Abstract

Introduction

In dental pulp tissues, stem and progenitor cells are essential regulators of pulpal tissues in response to pulpal inflammation, leading to healing/regeneration processes. When irritation or trauma occurs, stem cells in dental pulp tissues migrate to the injured site and differentiate into odontoblast-like cells, which form reparative dentin^1,2. Applying pulp-capping materials during a restorative operation stimulates dental stem cell responses and provides an appropriate microenvironment to initiate pulp-dentin tissue regeneration and repair.

Various pulp capping materials have been developed for use in current dental practice, such as calcium hydroxide (CH), mineral tricalcium aggregate (MTA), and calcium silicate-based materials^3,4. Recently, alternative materials like calcium phosphate-based materials (CaP) have been introduced as potential pulp-capping materials. CaP has also been proposed in several treatments, such as pulpotomy and dental pulp capping, as it can trigger reparative dentin formation^5–7. The key active components of CaP are calcium ions (Ca²⁺) and inorganic phosphate (P_i); these ions are the building blocks of hydroxyapatite mineral and play essential roles in the formation, maturation, repair, and regeneration of hard tissues such as bone and dentin^8–12.

Accumulating evidence over the last two decades supports P_i as more than an ionic component of hydroxyapatite in mineralized tissues. P_i acts as a signaling molecule for many types of mineralizing cells and their progenitors. P_i alters the expression of differentiation and mineralization-associated genes in mouse MC3T3-E1 pre-osteoblasts¹³, mesenchymal stem cells (MSC)¹⁴, dental pulp stem cells (DPSC)^15,16, and stem cells isolated from human exfoliated deciduous teeth (SHED)¹⁷. Evidence that P_i can inhibit osteoclastogenesis further implies a role in bone remodeling¹⁸, and positive effects of P_i on M2 macrophage polarization suggest an inhibitory role in inflammation¹⁹. Inherited and acquired disorders that reduce circulating P_i levels, such as nutritional rickets, X-linked hypophosphatemia (XLH), and vitamin D metabolism disorders, cause profound defects in odontoblast function and dentin mineralization²⁰. XLH predisposes to dentin hypomineralization, tooth fracture, pulp necrosis, and abscesses²¹.

Because of its cell signaling capabilities and pro-mineralization properties, P_i has been considered a potential bioactive agent to enhance pulp-dentin regeneration in regenerative endodontics^15–17. However, the capability of P_i to regulate SHED cell functions remains unclear and further experiments are necessary to support translational and clinical studies on pulp-dentin regeneration. We investigated the effects of P_i on SHED cell behaviors, including cell proliferation, migration, and differentiation. Underlying regulatory mechanisms were interrogated by RNA sequencing analysis.

Results

Isolation and confirmation of SHED cells

Isolated human dental pulp cells were characterized for mesenchymal stem cell characteristics. Cells expressed CD73, CD90, and CD105, while CD45 was not expressed (Fig. 1A). Under appropriate conditions, cells were able to differentiate into osteoblast-like cells and adipocyte-like cells (Fig. 1B and C). Increased calcium deposition occurred under osteogenic induction conditions (Fig. 1B). Furthermore, adipogenic induction medium promoted lipid droplet formation in the cytoplasm (Fig. 1C). These findings confirm that the isolated cells exhibited dental mesenchymal stem cell characteristics consistent with those reported for SHED cells^22,23.

Open in a separate window

Fig. 1

SHEDs characterization and effects of inorganic phosphate on cell functions. CD45, CD73, CD90, and CD105 expression were examined using flow cytometry (A). For differentiation ability assay, cells were maintained in an osteogenic or adipogenic induction medium. Mineral deposition and intracellular lipid accumulation were evaluated using Alizarin Red S staining on day 14 (B) and Oil Red O staining on day 20 (C), respectively. Cells cultured in a normal growth medium were used as the control (left panels). Cells were treated with 2.5 or 5 mM inorganic phosphate (P_i) for 1, 3, and 7 days. Cell viability was determined using MTT assay (D). Cells were treated with foscarnet for 30 min prior to P_i exposure and cell viability was examined using the MTT assay on day 7 (E). The mRNA expression of Ki67 was determined at 24 h after P_i exposure using real-time polymerase chain reaction (F and G). On day 3, cells were stained with propidium iodide to determine cell cycle progression by flow cytometry, and the quantitative measurement of proliferative cells was illustrated (H). Cells were stained with annexin V/ propidium iodide and further analyzed using flow cytometry. In order to elucidate cell apoptotic numbers, the percentage of cell death was quantified (I). Cell migration was examined using an in vitro scratch assay (J) and the percentage of area closure was calculated (K). ^*P<0.05 compared to the control, ^#P<0.05 compared to the P_i group.

