Evaluation of the cytotoxic effects of sodium hypochlorite on human dental stem cells

Purpose: To investigate the influence of sodium hypochlorite (NaOCl) on human dental stem cell proliferation and differentiation. Method: Dental pulp stem cells (DPSCs), periodontal ligament stem cell (PDLSCs), and gingival mesenchymal stem cells (GMSCs) were treated with NaOCl. Cell viability was evaluated with cellular counting kit-8 (CCK8), and cellular adenosine triphosphate (ATP) levels were analyzed by bromodeoxyuridine (BrdU) incorporation and subsequent flow cytometry. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to detect the expressions of differentiation markers. Results: The viability and ATP levels of all three stem cells types were impaired by NaOCl in a concentrationand time-dependent manners. However, the decrease ATP in GMSCs was less than the other two stem cell population (p < 0.05). NaOCl treatment significantly suppressed the proliferation of dental stem cells (p < 0.05). With regard to differentiation marker expression levels, the decrease in Stro-1 was greater in treatment groups when compared to control on Day 7, while increase in levels of dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and osteocalcin (OC) was smaller (p < 0.05). The expressional changes of Stro-1, DSPP, BSP, and OC were more prominent in DPSMs and PDLSCs than in GMSCs. Conclusion: NaOCl dose-dependently impairs the viability, proliferation and differentiation of dental stem cells. Thus, its toxicity to dental stem cells needs to be considered in clinical application.


INTRODUCTION
Sodium hypochlorite (NaOCl), usually used at concentrations from 0.5 to 5.25 %, is one of the most frequently used irrigants in regenerative endodontic procedures because of its bactericidal ability and excellent tissue dissolution capacity [1].Although it is effective and convenient, side effects have been extensively reported in both clinical cases and basic research studies [2].Previous investigations demonstrated that NaOCl negatively impacts dental stem cell survival and differentiation [3].Moreover, the viability of dental pulp stem cells (DPSCs) was significantly reduced by NaOCl in a dose-dependent manner [4].In vivo studies showed that DPSC differentiation into odontoblast cells was significantly impaired following exposure to 5.25 % NaOCl [5,6].Furthermore, 3 % NaOCl reduced the expression of DSPP by 50 %.
Although the effects of irrigants on APSCs have been extensively studied, the exact impacts of NaOCl on the proliferation and differentiation of DPSCs, PDLSCs, and GMSCs remains largely unknown.The aim of this study was to evaluate the effects of different NaOCl concentrations and treatment times on viability, proliferation, and differentiation of different dental stem cell types.

NaOCl treatment
After 24 h incubation, the cells were treated with different concentrations of NaOCl for various periods.The concentrations of NaOCl was set at 0, 0.005, 0.025, 0.05, and 0.1 mg/mL, and the time courses were 2, 4, 8, and 24 h.Each condition had three replicates.

Cell viability assay
Cell viability was assessed using Cell Counting Kit-8 (CCK-8) assays according to the manufacturer's protocol (Dojindo; Tokyo, Japan). 2 × 10 3 cells were seeded in 96-well plates and incubated at 37 °C for 2, 4, 8, or 24 h in a humidified chamber containing 5 % CO 2 .Then, 10 μL CCK-8 solution was added to each well, and the plates were incubated for 1 h at 37 °C.The absorbance of cells at 450 nm was measured in a microplate reader (Bio-Rad, USA).

Adenosine triphosphate (ATP) assay
ATP assay kits (Beyotime, China) were used to measure ATP levels according to the manufacturer's instruction.Dental stem cells treated with NaOCl were lysed with cell lysis buffer (200 μL), and centrifuged for 5 min at 4° C. Then 100 μL working buffer was added to measure the ATP level via luminometer.

Flow cytometry
DPSCs, PDLSCs, and GMSCs were placed in 96-well plates at 1× 10 5 /well.After 24 h, NaOCl was added to the cells at 0, 0.005, 0.01, 0.025, 0.05, or 0.1 mg/mL.Simultaneously, bromodeoxyuridine (BrdU; Sigma, USA) was added to the cells for 24 h, and flow cytometry was performed to evaluate proliferation.Cells were digested with 0.25 % EDTA-trypsin after three washes with phosphate-buffered saline (PBS), and then fixed with 4 % paraformaldehyde (Huanhai, China) overnight at 4 °C.Next, 0.1 % Triton X-100 (Sigma, USA) was used to permeabilise the cellular membrane at 4 °C for 10 min.After centrifugation, the cells were incubated with 300 µL DNase I at 37 °C for 30 min to denature the DNA.The cells were resuspended in 50 µL staining buffer and incubated with PE-Mouse anti-BrdU (Thermo Fisher Scientific, USA) and isotype control (Thermo Fisher Scientific) at 4 °C for 30 min in the dark.After washing out the staining buffer, the cells were resuspended in PBS and subjected to fluorescence detection.

