Biocompatibility of novel premixed putty calcium silicate-based cement on human dental pulp stem cells: An in vitro study

Abstract

Background:

Vital pulp therapy aims to preserve pulp vitality by promoting reparative dentin formation through bioactive materials that support human dental pulp stem cells (hDPSCs) proliferation.

Objectives:

This study compared the biocompatibility of premixed putty calcium-silicate-based cement (C-SbC) with mineral trioxide aggregate (MTA) on hDPSCs in terms of viability, proliferation, and mineralization potential.

Materials and Methods:

hDPSCs were divided and cultured in six different groups: MTA, premixed putty C-SbC (Bio-C Repair) at 1:1, 1:2, and 1:4 dilutions, and DMEM as control. Viability and proliferation were assessed using MTT and trypan blue staining after 24 h. Mineralization potential was evaluated qualitatively using Alizarin Red staining after 21 days. Statistical analysis was performed using the one-way analysis of variance and Bonferroni post hoc test (P < 0.05).

Results:

All the groups showed cell viability with no significant differences in proliferation potential at 24 h. However, cell counts indicated a significant increase in hDPSCs with all concentrations of premixed putty C-SbC compared to MTA (P < 0.05), with the 1:1 concentration (Group 4) showing the highest proliferation. Alizarin red staining revealed more mineral nodules in Group 4, indicating enhanced mineralization.

Conclusion:

Premixed putty C-SbC, particularly at a 1:1 concentration, demonstrated superior biocompatibility and mineralization potential compared to MTA, making it a promising material for vital pulp therapy.

INTRODUCTION

Mineral trioxide aggregate (MTA) is a bioactive tricalcium silicate used in vital pulp therapy for stimulating pulp repair and dentin formation.[1] Despite its biocompatibility for regular applications such as pulp capping, MTA has a long setting times as a drawback. To improve this matter, calcium silicate-based cement (C-SbC) were developed as a premixed putty, incorporating zirconium oxide for enhanced properties.[2,3] Research indicates that C-SbC promotes proliferation of human periodontal ligament stem cells (hPDLSCs). However, studies on its biocompatibility with human Dental Pulp Stem Cells (hDPSCs) are limited. This study aims to investigate the viability and proliferation of hDPSCs in response to C-SbC and MTA.[4,5]

MATERIALS AND METHODS

hDPSCs were obtained from previously approved samples (ethical approval no. 82/ethical approval/FKG UI/IX/2019, protocol no. 070940819). Following serum starvation for 24 h, 5 × 10³ cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Cat# 11885-084) supplemented with 10% exosome-depleted fetal bovine serum (FBS) (System Biosciences, California, USA) and 1% antibiotic to get 70%–80% confluency. The cells were divided into six experimental groups and control group. The experimental groups were treated with DMEM mixed with either MTA or premixed putty C-SbC at various dilutions (1:1, 1:2, 1:4). The control group received standard DMEM.

MTA was mixed according to the manufacturer’s instructions, hardened for 48 h then grounded into powder, sterilized, and diluted. Premixed putty C-SbC, containing various silicates and zirconium oxide, underwent similar processing. The MTT assay was used to analyze hDPSCs proliferation at 24 and 72 h postseeding by measuring absorbance at 570 nm after the addition of MTT reagent.

The proliferation of hDPSCs exposed to bioactive materials and nonexposed to bioactive materials (control group) were analyzed using the Cell Count Trypan Blue assay at 24 h after treatment. Then, at each observation time, 100 L of triple was added to the well and then incubated at 37°C for 12 min. Then, 100 L of phosphate-buffered saline (PBS) is added to neutralize the triple then centrifuge at 300GForce for 5 min. The supernatant then removed, and 1 mL of PBS was added. The cells were counted with trypan blue (10 L cells + 10 L trypan blue). Observation of cell count was done by intra-rater observer under microscope observation (ZEISS, Primovert Inverted Cell Culture Microscope) based on the comparison of the cell counts number with trypan blue with live cells that do not absorb color, and those that do not absorb blue in one visual field area was calculated three times. The percentage live cell count (%) = Live cell count/total cell count × 100% Qualitative Alizarin red test Alizarin red test samples of the control and experimental groups (MTA and premixed putty C-SbC groups) were incubated for 21 days to undergo Alizarin Red staining test (Sigma-Aldrich, Massachusetts, United States). This test was conducted as a qualitative analysis of the mineralization nodules released formation of hDPSCs that induced by the additional of biomaterials as supplemented conditioned media. In this test, differentiation media group with additional of 10 mM β glycerophosphate, 50 μg/ml ascorbic acid, and 10-8 M dexamethasone in the culture media was added as positive control. Statistical analysis: the quantitative data were statistically analyzed using the SPSS Statistics for Windows, version 22.0 (SPSS Inc., Chicago, Ill., USA). The number of proliferations of the control group and the test group was tested for normality and homogeneity. One-way analysis of variance (ANOVA) was performed, followed by a post hoc test using the Bonferroni. The Alizarin red qualitatively assessed the mineralization nodules formed by hDPSCs over 21 days with MTA and C-SbC. Differentiation media supplemented with β-glycerophosphate, ascorbic acid, and dexamethasone served as a positive control. Quantitative data were analyzed using the SPSS (version 22) through one-way ANOVA and Bonferroni post hoc tests, with significance set at P < 0.05.

