Skip to main content
Download PDF

Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells

BMC Oral Health volume 24, Article number: 148 (2024) Cite this article

Abstract

Background

This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs).

Methods

hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05.

Results

Treatment with TLR and CLR agonists was associated with an increase in hDPSCs’ colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs’ mineralisation.

Conclusions

The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway.

Peer Review reports

Introduction

Pulpitis is an inflammatory condition affecting the dental pulp, primarily caused by bacterial infection. Pattern recognition receptors (PRRs) recognise the invading pathogen via recognised pathogen-associated molecular patterns (PAMPs). Common PRRs comprised of toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs), and Absent in melanoma-2-like receptor (ALRs) [1]. Currently, there are 10 functional human TLRs identified. These receptors are categorised based on their cellular localisation and their specific recognition of PAMPs [2]. Upon the recognition of PAMPs, TLR initiates intricate signalling cascades that stimulate the expression of proinflammatory cytokines and activate immune responses.

Dental pulp stem cells (DPSCs) are specific mesenchymal stem cells (MSCs) that exist in human dental pulp tissues [3]. They exhibit remarkable regenerative potential attributed to their capacity for self-renewal and multi-lineage differentiation [4]. In response to pulpal injury or infection, DPSCs encounter microbes and their components, for example, gram-negative bacteria and fungi. TLRs recognise PAMPs of bacteria, viruses, and fungi, while CLRs mainly recognise PAMPs of fungi [1]. DPSCs expressed a variety of functional TLRs with upregulation of TLR2, TLR3, and TLR4 [5], which play a key role in the advancement of pulpal disease. It has become apparent that pulpal stimulation by TLR-specific agonists may significantly influence the migratory, proliferative, and differentiation potentials of the DPSCs [6].

Lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly(I:C)), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), Furfurman, and Zymosan have been reported as the specific ligand to activate TLRs-induced cellular signalling in hDPSCs. LPS mainly interacts with TLR4 [7]. Poly(I:C) binds with TLR3 to exert its mechanism of action [8]. Pam3CSK4 is recognised as a TLR1/2 agonist [9]. Zymosan collaborates with TLR2 to mediate the innate immune response [10]. Furfurman binds to Dectin-2 and Dectin-1. Several studies investigate the role of TLR agonists in the proliferation and osteoblastic differentiation of hDPSCs. For example, it has been reported that cell proliferation was decreased while osteogenic differentiation remained unaffected following treatment with LPS [11]. However, a contradictory result was observed in other studies [12, 13]. In addition, hDPSCs exhibit distinct inflammatory mediators in response to stimulation with bacterial LPS, Pam3CSK4, and poly(I:C), which affect the differentiation of hDPSC and coordinate tissue repair [14,15,16,17].

From the result of previous studies, it can be implied that different microbial compositions (bacteria and fungi) of the caries lesion may exert distinct influences on the inflammatory response of the pulp [18, 19]. However, there are some contradictory data regarding the roles of different TLRs in the differentiation of hDPSCs. In addition, the effects of Zymosan and Furfurman on the interactions of hDPSCs have only been investigated in a limited number of studies. Therefore, this study aimed to investigate the effect of various concentrations of TLR and CLR agonists (PG-LPS, E.coli LPS, Poly (I:C), Pam3CSK4, Furfurman, and Zymosan) on the colony-forming unit ability and osteogenic differentiation of hDPSCs. The null hypothesis is that TLR and CLR agonists do not affect osteogenic differentiation in hDPSCs.

Materials and methods

hDPSCs isolation

The cell isolation protocol was approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (Approval number 037/2021). The healthy teeth from the patients aged 18–30 years old, extracted according to the patient’s treatment plan (for example, embedded and impacted teeth), were obtained for cell isolation. The teeth with pathological conditions were excluded from cell collection. Cells were isolated using the explantation technique. Briefly, tissues were cut into small pieces and placed on 35-mm tissue culture plates, allowing cell outgrowth for 7 days. The isolated cells were cultured in a growth medium composed of Dulbecco’s modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum, 1% L-glutamine (Gibco, USA), 1% Antibiotic-Antimycotic (Gibco, USA) and incubated at 37 °C in 5% CO2. After reaching confluence, the cells were trypsinised and subcultured at a 1:3 ratio using trypsin/EDTA solution (Gibco, USA). The culture medium was changed every 48 h. Cells from passages 3–6 were employed in the present study.