Quantitative polymerase chain reaction (qPCR)
DPSCs, PDLSCs, and GMSCs were plated in 6well plates at 1×10 5 /well.After 24 h, the cells were treated with 0.025 mg/mL NaOCl for different time points.After three washes with PBS, the medium was replaced with differentiation-induction medium: αMEM plus 10 % FBS supplemented with dexamethasone (10 nM) (Sigma-Aldrich).The untreated groups were taken as control, and all conditions were tested in three replicates.Cells were collected in Trizol at day 0, 3, and 7. cDNA synthesis was performed with PrimeScriptRT reagent kit (Takara, Dalian, China) according to the manufacturer's instructions.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.Primer sequences were listed below:

Western blotting
Cells were washed three times with PBS and lysed in radioimmunoprecipitation assay buffer with protease inhibitors on ice for 10 min.After centrifugation at 12000 g for 10 min at 4 °C, the supernatant was electrophoresed on 12 % sodium dodecyl sulphate gels followed by transfer to polyvinylidene fluoride membranes.The membranes were blocked with 5 % skimmed milk for 2 h at room temperature, and then incubated with primary antibodies at 1:1000 for Stro-1 (Santa Cruz Biotechnology, USA), 1 DSPP (Abcam, UK) and OC (Sigma, USA).Mouse actin was used as the loading control.
After three 5-min washes in PBS-Tween (PBST), the membranes were incubated with secondary antibodies at ilution of 1:2000 for 1 h at room temperature.Protein signals were visualized by chemiluminescence after three 5-min washes in PBST.

Statistical analysis
Data are expressed as mean ± standard deviation.Statistical analyses comparing two groups were performed using Student's t-tests using SPSS 16.0 software (SPSS Inc., Chicago, IL).Comparisons between multiple groups were performed using one-way analysis of variance followed by least significant difference post hoc tests.Differences were considered statistically significant at p < 0.05.

NaOCl impairs dental stem cell viability
After NaOCl treatment, the viabilities of all the three dental stem cell types were significantly lower, especially those treated at high concentration for long incubation times.There was no significant change in DPSC viability at NaOCl concentration of 0.005 mg/mL when the cells were treated for 2 h, whereas viabilities decreased by about 20 and 40 % at 0.01 and 0.05 mg/mL NaOCl, respectively (Figure 1 A).At 24 h, DPSC viabilities decreased by 20, 30, 60, 70, and 80 % at concentrations of 0.005, 0.01, 0.025, 0.05, and 0.1 mg/mL, respectively.With regard to time, cell death rates of DPSCs after 2, 4, 8, and 24 h in 0.01 mg/mL NaOCl reached up to 20, 40, 60, and 70 %, respectively.Similar results were observed for PDLSCs and GMSCs.These findings demonstrate that dental stem cell viability decreases with higher concentrations and longer times of NaOCl treatment.

ATP levels in dental stem cells decreased following NaOCl treatment
As shown in Figure 2, ATP level was also downregulated by NaOCl treatment in concentration-and time-dependent manners (20 % at 0.005 mg/mL vs 50 % at 0.01 mg/mL in 24 h group, p < 0.05; 20 % for 2 h vs 80 % for 24 h at 0.01 mg/mL NaOCl, p < 0.05).The ATP decrease in GMSCs was much smaller than in the other two cell lines, especially at lower NaOCl concentrations.As an example, ATP levels in DPSCs and PDLSCs decreased by nearly 50 % at 0.01 mg/mL of NaOCl at 24 h, when compared to 20 % in GMSCs (p < 0.05).This suggests that GMSCs are more resistant than DPSCs and PDLSCs to NaOCl-induced ATP loss.

NaOCl significantly affected dental stem cell proliferation
To further investigate whether NaOCl attenuate stem cell proliferation, cells were incubated with BrdU and performed flow cytometry after NaOCl treatment.As shown in Figure 3 A, the percentages of proliferative cells was significantly decreased upon 0.1 mg/mL NaOCl treatment after 24 h (p < 0.05).The proliferation of cells treated with other concentrations are shown in Figure 3B.Proliferation of DPSCs, PDLSCs, and GMSCs decreased in an NaOCl concentrationdependent manner.Importantly, NaOCl induced more prominent growth inhibition in DPSCs and PDLSCs than in GMSCs (p < 0.05), indicating that DPSCs and PDLSCs were more vulnerable to NaOCl treatment.