RESULTS

The intra-rater reliability test, with an Interclass Correlation Coefficient of r = 0.995, confirmed reliable data on hDPSCs proliferation. The MTT assay showed no significant differences in hDPSCs viability among premixed putty C-SbC, MTA, and the control group at 24 h (P > 0.05). Normality testing confirmed a normal distribution (Shapiro–Wilk; P > 0.05), allowing for one-way ANOVA analysis, which revealed significant differences in hDPSCs proliferation among groups (P < 0.05), supported by the Cell Count Trypan Blue assay.

The post hoc test revealed significant differences in the live cell populations of hDPSCs at 24 h, with Group 1 differing from Group 4 and Group 3 differing from Group 6 (P < 0.05). However, there were no significant differences between Group 2 and Group 5 (P > 0.05). Qualitative analysis of hDPSCs using the trypan blue assay at 24 h showed that viable cells excluded the dye, while non-viable cells absorbed it. The highest viability was observed in the Pre-mixed Putty C-SbC 1:1 group, the lowest in the MTA 1:4 group, and intermediate levels in the control group [Figure 1a (a-c)].

Figure 1.

(a) Qualitative results of human dental pulp stem cells analyzed using the trypan blue cell count assay at 24 h of observation. Live cells did not absorb the dye, while dead cells absorbed the dye. (a) The highest cell count was observed in the Pre-mixed Putty C-SbC 1:1 group, (b) the lowest in the mineral trioxide aggregate 1:4 group, (c) and an intermediate result in the control group. (b) Alizarin red test results: Group 1 (A), Group 2 (B), Group 3 (C), Group 4 (D), Group 5 (E), Group 6 (F), control DMEM (G), and control differentiation medium (H)

Based on qualitative result of Alizarin test, it was showed that the highest number of nodule minerals was seen in Group 4 [Figure 1b-A], followed by Group 5 [Figure 1b-B], and the smallest number of nodule minerals in the Group 6 [Figure 1b-C]. While in the MTA test group, the highest number of mineral nodules was in Group 3 [Figure 1b-F] compared to Group 2 [Figure 1b-E] and Group 1 [Figure 1b-D]. This qualitative result of Alizarin red test was in accordance with the result of live cell population [Figure 1].

DISCUSSION

MTA was the first bioactive material in endodontics, promoting pulp tissue healing and reparative dentin formation.[1,2,3] However, MTA has drawbacks, including a long-setting time and potential tooth discoloration.[6,7] To address these issues, modified forms of MTA have emerged, including the recently developed premixed putty C-SbC. This ready-to-use material eliminates the need for powder and liquid manipulation, offering better consistency and ease of application.[6,8] In addition, C-SbC serves as a microbial barrier, stimulates tissue healing, and does not cause discoloration.[1,4] One of the differences in the composition of this premixed putty C-SbC compared to MTA is the addition of zirconium oxide to replace bismuth oxide as a radiopacifier. Zirconium oxide nanoparticles are the materials that can induce osteoblast cells proliferation. MTA radiopacifies in the form of bismuth oxide is a material that is more toxic than zirconium oxide.[6,9] This is related to reactive oxygen species (ROS) which can be triggered by a toxic substance so that it can change the biochemical balance of cells by producing irreversible oxidation species. At elevated levels, ROS can cause carcinogenesis, mitochondrial dysfunction, and cell death or apoptosis.[10,11]

This study aimed to evaluate the potential for increasing proliferation of hDPSCs by evaluating number of live cells population hDPSCs that culture with MTA and premixed putty C-SbC at concentrations of 1:1, 1:2, and 1:4 (divided into six groups). In the in vitro method, the extracts of materials without dilution and with dilution are used according to the recommendations of ISO 10993-5:2009 and refer to previous studies.[4,5,6] This is to adjust to the conditions when the material is applied to the tissue, because the compound can be dissolved continuously due to the presence of extracellular fluid, so that the local concentration is getting smaller. Therefore, this study was done with different dilutions, 1:1 as the same condition from manufacturer (3.5 mg/3.5 ml), 1:2 (3.5 mg/7 ml) and 1:4 (3.5 mg/3.5 ml). This was done to avoid the risk of cell death in dilution conditions that can cause cell toxicity. The potential in inducing proliferation was evaluated by two methods in this study, by MTT assay from relative formazan formation and Cell Count using trypan blue that analyzed the live cell population of hDPSCs.[12,13]

