Dopamine promotes osteogenic differentiation of PDLSCs by activating DRD1 and DRD2 during orthodontic tooth movement via ERK1/2 signaling pathway

Abstract

Introduction

Orthodontic tooth movement (OTM) involves complex interactions between mechanical forces and periodontal tissue adaptation, mainly mediated by periodontal ligament cells, including periodontal ligament stem cells (PDLSCs), osteoblasts, and osteoclasts. Dopamine (DA), a neurotransmitter known for its critical role in bone metabolism, is investigated in this study for its potential to enhance osteogenic differentiation in PDLSCs, which are pivotal in OTM. This study examined the potential of DA to facilitate OTM by binding to DA receptors (D1R and D2R) and activating the ERK1/2 signaling pathway. We propose that DA's interaction with these receptors on PDLSCs could enhance osteogenic differentiation, thereby accelerating bone remodeling and reducing the duration of orthodontic treatments, which offering a novel approach to improve clinical outcomes in orthodontic care.

Methods

This study utilized a rat OTM model, micro-CT, histological analyses, and in vitro assays to investigate dopamine's effect on osteogenesis. PDLSCs were cultured and treated with DA, and cytotoxicity, osteogenic differentiation, gene and protein expression assessed.

Results

Dopamine administration significantly increased trabecular bone density and osteogenic marker expression in an OTM rat model. In vitro, DA at 10 nM optimally promoted human PDLSCs osteogenesis without affecting proliferation. Blocking DA receptors or inhibiting the ERK1/2 pathway attenuated these effects, underscoring the importance of dopaminergic signaling in tension-induced osteogenesis during OTM.

Conclusion

Taken together, our study reveals that local dopamine administration at a concentration of 10 nM not only enhances tension-induced osteogenesis in vivo but also significantly promotes osteogenic differentiation of PDLSCs in vitro through D1 and D2 receptor-mediated ERK1/2 signaling pathway activation.

Highlights

• •During OTM, DA enhances bone formation on the tension side of rat molars.
• •DA at a concentration of 10 nM significantly promotes osteogenic differentiation in PDLSCs.
• •Inhibiting DA receptors reduces the osteogenic effect of DA on PDLSCs.
• •DA activates its receptors, facilitating osteogenesis in PDLSCs via ERK1/2 phosphorylation.

1. Introduction

OTM represents a complex biological process of periodontal tissue adaptation, driven by mechanical forces that orchestrate the interplay between osteoclastic resorption and osteoblastic deposition, encompassing metabolic shifts within the periodontal ligament and alveolar bone restructuring [1,2]. The quest to expedite bone remodeling in orthodontic treatment has led to the exploration of various modalities, including low-level laser therapy (LLLT), microcurrent (MC) applications, corticotomy, and targeted exosome delivery. Despite these advances, the imperative remains to identify a modality that reliably shortens treatment duration while maintaining safety and efficacy [[3], [4], [5]].

PDLSCs, a subset of the mesenchymal stem cell lineage, exhibit pluripotency with the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes under in vitro conditions [6]. Mechanical loading on PDLSCs induces a lineage commitment towards osteoclastogenesis in compression zones and osteoblastogenesis in tension zones, culminating in bone resorption and formation, respectively. Properly calibrated orthodontic forces have been demonstrated to enhance the osteogenic differentiation and proliferative capacity of PDLSCs [2,7,8].

