Abstract
Background/purpose
Various materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells.
Materials and methods
PD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain.
Results
The viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD.
Conclusion
PD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.
Keywords: Differentiation, Human dental pulp stem cells, Odontogenic differentiation, Polydatin
Introduction
Human dental pulp has a number of functions including dentin production, neural functions, and defense mechanisms.1 In response to various insults, pulp reacts by producing an inflammatory reaction. In particular, injuries resulting from bacterial infection of the pulp, if left untreated, will progress into an irreversible pulpal inflammation with eventual pulp necrosis.2,3
Traditionally, management of pulpal disease consisted of removal of the pulp or remnants thereof followed by cleaning the root canal and filling it with a biocompatible material. This process produced a predictable high success rate.4,5 However, certain situations, such as a necrotic pulp in an immature permanent tooth, require different management to stimulate continued root development, root wall thickening, and apical narrowing prior to root canal filling.6
In addition, irreversibly inflamed pulps have been traditionally treated with root canal therapy. Although direct pulp capping has been considered for a long time, earlier outcomes when calcium hydroxide (Ca (OH)2) was used were not encouraging.7 However, more recent studies utilizing calcium silicates have shown promising results when used on asymptomatic teeth.8
The biological principles involved in “regenerative endodontics” and “direct pulp capping” are essentially the same. Both procedures rely on recruiting undifferentiated mesenchymal stem cells to undergo differentiation into cells involved in the regeneration of lost dentin pulp complex tissues.9, 10, 11
Since formed dentin bridge is a primary indicator for successful pulp therapy; in vitro studies with stem cells are carried out to evaluate the ability of potential materials or therapeutic agents to induce stem cell differentiation into odontoblasts or odontoblast-like cells.12, 13, 14 Bone and dentine formation processes are related at the molecular level with similar non-collagenous proteins that direct the formation of calcified tissue. Bone markers such as osteocalcin (OC), osteopontin (OP), and alkaline phosphatase (ALP) can also indicate an odontogenic process as well. On the other hand, specific odontogenic markers that can be used to detect the differentiation of stem cells to odontoblasts or odontoblast-like cells include dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1).15,16
Despite significant success of regenerative endodontics procedures, certain important shortcomings are still evident, for example, the tissues produced in “regenerative endodontics” are not true pulp tissues, but rather a periodontal ligament-like, bone-like, or cementum-like tissues.17,18 In addition, formed dentin bridge at the site of vital pulp therapy procedures often contains atubular dentin, tunnel defects, and soft tissue inclusions.19, 20, 21 Furthermore, inflammation is often present, at least in the earlier stages, of vital pulp therapy using pulp capping materials.22,23
A material capable of promoting odontoblastic differentiation while reducing inflammatory process might prove advantageous in dental pulp regeneration/repair procedures. Polydatin (PD) a precursor of Resveratrol (RSV) is a traditional Chineses medicine with known anti-inflammatory and osteogenic properties.24,25 It is a naturally occurring product that was first extracted from the Japanese knotweed (Fallopia japonica) but is also present in other plants such as grapes, cocoa, and peanuts.24,25 PD (Fig. 1) has a molecular weight of 390.4 g/mol.24,26, 27, 28 Although PD and RSV have similar chemical structures and share many biological effects, PD has been reported to be more potent than RSV.27
Figure 1.
The chemical structure of Polydatin.28
PD has been reported to promote osteogenic differentiation of dental bud stem cells (DBSCs), while RSV has been shown to upregulate odontogenic and osteogenic messenger RNAs such as ALP, OC, DMP-1, and DSPP as well as mineralized nodule formation by hDPSCs.27,29
On the other hand, RSV had a protective effect against oxidative stress, while PD modulated the production of inflammatory cytokines associated with pulp capping materials application on bone marrow mesenchymal cells (BMSCs).30,31
To date, no study has evaluated the effect of PD on human dental pulp stem cells, therefore the aim of this study was to evaluate the effect of PD on the viability and differentiation of human dental pulp stem cells.
Materials and methods
Ethical approval
The research was approved by the Institutional Review Board (IRB) at King Saud University Medical City (KSUMC) with (IRB Project No. E−21-5997).