Mesenchymal stem cell characterisation

Cells at passages 2–3 were harvested. The single-cell suspension was prepared for fluorescence-conjugated antibody staining. Cell suspension was incubated with FITC conjugated anti-human CD44 (BD Bioscience, USA), PE-conjugated anti-human CD105 (Immuno Tools, Germany), FITC conjugated anti-CD90 (Abcam, USA), or PerCP-conjugated anti-CD45 (Abcam, USA) for 30 min. The surface marker staining was evaluated using a FACSCalibur flow cytometer with CellQuest software (BD Bioscience, USA). The mean fluorescence was evaluated.

For multipotential differentiation ability, cells were seeded in 24 well plates at the density of 25,000 cells per well in a normal growth medium (GM). After 24 h, the medium was changed to an osteogenic induction medium (OM) or an adipogenic induction medium (AM). The medium was replaced with the fresh medium every 48 h. The mineral deposition and intracellular lipid droplet accumulation were evaluated using Alizarin Red S staining on day 14 and Oil-Red O staining on day 16, respectively. The adipogenic medium contained 0.1 mg/ml insulin (Sigma-Aldrich, USA), 1 µM dexamethasone (Sigma-Aldrich, USA), 1 mM IBMX (Thermo Fisher Scientific, USA), and 0.2 mM indomethacin (Sigma-Aldrich, USA). An osteogenic differentiation medium comprised of growth medium supplemented with 5 mM β-glycerophosphate (Sigma-Aldrich, USA), 50 µg/ml L-ascorbic acid (Sigma-Aldrich, USA), and 250 nM dexamethasone (Sigma-Aldrich, USA).

Treatment protocol

The cells were treated with various concentrations (0.01-10 µg/ml) of TLR agonists: PG-LPS (Invivogen, CA, USA), E.coli LPS (Invivogen, CA, USA), Poly (I:C) (Invivogen, CA, USA), Pam3CSK4 (Invivogen, CA, USA), and Zymosan (Invivogen, CA, USA) or CLR agonist: Furfurman (Invivogen, CA, USA). In some experiments, cells were pretreated with 10 µM TRIF inhibitor or 10 µM AKT inhibitor (Calbiochem, USA) for 30 min prior to TLR agonists exposure.

Cell viability test

Cells (n = 4) were seeded in 24-well plates at a density of 25,000 cells per well and maintained in a normal growth medium for 1, 3, and 7 days. At the designated time points, the culture medium was removed, and the cells were incubated with 0.5 mg/ml MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) for 30 min at 37 °C in 5% CO2. Cells were then washed with phosphate-buffered saline to remove the MTT solution. The precipitated formazan crystals were solubilised in dimethyl sulfoxide (DMSO; Carlo Erba, 900 µl per well) and glycine buffer (pH = 10, 125 µl per well). The solubilised solution was collected, and the absorbance was measured at 570 nm using a microplate reader (Biotek ELX800, USA). The absorbance values were then normalised to the control condition at day 1.

Colony forming unit assay

Cells (n = 4) were seeded at a density of 500 cells in a 35 mm tissue culture plate and maintained in a growth medium for 14 days. The culture medium was changed every 2 days. At day 14, cells were fixed with 4% paraformaldehyde and washed with deionised water (DI). Colonies were stained with Coomassie blue (Sigma-Aldrich, USA).

Alkaline phosphatase (ALP) enzyme activity

Cells (n = 5) were seeded in 24-well plates at a density of 25,000 cells per well and maintained in a normal growth medium for 24 h. Subsequently, the medium was changed to an osteogenic induction medium. The culture medium was changed every 48 h. On day 7, cells were then fixed with 4% paraformaldehyde for 10 min and washed with DI, then incubated with NBT/BCIP solution (Roche, Germany) per well for 15 min at room temperature, according to the manufacturer’s protocol. After staining, cells were washed with DI and air-dried. The ALP-stained cells were observed using an inverted microscope (Olympus, USA).