Dopamine, a pivotal neurotransmitter within the central nervous system, extends its regulatory influence to encompass motor control, reward processing, and satiety. Beyond its neurological functions, dopamine exerts a significant impact on bone metabolism, modulating osteoblastic activity and bone matrix accrual [9,10]. Dopamine receptors, classified within the G protein-coupled receptor superfamily [11], are dichotomized into D1-like and D2-like subfamilies [12], with expression profiles confirmed on osteoblasts and bone marrow mesenchymal stem cells (BMMSCs) [13]. The synaptic release of dopamine and subsequent receptor engagement not only modulates neuronal activity but also plays a pivotal role in skeletal homeostasis [10]. Empirical evidence supports dopamine's role in enhancing osteogenic differentiation in osteoblastic cell lines and BMMSCs, an effect attenuated by DRD receptor antagonism [13,14], while genetic ablation of dopamine transporter genes is associated with compromised bone density and mechanical integrity in murine models [15]. The extracellular signal-regulated kinase (ERK) cascade is an integral signaling pathway implicated in dopamine biosynthesis, release, and receptor-mediated signal transduction, influencing dopamine synthase activity, modulating neurotransmitter release, and regulating receptor expression and downstream signaling [16]. Activation of the ERK1/2 pathway by D1 receptor agonists has been documented [17], however, it remains unclear whether the osteogenic differentiation of PDLSCs and the consequent bone formation in the tension zone are influenced by DA receptor engagement and the ERK1/2 signaling pathway.

In this study, we focus on the orthodontic tooth movement process, where local injection of dopamine (DA) in the periodontium can promote bone deposition on the tension side. This process is attributed to the binding of DA to D1 and D2 receptors (D1R and D2R), which through the ERK1/2 pathway, enhances osteogenic differentiation of PDLSCs. Unveiling this mechanism may provide a novel biological strategy for orthodontic treatment, facilitating tooth movement, reducing treatment duration, and potentially introducing new therapeutic approaches in the field of orthodontics.

2. Methods

2.1. Modulation of orthodontic tooth movement by dopamine treatment

The animal protocols for this study were authorized by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (SYSU-IACUC-2023-000887). Twenty 6-week-old male CD (SD)IGS rats were acquired from Besttest Biotechnology Co., Ltd. (Zhuhai, China). The experimental model for orthodontic tooth movement was established in accordance with previously described methodologies [18]. Briefly, following anesthesia, an orthodontic force of 50 g was applied by affixing a nickel-titanium coil spring between the incisors and the left maxillary first molar (Fig. 1a). The rats were randomly allocated into two groups: one group received subperiosteal injections of normal saline (OTM + Saline, n = 10), and the other group received subperiosteal injections of dopamine (OTM + DA, n = 10). In the dopamine-treated group, rats were administered a subperiosteal injection of 50 μL of 50 nM dopamine solution bi-daily post-modeling, while the control group received a corresponding volume of saline (Fig. 1b). Throughout the experimental duration, no adverse reactions or mortality were observed. On days 7 and 14 post-modeling, rats from each group (n = 5 per time point) were humanely euthanized, and samples of the left maxillary bone were harvested for subsequent micro-computed tomography (Micro-CT) and histological evaluations.

Fig. 1.

Subperiosteal injection of DA improved the microstructural parameters of trabecular bone on the tension side (red-marked regions). (a) Diagram illustrating the rat model of orthodontic tooth movement. (b) Illustration of the drug injection protocol and the timing of dopamine (DA) administration. (c) 2D micro-CT images and 3D reconstructions of maxillary specimens at days 7 and 14 post-orthodontic force application. (d) Detailed three-dimensional reconstruction of trabecular bone structure within the region of interest (ROI) at days 7 and 14 after DA injection. (e–g) Quantitative microstructural analysis of trabecular bone parameters, including bone mineral density (BMD), bone volume (BV), and the ratio of bone volume to tissue volume (BV/TV) at days 7 and 14 post-DA injection, showing significant enhancements in the dopamine-treated group. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; Scale bar = 1 mm).

2.2. Micro-CT analysis

We performed scanning and analysis of the samples using the Inveon Micro CT system (Siemens; 80 kv, 100 mA, 19 μm resolution) and its affiliated software. The region of interest (ROI) was set as the area of the alveolar bone at the distal cervical region of the mesial buccal root of the maxillary first molar (800 μm × 300 μm × 1000 μm). Additionally, microscopic structural analysis of the trabecular bone in ROI including bone volume (BV), bone volume to tissue volume ratio (BV/TV), and bone mineral density (BMD) were analyzed.