Effects of inorganic phosphate on SHED cell proliferation and cell death

Based on criteria established in previous studies, P_i levels used in the investigation were in a range of 1–8 mM^17,24. Exogenous Pi was required at concentrations higher than 2mM, up to 10mM, to modulate cellular behaviors^14,25,26. However, a report indicated that at least 4 to 6 mM Pi was required for optimal regulation of osteogenic potential in mesenchymal stem cells (MSC)¹⁴. Our previous report indicated that 5mM Pi was responsible for enhancing the osteogenic potential of SHED compared to controls receiving no additional P_i¹⁷. Here, the minimal and optimal requirements of 2.5 and 5 mM Pi were chosen to elucidate the cellular behaviors of SHED. Cell viability/proliferation was assessed using an MTT assay. On days 1 and 3, there were no differences in the number of viable cells between the 2.5 and 5 mM P_i groups. However, by day 7, treatment with 5 mM P_i resulted in fewer viable cells (Fig. 1D). To assess whether P_i import was required for effects on proliferation, cells were incubated for 30 min with foscarnet (Fos), a sodium-phosphate cotransporter inhibitor. Foscarnet pretreatment reversed the inhibitory effect of P_i on cell proliferation at day 7 (Fig. 1E). Expression of Ki67, a marker for cell proliferation, was significantly downregulated by 5 mM P_i, compared with the control at 24 h after treatment (Fig. 1F). Foscarnet pretreatment rescued the P_i-attenuated Ki67 expression in SHED cells (Fig. 1G). The cell cycle analysis was performed on day 3. The results demonstrate no differences in cell number in the G0/G1 phase between the control and P_i treatment groups. The 5 mM Pi treatment group slightly decreased the S phase population. However, there was no significant difference compared with the control (Fig. 1H).

Apoptosis was next assessed in SHED cells by phosphatidylserine exposure, followed by an assessment of membrane permeabilization by flow cytometry^27,28. Early and late apoptotic cell numbers were quantified (Fig. 1I). No differences in early apoptotic cell numbers were induced by either 2.5 or 5 mM P_i groups, compared to the control. However, both 2.5 and 5 mM P_i treated groups significantly increased numbers of late apoptotic cells compared to control.

Inorganic phosphate does not alter SHED cell migration ability

The in vitro scratch assay was performed to analyze cell migration. A linear gap was created through a confluent layer of SHED cells at the center of tissue culture wells. Cells were cultured in serum-free culture medium to restrain cell proliferation²⁹. Cells were then treated with 2.5 or 5 mM P_i. At 24 h, the width of the scratch area decreased to 77.33% in the control condition, while 2.5 and 5 mM P_i treatments showed reduced gap areas of 82.01% and 73.44%, respectively. However, there were no statistically significant differences among groups (Fig. 1J and K).

Inorganic phosphate promotes osteo/odontoblastic differentiation in SHED cells

Previous studies revealed that P_i enhanced the osteogenic potential of MC3T3-E1 pre-osteoblasts^13,18, DPSC¹⁶, and MSC¹⁴. SHED cells underwent an osteogenic induction in the presence of P_i to determine effects on in vitro mineralization capability. Addition of either 2.5 or 5 mM P_i increased mineral deposition compared to controls (Fig. 2A and C). Compared to controls, 5 mM P_i enhanced mRNA expression of DMP1 and DSPP, odontoblast-expressed genes encoding proteins essential for dentin mineralization (Fig. 2B). This phenomenon was attenuated by pre-treated cells with foscarnet (Fig. 2B).

Open in a separate window

Fig. 2

Inorganic phosphate induces mineralization by SHEDs. Cells were treated with 2.5 and 5 mM inorganic phosphate (P_i) and maintained in an osteogenic induction medium containing P_i for 14 days. Mineral deposition was measured using Alizarin Red S staining (A). The graph demonstrates the absorbance of eluted dye at 570 nm and normalized to the control (B). DMP1 and DSPP mRNA expression levels were assayed using real-time polymerase chain reaction at day 7 (C). Cells were pretreated with foscarnet, p38 inhibitor, ERK inhibitor, or JNK inhibitor for 30 min prior to Pi exposure. Cells were maintained in an osteogenic induction medium for 14 days. The mineral deposition was measured using Alizarin Red S staining (D). The graph shows the absorbance of eluted dye measured at 570 nm and normalized to the control. ^*P<0.05 compared to the control.

To investigate the potential mechanisms for regulation of mineralization, cells were pretreated with foscarnet, p38 inhibitor, ERK inhibitor, or JNK inhibitor for 30 min prior to P_i exposure. Cells were then maintained in osteogenic induction for 14 days in the presence of P_i and inhibitors. P_i-induced mineralization was attenuated when cells were treated with foscarnet or p38 inhibitor, but not in the presence of ERK or JNK inhibitors (Fig. 2D). Interestingly, the ERK inhibitor promoted increased mineralization compared to the controls.

Inorganic phosphate attenuates adipogenic differentiation of SHED cells

Increased P_i appeared to increase the expression of odontoblast markers and in vitro mineral deposition by SHED cells. We tested the effects of Pi on adipogenic differentiation to better understand the impact on cell fate. SHED cells were incubated in an adipogenic induction medium in the presence of 2.5 and 5 mM P_i. Adipocyte differentiation-related genes, including PPARγ, LPL, and C/EBP-α, were analyzed on day 8. PPARγ mRNA levels were significantly downregulated by 5 mM P_i treatment compared to controls (Fig. 3A), though P_i did not affect LPL and C/EBP-α mRNA expression (Fig. 3B and C). Intracellular lipid accumulation was observed using Oil Red O staining at day 20. Reduced lipid accumulation was noted under P_i treatment (Fig. 3D).