Mineralisation assay

Cells (n = 4) were seeded in 24-well plates at a density of 25,000 cells per well and maintained in a normal growth medium for 24 h. Subsequently, the medium was changed to an osteogenic induction medium. The culture medium was changed every 48 h. Cells were then fixed with 4% paraformaldehyde for 10 min and washed with DI. Cells were stained at room temperature with a 0.5% (w/v) Alizarin Red S solution with gentle agitation. The excess staining was washed with DI. The staining was observed using an inverted microscope (Olympus, USA). The stain was solubilised in 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate (Sigma-Aldrich, USA). The absorbance was measured at 570 nm using a microplate reader (Biotek ELX800, USA). The absorbance values were then normalised to the control condition. As for Von Kossa staining, cells (n = 4) were fixed with 4% paraformaldehyde for 10 min and washed with DI. At room temperature, the cells were stained with a 5% silver nitrate solution under ultraviolet light for 5 min. The stained cells were observed using an inverted microscope (Olympus, USA).

Quantitative polymerase chain reaction (qPCR)

Cells (n = 4) were seeded in 24-well plates at a density of 25,000 cells per well and maintained in a normal growth medium for 24 h. Subsequently, the medium was changed to an osteogenic induction medium. The culture medium was changed every 48 h. Total RNA was extracted with RiboEx™ reagents (GeneAll, Korea) on day 7. The quality and quantity of isolated RNA was evaluated using Nanodrop. The cDNA was synthesised with iScript RT Supermix (Bio-Rad, USA) from 1 µg of the isolated RNA. For quantification of mRNA expression, qPCR amplification was performed using CFX Connect™ Real-Time PCR (Bio-Rad, USA), and PCR products were detected using FastStart Essential DNA Green Master (Bio-Rad, USA). The amplification profile was 95 °C/20 s, 60 °C/20 s, and 72 °C/20 s for 45 cycles. A melting curve analysis was performed for the evaluation of PCR product specificity. Target gene expression was normalised by the levels of GAPDH. The forward and reverse primers are in Table S1.

Statistical analysis

Data were analysed, and graphs were generated using GraphPad Prism 8 (GraphPad Software, CA, USA). Data were expressed as the mean ± standard error of the mean (SEM). In each experiment, cells from at least four different donors were used without pooling. The statistical comparison was performed using the Kruskal-Wallis test, followed by a pairwise comparison test for three or more group comparisons. A pairwise comparison was performed between the treatment groups and the control group. For a two-group comparison, the Mann-Whitney U test was used. A value of p < 0.05 was considered statistically significant. Each dot on the graph represented each normalisation of the raw data value.

Results

Stem cell characterisation

The cells harvested from dental pulp tissue exhibited a spindle-shaped and fibroblast-like morphology. Surface protein analysis confirmed the presence of mesenchymal stem cell-related surface markers (CD44, CD90, and CD105). In contrast, the hematopoietic cell marker, CD45, was notably absent (Fig. 1A). Differentiation ability towards osteogenic and adipogenic lineages was performed. In the osteogenic induction medium, mineral deposition manifested as distinct red nodules stained by Alizarin Red S staining (Fig. 1B). The cells maintained in the adipogenic induction medium demonstrated an intracellular lipid accumulation, visualised through Oil Red O staining (Fig. 1C).

Fig. 1
figure 1

Characterisation of cells harvested from dental pulp tissues. Stem cell surface markers were examined using flow cytometry (A). The differentiation ability towards osteogenic (B) (scale bar, 300 μm) and adipogenic lineages (C) (scale bar, 30 μm) was examined using Alizarin Red S staining and Oil Red O staining, respectively. Effect of various TLR agonists on cell proliferation of hDPSCs. Cells were treated with PG-LPS, E.coli LPS, Poly (I:C), Pam3CSK4, Furfurman, and Zymosan (0, 0.01, 0.1, 1, and 10 µg/ml) for 1, 3, and 7 days. Cell proliferation was observed using an MTT assay (D). Cells were treated with TLR agonists at 1 and 10 µg/ml and cultured for 14 days. Colony formation was examined using Coomassie blue staining (E). The relative absorbance of the solubilised dye was evaluated (F). Bars indicate a statistically significant difference between groups (p < 0.05). GM; normal growth medium, OM; osteogenic induction medium, AM; adipogenic induction medium