Micro-CT system (Siemens) operating at 80 kV and 100 mA with a resolution of 19 μm. The associated proprietary software was utilized for image acquisition and analysis. The region of interest (ROI) was delineated as the alveolar bone area located at the distal cervical region of the mesial buccal root of the maxillary first molar, measuring 800 μm × 300 μm × 1000 μm. Quantitative assessment of the trabecular bone microarchitecture within the ROI was performed, including the determination of bone volume (BV), the ratio of bone volume to tissue volume (BV/TV), and bone mineral density (BMD).

2.3. Histological and immunohistochemical analysis

For the purpose of histological and immunohistochemical (IHC) examination, the decalcified and dehydrated maxillary samples, embedded in paraffin, were sectioned into 5 μm slices following a 6-week decalcification period. Subsequent to sectioning, hematoxylin and eosin (H&E) staining was performed, and the samples were imaged. For the IHC analysis, sections were incubated with anti-OSX antibody (1:200, DF7731, Affinity Biosciences, OH, USA) at 4 °C. Quantification of immunostaining was achieved by measuring the integrated optical density (IOD) and the area of positive expression using Image-Pro Plus software. The mean optical density (MOD) was calculated using the formula: MOD = IOD/Area.

2.4. Isolation, culture and characterization of PDLSCs

The protocols for obtain and culture of PDLSCs were approved by the respective Institutional Review Boards and followed ethical guidelines. PDLSCs were isolated from periodontal ligament tissue of premolars extracted for orthodontic reasons from healthy adolescents aged 10–18 years in the stomatology department of the Third Affiliated Hospital of Sun Yat-sen University. This study was approved by the ethics committee of the Third Affiliated Hospital of Sun Yat-sen University. All participants were informed the experimental principle and signed the informed consent at the beginning of the study. The teeth were rinsed with PBS containing antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). The periodontal ligament tissue was gently scraped from the middle third of the tooth root and then digested with a solution of 3 mg/ml collagenase type I for 45 min to 1 h at 37 °C. After digestion, the cells were seeded into culture flasks containing Dulbecco's modification of MEM alpha modified Eagle's medium, supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂, and the culture medium was replaced every three days. Cells from the 3rd to 5th passages were used for subsequent experiments. The protocols for isolation and culture of PDLSCs were approved by the respective Institutional Review Boards and followed ethical guidelines.

Flow cytometry was used to identify the expression of cell surface markers CD146, CD105, CD90, CD73, CD44 on PDLSCs, while confirming the absence of hematopoietic markers CD45 and CD34. This established the mesenchymal stem cell identity of the isolated PDLSCs. Fluorescein isothiocyanate- (FITC-) conjugated mouse IgG (Invitrogen, USA) served as a negative control to ensure the specificity of the staining.

2.4.1. Cell Counting Kit-8 (CCK-8) assay

The cytotoxicity of DA on PDLSCs was evaluated using the Cell Counting Kit- 8 (CCK-8) assay. Initially, PDLSCs were seeded in 96-well plates at a density of 3 × 10³ cells per well and incubated for 24 h. Subsequently, the cells were treated with DA at concentrations of 0, 10 nM, 25 nM, and 50 nM. After treatment periods of 3, 5, and 7 days, the culture medium was replaced with 100 μL of 10% CCK-8 solution (Dojindo, Japan) and incubated at 37 °C for an additional 2 h. The optical density (OD) at 450 nm was measured using an ELX-808 Absorbance Microplate Reader (BioTek, Winooski, VT). The mean OD value of triplicate wells was calculated for each treatment group, and the assay was independently repeated three times to ensure reliability.