Cells and cell culture condition
Cell expansion
Human dental pulp stem cells (hDPSCs) were purchased from Axol Bioscience LTD, (Catalog number ax3901, Axol, Cambridge, UK). Cells were expanded and cultured in alpha-modified minimum essential medium (α-MEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% penicillin/streptomycin, and 1% MEM Non-Essential Amino Acids Solution (NEAA), hereafter referred to as supplemented α-MEM. All cells were cultured at 37 °C at 5% CO2 and 95% humidity. After reaching 90% confluency cells were transferred to 96, 24, and 12 well plates with the supplemented α-MEM in preparation for the consequent experiments. Passages 3–5 of hDPSCs were used in the experiments.
Material preparation
Solution preparation
Polydatin
Polydatin (PD) (purchased from Shandong Zhi Shang Chemical LTD, Jinan, China, CAS 27208-80-6), was dissolved in 0.1% Dimethyl sulfoxide (DMSO) according to the manufacturer's instructions. PD effect on viability of hDPSCs was performed using 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM compared to the control group with DMSO.27 PD was re-added with each medium change.
Osteogenic differentiation medium
Osteogenic differentiation medium (OM) consists of (α-MEM) supplemented with 10% FBS, 1% Pen-strep, 50 μg/mL l-ascorbic acid (Wako Chemicals, Neuss, Germany), 10 nmol/L calcitriol (1α,25-dihydroxy vitamin D3 (Sigma–Aldrich, Burlington, MA, USA), 10 mmol/L β-glycerophosphate (Sigma–Aldrich), and 10 nmol/L dexamethasone (Sigma–Aldrich).
Cellular viability of human dental pulp stem cells exposed to polydatin
Alamarblue™ assay
The effect of PD on the viability of hDPSCs was evaluated at 5 concentrations 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM on days 1, 3, and 7 using the AB assay (Thermo Fisher Scientific) as prescribed previously.32,33 Cells were seeded in 96-well plates at a concentration of 5 × 103 cells/well. At the end of each time point, 10% AlamarBlue™ reagent (Bio-Rad Inc., Hercules, CA, USA) was added to each well. After 1 h of incubation, fluorescence was measured at an excitation and emission wavelength of 530 nm and 590 nm, respectively, using a fluorescence reader SpectraMax® M5/M5e Multimode Plate Reader (Molecular Devices, San Jose, CA, USA). Data was collected using the SoftMax® Pro 6 Microplate Data Acquisition and Analysis Software (Molecular Devices). The experiment was quadrupled in triplicates.34
Evaluating odontogenesis
Alkaline phosphatase activity and stain
Dental pulp stem cells were seeded in a 24 well-plate in α-MEM with a density of 1 × 105 cells/well for alkaline phosphatase (ALP) activity and 12 well plate with a density of 2 × 105 cells/well For ALP staining. After cells reached confluency of 70–80%, PD was added to each group with α-MEM for 24 h. The next day, cells were cultured with OM supplemented with 0.01 μM, 0.1 μM, or 1 μM PD. The expression of the biochemical marker for ALP activity and stain was assessed on days 7 and 14.12,13 Briefly Alkaline Phosphatase was stained using (Sigma–Aldrich) following manufacturers’ protocol. Samples were viewed by light microscopy for the detection of ALP stain and images were evaluated by two blinded examiners. On the other hand, Alkaline Phosphatase Diethanolamine Activity Kit (Thermo Fisher Scientific) was used to quantify ALP activity by absorbance level measured at 405 nm by SpectraMax M5/M5e Multimode Plate Reader (Molecular Devices). The ALP assay was duplicated in triplicates. ALP activity was expressed as units/mg protein normalized to the number of cells.