Full size image

TLR and CLR agonists reduced hDPSCs cell viability but enhanced colony-forming unit ability

MTT assays were performed with 0.01, 0.1, 1, and 10 µg/mL of PG-LPS, E.coli LPS, Poly (I:C), Pam3CSK4, Furfurman, and Zymosan at day 1, 3 and 7. The results showed that cell viability was significantly decreased when cells were treated with high concentration of E.coli LPS, Poly (I:C), Pam3CSK4, and Furfurman at day 7 (p < 0.05) (Fig. 1D). Poly (I:C), Pam3CSK4, and Furfurman (1 and 10 µg/ml) significantly reduced cell viability of hDPSCs at day 3 (p < 0.05). However, there was no statistically significant difference in cell viability when cells were treated with PG-LPS and Zymosan.

On the contrary, TLR and CLR agonists markedly promoted the colony-forming unit ability of hDPSCs. TLR and CLR agonist-treated hDPSCs (1 µg/ml) displayed a significant increase in colony-forming efficiency compared to the control group, except for Poly (I:C) and Pam3CSK4 (p < 0.05). Moreover, E.coli LPS, Pam3CSK4, and Zymosan-treated cells at 10 µg/ml exhibited a statistically significant higher colony-forming efficiency in comparison with the control group (p < 0.05). No significant difference was observed between the 1 µg/ml and 10 µg/ml concentrations (Fig. 1E and F).

TLR and CLR agonists impaired the osteogenic differentiation of hDPSCs

To investigate the effect of TLR and CLR agonists on osteogenic differentiation, hDPSCs were treated with PG-LPS, E.coli LPS, Poly (I:C), Pam3CSK4, Furfurman, or Zymosan at concentrations of 1 and 10 µg/ml in osteogenic medium. ALP enzymatic activity was downregulated in a dose-dependent manner at day 7 (Fig. 2A). A significant decrease in ALP enzymatic activity was observed in the PG-LPS-, E.coli LPS-, Pam3CSK4-, and Furfurman-treated groups at a concentration of 10 µg/ml compared with the control (p < 0.05) (Fig. 2B). Further, mineralisation was dramatically inhibited in a dose-dependent manner at day 14 (Fig. 2C and D). A significant downregulation of mineral deposition was noted in the PG-LPS-, E.coli LPS-, Pam3CSK4-, Furfurman, and Zymosan-treated groups (p < 0.05). Moreover, 10 µg/ml of all treatments significantly decreased the mRNA expression of key osteogenesis-related regulators and effector genes (ALP, COL1A1, RUNX2, OSX, and DMP) as compared with the control (p < 0.05) (Fig. 3).

Fig. 2
figure 2

Effect of TLR and CLR agonists on the osteogenic differentiation of hDPSCs. Cells were treated with TLR or CLR agonists at 1 and 10 µg/ml in an osteogenic induction medium (OM). Alkaline Phosphatase Activity (ALP) activity was examined on day 7 (A). The relative absorbance of the solubilised ALP staining was measured (B). The mineralisation was shown by Alizarin Red S staining and Von Kossa staining on day 14 (C). The relative absorbance of the solubilised alizarin red dye was measured at 570 nm (D). Bars indicate a statistically significant difference between groups (p < 0.05)

Full size image
Fig. 3
figure 3

Effect of TLR agonists on the osteogenic differentiation of hDPSCs. Cells were treated with TLR agonists at 1 and 10 µg/ml in an osteogenic induction medium (OM). The mRNA expression of osteogenic-related genes was examined using qPCR. Bars indicate the statistically significant difference between groups (p < 0.05)

Full size image

TRIF inhibition rescued TLR and CLR agonist-attenuated mineralisation in hDPSCs

To investigate the potential pathways involved in TLRs/CLRs-attenuated osteogenic differentiation of hDPSCs, cells were pretreated with an Akt inhibitor or TRIF inhibitor 30 min prior to TLR/CLR agonist exposure (10 µg/ml). The result showed that pretreatment with a TRIF inhibitor prior to TLR/CLR agonist exposure abolished the TLR/CLR agonist-attenuated mineralisation (Fig. 4A and B). Pretreatment with an Akt inhibitor failed to rescue the effects of TLR/CLR agonist-attenuated mineralisation.