2.5. Alizarin Red staining

For the induction of osteogenic differentiation, PDLSCs were cultured in 6-well plates at a density of 40,000 cells per well and in 12-well plates at 20,000 cells per well. Upon reaching 80–90% confluence, the standard culture medium was substituted with an osteogenic differentiation medium which was refreshed every 2–3 days. After a 14-day period of osteogenic induction, the medium was discarded, and the cells were rinsed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich, Germany) for 20 min, and stained with Alizarin Red Solution (Cyagen, China) for 10 min. Following staining, the cells were washed thrice with PBS. The formation of calcium complexes during the chelation process was then visualized using an OLYMPUS IX71 microscope.

2.6. Quantitative real-time reverse transcription polymerase chain reaction (qRT- PCR)

Total RNA was extracted from PDLSCs using RNA-Quick Purification Kit (Esunbio, China). The purity and concentration of the isolated RNA were determined by UV spectrophotometry, with only samples exhibiting an A₂₆₀: A₂₈₀ ratio of ≥ 1.9 being selected for subsequent procedures. A PrimeScript RT Reagent Kit (Takara, Japan) was utilized to reverse transcribe 0.5 μg of mRNA from these samples into cDNA, in accordance with the provided manufacturer's protocol. The cDNA was then subjected to quantitative real-time PCR (qRT-PCR) using FastStart Universal SYBR Green Master Mix (ROX, USA) on an ABI PRISM 7500 sequence detection system (Applied Biosystems, USA). The primer sequences for qRT-PCR are listed in Table 1.

Table 1.

Primer sequences used in quantitative real-time reverse transcription polymerase chain reaction.

Gene Target	Sequence
COL1A1	Forward: 5′-TGTTGGTCCTGCTGGCAAGAATG-3′ Reverse: 5′-GTCACCTTGTTCGCCTGTCTCAC-3′
RUNX2	Forward: 5′-TCCGCCACCACTCACTACCAC-3′ Reverse: 5′-GGAACTGATAGGACGCTGACGAAG-3′
OSX	Forward 5′-GCGGCAAGGTGTATGGCAAGG-3′ Reverse 5′-GCAGAGCAGGCAGGTGAACTTC-3′
ALP	Forward: 5′-TATGGCTCACCTGCTTCACGG-3′ Reverse: 5′-GCTGTCCATTGTGGGCTCTTG-3
GAPDH	Forward 5′-GAGTCCACTGGCGTCTTCAC- 3′ Reverse 5′-TTCACACCCATGACGAACAT-3′

2.7. Western Blot analysis

Total protein was isolated from PDLSCs utilizing RIPA lysis buffer (Beyotime, China) enhanced with a 1% protease inhibitor cocktail and 1% phosphatase inhibitors (Cwbio, China). The cell lysates were chilled on ice for half an hour before being centrifuged at a force of 14, 000 g for a quarter-hour to precipitate any cellular debris. The protein yield was quantified employing a BCA Protein Assay Kit (Pierce, USA) in strict accordance with the supplier's protocol. Subsequently, 20 − 30 μg of the proteins were separated by electrophoresis on 12% SDS-PAGE gels and electrotransferred onto PVDF membranes (Millipore). To block nonspecific sites, the membranes were incubated with 5% BSA or skim milk in TBST at ambient temperature for 1 h. The membranes were then probed with primary antibodies against COL1A1, ALP, RUNX2, Osterix (OSX), OPN and GAPDH from Affinity Biosciences (China), and p-ERK and ERK from Selleck (USA) Group, each at a 1:1000 dilution, and incubated at 4 °C overnight. Following thorough washing, the membranes were treated with horseradish peroxidase-linked secondary antibodies (Beyotime, China, 1:1000) for 1 h at room temperature. The bound antibodies were detected using an ECL detection system (Millipore, Germany), and the intensity of the bands was analyzed quantitatively with ImageJ software.