Quantitative reverse transcription polymerase chain reaction
Dental pulp stem cells were seeded in 12 well plate in OM with PD with a density of 2 × 105 cells/well. On day 7 cells were collected and RNA lysis buffer was added to the cell pellet. RNA was isolated using RNeasy mini kit (RNeasy; Qiagen, Hilden, Germany) then quantified using Nanodrop spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific). The extracted RNA was reverse transcribed by High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) and cDNA was synthesized by Multigene thermocycler (Labnet International, Inc., Edison, NJ, USA). Thereafter, mRNA expression was analyzed using Fast SYBR™ Green PCR Master Mix (Thermo Fisher Scientific). The primer sequences for ALP, Runt-related transcription factor-2 (RUNX-2), OC, OP, osteonectin (ON), DSPP, and DMP-1 (Oligo™, Seoul, South Korea) are listed in Table 1. Groups were run in duplicates, cycle threshold (Ct) values were obtained after 45 amplification cycles. The Ct values for tested genes were normalized to the endogenous control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values. The comparative delta–delta Ct method (2−ΔΔ CT) was used to normalize the data based on the endogenous reference (GAPDH) to be expressed as the relative fold change in percentage compared to the values obtained from the control group.35
Table 1.
List of primers sequences for ALP, RUNX-2, OC, OP, ON, DSPP, and DMP-1.
| Gene | Sequence (5′-3′) |
|---|---|
| GAPDH | Sense (Forward primer) 5′-CTGGTAAAGTGGATATTGTTGCCAT-3′ Antisense (Reverse primer) 5′-TGGAATCATATTGGAACATGTAAACC-3′ |
| ALP | Sense (Forward primer) 5′-GACGGACCCTCGCCAGTGCT-3′ Antisense (Reverse primer) 5′-AATCGACGTGGGTGGGAGGGG-3′ |
| RUNX-2 | Sense (Forward primer) 5′-ACGIGGCTAAGAATGTCATC-3′ Antisense (Reverse primer) 5′-CTGGTAGGCGATGTCCTTA-3′ |
| OC | Sense (Forward primer) 5′-GGCAGCGAGGTAGTGAAGAG-3′ Antisense (Reverse primer) 5′-CTCACACACCTCCCTCCTG-3′ |
| OP | Sense (Forward primer) 5′-CAGTTCAGAAGAGGAGG-3′ Antisense (Reverse primer) 5′-TCAGCCTCAGAGTCTTCATC- 3′ |
| ON | Sense (Forward primer) 5′-GAGGAAACCGAAGAGGAGG-3′ Antisense (Reverse primer) 5′-GGGGTGTTGTTCTCATCCAG-3′ |
| DSPP | Sense (Forward primer) 5′-GCCATTCCAGTTCCTCAACTT-3′ Antisense (Reverse primer) 5′-CATGCACCAGGACACCACTT-3′ |
| DMP-1 | Sense (Forward primer) 5′-CGGTTCCTGGAATACTGACC-3′ Antisense (Reverse primer) 5′-TCTTTGGCTGTGTTCTGGTG-3′ |
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), osteopontin (OP), osteonectin (ON), dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1).
Evaluation of calcified deposits
Alizarin red staining
Alizarin red staining was performed on days 14 and 21 according to previously published studies.12,13 Briefly, hDPSCs were exposed to PD in a 12-well plate in OM with a density of 2 × 105 cells/well. Wells were rinsed then fixed with 4% paraformaldehyde followed by incubation with 2% Alizarin red stain (Cat. No. 0223, ScienCell Research Laboratories, Carlsbad, CA, USA) for 30 min at room temperature and washed with dH2O 4 times for 5 min each. Light microscope Pictures were registered and evaluated by two blinded examiners for each group. Experiments were duplicated in triplicates.
Statistical analysis
Data was expressed as the mean ± standard deviation (SD) of the number of observations. Treatment groups were normalized to the control group. Distribution was examined for normality using the Shapiro–Wilk test. When data is normally distributed, a parametric test (one-way ANOVA) was performed. Equality of variances verified using Levene test, if assumption is violated, Welch ANOVA with Games-Howell post-hoc tests was used. Statistically significant if P-values <0.05. Analysis was performed using IBM SPSS Statistics software (version 29). Graphs were designed using GraphPad Prism 10 software (GraphPad Software, San Diego, CA, USA).