Fig. 4
figure 4

To investigate the potential pathways involved in TLRs-attenuated osteogenic differentiation of hDPSCs, cells were pretreated with an Akt inhibitor or TRIF inhibitor 30 min prior to TLR agonist exposure and maintained in osteogenic induction medium (OM). Mineralisation was examined by Alizarin Red S staining (A). The relative absorbance of the solubilised alizarin red dye was measured at 570 nm (B). Bars indicate the statistically significant difference between groups (p < 0.05)

Full size image

Discussion

The growing interest in utilising hDPSCs for cellular therapy highlights the need to consider the potential impact of microbial components on hDPSCs growth and differentiation. Exposure to environments enriched with bacterial endotoxins may have various effects on hDPSCs properties, including self-renewal, differentiation potential, cytokine production, and extracellular matrix composition [20]. Our results demonstrated that exposure of hDPSCs to TLR/CLR agonists negatively influences osteogenic differentiation.

LPS from gram-negative bacteria mainly interacts with TLR4 [7]. Poly(I:C), a synthetic analogue of dsRNA, binds with TLR3 to exert its mechanism of action [8]. Pam3CSK4, a synthetic triacylated lipopeptide, is recognised as a TLR1/2 ligand [9]. Furfurman binds to CLRs, namely Dectin-2 and Dectin-1 (high concentration). Zymosan collaborates with TLR2 to mediate the innate immune response [10] and it is also able to bind to Dectin-1. With the complex nature of binding efficacy, the interpretation of the results should be done with caution because the phenomenon could occur from the activation of multiple types of receptors.

Cell proliferation plays an important role in tissue repair, which is significantly influenced by the engagement of specific PRRs agonists. Previous research reported the contradictory effect of TLR agonists on cell proliferation, encompassing instances of both stimulation [21, 22] and reduction in proliferative capacity [23], while other studies have not observed any significant effect [24,25,26]. In the present study, E.coli LPS, Poly (I:C), Pam3CSK4, and Furfurman in tested concentrations did not affect the cell viability at day 1; however, hDPSCs cell viability was attenuated as compared with the control at later time points.

The present study showed that TLR and CLR agonist-treated cells enhanced the colony formation ability of hDPSCs. Our results were inconsistent with previous studies, which showed that dental pulp stem cells (hDPSCs) derived from inflamed tooth pulp exhibit a diminished ability to form cell colonies [27, 28]. Moreover, the results of the clonogenic assay give the opposite results of the MTT assay. The possible explanation includes variations in the testing methodology. The clonogenic assay evaluates cell survival by measuring the ability of a single cell to form a colony. In contrast, the MTT assay measures mitochondrial activity, referring to cell metabolic activity. This method is generally used to determine the increase in cell number. However, it is only considered to be an indirect approach. The reduction of cellular metabolic activities can result from several factors, including cell death. The colony-forming unit assay provides information regarding the cells’ ability to survive and grow but at a single-cell level. Further, the cell cycle should be used to evaluate the role of these TLR agonists on cell proliferation.

