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. 2026 Jul 1;21(3):1948–1957. doi: 10.1016/j.jds.2025.07.3564

Dual effects of 1α,25-dihydroxyvitamin D3 on inflammation and osteogenic differentiation in lipopolysaccharide-stimulated dental pulp stem cells

Seong Eun Song a,†, Giyoung Yun b,†, Cheul-Hong Kim b,c, Ji-Young Yoon b,c, Hee Young Kim d,e, Hyae Jin Kim d,f, Eun-Jung Kim b,c,*
PMCID: PMC13536342  PMID: 42689044

Abstract

Background/purpose

Vitamin D is traditionally known for bone metabolism and also exerts immunomodulatory effects. Dental pulp stem cells (DPSCs) are essential for pulp repair but are impaired under lipopolysaccharide (LPS)-induced inflammation. Therefore, we examined the dual effects of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on inflammation and osteogenic differentiation in DPSCs.

Materials and methods

Human DPSCs were pretreated with 1α,25(OH)2D3 (0.001–1 μM) and stimulated with LPS (1 μg/mL). Thereafter, cell viability was assessed using the 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide assay. Expression of interleukin (IL)-1β, IL-8, and cyclooxygenase-2 (COX-2) was measured using real-time quantitative PCR and Western blot. Moreover, osteogenic activity was evaluated via alkaline phosphatase (ALP) staining. Mitogen-activated protein kinase signaling was analyzed via Western blot, and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) inhibitors were applied to confirm mechanistic involvement.

Results

The 1α,25(OH)2D3 did not affect DPSC viability under inflammatory conditions. However, the compound markedly suppressed LPS-induced IL-8 and COX-2 expression, with modest effects on IL-1β. LPS markedly reduced ALP activity, which was dose-dependently restored by 1α,25(OH)2D3. Mechanistically, 1α,25(OH)2D3 induced transient phosphorylation of ERK and JNK but not that of p38. Furthermore, Inhibition of ERK or JNK abrogated the osteogenic rescue, thereby confirming their essential roles.

Conclusion

The 1α,25(OH)2D3 exerts dual actions in LPS-stimulated DPSCs by attenuating inflammatory mediators and promoting osteogenic differentiation through ERK/JNK signaling. These findings suggest its promising potential as a therapeutic adjunct in regenerative endodontics under inflamed conditions.

Keywords: c-Jun N-Terminal kinase, Dental pulp stem cell, Extracellular signal-regulated kinase, Inflammation, Osteogenic differentiation, Vitamin D

Introduction

Vitamin D is a secosteroid hormone traditionally recognized for its role in calcium homeostasis and bone metabolism and has emerged as a critical modulator of immune responses.1 The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exerts potent anti-inflammatory effects by suppressing the expression of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α, while simultaneously promoting the expression of anti-inflammatory mediators, such as IL-10.2,3 Notably, vitamin D signaling inhibits nuclear factor-kappa B (NF-κB) activation and reduces toll-like receptor (TLR)-mediated inflammatory responses, which underscores its potential therapeutic value in inflammatory and autoimmune conditions.4,5 These findings have been extended to various tissue models, including periodontal and dental pulp tissues, where vitamin D supplementation attenuated inflammation-induced damage and supported tissue homeostasis.6

Dental pulp stem cells (DPSCs) are derived from the neural crest lineage and reside within the dental pulp tissue. The cells possess robust regenerative and immunomodulatory properties that are critical for maintaining pulpal homeostasis.7,8 Under normal conditions, DPSCs contribute to tissue repair by differentiation into odontoblast-like and osteoblast-like cells.7 However, under pathological conditions, particularly bacterial infections with lipopolysaccharide (LPS) release, DPSCs become exposed to a pro-inflammatory microenvironment that disrupts their regenerative potential.9,10 Consequently, chronic or unresolved inflammation in the pulp may lead to irreversible pulpitis, necrosis, and eventual tooth loss.11 Thus, strategies that modulate inflammation while preserving or enhancing DPSC differentiation are of substantial clinical relevance in endodontic and regenerative therapies.12 Despite growing interest in immunomodulatory agents, the potential of vitamin D to regulate inflammation-induced dysfunction in DPSCs remains underexplored.13