Results
Viability of cells
The viable hDPSCs exposed 0.01 μM PD concentration was slightly higher than the control on day 1 and 7 and significantly higher on day 3 (P = 0.043). Similarly, 0.1 μM PD concentration had significantly increased viable cells on day 7 (P = 0.010). Whilst 1 μM PD concentration was similar to the control group in all days. Conversely, 10 and 100 μM PD had significantly decreased viable cells on Days 1, 3, and 7 compared to the control group (Fig. 2).
Figure 2.
Viability of hDPSCs on Days 1,3, and 7. Data presented as mean ± SD. ∗ Indicates a statistically significant difference P < 0.05 compared to the control group.
Alkaline phosphatase assay and stain
Alkaline phosphatase activity of hDPSCs on day 7 was significantly increased with all the PD concentrations compared to the control 0.01 μM (P = 0.003), 0.1 μM (P = 0.010), and 1 μM (P = 0.005). The highest ALP activity among the three concentrations was with 0.1 μM followed by 1 μM and 0.01 μM. On day 14, 0.01 μM had the highest activity followed by 0.1 μM, both were significantly higher than the control (P = 0.007, P = 0.005), respectively. ALP-stained wells showed denser stain of treated wells compared to the control group with slight differences between treated groups. Light microscope pictures of stained wells and ALP assay graph are shown in (Fig. 3).
Figure 3.
(A, C) ALP staining photomicrograph and ALP assay optical density (OD) in percentage change compared to the control group on day 7; (B, D) ALP staining photomicrograph and ALP assay in percentage change compared to control group on day 14. Data are presented as mean ± SD. Scale bar 200 μm; magnification 10x. ∗ Indicates a statistically significant difference at P < 0.05 compared to the control group.
Quantitative reverse transcription polymerase chain reaction
PD concentration 0.1 μM demonstrated the highest mean value compared to the control group for the following messenger RNA (mRNA): ALP, RUNX-2, OC, OP, ON. DSPP expression was highest with 0.01 μM, while DMP-1 expression was highest with 1 μM PD, however, the differences were not statistically significant (Fig. 4).
Figure 4.
RT-qPCR of 0.01,0.1.1 μM PD on day 7 of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), osteopontin (OP), osteonectin (ON), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) mRNA. Data are presented as mean, minimum, and maximum fold change increase of mRNA compared to the control group.
Alizarine red stain
On day 14, hDPSCs stained with alizarin red, showed slight increase in the size of calcified nodules in the PD treated groups compared to the control, however, similar staining intensity was observed for all PD treated groups. On day 21, PD groups showed an evident increase in the calcified nodules number, color, and size compared to the control group. Calcified nodules seen in 0.1 μM were more prevalent on day 21 than other groups (Fig. 5).
Figure 5.
(A) Alizarin red stain photomicrograph on day 14; (B) Alizarin red stain photomicrograph on day 21. Scale bar 200 μm; magnification 10x.
Discussion
The biological principle involved in “regenerative endodontics” and “direct pulp capping” rely on recruiting undifferentiated mesenchymal stem cells to undergo differentiation into cells that are involved in the regeneration of lost dentin pulp complex tissues.10,11,36 This is the first study to investigate the effect of PD on hDPSCs in terms of viability and odontogenic differentiation. The findings suggest that PD promotes the viability of hDPSCs and induces odontogenic differentiation and mineralized nodule formation. Cell viability and mineralized tissue deposits are critical for dental applications aiming to replace missing pulp–dentin complex tissues.
The literature is limited on work studying odontogenic/osteogenic differentiation of human stem cells in response to PD and its derivative resveratrol.27,29,37 However, the findings of the published papers are in line with our findings suggesting that PD promoted the production of odontogenic/osteogenic markers. In addition, viability of hDPSCs in the presence of PD was found to be dose-dependent where lower concentrations promoted viability, whilst higher concentrations (10 and 100 μM) reduced cell viability, this finding is in agreement with Di Benedetto et al. (2018) and Zhan et al. (2020) using DBSCs and BMSCs.27,38
Although mRNA levels provide evidence of odontogenic differentiation, the ultimate indicator for odontogenic differentiation is the formation of mineralized nodules and the production of molecules involved in mineralization such as ALP. In this study, ALP levels were significantly elevated with observed increased mineralized deposits in agreement with previous work on DBSCs.27
The findings of the current in-vitro study were shown on one type of stem cells with controlled variables, therefore, the effect on other types of stem cells as well as in vivo conditions need to be investigated.