Open in a separate window

Fig. 3

Inorganic phosphate attenuates adipocyte differentiation of SHEDs. Cells were treated with 2.5 and 5 mM P_i and maintained in adipogenic induction medium. PPAR-γ, C/EBP-α, and LPL mRNA expression levels were analyzed on day 8 using real-time polymerase chain reaction (A-C). The intracellular lipid accumulation was determined using Oil Red O staining at day 20 (D). ^*P<0.05 compared to the control.

Differential transcriptomic profiling after induction of pi

Selective gene analyses revealed P_i regulates odontoblastic and adipogenic genes in SHED cells. In order to identify underlying regulatory mechanisms, SHED cells were treated with 5 mM P_i and total RNA was collected and subjected to RNA sequencing analysis for differential mRNA expression profiling. The top 50 differentially expressed genes identified from P_i treatment were illustrated as a heat map (Fig. 4A). Volcano plots demonstrated the distribution of expressed genes (Fig. 4C). Gene ontology analysis of the differentially expressed genes was enriched for biological process (red bar), cellular component (blue bar), and molecular function (green bar) (Fig. 4C).

Open in a separate window

Fig. 4

Transcriptomic profile of inorganic phosphate-treated SHEDs. Cells were treated with 5 mM P_i for 24 h. RNA was extracted and subjected to RNA sequencing analysis for differential gene expression. The top 50 significantly expressed genes were included in the heat map (A). The distribution of all significantly differential genes was shown in the volcano plot (B). Differentially expressed genes were investigated in GO term analysis (C). Functional and pathway enrichments were analyzed in KEGG (D) and Reactome pathway database (E). Control1 and Control2 as representatives for Control condition from 2 different donors, Pi1 and Pi2 as representatives for 5mM Pi treated conditions from 2 different donors, ^*P<0.05 compared to the control.

From pathway enrichment analysis by KEGG, The significant genes were identified to be enriched in the VEGF signaling pathway and MAPK signaling pathway (Fig. 4D). For Reactome enrichment analysis, the upregulated genes were enriched in several pathways (Fig. 4E), including PTK6 promotes HIF1A stabilization, MET activates PI3K/AKT signaling, activated NTRK3 signals through PI3K, while the downregulated genes were organized in pathways, including ESR-mediated signaling, signaling by receptor tyrosine kinase (Fig. 4E).

Discussion

P_i is a free form of phosphorus complex existing in extracellular fluid⁹ and plays essential roles in bone and tooth development^30,31. P_i concentrations are regulated at both the systemic and local levels. Systemically, the gut-kidney-bone axis controls circulating P_i levels through endocrine mechanisms employing vitamin D, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23)^20,32. The normal circulating range of P_i typically stays within 0.5–1.5 mM³³. At the local cell and tissue levels, P_i levels are controlled by a host of enzymes and transporters that work cooperatively and antagonistically with one another. Tissue non-specific alkaline phosphatase (TNAP) is a cell membrane-bound enzyme that generates P_i ions from inorganic pyrophosphate and other phosphorylated substrates³⁴. Orphan phosphatase 1 (PHOSPHO1) generates P_i within the confines of matrix vesicles, which promotes the initiation of mineralization in some calcified tissues³⁵. P_i transporters regulate the import of Pi into cells and play roles in the sensing and signaling capabilities of P_i in mineralized tissue cells³⁶. Dysregulation of either systemic or local P_i metabolism results in dramatic effects on cell function and tissue mineralization^21,32,37.

P_i was shown in prior publications to induce signaling in a range of mineralized tissue cells^{13,25,38–40}. In the present study, we investigated effects of P_i at concentrations of 2.5 and 5 mM, consistent with the range of previous publications^14,16,17,26. While some cell populations were reported to have sensitivity to high P_i^41,42, these concentrations were non-toxic to SHED cells. Similarly, a recent report demonstrates that mesenchymal stem cells survive in the presence of 10 mM Pi¹⁴.

In addition to being a regulator of mineralization, P_i is necessary for numerous cell functions, including cell growth and proliferation, through the phosphorylation of intracellular enzymes. In non-mineralizing cells, the increased extracellular P_i levels promoted P_i influx into the cell, resulting in increased cell proliferation⁴³. P_i activates AKT phosphorylation and the ERK signaling cascade, leading to the enhancement of cell proliferation⁴⁴. Some studies indicate that PiT1, a sodium-P_i co-transporter, participates in the enhancement of cell proliferation. PiT1 silencing decreased P_i influx, leading to decreased HeLa cell proliferation. However, cell proliferation inhibition of PiT1 silencing does not depend on the P_i transport function; the functional deletion of the P_i transporter activated the P38 signaling pathway^45,46.