Recent studies have yielded contradictory findings regarding the impact of TLR and CLR agonists on cell differentiation. Previous studies reported that E.coli LPS-mediated TLR4 activation promoted osteogenic differentiation as indicated by an increase in mineralised nodule formation and odontoblastic-related mRNA expression [13, 24]. On the contrary, other studies demonstrated that PG-LPS and E.coli LPS exhibited inhibitory effects on osteoblastic differentiation of human periodontal ligament stem cells and murine dental pulp-derived cell lines [21, 26, 29, 30]. Further, exposure of dental pulp cells to the cell wall mannan of Candida species, a CLR ligand, led to the reduction of mineralisation [18]. In accordance with these latter studies, our result showed that different concentrations of TLR and CLR agonists used in the present study (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan) impaired the osteogenic differentiation of hDPSCs, as evidenced by the reduction in both ALP activity and mineralised nodule formation. Moreover, the mRNA expression levels of osteogenesis marker genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP) were downregulated. This data discrepancy could be attributed to the concentration of TLR and CLR agonists used in the present study.

The stimulation of TLR agonists triggers the activation of two major downstream signalling pathways: the myeloid differentiation factor 88- (MyD88-) dependent and TIR (Toll-interleukin receptor) domain-containing adapter-inducing interferon- (IFN-) β- (TRIF-) dependent pathways [31]. In addition to the TRIF-dependent pathway, other downstream signalling pathways, such as PI3K-Akt, have also been reported to play an important role in the osteogenic differentiation of human dental pulp cells and murine tendon stem cells [13, 32]. Thus, in the present study, we employed the pharmacologic inhibitor of TRIF or Akt to investigate the effect of TRIF/Akt signalling on the osteogenic differentiation of hDPSCs after TLR or CLR agonist stimulation. Our result demonstrated that TRIF inhibition rescued the inhibitory effects of TLR and CLR agonists on the osteogenic differentiation by hDPSCs. These findings imply that TRIF plays a crucial role during the differentiation phases of hDPSCs within the infected dental pulp.

The limitation of this study is primary cell multiplicity, which may react differently despite similar chemical substances being exposed. Furthermore, in vitro cellular responses may differ when cultured in vivo. Hence, it is essential to investigate animal models to obtain more efficient data on TLR and CLR agonists attenuated osteogenic differentiation.

Conclusion

The present study demonstrated that TLR and CLR activation promoted colony formation. Further, the osteogenic differentiation was attenuated in a dose-dependent manner. This inhibitory effect of TLR activation on osteogenic differentiation occurs through TRIF but not Akt signalling. This knowledge elucidates the role of various TLR and CLR agonists on hDPSCs, which potentially advances our comprehension of the management strategies for the diseased pulp conditions.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

References

  1. Li D, Wu M. Pattern recognition receptors in health and diseases. Signal Transduct Target Ther. 2021;6(1):291.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 2004;5(10):987–95.

    Article  PubMed  Google Scholar 

  3. Rodriguez-Lozano FJ, Bueno C, Insausti CL, Meseguer L, Ramirez MC, Blanquer M, et al. Mesenchymal stem cells derived from dental tissues. Int Endod J. 2011;44(9):800–6.

    Article  PubMed  Google Scholar 

  4. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009;88(9):792–806.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Fawzy El-Sayed KM, Klingebiel P, Dörfer CE. Toll-like receptor expression Profile of Human Dental Pulp Stem/Progenitor cells. J Endod. 2016;42(3):413–7.

    Article  PubMed  Google Scholar 

  6. DelaRosa O, Dalemans W, Lombardo E. Toll-like receptors as modulators of mesenchymal stem cells. Front Immunol. 2012;3.

  7. Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine. 2008;42(2):145–51.

    Article  PubMed  Google Scholar 

  8. Kumar A, Zhang J, Yu FS. Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology. 2006;117(1):11–21.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Funderburg NT, Jadlowsky JK, Lederman MM, Feng Z, Weinberg A, Sieg SF. The toll-like receptor 1/2 agonists Pam(3) CSK(4) and human β-defensin-3 differentially induce interleukin-10 and nuclear factor-κB signalling patterns in human monocytes. Immunology. 2011;134(2):151–60.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Dillon S, Agrawal S, Banerjee K, Letterio J, Denning TL, Oswald-Richter K, et al. Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest. 2006;116(4):916–28.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Shayegan A, Zucchi A, De Swert K, Balau B, Truyens C, Nicaise C. Lipoteichoic acid stimulates the proliferation, migration and cytokine production of adult dental pulp stem cells without affecting osteogenic differentiation. Int EndodJ. 2021;54(4):585–600.