Pulpal inflammation is a feature of pulp disease as well as a response to agents applied to the pulp to promote hard tissue repair.22,23 PD has been shown to affect the expression of inflammatory cytokines in a manner favorable to reducing the inflammatory response.26 The beneficial effect of PD on hDPSCs in terms of viability and mineralized tissue deposition might be augmented by the inflammation reducing properties making it a potential therapeutic agent for dental pulp procedures requiring hard tissue barrier formation, however, further work is required to determine the anti-inflammatory properties of PD on hDPSCs.
In conclusion, PD promoted cell viability and increased expression of markers associated with odontogenic differentiation, as well as mineralized tissue deposition in hDPSCs. The findings suggest that PD merits further investigation for its use as a therapeutic agent in endodontic applications that require dentin bridge formation.
Declaration of competing interest
The authors have no conflicts of interest relevant to this article.
Acknowledgment
The authors would like to thank College of Dentistry Research Center and Deanship of Scientific Research at King Saud University, Saudi Arabia for funding this research project.
References
- 1.Yu C., Abbott P. An overview of the dental pulp: its functions and responses to injury. Aust Dent J. 2007;52:S4–S6. doi: 10.1111/j.1834-7819.2007.tb00525.x. [DOI] [PubMed] [Google Scholar]
- 2.Kakehashi S., Stanley H.R., Fitzgerald R.J. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol. 1965;20:340–349. doi: 10.1016/0030-4220(65)90166-0. [DOI] [PubMed] [Google Scholar]
- 3.Lin L.M., Ricucci D., Saoud T.M., Sigurdsson A., Kahler B. Vital pulp therapy of mature permanent teeth with irreversible pulpitis from the perspective of pulp biology. Aust Endod J. 2020;46:154–166. doi: 10.1111/aej.12392. [DOI] [PubMed] [Google Scholar]
- 4.Ng Y.L., Mann V., Rahbaran S., Lewsey J., Gulabivala K. Outcome of primary root canal treatment: systematic review of the literature - Part 1. Effects of study characteristics on probability of success. Int Endod J. 2007;40:921–939. doi: 10.1111/j.1365-2591.2007.01322.x. [DOI] [PubMed] [Google Scholar]
- 5.Ricucci D., Russo J., Rutberg M., Burleson J.A., Spngberg L.S.W. A prospective cohort study of endodontic treatments of 1,369 root canals: results after 5 years. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112:825–842. doi: 10.1016/j.tripleo.2011.08.003. [DOI] [PubMed] [Google Scholar]
- 6.Law A.S. Considerations for regeneration procedures. J Endod. 2013;39:44–56. doi: 10.1016/j.joen.2012.11.019. [DOI] [PubMed] [Google Scholar]
- 7.Barthel C.R., Rosenkranz B., Leuenberg A., Roulet J.F. Pulp capping of carious exposures: treatment outcome after 5 and 10 years: a retrospective study. J Endod. 2000;26:525–528. doi: 10.1097/00004770-200009000-00010. [DOI] [PubMed] [Google Scholar]
- 8.Bogen G., Kim J.S., Bakland L.K. Direct pulp capping with mineral trioxide aggregate: an observational study. J Am Dent Assoc. 2008;139:305–315. doi: 10.14219/jada.archive.2008.0160. [DOI] [PubMed] [Google Scholar]
- 9.Cooper P.R., Takahashi Y., Graham L.W., Simon S., Imazato S., Smith A.J. Inflammation-regeneration interplay in the dentine-pulp complex. J Dent. 2010;687–97 doi: 10.1016/j.jdent.2010.05.016. [DOI] [PubMed] [Google Scholar]