The present study demonstrated that SHED cell number was decreased in P_i-treated groups at day 7, compared with the control. Furthermore, reduction of Ki67 expression was also noted in the 5 mM P_i-treated cells. These effects were rescued when cells were pretreated with foscarnet, an inhibitor of P_i transport. However, there was no significant difference in the S or G2/M populations in those cells treated with P_i, compared with the control. Consistent with mineralizing cell, the impairment of proliferative activity in cells exposed to P_i has previously been reported in such as osteoblast and chondroblast^24,41. Further, our present study found the influence of Pi on cell apoptosis. P_i-induced mineralizing cell apoptosis occurs during the resorptive activity of osteoclasts, leading to the release of P_i⁴⁷. Mechanistically, P_i-induced bone cell apoptosis promotes Ca²⁺ influx, resulting in mitochondria dysfunction and increasing levels of reactive oxygen species (ROS)^47–50.

Our results showed that P_i promoted mineral deposition and induced DSPP and DMP1 mRNA expression in SHED, similar to previous reports^16,51. DMP1 and DSSP have been used as markers for odonto/osteogenic differentiation^52,53. The increased mineralization seen in the present study may not be from the direct precipitation of the binding between phosphate ions and calcium ions available in the medium. Previous reports demonstrated that culture medium supplemented with inorganic phosphate did not show increased mineralization in the condition without cells. In this regard, adding up to 10 mM Pi in the culture medium did not mark an increase in alizarin red S staining in the condition without cells¹⁴. This evidence implies that the significant increase of mineralization upon treated cells with inorganic phosphate occurred via biological processes. This observation was noted in other publications on other cell types^14,54. However, several biological hypotheses need further clarification, for example, the possible calcium depletion effects on cell responses and the change of cell behaviors due to the potential apoptotic effect of Pi.

This finding supports that P_i induces odonto/osteogenic differentiation in SHED. The mitogen-activated protein kinases (MAPKs) signaling is indeed one of remarkable pathway for growth factor-induced osteogenic differentiation⁵⁵. A member family of MAPK, including extracellular signal-related kinases (ERKs), c-Jun N terminal kinases (JNKs), and P38 can be activated by MAP3K, upstream MAPK activator, phosphorylating MAPK family⁵⁶.

As for Pi activation-regulated pathways, Pi induces the ERK1/2 signaling pathway in the upregulation of OPN, matrix Gal protein, and DMP1 by MC3T3-E1 pre-osteoblasts and dental pulp stem cells (DPSC)^13,16. Our study demonstrated that P38 inhibition attenuated the effects of Pi-induced mineralization in SHEDs. P38 was shown to be part of osteogenic differentiation inductive pathways in mesenchymal stem cells⁵⁷. ERK and JNK inhibitors did not reverse Pi-induced mineralization, whereas ERK inhibition enhanced Pi-induced mineralization in SHED, consistent with previous study⁵⁸. Since ERKs and P38 pathways are a downstream of MAP3K, we speculate that the ERK inhibition may increase P38 activation, resulting in the enhancement of mineral deposition in SHEDs by an increase of P38 activation. Here, this finding demonstrated that Pi is responsible for promoting mineral deposition in SHEDs though P38 and ERK signalling pathway. However, the further study on targeting to P38 and ERK pathway to enhance osteogenic potential in SHEDs needed investigation.

During the adipogenic differentiation process, MSC expresses specific gene markers, including PPAR-γ, C/EBP-α, and LPL⁵⁹. In our study, P_i treatment inhibited the expression of PPAR-γ and reduced intracellular lipid droplet formation. A previous study found that adipogenic potential was regulated by the MAPK family. ERK and JNK signaling have been demonstrated as activators, while P38 signaling has been shown to inhibit adipogenesis⁶⁰. This finding implicates that, in the presence of elevated P_i, SHED cells are induced to commit toward osteogenic differentiation rather than adipogenic differentiation.

We used transcriptomic analysis to further investigate signalling effects of P_i on SHED cell differentiation. The expression of RELB gene was downregulated in Pi treated group. A recent report has showed that abolished expression of RelB have promoted cell apoptosis⁶¹ and inhibited cell proliferation⁶², similar to our finding. Additionally, Reactome enrichment analysis showed that significantly upregulated genes were enriched in signaling by MAPK mutant, which is relevant to DUSP6. DUSP6 is well-recognized as MKP3, a specific phosphatase for ERK signaling in skeleton development⁶³. Additionally, DUSP6 has played an essential role in maintaining phosphorylated P38 signaling⁶⁴. A study has revealed that the DUSP6 depletion in mouse models caused dwarf-consisted skeleton abnormalities⁶⁵. In this regard, inducible DUSP6 expression has been reported to inhibit cell proliferation in hair follicle stem cells⁶⁴ and induce cell apoptosis⁶⁶. These findings were consistent with the in vitro investigation demonstrating that P_i promoted mineralization via the P38 signaling pathway but induced apoptosis in SHED.

As for dental materials, P_i is based on most pulp capping materials, along with calcium. Less Pi release may not be enough to influence neighboring cells. Recently, calcium phosphate-based material, an alternative dental material, can provide persistently calcium and phosphate ions^8–12. Both calcium and phosphate ions were reported to be key modulators for osteogenic differentiation. Consistent with our observation, Pi was shown to have influences in the induction of early-stage osteogenic inducer genes, for example, the DMP1 gene⁶⁷. On the contrary, calcium ion has a role in promotion of late-stage osteogenic genes^68,69. We speculate that the presence of both calcium and phosphate ions may function in different steps of tissue mineralization and also accelerate mineralized tissue repair.