    Article  Google Scholar 

  12. Rothermund K, Calabrese TC, Syed-Picard FN. Differential effects of Escherichia coli– Versus Porphyromonas gingivalis–derived lipopolysaccharides on Dental Pulp Stem Cell differentiation in Scaffold-free Engineered tissues. J Endod. 2022;48(11):1378–86e2.

    Article  PubMed  PubMed Central  Google Scholar 

  13. He W, Wang Z, Zhou Z, Zhang Y, Zhu Q, Wei K, et al. Lipopolysaccharide enhances Wnt5a expression through toll-like receptor 4, myeloid differentiating factor 88, phosphatidylinositol 3-OH Kinase/AKT and nuclear factor Kappa B pathways in Human Dental Pulp Stem cells. J Endod. 2014;40(1):69–75.

    Article  PubMed  Google Scholar 

  14. Ha Y-E, Ju So Y, Im J, Yun C-H, Park J-C, Hyun Han S. TLR3 recognition of viral double-stranded RNA in human dental pulp cells is important for the innate immunity. Int Immunopharmacol. 2023;119:110161.

    Article  PubMed  Google Scholar 

  15. Sabatini C, Ayenew L, Khan T, Hall R, Lee T. Dental Pulp Cell Conditioning through Polyinosinic-Polycytidylic Acid activation of toll-like receptor 3 for amplification of trophic factors. J Endod. 2022;48(7):872–9.

    Article  PubMed  Google Scholar 

  16. Tomic S, Djokic J, Vasilijic S, Vucevic D, Todorovic V, Supic G, Colic M. Immunomodulatory properties of mesenchymal stem cells derived from Dental Pulp and Dental follicle are susceptible to activation by toll-like receptor agonists. Stem Cells Dev. 2010;20(4):695–708.

    Article  Google Scholar 

  17. Takegawa D, Nakanishi T, Hirao K, Yumoto H, Takahashi K, Matsuo T. Modulatory roles of Interferon-γ through Indoleamine 2, 3-dioxygenase induction in Innate Immune response of Dental Pulp cells. J Endod. 2014;40(9):1382–7.

    Article  PubMed  Google Scholar 

  18. Bulanawichit W, Nguyen TNY, Ritprajak P, Nowwarote N, Osathanon T. Cell Wall Mannan of Candida attenuates osteogenic differentiation by Human Dental Pulp cells. J Endod. 2023;49(2):190–7.

    Article  PubMed  Google Scholar 

  19. Veerayutthwilai O, Byers MR, Pham TTT, Darveau RP, Dale BA. Differential regulation of immune responses by odontoblasts. Oral Microbiol Immunol. 2007;22(1):5–13.

    Article  PubMed  Google Scholar 

  20. Li D, Fu L, Zhang Y, Yu Q, Ma F, Wang Z, et al. The effects of LPS on adhesion and migration of human dental pulp stem cells in vitro. J Dent. 2014;42(10):1327–34.

    Article  PubMed  Google Scholar 

  21. Kato H, Taguchi Y, Tominaga K, Umeda M, Tanaka A. Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells. Arch Oral Biol. 2014;59(2):167–75.

    Article  PubMed  Google Scholar 

  22. Yu S, Cho HH, Joo HJ, Bae YC, Jung JS. Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells. Mol Cell Biochem. 2008;317(1):143–50.

    Article  PubMed  Google Scholar 

  23. Liu Y, Gao Y, Zhan X, Cui L, Xu S, Ma D, et al. TLR4 activation by Lipopolysaccharide and Streptococcus mutans induces Differential Regulation of Proliferation and Migration in Human Dental Pulp Stem cells. J Endod. 2014;40(9):1375–81.

    Article  PubMed  Google Scholar 

  24. He W, Wang Z, Luo Z, Yu Q, Jiang Y, Zhang Y, et al. LPS promote the Odontoblastic differentiation of Human Dental Pulp Stem cells via MAPK signaling pathway. J Cell Physiol. 2015;230(3):554–61.