2.8. Statistical analysis

Statistical analyses were carried out using the GraphPad Prism software package (version 25.0). Each experiment was replicated a minimum of three times to ensure reliability. The data were rigorously tested for normality and variance homogeneity and are reported as the mean ± standard error of the mean (SEM). For comparisons involving two groups, an independent samples t-test was performed. For comparisons involving more than two groups, a one-way analysis of variance (ANOVA) was conducted, with subsequent Bonferroni post hoc tests applied to assess the significance of multiple comparisons. Differences were considered statistically significant at a p-value less than 0.05.

3. Results

3.1. Dopamine enhance tension-induced osteogenesis in vivo

Fig. 1a and b respectively depict the rat model for orthodontic tooth movement and the mouse subperiosteal injection model. Fig. 1c illustrates the post-modeling micro-CT scan images, showing a gap between the molars at 7d and 14d, indicating successful modeling, and highlighting the ROI for trabecular bone structure three-dimensional reconstruction and trabecular bone microstructure analysis through red-marked regions. The three-dimensional reconstruction of trabecular bone structure within ROI revealed a more compact architecture in the dopamine-injected group (Fig. 1d). Additionally, microscopic structural analysis of the trabecular bone in ROI indicated enhancements in bone volume (BV), bone volume to tissue volume ratio (BV/TV), and bone mineral density (BMD) in the dopamine-treated group (Fig. 1e–g). H&E staining at day 7 and day 14 in the control group revealed the elongation of the periodontal ligament under tensile force (Fig. 2a). Additionally, immunohistochemical staining demonstrated an upregulation of the OSX in the dopamine-injected group (Fig. 2b and c). These findings collectively indicate that during orthodontic tooth movement, the injection of dopamine can enhance tension-induced osteogenesis.

Fig. 2.

Subperiosteal injection of DA enhanced the tension-induced osteogenesis in vivo. (a) Hematoxylin and eosin staining of the tension side, with 400x magnification, displaying tissue morphology. (b) Immunohistochemical staining for osterix (OSX) at 400x magnification, indicating osteogenic activity. (c) Quantitative analysis of OSX expression on the tension side. AB, alveolar bone; R, root; PDL, periodontal ligament. Statistical significance was assessed using unpaired one-way ANOVA. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; Scale bar = 50 μ m).

3.2. Low concentration of DA facilitates PDLSCs osteogenic differentiation

Flow cytometric analysis showed high expression of mesenchymal stem cell markers in the cells: CD146 (83.4%), CD105 (99.8%), CD90 (99.4%), CD73 (99.9%), CD44 (100%), and, while negative markers CD45 (3.16%) and CD34 (5.70%) were minimally expressed (Fig. 3a). This confirms the mesenchymal identity and purity of the isolated PDLSCs.

Fig. 3.

A low concentration of DA facilitates PDLSCs osteogenic differentiation. (a) Flow cytometry analysis of surface markers of PDLSCs. (b) Quantitative RT-PCR analysis showing the expression of osteogenic gene expression in PDLSCs undergoing osteogenic differentiation treated with a low concentration of DA at day 7. (c) CCK-8 assay evaluating the effects of DA on PDLSC proliferation at days 3, 5, and 7. (d) Alizarin Red S staining of PDLSCs during late-stage osteogenic differentiation under various concentrations of DA (0, 10, 25, 50 nM). (e, f) Western Blot analysis of osteogenic protein expression in PDLSCs during osteogenic differentiation induced by different concentrations of DA (0, 10, 25, 50 nM). (g) Alizarin Red S staining demonstrating the osteogenic promotion by 10 nM DA in PDLSCs. Statistical significance was determined using unpaired one-way ANOVA for multiple-group comparisons. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

In our investigation, we initially uncovered that dopamine, at nanomolar concentrations, exerts a positive effect on the osteogenic differentiation of PDLSCs at the genetic level, as evidenced by our gene expression analysis (Fig. 3b). To further elucidate the osteoinductive properties of DA, we assessed its impact at concentrations of 10 nM, 25 nM, and 50 nM. Our findings, as determined by the CCK-8 assay, indicated that DA does not adversely affect the proliferation of PDLSCs within this concentration range (Fig. 3c). Notably, ARS staining and Western blot analyses revealed that a concentration of 10 nM DA markedly promotes osteogenic differentiation, surpassing the efficacy observed at higher concentrations (Fig. 3d, e, f). These observations are pivotal as they highlight the potential of DA, especially at 10 nM, to significantly enhance the osteogenic capacity of PDLSCs within an osteogenic induction environment (Fig. 3g).