- 10.Cooper P.R., Holder M.J., Smith A.J. Inflammation and regeneration in the dentin-pulp complex: a double-edged sword. J Endod. 2014;40:S46–S51. doi: 10.1016/j.joen.2014.01.021. [DOI] [PubMed] [Google Scholar]
- 11.Eramo S., Natali A., Pinna R., Milia E. Dental pulp regeneration via cell homing. Int Endod J. 2018;51:405–419. doi: 10.1111/iej.12868. [DOI] [PubMed] [Google Scholar]
- 12.Seo M.S., Hwang K.G., Lee J., Kim H., Baek S.H. The effect of mineral trioxide aggregate on odontogenic differentiation in dental pulp stem cells. J Endod. 2013;39:242–248. doi: 10.1016/j.joen.2012.11.004. [DOI] [PubMed] [Google Scholar]
- 13.Luo Z., Kohli M.R., Yu Q., Kim S., Qu T., He W.X. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways. J Endod. 2014;40:937–942. doi: 10.1016/j.joen.2013.11.022. [DOI] [PubMed] [Google Scholar]
- 14.Agrafioti A., Taraslia V., Chrepa V., et al. Interaction of dental pulp stem cells with biodentine and MTA after exposure to different environments. J Appl Oral Sci. 2016;24:481–486. doi: 10.1590/1678-775720160099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Linde A., Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med. 1994;4:679–728. doi: 10.1177/10454411930040050301. [DOI] [PubMed] [Google Scholar]
- 16.Huang B., Sun Y., MacIejewska I., et al. Distribution of SIBLING proteins in the organic and inorganic phases of rat dentin and bone. Eur J Oral Sci. 2008;116:104–112. doi: 10.1111/j.1600-0722.2008.00522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Martin G., Ricucci D., Gibbs J.L., Lin L.M. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod. 2013;39:138–144. doi: 10.1016/j.joen.2012.09.015. [DOI] [PubMed] [Google Scholar]
- 18.Arslan H., Şahin Y., Topçuoğlu H.S., Gündoğdu B. Histologic evaluation of regenerated tissues in the pulp spaces of teeth with mature roots at the time of the regenerative endodontic procedures. J Endod. 2019;45:1384–1389. doi: 10.1016/j.joen.2019.07.016. [DOI] [PubMed] [Google Scholar]
- 19.Nowicka A., Wilk G., Lipski M., Kołecki J., Buczkowska-Radlińska J. Tomographic evaluation of reparative dentin formation after direct pulp capping with Ca(OH)2, MTA, biodentine, and dentin bonding system in human teeth. J Endod. 2015;41:1234–1240. doi: 10.1016/j.joen.2015.03.017. [DOI] [PubMed] [Google Scholar]
- 20.Mehrvarzfar P., Abbott P.V., Mashhadiabbas F., Vatanpour M., Tour Savadkouhi S. Clinical and histological responses of human dental pulp to MTA and combined MTA/treated dentin matrix in partial pulpotomy. Aust Endod J. 2018;44:46–53. doi: 10.1111/aej.12217. [DOI] [PubMed] [Google Scholar]
- 21.Trongkij P., Sutimuntanakul S., Lapthanasupkul P., Chaimanakarn C., Wong R.H., Banomyong D. Pulpal responses after direct pulp capping with two calcium-silicate cements in a rat model. Dent Mater J. 2019;38:584–590. doi: 10.4012/dmj.2018-225. [DOI] [PubMed] [Google Scholar]
- 22.Sawicki L., Pameijer C.H., Emerich K., Adamowicz-Klepalska B. Histological evaluation of mineral trioxide aggregate and calcium hydroxide in direct pulp capping of human immature permanent teeth. Am J Dent. 2008;21:262–266. [PubMed] [Google Scholar]
- 23.Ozdemir Y., Kutukculer N., Topaloglu-Ak A., Kose T., Eronat C. Comparative evaluation of pro-inflammatory cytokine levels in pulpotomized primary molars. J Oral Sci. 2015;57:145–150. doi: 10.2334/josnusd.57.145. [DOI] [PubMed] [Google Scholar]
- 24.Ribeiro de Lima T., Waffo-Téguo P., Teissedre P L., et al. Determination of Stilbenes (trans-Astringin, cis- and trans-Piceid, and cis- and trans-Resveratrol) in Portuguese Wines. J Agric Food Chem. 1999;47:2666–2670. doi: 10.1021/jf9900884. [DOI] [PubMed] [Google Scholar]
