperature. Before visualizing, the unbound stains were removed by extensive washing in PBS.

2.6

Western blot analysis

Cell pellets were re-suspended in RIPA buffer (containing 1% Triton X-100 and 1% cocktail proteinase inhibitors) and the protein content measured using DC™ Protein Assay. Intracellular expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1) and vascular endothelial growth factor (VEGF) was evaluated by Western blotting using indicated antibodies (Tables S1) on a PVDF membrane. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Cell culture supernatant was also collected and analyzed for DSPP and DMP-1 secretion, Coomassie blue was used to visualize sample separation. The band densities were quantified by ImageJ software.

2.7

Immunocytochemistry

4% paraformaldehyde in PBS was used to fix the cells. Subsequently, cells were permeabilized with 0.1% Triton X-100 for 10 min, and incubated with 10% goat serum for 30 min to block nonspecific binding. Following 1 h incubation in primary antibody (Table S1) at room temperature, cells were washed and incubated with secondary antibody for 1 h and counter stained with DAPI. Finally, the cells were mounted on slides and viewed under confocal microscopy (LSM710, Zeiss). The images were then analyzed with ImageJ software.

2.8

Quantitative real-time polymerase chain reaction-qPCR

Message RNA (mRNA) was isolated from cells with Dynabeads ^® (Thermo Fisher Scientific) and reverse-transcribed into cDNA using Transcriptor cDNA Synthesis Kit (Roche). The forward and reverse primers used for PCR were designed by Roche universal probe library according to the complementary DNA sequences available in GenBank (Table S2). qPCR amplifications were performed on LightCycler 480 qPCR system (Roche) using LightCycler ^® 480 SYBR Green I Master. For each PCR reaction, 2 μl of 10 fold-diluted cDNA was used for amplification of the target genes. The thermal cycling conditions were as follows: 95 °C for 5 min and 55 cycles of 95 °C for 10 s, 60 °C for 6 s, 72 °C for 6 s, 76 °C for 1 s. Relative expression levels of runt-related transcription factor 2 (RUNX2), osteonectin (ON), osteocalcin (OCN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), and bone morphogenetic protein 2 (BMP-2) were determined with reference to GAPDH .

2.9

Alizarin Red S staining

After 3–5 weeks culture, cells were fixed in 4% glutaraldehyde for 30 min and stained with 2% Alizarin Red S (Sigma–Aldrich) for 1 h at room temperature. After three washes in dH ₂ O to remove unbound stain, samples were air-dried and photographed. For quantification, the bound stain was extracted with 10% cetylpyridinum chloride, and the absorbance of solution was measured at 560 nm.

2.10

Scanning electron microscope (SEM)

Cells were fixed in 2.5% glutaraldehyde for 1 h and dehydrated in graded ethanol (50%, 70%, 80%, 90%, 95%, and 100%), followed by critical-point drying with hexamethyldisilazane. The samples were coated with carbonate and viewed using SEM with an energy-dispersive X-ray (EDX) detector operated from 10–20 kV to scan and analyze mineralized nodule formation and composition.

2.11

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5. All data were collected from three independent experiments and presented as mean ± standard deviation (SD). Differences were considered significant at p < 0.05, determined using one-way ANOVA with Bonferroni’s post hoc multiple comparison test.