Collectively, our data suggest that DA, at an optimally determined concentration of 10 nM, acts as a potent osteoinductive agent, thereby presenting a promising avenue for enhancing in vitro osteogenic differentiation of PDLSCs.

3.3. Blocking the DA receptor inhibits PDLSC differentiation and DA effects

In our comprehensive study examining the role of dopaminergic signaling in the osteogenic differentiation of PDLSCs, we specifically investigated the contribution of D1 and D2 receptors. To determine the optimal concentration for receptor antagonism, we conducted a titration experiment using a concentration gradient ranging from 0.5 μM to 2 μM for the D1 receptor antagonist SCH23390 and the D2 receptor antagonist Haloperidol. Cell viability was meticulously evaluated on days 3, 5, and 7 employing the CCK-8 assay. Our data revealed that within the tested concentration range, there was no discernible inhibitory effect on PDLSC proliferation (Fig. 4a and b). Subsequent Alizarin Red S staining allowed us to pinpoint the optimal antagonist concentration at 2 μM for the downstream experiments Fig. 4c and d.

Fig. 4.

Inhibition of DA-induced osteogenic differentiation in PDLSCs by receptor antagonism. (a, b) CCK-8 assay shows no significant effect on PDLSC proliferation with D1 receptor antagonist SCH23390 and D2 receptor antagonist Haloperidol at concentrations from 0.5 μM to 2 μM. (c, d) Alizarin Red S staining determines 2 μM as the optimal antagonist concentration for further studies. (e) Alizarin Red S staining during PDLSCs osteogenic differentiation stimulated with DA, SCH23390, and Haloperidol. (f) Quantitative RT-PCR analysis of osteogenic gene expression during PDLSCs osteogenic differentiation stimulated with DA, SCH23390, and Haloperidol. (g, h) Western Blot analysis of osteogenic protein expression during PDLSCs osteogenic differentiation stimulated with DA, SCH23390, and Haloperidol. Statistical significance was determined using unpaired one-way ANOVA for multiple-group comparisons. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Upon a cultivation period extending to 14 days, it was observed that DA substantially augmented osteogenic differentiation in PDLSCs. This facilitation of osteogenesis by DA was significantly inhibited when either SCH23390 or Haloperidol was administered alongside DA (Fig. 4e), highlighting their roles as DA receptor antagonists in obstructing DA's pro-osteogenic effects. Further elucidation of the molecular underpinnings, through qPCR and Western blot analyses, demonstrated that the blockade of both D1 and D2 receptors impeded the DA-mediated osteogenic differentiation (Fig. 4f, g, h).

Taken together, our findings provide compelling evidence that both D1 and D2 receptors are instrumental in mediating the osteogenic effects of DA on PDLSCs. These receptors exert distinct modulatory influences on specific osteogenic markers, underscoring the complexity of dopaminergic signaling pathways in the regulation of stem cell osteogenesis.

3.4. Inhibition of ERK1/2 signaling attenuates DA-driven osteogenesis via Runx2 and OPN downregulation

Building upon the premise that activation of the ERK1/2 signaling pathway is crucial for osteogenic differentiation [14], we posited that a similar mechanism might be applicable to PDLSCs. The Western blot analysis revealed that DA indeed promoted the phosphorylation of ERK1/2, and this phosphorylation was diminished following the inhibition of both D1 and D2 receptors, leading us to speculate that DA may enhance ERK1/2 phosphorylation through either D1R or D2R (Fig. 5a and b).