Mesenchymal stem cells (MSC) possess the self-renewal and multipotential differentiation ability. Numerous surface markers have been proposed to identify the MSC population, including CD105, CD90, CD73, CD44, CD166, CD29, STRO-1, CD146, and CD271⁷⁰. However, the International Society for Cellular Therapy set three minimum criteria to identify MSC²². First, MSC must be the adherent cells on tissue culture surfaces. Second, MSC must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules. Third, MSC must possess multipotential differentiation. In the present study, we evaluated the expression of CD105, CD90, and CD73, which are considered the minimum and common MSC surface markers⁷⁰. In addition, we demonstrated the mineral deposition and intracellular lipid accumulation ability after cultured in osteogenic and adipogenic induction medium, respectively. Together, our data illustrated the MSC characteristics of the cells isolated from the remaining dental pulp tissues of the exfoliated deciduous teeth.

In summary, our data derived from in vitro experiments and mRNA expression profiles demonstrated strong evidence that P_i regulates cell proliferation and differentiation via different mechanisms in SHEDs. The present study reveals the effects of P_i induced on the osteo/odontogenic ability of SHEDs via the P38 signaling (Fig. 5). Our finding contributes insight into the mechanism of P_i on SHED in regulating regenerative potentials. Additional experiments are needed to investigate translational implications and clinical potential for P_i to positively promote SHED cell differentiation and tertiary dentin formation after dental procedures.

Open in a separate window

Fig. 5

Schematic diagram of inorganic phosphate’s effects on regenerative responses and multipotency of SHEDs. The presence of P_i regulated SHED cell number via the promotion of cell apoptosis. Additionally, P_i reduced the Ki67 gene expression. P_i induced the gene expression of DMP1 and DSPP to undergo osteogenic induction. Also, P_i enhanced the osteogenic potential of SHEDs via the p38 pathway. Created with Biorender.com.

Materials & methods

Electronic supplementary material

Below is the link to the electronic supplementary material.

Acknowledgements

Author contributions

R.S. data acquisition, data analysis, data interpretation, drafted, and critically revised the manuscript; N.N. contributed to data acquisition, data analysis, data interpretation, and critically revised the manuscript; C.K. contributed to data analysis, data interpretation, drafted, and critically revised the manuscript; W.S. contributed to resources, data interpretation and critically revised the manuscript; B.L.F contributed to data interpretation and critically revised the manuscript; H.E. and T.O. contributed to the study conceptualization, experimental design, and data interpretation and critically revised the manuscript. All authors critically revised the paper and gave final approval for publication.

Data availability

The datasets generated during and/or analyzed during the current study are available in the NCBI’s Gene Expression Omnibus (GSE266257). All data generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Footnotes

Contributor Information

Hiroshi Egusa, Email: pj.ca.ukohot@2e.asuge.ihsorih.

Thanaphum Osathanon, Email: ht.ca.aluhc@o.muhpanaht.

References

1. Dong, Q. et al. Dental Pulp Stem cells: isolation, characterization, expansion, and Odontoblast differentiation for tissue Engineering. Methods Mol. Biol. 1922, 91–101 (2019). [Abstract] [Google Scholar]

2. Xiao, X. et al. Characterization of odontogenic differentiation from Human Dental Pulp Stem cells using TMT-Based proteomic analysis. Biomed. Res. Int. 2020, 3871496 (2020). [Europe PMC free article] [Abstract] [Google Scholar]

3. Yaemkleebbua, K., Osathanon, T., Nowwarote, N., Limjeerajarus, C. N. & Sukarawan, W. Analysis of hard tissue regeneration and wnt signalling in dental pulp tissues after direct pulp capping with different materials. Int. Endod J. 52 (11), 1605–1616 (2019). [Abstract] [Google Scholar]

4. Manaspon, C. et al. Human dental pulp stem cell responses to different dental pulp capping materials. BMC Oral Health. 21 (1), 209 (2021). [Europe PMC free article] [Abstract] [Google Scholar]

5. Davaie, S., Hooshmand, T. & Ansarifard, S. Different types of bioceramics as dental pulp capping materials: a systematic review. Ceram. Int. 47 (15), 20781–20792 (2021). [Google Scholar]

6. TOBIAS, R. S. & PLANT, C. G. Reduction in pulpal inflammation beneath surface-sealed silicates. Int. Endod. J. 15 (4), 173–180 (1982). [Abstract] [Google Scholar]

7. Huang, H. et al. Calcium phosphate cement promotes odontoblastic differentiation of Dental Pulp cells in Vitro and in vivo. Coat. [Internet] ; 12(4). (2022).