LPS is an endotoxin derived from gram-negative bacteria, such as Porphyromonas gingivalis, activates TLR4 or TLR2 on DPSCs and gingival fibroblasts, thereby initiating downstream NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. This activation upregulates pro-inflammatory cytokines, such as IL-6 and IL-8, and matrix metalloproteinases and other mediators that disrupt tissue integrity and promote alveolar bone resorption.14,15 In human DPSCs, LPS stimulation through TLR4/myeloid differentiation primary response 88-dependent cascades increased IL-8 expression and triggered p38 MAPK phosphorylation, which exacerbates inflammation and also paradoxically modulates odontogenic differentiation.16,17 Thus, applying LPS in vitro simulates the inflammatory milieu seen in periodontitis or pulpitis and also provides a mechanistic model to evaluate the mechanism by which vitamin D attenuates inflammatory signaling while supporting osteo/odontogenic differentiation under pathophysiological conditions.

Therefore, this study aimed to investigate the dual effects of 1α,25(OH)2D3 on LPS-induced inflammatory responses and osteogenic differentiation in DPSCs. Specifically, the study hypothesized that 1α,25(OH)2D3 attenuates pro-inflammatory cytokine expression and modulates MAPK signaling activation, while concurrently promoting or preserving the osteogenic potential of DPSCs under inflammatory conditions. Understanding these combined effects may provide valuable insights into the therapeutic utility of 1α,25(OH)2D3 in inflammation control and tissue regeneration for dental applications.

Material and methods

Cell culture and treatment

DPSCs were purchased from Lonza Bioscience (PT-5025, Basel, Switzerland) and cultured in alpha modified Eagle’s medium (α-MEM) supplemented with 10 % (v/v) fetal bovine serum (Gibco, Waltham, MA, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Biowest, Nuaillé, France). DPSCs were maintained in a humidified atmosphere at 5 % CO2 and 37 °C. DPSCs were seeded in a culture plate and incubated at 37 °C for 24 h. Thereafter, the cells were pretreated with 1α,25(OH)2D3 (0.001–1 μM; Sigma–Aldrich, St. Louis, MO, USA) for 2 h and exposed to LPS (1 μg/mL; Sigma–Aldrich) for 24 h. In pathway inhibition experiments, DPSCs were pretreated with the MAPK inhibitors U0126 (extracellular signal-regulated kinase [ERK]1/2; 20 μM; Cell Signaling Technology, Danvers, MA, USA) and SP600125 (c-Jun N-terminal kinase [JNK]; 25 μM; Tocris Bioscience, Bristol, UK) 90 min before treatment with 1α,25(OH)2D3.

Cell viability assay

The viability and proliferation of cells were determined using Thiazolyl Blue Tetrazolium Bromide (MTT; Biosesang, Seongnam, Korea). First, DPSCs were grown from 80 to 90 % confluence in 96-well plates and then treated with 1α,25(OH)2D3 (0.001–1 μM) and LPS (1 μg/mL). Subsequently, MTT solution (100 μL) was added to each well, and the plate was incubated at 37 °C for 4 h. Absorbance was measured at 570 nm using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate.

Real-time quantitative PCR

Collected DPSC pellets were incubated with RiboEx reagent (0.5 mL; GeneAll, Seoul, Korea) in accordance with the manufacturer’s instructions. Reverse transcriptions were performed using a HiSenScript™ RH [-] RT PreMix Kit (INtRON Biotechnology, Seongnam, Korea). Quantitative PCR was conducted with SYBR Green Q-PCR Master Mix (Applied Biosystems, Waltham, MA, USA) on an Applied Biosystems 7500 Fast Real-time PCR system. Real-time PCR conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 15 s, and 60 °C for 30 s. Specific primer sequences for the quantitative real-time PCR assay are shown in Table 1. Each sample was analyzed in triplicate and normalized using glyceraldehyde 3-phosphate dehydrogenase. The relative expression of a gene was analyzed using the 2−ΔΔCt method.

Table 1.

Real-time PCR primer sequences.