Fig. 5.

Impact of ERK1/2 signaling inhibition on DA-induced osteogenesis in PDLSCs. (a, b) Western blot analysis demonstrates DA-promoted ERK1/2 phosphorylation and its attenuation by D1 and D2 receptor antagonists. (c, d) Western blot analysis demonstrates osteogenic differentiation is reduced by D1R antagonist or ERK1/2 inhibitor alone, with a greater reduction when combined. (e, f) Western blot analysis demonstrates osteogenic differentiation is reduced by D2R antagonist or ERK1/2 inhibitor alone, with a greater reduction when combined. Statistical significance was determined using unpaired one-way ANOVA for multiple-group. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

To further substantiate the link between DA-induced osteogenic differentiation and the activation of the ERK1/2 signaling pathway, we introduced both DA receptor antagonists and the ERK1/2 pathway inhibitor PD0325901. We observed that the downregulation of Runx2 and OPN expression levels occurred with the use of either the D1R antagonist or the ERK1/2 pathway inhibitor alone. Notably, the simultaneous application of the D1R antagonist and the ERK1/2 pathway inhibitor resulted in a more pronounced decrease in the expression levels of Runx2 and OPN than the use of either inhibitor alone (Fig. 5c and d). A similar outcome was observed when the D2R antagonist was applied (Fig. 5e and f), suggesting that upon binding of DA to its receptors, the ERK1/2 signaling pathway may play a predominant role in osteogenic differentiation.

In summary, our findings indicate that the blockade of the ERK1/2 signaling pathway abrogates DA-induced Runx2 and OPN transcriptional activity, leading to the inhibition of osteogenic differentiation in PDLSCs. These comprehensive results underscore the pivotal role of the ERK1/2 signaling pathway in mediating DA-induced osteogenic differentiation, potentially through the modulation of Runx2 and OPN transcriptional activity.

4. Discussion

Due to aesthetic demands, an increasing number of individuals are undergoing orthodontic treatment. How to achieve safe and efficient orthodontic tooth movement has become a focal point of research in orthodontic therapy. Our investigation delves into the intricate biological underpinnings of OTM, with a particular emphasis on the role of DA in the osteogenic differentiation of PDLSCs mediated by the ERK1/2 signaling cascade. OTM is a complex biological process that involves the conversion of mechanical loads into biological signals by cells within the periodontal ligament and alveolar bone, such as osteoblasts, osteocytes, and osteoclasts. The interaction between cell membrane receptors and ligands plays a pivotal role in signal transduction and serves as a target for the discovery of novel anabolic agents for bone enhancement [19]. OTM is characterized by bone deposition and resorption at sites of tension and pressure, respectively [8,20]. PDLSCs, a type of mesenchymal stem cell, possess the potential to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and may play a crucial role in periodontal and skeletal remodeling during OTM due to their sensitivity to mechanical loads [6,21].

Prolonged orthodontic treatment durations are associated with an increased incidence of adverse effects, including periodontitis and root resorption. With the growing population of adult orthodontic patients, here is a burgeoning demand for reduced treatment times, enhanced periodontal health, and sustained long-term stability [22,23]. Meta-analyses by Dab et al. and Kamal et al. have shown that surgical procedures such as corticotomy can accelerate OTM [22,23]. Zaniboni et al. found that adjunctive therapies, including low-level laser therapy (LLLT) and low-intensity electrical stimulation (microcurrent-MC), enhance bone remodeling during orthodontic treatment [3]. Liu et al. and Zhu et al. demonstrated that periodontal injection of exosomes and parathyroid hormone (PTH), respectively, can expedite the bone remodeling process during OTM [5,24]. Oortgiesen et al. reported that the combination of locally applied FGF-2 and injectable CaP can also enhance periodontal regeneration [25]. However, we are still in pursuit of a method that is both safer and more effective.