8. Arkady, M. Inorganic phosphate: the backbone of life. In: (eds Sadia, A., Mohammad Shaheer, A. & Hyung-Shik, S.) Functional Phosphate Materials and Their Applications. Rijeka: IntechOpen; p. (2023). Ch. 2. [Google Scholar]

9. Iheagwara, O. S., Ing, T. S., Kjellstrand, C. M. & Lew, S. Q. Phosphorus, phosphorous, and phosphate. Hemodial. Int. 17 (4), 479–482 (2013). [Abstract] [Google Scholar]

10. Carpenter, T. et al. Principles of Bone Biology. (2020).

11. Bottini, M. et al. Matrix vesicles from chondrocytes and osteoblasts: their biogenesis, properties, functions and biomimetic models. Biochim. et Biophys. Acta (BBA)-General Subj. 1862 (3), 532–546 (2018). [Europe PMC free article] [Abstract] [Google Scholar]

12. Michigami, T. & Ozono, K. Roles of phosphate in Skeleton. Front. Endocrinol. 10, 180 (2019). [Europe PMC free article] [Abstract] [Google Scholar]

13. Beck, G. R. Jr., Zerler, B. & Moran, E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc. Natl. Acad. Sci. U S A. 97 (15), 8352–8357 (2000). [Europe PMC free article] [Abstract] [Google Scholar]

14. Lin, H. et al. Effect of inorganic phosphate on migration and osteogenic differentiation of bone marrow mesenchymal stem cells. BMC Dev. Biol. 21 (1), 1 (2021). [Europe PMC free article] [Abstract] [Google Scholar]

15. Jomana, A. & Laisheng, C. Inorganic phosphate effect of on Human Dental Pulp cell cultures. Int. J. Mater. Sci. Appl. 8 (3), 40–46 (2019). [Google Scholar]

16. Tada, H., Nemoto, E., Foster, B. L., Somerman, M. J. & Shimauchi, H. Phosphate increases bone morphogenetic protein-2 expression through cAMP-dependent protein kinase and ERK1/2 pathways in human dental pulp cells. Bone. 48 (6), 1409–1416 (2011). [Abstract] [Google Scholar]

17. Nowwarote, N., Sukarawan, W., Pavasant, P., Foster, B. L. & Osathanon, T. Basic fibroblast growth factor regulates phosphate/pyrophosphate regulatory genes in stem cells isolated from human exfoliated deciduous teeth. Stem Cell Res. Ther. 9 (1), 345 (2018). [Europe PMC free article] [Abstract] [Google Scholar]

18. Kanatani, M., Sugimoto, T., Kano, J., Kanzawa, M. & Chihara, K. Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity. J. Cell. Physiol. 196 (1), 180–189 (2003). [Abstract] [Google Scholar]

19. Gu, Y. et al. A biphasic calcium phosphate cement enhances dentin regeneration by dental pulp stem cells and promotes macrophages M2 phenotype in vitro. Tissue Eng. Part A. 27 (17–18), 1113–1127 (2021). [Abstract] [Google Scholar]

20. Foster, B. L., Chu, E. Y. & Hujoel, P. P. Vitamin D in dentoalveolar and oral health. In: (eds Hewison, M., Bouillon, R., Giovannucci, E., Meyer, M. & Welsh, J.) Feldman and Pike’s Vitamin D. 1. 5th ed. London: Academic; 453–484. (2023). [Google Scholar]

21. Clayton, D. et al. Mineralization defects in the primary dentition Associated with X-Linked hypophosphatemic rickets. JBMR Plus. 5 (4), e10463 (2021). [Europe PMC free article] [Abstract] [Google Scholar]

22. Dominici, M. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8 (4), 315–317 (2006). [Abstract] [Google Scholar]

23. Rossato, C. et al. Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis. Biologicals 49, (2017). [Abstract]

24. Ali Akbari Ghavimi, S. et al. Calcium and phosphate ions as simple signaling molecules with versatile osteoinductivity. Biomed. Mater. 13 (5), 055005 (2018). [Abstract] [Google Scholar]

25. Beck, G. R. Jr., Moran, E. & Knecht, N. Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp. Cell. Res. 288 (2), 288–300 (2003). [Abstract] [Google Scholar]

26. Rahabi-Layachi, H., Ourouda, R., Boullier, A., Massy, Z. A. & Amant, C. Distinct effects of Inorganic phosphate on cell cycle and apoptosis in human vascular smooth muscle cells. J. Cell. Physiol. 230 (2), 347–355 (2015). [Abstract] [Google Scholar]

27. Lee, S. H., Meng, X. W., Flatten, K. S., Loegering, D. A. & Kaufmann, S. H. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell. Death Differ. 20 (1), 64–76 (2013). [Europe PMC free article] [Abstract] [Google Scholar]

28. De Schutter, E., Cappe, B., Wiernicki, B., Vandenabeele, P. & Riquet, F. B. Plasma membrane permeabilization following cell death: many ways to dye! Cell. Death Discovery. 7 (1), 183 (2021). [Europe PMC free article] [Abstract] [Google Scholar]

29. Rodriguez, L. G., Wu, X. & Guan, J. L. Wound-healing assay. Methods Mol. Biol. 294, 23–29 (2005). [Abstract] [Google Scholar]

30. Murshed, M., Harmey, D., Millan, J. L., McKee, M. D. & Karsenty, G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19 (9), 1093–1104 (2005). [Europe PMC free article] [Abstract] [Google Scholar]

31. Foster, B. L. et al. Phosphate: known and potential roles during development and regeneration of teeth and supporting structures. Birth Defects Res. Part. C: Embryo Today: Reviews. 84 (4), 281–314 (2008). [Europe PMC free article] [Abstract] [Google Scholar]