Gene Forward primer (5′→3′) Reverse primer (5′→3′)
IL-1β ATG CAC CTG TAC GAT CAC TG ACA AAG GAC ATG GAG AAC ACC
IL-8 CTG CGC CAA CAC AGA AAT TA ATT GCA TCT GGC AAC CCT AC
COX-2 CGG TGA AAC TCT GGC TAG ACA G GCA AAC CGT AGA TGC TCA GGG a
GAPDH ACA TCG CTC AGA CAC CAT G TGT AGT TGA GGT CAA TGA AGG G
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IL-1β, interlukin-1β; IL-8, interlukin-8; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Western blot

The treated DPSCs were lysed with passive lysis buffer (Promega, Madison, WI, USA) supplemented with a protease inhibitor cocktail (GenDEPOT, Katy, TX, USA) and incubated for 45 min on ice. Thereafter, protein samples (20 μg protein/well) were separated via 10 % or 15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5 % skim milk, the blots were incubated with primary and secondary antibodies. The following antibodies were used: IL-1β (1:500), IL-8 (1:1000), COX-2 (1:1000), phospho-ERK (1:1000), ERK (1:1000), phospho-JNK (1:1000), JNK (1:1000), phospho-p38 (1:1000), and p38 (1:1000) (all from Cell Signaling Technology); and β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Chemiluminescence was detected using an Image Quant LAS4000 Image Analyzer (GE Healthcare, Chicago, IL, USA). For image analysis, relative protein expression levels were quantified using ImageJ software (NIH, Bethesda, MD, USA). β-actin was used as an internal control.

Alkaline phosphatase (ALP) staining

For ALP staining, DPSCs in the positive control group were cultured in osteogenic differentiation medium (ODM; α-MEM supplemented with 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid, and 100 nM dexamethasone; all from Sigma–Aldrich) for 4 or 7 days, with or without 1α,25(OH)2D3 (0.001–1 μM) and LPS (1 μg/mL). After 4 and 7 days of culture, the cells were stained with an Alkaline Phosphatase Staining Kit II (Reprocell, Yokohama, Japan) according to the manufacturer’s protocol. The stained cells were washed with DPBS and assessed using a light microscope. For quantification, the intensity of stained cells was measured using ImageJ software (NIH).

Statistical analysis

The statistical analyses were performed using the Student’s t-test. Results are presented as the mean ± standard error of the mean (SEM). All experiments were performed separately in triplicate, and P < 0.05 was considered statistically significant.

Results

DPSC viability under inflammatory conditions

To determine the biocompatibility of 1α,25(OH)2D3 under inflammatory stress, DPSC viability was assessed after 1α,25(OH)2D3 (0.001–1 μM) and LPS treatment. The 1α,25(OH)2D3 treatment (0.001–1 μM) did not cause cytotoxic effects in DPSCs, even in the presence of LPS, thereby indicating that 1α,25(OH)2D3 is well tolerated in this inflammatory microenvironment (Fig. 1A and B).

Figure 1.

Figure 1

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Effects of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on the viability and proliferation of dental pulp stem cells (DPSCs) under LPS-induced inflammatory conditions. (A) Representative images of MTT assay after 24 h treatment with LPS (1 μg/mL), and increasing concentrations of 1α,25(OH)2D3 (0.001–1 μM). (B) Quantitative analysis of cell proliferation assessed by MTT assay for three consecutive days. Data are presented as mean ± SEM from three independent experiments. DPSCs, dental pulp stem cells; LPS, lipopolysaccharide.

Expression of pro-inflammatory mediators in DPSCs

To assess the anti-inflammatory effects of 1α,25(OH)2D3 on LPS-stimulated DPSCs, IL-1β, IL-8, and COX-2 mRNA expression was examined using RT-qPCR following pretreatment with 1α,25(OH)2D3 (0.001–1 μM). As shown in Fig. 2, LPS stimulation markedly upregulated IL-1β, IL-8, and COX-2 mRNA expression compared to that in the untreated control group. Moreover, 1α,25(OH)2D3 treatment exhibited an overall dose-dependent trend in reducing inflammatory marker expression; however, the degree and statistical significance differed among targets. For IL-1β, a pronounced reduction was observed only at the highest concentration (Fig. 2A), whereas IL-8 mRNA levels were substantially suppressed at both 0.1 and 1 μM 1α,25(OH)2D3 (Fig. 2B), suggesting a more robust regulatory effect. Similarly, COX-2 mRNA expression was markedly decreased only at 1 μM, despite a non-noteworthy downward trend at lower concentrations (Fig. 2C). Collectively, 1α,25(OH)2D3 modulated transcriptional activation of key inflammatory mediators in LPS-treated DPSCs in a concentration-dependent manner.

Figure 2.