MTT-assay test is used to measure the cellular metabolic activity which is an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric test based on the reduction of the yellow tetrazolium salt (MTT-Assay) to purple formazan crystals because of metabolic active cells that are still alive. The results of the MTT assay test showed that there was no significant difference in the potential for increasing proliferation of hDPSCs in MTA and premixed Putty C-SbC with various concentrations in 24-h observation compared to control (P < 0.05) [Figure 2]. These results are in line with a previous study by Ghilotti et al.[4] which compared MTA and modified MTA or premixed putty C-SbC using hDPSCs cells.[4,14] The MTT Assay results obtained were not significantly different. Based on ISO 10993-5:2009, it is said that the percentage is <70% compared to blank cells, so the material has a cytotoxic potential. Therefore, based on these results, it can be proven that all the biomaterials added to the culture media in this study were non-toxic to cells [Figure 2]. There were significant differences in the potential for increasing proliferation of hDPSCs on various culture media within 24 h [Figures 1 and 2]. The highest mean live cell population in 24-h observation was in Group 4 [Figures 1 and 2]. This shows that modified MTA has the potential to support the potential in inducing proliferation of hDPSCs, especially at 24 h, which is the time for cells to cycle through the G1-S-G2-M phase. After 24 h, the cell has gone through its cycle and will determine its cell fate to the cell cycle, undergo apoptosis, or continue to differentiate.[12,15]

Figure 2.

(a) Mean value of relative viability rate human dental pulp stem cells (hDPSCs) cultured in premixed Putty calcium-silicate-based cement from formazan relative formation (MTT Assay) in 24 h and 72 h of observation. (b) Live cell population (%) of hDPSCs in six experimental culture media and a control media groups after 24 h of observation. The highest % live cell population was in Premixed Putty mineral trioxide aggregate 1:1 group (99.56%). hDPSCs: Human dental pulp stem cells, C-SbC: Calcium-silicate-based cement, MTT: Methyl Thiazolyl Tetrazolium, MTA: Mineral trioxide aggregate

Post hoc test comparison of MTA and premixed putty MTA with various concentrations of dilution within 24 h showed a significant difference in Group 4, Group 5, and Group 6 (P < 0.05). The average potential for increased proliferation potential of premixed putty MTA was higher than that of MTA in all concentration dilution groups.[13,14]

Ghilotti et al.[4] revealed that premixed putty MTA (Bio-C Repair^®, Angelus, Brazil) was mostly composed of carbon (34.81%) and oxygen (34.51%), with a lower concentration of calcium compared to the other biomaterials. On the contrary, Camilleri et al.[2] stated that the amount of calcium related to the bioactivity of the material. This is not in line with the result of the study that showed superior proliferation potential ability of premixed putty C-SbC compared to white MTA, this also showed that even premixed putty of C-SbC has lower content of calcium despite has better bioactivity compared to white MTA. On the other hand, study by Sanaee et al.[13] also proved that the amount of carbon in MTA was in correlation with its physical properties, this is related to the formation of multi walled carbon nanotubules that can induce the compressive strength of Portland cement as the basic content of MTA or its modification.[10,15,16]

This study was limited to in vitro assessments using a single cell type (hDPSCs) and did not evaluate the long-term behavior of the materials under the in vivo conditions. The assessment of proliferation was limited to a 24-h period, which may not adequately reflect the long-term effects of the materials on cell behavior. Future studies incorporating animal models and clinical trials are necessary to confirm the in vivo efficacy and safety of premixed putty C-SbC in vital pulp therapy.

In the era of C-SbC or known as premixed putty C-SbC or bioceramic cement, lower concentration of calcium was replaced by higher amount of zirconium oxide. Silva et al.[6] reported that modified MTA or calcium silicate base cement containing higher zirconium oxide can induce cell proliferation.[8,17] In line with the research of Chen et al.[14] that proved zirconium ion can also increase cell proliferation of human osteoblast cells by the bone morphogenetic protein (BMP) pathway signals via receptor-regulated mothers against decapentaplegic homologs (SMAD). The result of qualitative Alizarin red test also proved that the premixed putty C-SbC groups has more mineral nodules compare to the MTA groups, thought that higher released of zirconium ion can increase mineralization potential of hDPSCs [Figure 2a]. Other study also proved that better viability, cell migration activity, and osteogenic potential of hPDLSCs of Bio-C-sealer with the higher amount of Zirconium oxide compared to other sealer.[14,18] This result of this study was in line with the previous study that proven the superior biocompatibility of premixed putty C-SbC compared to MTA.

CONCLUSION

Premixed putty C-SbC has superior potential to induce proliferation and mineralization potential of hDPSCs compared to MTA. This study proved that C-SbC premixed putty 1:1 (Group 4) dilution group has the most superior biocompatibility than other groups. Further study needs to be conducted by in vivo study.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was privately funded by the investigator.