32. Foster, B. L., Nociti, F. H. Jr. & Somerman, M. J. The rachitic tooth. Endocr. Rev. 35 (1), 1–34 (2014). [Europe PMC free article] [Abstract] [Google Scholar]

33. Serna, J. & Bergwitz, C. Importance of dietary phosphorus for bone metabolism and healthy aging. Nutrients. 12 (10), 3001 (2020). [Europe PMC free article] [Abstract] [Google Scholar]

34. Millan, J. L. & Whyte, M. P. Alkaline phosphatase and Hypophosphatasia. Calcif Tissue Int. 98 (4), 398–416 (2016). [Europe PMC free article] [Abstract] [Google Scholar]

35. Millan, J. L. The role of phosphatases in the initiation of skeletal mineralization. Calcif Tissue Int. 93 (4), 299–306 (2013). [Europe PMC free article] [Abstract] [Google Scholar]

36. Figueres, L., Beck-Cormier, S., Beck, L. & Marks, J. The complexities of Organ crosstalk in phosphate homeostasis: time to put phosphate sensing back in the limelight. Int. J. Mol. Sci. 22(11), (2021). [Europe PMC free article] [Abstract]

37. Mohamed, F. F. et al. Perspective on dentoalveolar manifestations resulting from PHOSPHO1 loss-of-Function: a form of Pseudohypophosphatasia? Front. Dent. Med. 3, (2022). [Europe PMC free article] [Abstract]

38. Beck, G. R. Jr. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J. Cell. Biochem. 90 (2), 234–243 (2003). [Abstract] [Google Scholar]

39. Foster, B. L. et al. Phosphate: known and potential roles during development and regeneration of teeth and supporting structures. Birth Defects Res. C Embryo Today. 84 (4), 281–314 (2008). [Europe PMC free article] [Abstract] [Google Scholar]

40. Foster, B. L. et al. Regulation of cementoblast gene expression by inorganic phosphate in vitro. Calcif Tissue Int. 78 (2), 103–112 (2006). [Abstract] [Google Scholar]

41. Meleti, Z., Shapiro, I. M. & Adams, C. S. Inorganic phosphate induces apoptosis of osteoblast-like cells in culture. Bone. 27 (3), 359–366 (2000). [Abstract] [Google Scholar]

42. Mansfield, K., Rajpurohit, R. & Shapiro, I. M. Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J. Cell. Physiol. 179 (3), 276–286 (1999). [Abstract] [Google Scholar]

43. Tan, X., Xu, X., Zeisberg, E. M. & Zeisberg, M. High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation. Biochem. Biophys. Res. Commun. 472 (3), 459–464 (2016). [Abstract] [Google Scholar]

44. Chang, S-H. et al. Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells. Am. J. Respir. Cell Mol. Biol. 35 (5), 528–539 (2006). [Europe PMC free article] [Abstract] [Google Scholar]

45. Beck, L. et al. Identification of a Novel function of PiT1 critical for cell proliferation and Independent of its phosphate transport Activity*♦. J. Biol. Chem. 284 (45), 31363–31374 (2009). [Europe PMC free article] [Abstract] [Google Scholar]

46. Kongsfelt, I. B., Byskov, K., Pedersen, L. E. & Pedersen, L. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion. Exp. Cell. Res. 326 (1), 57–67 (2014). [Abstract] [Google Scholar]

47. Goretti Penido, M. & Alon, U. S. Phosphate homeostasis and its role in bone health. Pediatr. Nephrol. 27 (11), 2039–2048 (2012). [Europe PMC free article] [Abstract] [Google Scholar]

48. Lacerda-Abreu, M. A. & Meyer-Fernandes, J. R. Extracellular inorganic phosphate-Induced release of reactive oxygen species: roles in physiological processes and Disease Development. Int. J. Mol. Sci. 22(15), (2021). [Europe PMC free article] [Abstract]

49. Schuster, S. M. & Olson, M. S. The regulation of pyruvate dehydrogenase in isolated beef heart mitochondria. The role of calcium, magnesium, and permeant anions. J. Biol. Chem. 249 (22), 7159–7165 (1974). [Abstract] [Google Scholar]

50. Okamoto, T., Taguchi, M., Osaki, T., Fukumoto, S. & Fujita, T. Phosphate enhances reactive oxygen species production and suppresses osteoblastic differentiation. J. Bone Min. Metab. 32 (4), 393–399 (2014). [Abstract] [Google Scholar]

51. Merametdjian, L. et al. Expression of phosphate transporters in optimized cell culture models for dental cells biomineralization. Bull. Group. Int. Rech Sci. Stomatol. Odontol. 53 (1), e16 (2016). [Abstract] [Google Scholar]

52. Ching, H. S., Luddin, N., Rahman, I. A. & Ponnuraj, K. T. Expression of Odontogenic and osteogenic markers in DPSCs and SHED: a review. Curr. Stem Cell. Res. Ther. 12 (1), 71–79 (2017). [Abstract] [Google Scholar]