NaOCl impaired dental stem cell differentiation
Next, the expression of Stro-1 was measured, a pluripotent marker for dental stem cells, and the differentiation markers DSPP, BSP, and OC to verify whether NaOCl impacted cell differentiation.As shown in Figure 4, significantly less Stro-1 was expressed in the experimental groups relative to the untreated groups (25 % vs 10 % for DPSCs and PDLSCs at D0, p < 0.05).Similar to its effect on cell proliferation, NaOCl caused more prominent differentiation defect in DPSCs and PDLSCs than in GMSCs (p < 0.05).Similarly, DSPP, BSP, and OC levels were upregulated in a time-dependent manner.In DPSCs and PDLSCs, the downregulation in gene expression was much slower in the experimental groups than the control groups (50 vs 90 % for DPSCs at D7 and 50 vs 90 % for PDLSCs, p < 0.05), the changes of these three markers were faster in GMSCs.Only DSPP significantly decreased in treated cells (70 vs 90 %, p < 0.05), while BSP levels remained unchanged (70 vs 70 %, p > 0.05) and OC levels were enhanced in the experimental groups (70 vs 80 %, p < 0.05), again suggesting less of an impact on GMSC proliferation.NaOCl exert antibacterial function through multiple ways.For instance, NaOCl can reduce surface tension of solution by functioning as a fat solvent [18].It can also degrade dentin collagen by breaking the bonds between carbon atoms, thus disorganizing the protein primary structure [2].Moreover, NaOCl denatures bacterial enzymes by releasing chlorine, a strong oxidant that can bind to the amino groups to generate chloramines, thus interfering with bacterial metabolism [1,18].: 0 mg/mL NaOCl; :0.025 mg/mL NaOCl Despite its antibacterial function, NaOCl can also induce some side effects such as canal tissue damage Furthermore, an additional chelating agent is needed to remove the smear layer [19].Severe NaOCl-induced necrosis has been observed both in preclinical and clinical studies and is most likely to occur in maxillary teeth [20][21][22].NaOCl was also reported to induce acute inflammation in vital tissues [23].Although 6 % NaOCl conditioned with dentin was reported to prevent DPSC differentiation both in vivo and in vitro [5,24], there have been few reports on the effect of NaOCl on PDLSCs and GMSCs, especially with regard to differentiation.
In the present study, the impact of NaOCl on GMSC proliferation and differentiation was investigated.The results showed that NaOCl negatively affected dental mesenchymal stem cell survival and differentiation, and dental stem cell cytotoxicity was directly correlated with the concentration and treatment time.These results are consistent with previous studies, suggesting that cell viability and differentiation decrease along with a higher NaOCl concentration and longer treatment [3].The present research firstly demonstrated that NaOCl affects the proliferation and differentiation of PDLSCs and GMSCs.It is noteworthy that the change in ATP level and differentiation markers expression levels was less significant for GMSCs than the other two stem cells lines, indicating stronger tolerance of GMSCs to differentiation-induced stimuli.This might be attributable to their resistance to inflammatory stimuli.
A study demonstrated that GMSCs isolated from inflamed tissues displayed a normal phenotypic profile and developmental potential similar to cells obtained from healthy gingival tissues [25].However, further investigation is needed to clarify the exact reasons for these observations.Taken together, a deteriorative effect of NaOCl on dental stem cell proliferation and differentiation was revealed in this study.It should therefore be used with caution during endodontic therapy.

CONCLUSION
NaOCl dose-dependently impairs the viability, proliferation and differentiation of dental stem cells.Thus, its toxicity to dental stem cells needs to be considered in clinical application.

Figure 3 :
Figure 3: Flow cytometry data for DPSC, PDLSC, and GMSC proliferation.(A) The proliferation of three stem cell lines was assayed by fluorescence microscope (left) and flow cytometry (right).(B) Detained relative proliferation of the three stem cell lines treated with the 5 NaOCl concentrations in (A); **p < 0.01,*** p < 0.001; △: GMSCs; □: PDLSCs; ▽ : DPSCs DISCUSSION Dental pulp stem cells, gingival mesenchymal stem cells, and periodontal ligament stem cells are similar to bone marrow mesenchymal stem cells in that they possess powerful proliferation and differentiation abilities, making them valuable resources for both endodontic and periodontal regeneration [8].NaOCl was recently recommended as an effective irrigant and is extensively used in dentistry due to its functions in microbial control and tissue dissolution [17].