Zinc bioglasses regulate mineralization in human dental pulp stem cells

Abstract

Objective

A promising strategy in regenerative endodontics is the combination of human dental pulp stem cells (hDPSCs) with an appropriate biomaterial substrate. The effects of zinc and zinc containing bioactive glasses (ZnBGs) on hDPSCs have been characterized in this study.

Methods

ZnBGs were designed and produced. Then the odontogenic differentiation and mineralization potential of hDPSCs upon ZnBGs treatment were investigated.

Results

Free Zn ions (0–5 ppm) enhanced proliferation and alkaline phosphatase (ALP) activity of hDPSCs. Further, ZnBGs conditioned medium (ZnBG-CM) increased the production and secretion of odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1). In addition, we identified that mRNA expression of the osteogenic markers RUNX2, OCN, BSP, BMP-2, MEPE and ON was increased following treatment with ZnBG-CM. Long term treatment with ZnBG-CM increases the formation rate of mineralized nodules (similar to hydroxyapatite, Ca:P = 1.6), as confirmed by scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM-EDX). Lastly, the administration of ZnBG-CM induces VEGF expression.

Significance

These findings implicate that ZnBG would be beneficial in regenerative endodontics and could influence the way present Zn containing clinical products are used.

Introduction

The high incidence of tooth decay and tooth loss combined with the limited regeneration capacities of dental tissues necessitates the development of novel strategies for dental practices . Human dental pulp stem cells (hDPSCs) are a mesenchymal stem cell (MSC) population . Analogous to MSCs derived from bone marrow (BMSCs), hDPSCs can form alizarin red-positive mineralized deposits, physiologically similar to hydroxyapatite (HA) . Thus, experiments have been conducted to evaluate the value of hDPSCs in the repair of bone defects . However, numerous studies have demonstrated that hDPSCs have a greater ability to differentiate into odontoblasts compared to BMSCs . Co-transplanting cultured hDSPCs with HA/TCP particles, into immunocompromized mice, led to the vascularisation of a fibrous pulp tissue surrounded by a layer of odontoblast-like cells, cells had extended tubular structures that resembling a dentin-like structure . In contrast, a lamellar bone structure with associated adipose tissue was formed by human BMSCs under the same experimental condition . These findings clearly demonstrate distinct tissue regeneration capacities of hDPSCs and BMSCs, which appear to have maintained a level of epigenetic memory of their tissue origin . Somewhat unsurprisingly then, hDPSCs is the preferred derivation of cells for dental tissue engineering.

The fundamental concept in hard tissue engineering lies in combining a scaffold with living cells to repair damaged tissue . Scaffolds provide a suitable environment for cell attachment, proliferation, differentiation and to promote bio-mineralization . A significant challenge regarding scaffold usage is the choice of a suitable material. A broad range of materials, natural or synthetic polymers and inorganics, have been designed as scaffolds for dentin-pulp regeneration . Bioactive glasses (BGs) are a promising choice for repair and reconstruction of hard tissues, such as bone and dentin . BGs can form hydroxyapatite (HA) quickly, is biocompatible and can deliver ‘therapeutic ions’ .

In dental practice, the use of zinc (Zn) is mostly favored owing to its bacteriostatic and cariostatic properties . For example, Zn oxide eugenol (ZOE), Zn oxyphosphate (ZOP), and Zn polycarboxylate (ZPC) are widely used. However, despite the clinical advantages, the dentinogenic benefits of Zn have yet to be determined. Accordingly, we have focused on Zn and Zn containing BGs (ZnBGs) to characterize their effects on hDPSCs.

Materials and methods

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).

Fig 1
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.

Fig. 2
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.

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.

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.

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).

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.

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.

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.

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 .

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.

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.

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.

Materials and methods

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).

Fig 1
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.

Fig. 2
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.

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.

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.

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).

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.

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.

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.

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 .

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.

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.

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.

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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Zinc bioglasses regulate mineralization in human dental pulp stem cells
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