    Article  PubMed  Google Scholar 

  25. Hwa Cho H, Bae YC, Jung JS. Role of toll-like receptors on human adipose‐derived stromal cells. Stem Cells. 2006;24(12):2744–52.

    Article  PubMed  Google Scholar 

  26. Kukolj T, Trivanović D, Djordjević IO, Mojsilović S, Krstić J, Obradović H, et al. Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling. J Cell Physiol. 2018;233(1):447–62.

    Article  PubMed  Google Scholar 

  27. Wang Z, Pan J, Wright JT, Bencharit S, Zhang S, Everett ET, et al. Putative stem cells in Human Dental Pulp with Irreversible Pulpitis: an exploratory study. J Endod. 2010;36(5):820–5.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Arora S, Cooper PR, Friedlander LT, Seo B, Rizwan SB, Rich AM, Hussaini HM. Potentiality and inflammatory marker expression are maintained in Dental Pulp cell cultures from Carious Teeth. Int J Mol Sci [Internet]. 2022; 23(16).

  29. Wang W, Yuan C, Geng T, Liu Y, Zhu S, Zhang C, et al. Lipopolysaccharide inhibits osteogenic differentiation of periodontal ligament stem cells partially through toll-like receptor 4-mediated ephrinB2 downregulation. Clin Oral Investig. 2020;24(10):3407–16.

    Article  PubMed  Google Scholar 

  30. Nomiyama K, Kitamura C, Tsujisawa T, Nagayoshi M, Morotomi T, Terashita M, Nishihara T. Effects of Lipopolysaccharide on newly established Rat Dental pulp–derived cell line with Odontoblastic Properties. J Endod. 2007;33(10):1187–91.

    Article  PubMed  Google Scholar 

  31. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol. 2004;4(7):499–511.

    Article  PubMed  Google Scholar 

  32. Liu J, Chen L, Tao X, Tang K. Phosphoinositide 3-kinase/Akt signaling is essential for prostaglandin E2-induced osteogenic differentiation of rat tendon stem cells. BBRC. 2013;435(4):514–9.

    PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Oral Biology Research Center for facility support.

Funding

The present study was supported by the National Research Council of Thailand (N41A640135) and the Faculty Research Fund, Faculty of Dentistry, Chulalongkorn University.

Author information

Authors and Affiliations

  1. Center of Excellence for Dental Stem Cell Biology, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

    Wajathip Bulanawichit, Chatvadee Kornsuthisopon, Ajjima Chansaenroj & Thanaphum Osathanon

  2. Department of Operative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

    Chanakarn Sinsareekul

  3. Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

    Vorapat Trachoo

  4. Centre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, INSERM UMR1138, Molecular Oral Pathophysiology and Department of Oral Biology, Faculty of Dentistry, Université Paris Cité, Paris, France

    Nunthawan Nowwarote

Authors
  1. Wajathip Bulanawichit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chanakarn Sinsareekul
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chatvadee Kornsuthisopon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ajjima Chansaenroj
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vorapat Trachoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nunthawan Nowwarote
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thanaphum Osathanon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Wajathip Bulanawichit and Chatvadee Kornsuthisopon: Data curation and Writing-Original draft. Chanakarn Sinsareekul and Ajjima Chansaenroj: Formal analysis, Visualization, Investigation, and Writing - Review & Editing. Vorapat Trachoo: Resources and Writing - Review & Editing. Nunthawan Nowwarote and Thanaphum Osathanon: Conceptualization, data curation, supervision, and Writing - Review & Editing. All authors approve the submission.

Corresponding authors

Correspondence to Nunthawan Nowwarote or Thanaphum Osathanon.

Ethics declarations

Ethics approval and consent to participate

The study was approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (Approval number 037/2021). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained.

Consent for publication

Not applicable.

Competing interests

The authors deny any conflicts of interest related to this study.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bulanawichit, W., Sinsareekul, C., Kornsuthisopon, C. et al. Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells. BMC Oral Health 24, 148 (2024). https://doi.org/10.1186/s12903-024-03894-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12903-024-03894-7

Keywords

BMC Oral Health

ISSN: 1472-6831

Contact us