DA is a crucial neurotransmitter involved in various physiological processes, including bone metabolism [11,14,17,26]. Research has indicated that patients with Parkinson's disease (PD) exhibit reduced levels of DA and, in comparison to healthy individuals, present with diminished bone density, particularly in critical regions such as the hip, lumbar spine, and femoral neck, thereby facing an elevated risk of fractures [[27], [28], [29], [30]]. DA has been shown to promote bone formation and inhibit bone resorption, with a decline in DA levels correlating with accelerated bone resorption, increased bone tissue destruction, and an augmented risk of osteoporosis [27,[31], [32], [33], [34]]. The multitude of physiological functions controlled by dopamine in the brain and peripheral tissues is mediated through D1, D2, D3, D4, and D5 dopamine G protein-coupled receptors (GPCRs) [35]. Dopamine receptors are associated with alcoholism, substance abuse, PTSD, and other conditions [36]. Additionally, there is a certain correlation between dopamine receptors and skeletal health [31], with the balance of signaling between D1-like and D2-like receptors seemingly playing a role in the fine-tuning of bone remodeling [37]. Multiple studies have indicated that the introduction of dopamine receptor agonists can improve osteopenia, whereas the introduction of dopamine receptor antagonists leads to an increase in bone resorption [[38], [39], [40]].

In our research, we established a rat model of OTM over 7 and 14 days and administered DA locally to the periodontium of rat molars. Our findings revealed an augmentation in trabecular bone density and an upregulation of osteogenic protein OSX expression in the experimental group relative to controls, suggesting that local DA injection bolsters bone deposition on the tension side of moving teeth. To further validate the osteogenic effects of dopamine on the tension side, we conducted in vitro experiments using PDLSCs. We observed that low concentrations of DA upregulated the expression of osteogenic genes such as OSX, COL1A1, and ALP. After screening for optimal concentrations, we selected 10 nM dopamine for subsequent experiments. Alizarin Red staining, used to assess osteoblast-like cells derived from PDLSCs, showed that 10 nM DA promotes osteogenic differentiation of PDLSCs. This effect was antagonized by inhibitors of DRD1 or DRD2 receptors, suggesting that DA enhances osteogenesis through receptor binding.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway is implicated in a plethora of cellular activities, including mitosis, metabolism, movement, survival, apoptosis, and differentiation, mediated by the phosphorylation of a diverse array of substrates such as phospholipases, transcription factors, and cytoskeletal proteins [41]. The ERK/MAPK signaling pathway is essential for bone formation in bone development and homeostasis, with activation of the ERK/MAPK pathway being associated with proliferation, differentiation, gene, and protein expression in primary osteoblasts [42,43] Previous studies have reported that MAPK/ERK induces the activation of Runx2, suggesting a positive effect of the MAPK/ERK pathway on osteogenesis [44,45]. Roof et al. reported that dopamine-mediated D2R activation leads to ERK stimulation and helps maintain prolactin homeostasis [46]. Wang et al. found that compared to P38, MAPK, and JNK, the phosphorylation of ERK1/2 leading to upregulated transcriptional activity of Runx2 and promoting osteogenic differentiation [14]. We hypothesized that PDLSCs would respond similarly to DA stimulation. Our results indicate that DA promotes the phosphorylation of ERK1/2, which is significantly inhibited by dopamine receptor antagonists. Additionally, the use of either D1R or D2R inhibitors or ERK1/2 pathway inhibitors alone suppressed osteogenic differentiation, and the combined use of DRD inhibitors and ERK1/2 pathway inhibitors resulted in a more significant reduction in osteogenic differentiation than the use of either inhibitor alone.

In conclusion, our study demonstrates for the first time that local injection of DA can accelerate OTM in rats, and appropriate concentrations of DA can activate D1 and D2 receptors on PDLSCs, further promoting osteogenesis through the activation of the ERK1/2 signaling pathway. This provides a potential direction for safely and effectively accelerating the OTM process in orthodontic treatment.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.