- 25.Hurst W.J., Glinski J.A., Miller K.B., Apgar J., Davey M.H., Stuart D.A. Survey of the trans-resveratrol and trans-piceid content of cocoa-containing and chocolate products. J Agric Food Chem. 2008;56:8374–8378. doi: 10.1021/jf801297w. [DOI] [PubMed] [Google Scholar]
- 26.Lanzilli G., Cottarelli A., Nicotera G., Guida S., Ravagnan G., Fuggetta M.P. Anti-inflammatory effect of resveratrol and polydatin by in vitro IL-17 modulation. Inflammation. 2012;35:240–248. doi: 10.1007/s10753-011-9310-z. [DOI] [PubMed] [Google Scholar]
- 27.Di Benedetto A., Posa F., De Maria S., et al. Polydatin, natural precursor of resveratrol, promotes osteogenic differentiation of mesenchymal stem cells. Int J Med Sci. 2018;15:944–952. doi: 10.7150/ijms.24111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.National Center for Biotechnology Information . 2023. "PubChem compound summary for CID 5281718, Polydatin" PubChem.https://pubchem.ncbi.nlm.nih.gov/compound/Piceid Available at: Data Accessed. [Google Scholar]
- 29.Kim J.J., Kim S.J., Kim Y.S., Kim S.Y., Park S.H., Kim E.C. The role of SIRT1 on angiogenic and odontogenic potential in human dental pulp cells. J Endod. 2012;38:899–906. doi: 10.1016/j.joen.2012.04.006. [DOI] [PubMed] [Google Scholar]
- 30.Chen M., Hou Y., Lin D. Polydatin protects bone marrow stem cells against oxidative injury: involvement of Nrf 2/ARE pathways. Stem Cell Int. 2016;1:1–10. doi: 10.1155/2016/9394150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Çelik N., Işcan Yapar M., Taghizadehghalehjoughi A., Nalcı K.A. Influence of resveratrol application with pulp-capping materials on the genetic expression levels of stem cells. Int Endod J. 2020;53:1253–1263. doi: 10.1111/iej.13345. [DOI] [PubMed] [Google Scholar]
- 32.Pramono S., Pugdee K., Suwanprateep J., Koontongkaew S. Sandblasting and fibronectin-derived peptide immobilization on titanium surface increase adhesion and differentiation of osteoblast-like cells (MC3T3-E1) J Dent Sci. 2016;11:427–436. doi: 10.1016/j.jds.2016.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Chen C.S., Lee S.S., Yu H.C., Huang F.M., Chang Y.C. Effects of nicotine on cell growth, migration, and production of inflammatory cytokines and reactive oxygen species by cementoblasts. J Dent Sci. 2015;10:154–160. [Google Scholar]
- 34.Bopp S.K., Lettieri T. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol. 2008;8:8. doi: 10.1186/1471-2210-8-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Schmittgen T.D., Livak K.J. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101–1108. doi: 10.1038/nprot.2008.73. [DOI] [PubMed] [Google Scholar]
- 36.Farges J.C., Alliot-Licht B., Renard E., et al. Dental pulp defence and repair mechanisms in dental caries. Mediat Inflamm. 2015;1:230–251. doi: 10.1155/2015/230251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Feng G., Zheng K., Song D., et al. SIRT1 was involved in TNF-α-promoted osteogenic differentiation of human DPSCs through Wnt/β-catenin signal. In Vitro Cell Dev Biol Anim. 2016;52:1001–1011. doi: 10.1007/s11626-016-0070-9. [DOI] [PubMed] [Google Scholar]
- 38.Zhan J., Li X., Luo D., et al. Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: involvement of Nrf2 signalling pathway. J Cell Mol Med. 2020;24:5317–5329. doi: 10.1111/jcmm.15187. [DOI] [PMC free article] [PubMed] [Google Scholar]