53. Lim, D., Wu, K. C., Lee, A., Saunders, T. L. & Ritchie, H. H. DSPP dosage affects tooth development and dentin mineralization. PLoS One. 16 (5), e0250429 (2021). [Europe PMC free article] [Abstract] [Google Scholar]

54. Jono, S. et al. Phosphate regulation of vascular smooth muscle cell calcification. Circ. Res. 87 (7), E10–E17 (2000). [Abstract] [Google Scholar]

55. Tsang, E. J., Wu, B. & Zuk, P. MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro. Connect. Tissue Res. 59 (2), 129–146 (2018). [Europe PMC free article] [Abstract] [Google Scholar]

56. Yang, X. et al. MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling. J. Orthop. Translat. 38, 98–105 (2023). [Europe PMC free article] [Abstract] [Google Scholar]

57. Doan, T. K. P. et al. Inhibition of JNK and ERK pathways by SP600125- and U0126-enhanced osteogenic differentiation of bone marrow stromal cells. Tissue Eng. Regenerative Med. 9 (6), 283–294 (2012). [Google Scholar]

58. Kono, S. et al. Erk pathways negatively regulate matrix mineralization. Bone. 40 (1), 68–74 (2007). [Abstract] [Google Scholar]

59. Robert, A. W., Marcon, B. H., Dallagiovanna, B. & Shigunov, P. Adipogenesis, Osteogenesis, and chondrogenesis of human mesenchymal Stem/Stromal cells: a comparative Transcriptome Approach. Front. Cell. Dev. Biology  8, (2020). [Europe PMC free article] [Abstract]

60. Qi, R. et al. Expressions and Regulatory effects of P38/ERK/JNK mapks in the adipogenic trans-differentiation of C2C12 myoblasts. Cell. Physiol. Biochem. 44 (6), 2467–2475 (2017). [Abstract] [Google Scholar]

61. Wang, M. et al. RelB sustains endocrine resistant malignancy: an insight of noncanonical NF-κB pathway into breast Cancer progression. Cell. Communication Signal. 18, 1–17 (2020). [Europe PMC free article] [Abstract] [Google Scholar]

62. Thakar, N. Y., Ovchinnikov, D. A., Hastie, M. L., Gorman, J. & Wolvetang, E. J. RELB alters proliferation of human pluripotent stem cells via IMP3-and LIN28-mediated modulation of the expression of IGF2 and other cell-cycle regulators. Stem. Cells. Dev. 24 (16), 1888–1900 (2015). [Abstract] [Google Scholar]

63. Ma, J., Yu, X., Guo, L. & Lu, S. H. DUSP6, a tumor suppressor, is involved in differentiation and apoptosis in esophageal squamous cell carcinoma. Oncol. Lett. 6 (6), 1624–1630 (2013). [Europe PMC free article] [Abstract] [Google Scholar]

64. Wang, Q. et al. DUSP6 inhibits the proliferation of hair follicle stem cells (HFSCs) in vitro. Animal Biotechnol. 34 (4), 1223–1231 (2023). [Abstract] [Google Scholar]

65. Li, C., Scott, D. A., Hatch, E., Tian, X. & Mansour, S. L. Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development (2007). [Europe PMC free article] [Abstract]

66. Purcell, N. H. et al. Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo. Proceedings of the National Academy of Sciences. 104(35), 14074-9 (2007). [Europe PMC free article] [Abstract]

67. Narayanan, K. et al. Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca2+store. J. Biol. Chem. 278 (19), 17500–17508 (2003). [Abstract] [Google Scholar]

68. An, S. et al. Short-term effects of calcium ions on the apoptosis and onset of mineralization of human dental pulp cells in vitro and in vivo. Int. J. Mol. Med. 36 (1), 215–221 (2015). [Europe PMC free article] [Abstract] [Google Scholar]

69. An, S., Gao, Y., Ling, J., Wei, X. & Xiao, Y. Calcium ions promote osteogenic differentiation and mineralization of human dental pulp cells: implications for pulp capping materials. J. Mater. Science: Mater. Med. 23 (3), 789–795 (2012). [Abstract] [Google Scholar]

70. Fonseca, L. N. et al. Cell surface markers for mesenchymal stem cells related to the skeletal system: a scoping review. Heliyon. 9 (2), e13464 (2023). [Europe PMC free article] [Abstract] [Google Scholar]

71. Kornsuthisopon, C. et al. Human dental pulp stem cells derived extracellular matrix promotes mineralization via Hippo and wnt pathways. Sci. Rep. 14 (1), 6777 (2024). [Europe PMC free article] [Abstract] [Google Scholar]

72. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25 (4), 402–408 (2001). [Abstract] [Google Scholar]

73. Andrews, S. FastQC: a quality control tool for high throughput sequence data. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc. (2018).

74. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14 (4), R36 (2013). [Europe PMC free article] [Abstract] [Google Scholar]

75. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7 (3), 562–578 (2012). [Europe PMC free article] [Abstract] [Google Scholar]

76. Xia, J., Gill, E. E. & Hancock, R. E. NetworkAnalyst for statistical, visual and network-based meta-analysis of gene expression data. Nat. Protoc. 10 (6), 823–844 (2015). [Abstract] [Google Scholar]