Figure 2

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1α,25(OH)2D3 reduces LPS-induced mRNA expression of inflammatory mediators in DPSCs. DPSCs were pretreated with 1α,25(OH)2D3 (0.001–1 μM) for 2 h and subsequently exposed to LPS (1 μg/mL) for 24 h. Relative mRNA expression levels of IL-1β (A), IL-8 (B), and COX-2 (C) were determined by real-time quantitative PCR (RT-qPCR) and normalized to GAPDH. Data are shown as mean ± SEM of triplicate experiments. **P < 0.01, ***P < 0.001 compared with control group, #P < 0.05, ##P < 0.01 compared with LPS group. LPS, lipopolysaccharide; DPSCs, dental pulp stem cells; IL-1β, interlukin-1β; IL-8, interlukin-8; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Expression of inflammatory proteins in DPSCs

To further validate the anti-inflammatory effects of 1α,25(OH)2D3 at the translational level, IL-1β, IL-8, and COX-2 protein expression was examined in LPS-stimulated DPSCs using Western blot analysis. As shown in Fig. 3A, LPS exposure substantially upregulated the expression of all three inflammatory markers compared to that in the untreated control group. Although 1α,25(OH)2D3 treatment did not markedly suppress IL-1β protein levels at any concentration tested (Fig. 3B), a consistent and concentration-dependent reduction was observed for IL-8 and COX-2 (Fig. 3C and D). Notably, IL-8 protein expression was considerably decreased by 1α,25(OH)2D3 at concentrations of 0.1 and 1 μM, indicating a robust anti-inflammatory effect at the post-transcriptional level (Fig. 3C). Similarly, COX-2 protein levels were considerably reduced at 1 μM 1α,25(OH)2D3, with a downward trend evident at lower concentrations (Fig. 3D). These findings corroborate the transcriptional data and further suggest that 1α,25(OH)2D3 exerts differential regulatory effects on inflammatory mediators, with IL-8 and COX-2 being more responsive targets than IL-1β at the protein level.

Figure 3.

Figure 3

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1α,25(OH)2D3 suppresses LPS-induced protein expression of inflammatory markers in DPSCs. Western blot analysis of IL-1β, IL-8, and COX-2 (A) and densitometric quantification (B–D) revealed that 1α,25(OH)2D3 attenuated LPS-induced upregulation of IL-8 and COX-2 in a concentration-dependent manner, while effects on IL-1β were less pronounced. β-actin was used as a loading control. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 compared with control group, #P<0.05, ##P<0.01 compared with LPS group. LPS, lipopolysaccharide; DPSCs, dental pulp stem cells; IL-1β, interlukin-1β; IL-8, interlukin-8; COX-2, cyclooxygenase-2.

ALP activity in LPS-treated DPSCs

To evaluate the effects of 1α,25(OH)2D3 on the osteogenic differentiation of DPSCs under inflammatory conditions, ALP staining was performed 4 and 7 days after osteogenic induction in the presence or absence of LPS and 1α,25(OH)2D3. As shown in Fig. 4A and B, ODM increased ALP activity compared to that in undifferentiated control cells. However, LPS (1 μg/mL) markedly suppressed ALP staining intensity at both time points. This indicated inhibition of early osteogenic commitment via inflammatory stimulation. Notably, pretreatment with 1α,25(OH)2D3 reversed the LPS-induced suppression of ALP activity in a dose-dependent manner. On day four, all tested 1α,25(OH)2D3 concentrations led to robust concentration-dependent increases in the ALP-positive area, and on day seven, 1α,25(OH)2D3 concentrations ≥0.01 μM substantially restored ALP staining levels, with 1 μM showing the greatest enhancement. Overall, 1α,25(OH)2D3 counteracted the negative effects of LPS on osteogenic differentiation and also promoted early osteoblastic activity in a concentration-dependent manner.

Figure 4.

Figure 4

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1α,25(OH)2D3 restores and enhances ALP activity in LPS-treated DPSCs. Cells were cultured in osteogenic differentiation medium (ODM) with or without LPS (1 μg/mL) and 1α,25(OH)2D3 (0.001–1 μM). Representative ALP staining images at day 4 and day 7 (A) (scale bar = 100 μm), and quantification of ALP-positive area (B) demonstrate that 1α,25(OH)2D3 reverses the inhibitory effect of LPS and promotes osteogenic differentiation in a time- and dose-dependent manner. *P < 0.05, **P < 0.01 compared with ODM group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with LPS group. LPS, lipopolysaccharide; DPSCs, dental pulp stem cells; ALP, alkaline phosphatase.

Transient activation of ERK and JNK pathways in DPSCs

To assess the influence of 1α,25(OH)2D3 on MAPK signaling in DPSCs, phosphorylation of ERK, JNK, and p38 were analyzed after 1 μM 1α,25(OH)2D3 treatment for 60 min. As shown in Fig. 5A–D, both p-ERK and p-JNK levels peaked considerably at 5 min post-treatment, followed by a gradual decline to baseline levels by 30–60 min. This transient activation suggests that 1α,25(OH)2D3 rapidly stimulates ERK and JNK signaling, potentially initiating downstream transcriptional responses. In contrast, p38 phosphorylation remained relatively unchanged throughout the time course. Altogether, 1α,25(OH)2D3 selectively and temporally activated MAPK pathways, particularly ERK and JNK, in the early phase of signaling.

Figure 5.

Figure 5

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1α,25(OH)2D3 induces transient activation of MAPK pathways in DPSCs. Western blot analysis of phosphorylated and total ERK, JNK, and p38 following treatment with 1 μM 1α,25(OH)2D3 for 0–60 min (A), with quantitative analysis of ERK (B), JNK (C), and p38 (D) phosphorylation. Results indicate rapid and transient activation of ERK and JNK, peaking at 5 min, with no significant change in p38. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with time 0. MAPK, mitogen-activated protein kinases; DPSCs, dental pulp stem cells; ERK, extracellular signal regulated kinase; JNK, c-Jun N-terminal kinase.

ERK and JNK signaling in osteogenic activation

To determine whether ERK and JNK pathways are involved in the pro-osteogenic effects of 1α,25(OH)2D3, DPSCs were co-treated with 1α,25(OH)2D3 (1 μM) and either the ERK inhibitor (U0126) or the JNK inhibitor (SP600125) under LPS-stimulated conditions. Consistently, LPS markedly reduced ALP staining in DPSCs, whereas 1α,25(OH)2D3 (1 μM) pretreatment notably restored ALP activity on days four and seven (Fig. 6A and B). However, this effect was markedly suppressed by U0126 or SP600125, with both inhibitors markedly reducing the ALP-positive area compared to that under the 1α,25(OH)2D3 treatment. Therefore, activation of both ERK and JNK pathways is required for 1α,25(OH)2D3-induced enhancement of osteogenic differentiation under inflammatory conditions.

Figure 6.

Figure 6

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ERK and JNK pathways are required for 1α,25(OH)2D3–mediated osteogenic activation in LPS-stimulated DPSCs. Cells were cultured in ODM with LPS (1 μg/mL) in the presence of 1 μM 1α,25(OH)2D3, with or without ERK inhibitor (U0126) or JNK inhibitor (SP600125). Representative ALP staining images at day 4 and day 7 (A) (scale bar = 100 μm), and quantification of ALP-positive area (B) show that inhibition of ERK or JNK significantly reduced the pro-osteogenic effects of 1α,25(OH)2D3. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with ODM group, ##P < 0.01, ###P < 0.001 compared with LPS group, †††P < 0.001 compared with 1α,25(OH)2D3 group. LPS, lipopolysaccharide; DPSCs, dental pulp stem cells; ERK, extracellular signal regulated kinase; JNK, c-Jun N-terminal kinase.

Discussion

Owing to its well-established roles in dental and skeletal biology, the active form of vitamin D (1α,25(OH)2D3) was selected for this study. Vitamin D is essential for systemic calcium and phosphate homeostasis and regulates odontogenesis and alveolar bone metabolism.18 Furthermore, vitamin D deficiency is associated with enamel hypoplasia, delayed tooth eruption, and increased susceptibility to dental caries and periodontitis.19,20 Moreover, vitamin D plays a key role in osteoblastic differentiation, matrix mineralization, and maintenance of alveolar bone integrity—factors critical for both tooth development and periodontal stability.21 In terms of regenerative dentistry, vitamin D has gained attention as a potential adjunct to enhance the healing of inflamed or damaged tissues, particularly through its dual actions in immunomodulation and osteoinduction.22,23 Thus, the use of 1α,25(OH)2D3 in this study is biologically relevant and clinically meaningful, as it reflects a growing interest in harnessing its regenerative potential in inflamed dental tissues, such as the pulp and periodontal complex.

The study data underscore the critical contribution of ERK and JNK pathways to the 1α,25(OH)2D3-induced osteogenic response in an inflammatory DPSC environment. The observed transient phosphorylation of ERK and JNK, peaking at 5 min post-1α,25(OH)2D3 treatment and subsequently declining, suggests a rapid and time-sensitive activation pattern. This dynamic is consistent with 1α,25(OH)2D3 acting as a signaling initiator, thereby triggering early intracellular cascades necessary for initiating osteogenic differentiation rather than maintaining prolonged signaling.24,25 This rapid response likely represents the previously described nongenomic action of 1α,25(OH)2D3, where early ERK or JNK activation primed transcription factors, such as runt-related transcription factor 2 (Runx2) and c-Jun, leading to ALP induction and mineralization-related gene expression.3,26,27 In this study, the p38 pathway did not exhibit substantial modulation, supporting the notion that 1α,25(OH)2D3 selectively engages ERK and JNK signaling for osteogenic programming. Collectively, these results reveal the mechanism by which 1α,25(OH)2D3 simultaneously resolves inflammation and facilitates tissue regeneration by leveraging early MAPK signaling cues in DPSCs.

The dual role of 1α,25(OH)2D3 in regulating both inflammation and osteogenic differentiation has been highlighted in various mesenchymal stem cell populations. Nevel et al.28 reported that 1α,25(OH)2D3 suppressed proinflammatory cytokine expression while promoting osteogenic differentiation in periodontal ligament cells (PDLC). Furthermore, Qi et al.29 showed that 1α,25(OH)2D3 downregulates cytokines, such as IL-6, IL-8, and IL-1β in TNF-α-induced inflammation of PDLSCs and promoted ALP, Runx2, bone morphogenetic protein 2, and bone gamma-carboxyglutamate protein expression. Collectively, these findings suggest that 1α,25(OH)2D3 exerts a context-dependent but often synergistic effect on immune suppression and osteogenic commitment in stem cells via modulation of inflammatory and differentiation-related signaling pathways.

However, not all studies uniformly support this dual action. For instance, Wasnik et al.30 reported that high-dose 1α,25(OH)2D3 administered during the early pro-inflammatory phase of fracture healing in mice delayed bone regeneration by prolonging M1 macrophage polarization and impairing the transition to a regenerative milieu. Similarly, Behm et al.31 showed that the effects of 25-hydroxyvitamin D3 (metabolic precursor of 1α,25(OH)2D3) on human PDLSCs were highly dependent on cytokine type and duration. In some inflammatory settings, 1α,25(OH)2D3 did not elicit notable suppression of pro-inflammatory mediators or enhancement of osteogenesis. These discrepancies highlight the complexity of 1α,25(OH)2D3 signaling in tissue regeneration and the necessity of considering inflammatory heterogeneity, cellular context, and treatment timing when evaluating its therapeutic application.32,33 Despite these variations, the study findings verified that 1α,25(OH)2D3 simultaneously attenuates inflammation and promotes early osteogenic commitment in dental stem cells under defined conditions.

The study findings showed the potential of 1α,25(OH)2D3 as a therapeutic adjunct for enhancing dental pulp regeneration under inflammatory conditions via its dual capacity to modulate immune responses and promote early osteogenic commitment. Despite these promising in vitro results, translational applicability remains to be established. In addition, MAPK activation was evaluated only under basal conditions, and the phosphorylation status of ERK, JNK, and p38 in the presence of LPS was not directly assessed, which should be considered in the interpretation of our findings. Accordingly, future studies should include in vivo validation to determine long-term regenerative efficacy, as well as more detailed mechanistic analyses focusing on inflammatory and osteogenic pathways such as the TLR4–NF-κB axis and late-stage differentiation markers, such as Runx2, osteocalcin, and matrix mineralization, which are critical determinants of functional pulp–dentin regeneration.

In conclusion, this study suggests that 1α,25(OH)2D3 exerts dual beneficial effects in LPS-stimulated dental pulp stem cells by attenuating inflammatory responses and promoting osteogenic differentiation. These actions may be partially supported by transient activation of ERK and JNK MAPK signaling pathways, although further mechanistic validation is required. The ability of 1α,25(OH)2D3 to improve ALP activity under inflammatory conditions indicates its potential as a regenerative adjunct in inflamed pulp tissue.

Acknowledgments

This work was supported by a New Faculty Research Grant of Pusan National University, 2023.

Funding Statement

This work was supported by a New Faculty Research Grant of Pusan National University, 2023.

Footnotes

Declaration of competing interest: The authors have no conflicts of interest relevant to this article.

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