2.1 Rats orthodontic tooth movement model

We utilized 20 seven-week-old male Wistar rats (Charles River) weighing around 220 g. Rats were maintained under a 12-h day-night cycle with unrestricted access to food and water. We ligatured the left maxillary first molar of each rat to their upper incisor using a nickel-titanium closed-coil spring secured by a 0.25 mm stainless steel thread (TOMY, Japan). Approximately 20 g of force was applied. The rats were divided into five random groups based on the duration of dental device maintenance (1, 3, 7, 14 and 28 days), with each group comprising four independent samples for statistical analysis. The right side was taken as the control. After the injection of 3% pentobarbital sodium, the left tooth as a model of tooth movement and their alveolar bone were collected and fixed in 10% formalin for 24 h. The bone tissues were decalcified in 14% ethylenediaminetetraacetic acid (EDTA) for 3 months. The paraffin-embedded tissues were cut into 5 μm thick slices and then dehydrated with an alcohol gradient.

2.2 Cell culture

RAW264.7 cells simulating M0 macrophages in dental pulp were obtained from Procell Life Science and cultured in DMEM high glucose medium (10% FBS, 1% penicillin/streptomycin) at 37°C with 5% CO₂. Logarithmically growing cells were chosen for experiments under normal (37°C, 5% CO₂, 21% O₂) and hypoxic conditions (2% O₂) using a C-chamber incubator (ProOx P110 O₂ Controllers, BioSperix). Mouse Dental Pulp Cells (MDPCs) representing pulp tissue cells were isolated from maxillary bilateral first molars were extracted from 6- to 8-week-old mice. MDPCs were cultured with normal conditions (37°C, 5% CO₂, 21% O₂) and hypoxic conditions (2% O₂) for 24 h using a C-chamber incubator (ProOx P110 O₂ Controllers, BioSperix). To assess the anti-inflammatory effects of M2 polarization of macrophages under hypoxia, we used trametinib to inhibit their M2 polarization to study its effect on MDPCs. The M2 inhibition group was treated with 10 nM trametinib under 37°C, 5% CO₂, 2% O₂ for 24 h, and which were maintained using the same C-chamber incubator (ProOx P110 O₂ Controllers, BioSperix).

2.3 RNA Extraction and Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted using TRIzol (Takara, Japan) following the manufacturer's instructions. The cDNA reverse-transcription process utilized the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). Following cDNA synthesis, qRT-PCR was performed in triplicate on the LightCycler 480 system (Roche Diagnostics) using TB Green Premix Ex Taq II (Takara). Data were analysed using the 2^−△△Ct method, with GAPDH as the internal standard.

All qRT-PCR primer sequences used in this study are listed in Table 1.

2.4 HE Staining

For HE staining, sections were dewaxed in graded alcohol, then subjected to staining using a solution comprising haematoxylin for cell nuclei and eosin for cytoplasm. Subsequent dehydration, transparency and sealing with neutral glue were carried out. Finally, specimens were visualized using a BX51 fluorescence microscope.

2.5 Immunohistochemistry

To assess macrophage polarization in the dental pulp during orthodontic force application, we examined the expression of macrophage polarization markers, iNOS and Arg-1, in the pulp of the first left maxillary molar. Sections for IHC were deparaffinized and subjected to antigen retrieval using 0.25% trypsin (Solarbio) for 30 min at 37°C. This was followed by a 15-min treatment with 3% hydrogen peroxide and a 1-h blocking step with 5% normal goat serum (Solarbio) at room temperature. Primary antibodies were then applied to the sections overnight at 4°C. Subsequently, secondary antibodies were added, and the sections were counterstained with haematoxylin (Solarbio). Quantification was performed using ImageJ software.

All antibodies used for immunohistochemical staining in this study are listed in Table 2.

2.6 Western blot

For Western blot analysis, RAW264.7 cells were lysed in 1× ice-cold RIPA lysis buffer (NCM Biotech, WB3100) with EDTA-Free Protease Inhibitor Cocktail (NCM Biotech, P001) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, P5726). After centrifugation, protein concentration was quantified using a BCA assay kit (Beyotime, P0012S) according to the manufacturer's instructions. Equal amounts of samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore). Following a 2-h blocking step with 5% non-fat milk at room temperature, membranes were incubated with primary antibodies at 4°C overnight. After washing with TBS-T, membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature, followed by further washes with TBS-T. Protein bands were visualized using an Amersham Imager 600 (General Electric) with ECL Western Blotting Substrate (Biosharp, China). Densitometric quantification of protein bands was performed using ImageJ (National Institutes of Health).

All antibodies used for Western Blot in this study are listed in Table 2.

2.7 Immunofluorescence

MDPCs fixed with 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 in PBS for 20 min, rinsed with TBS and blocked with PBS containing 5% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h. The cells were then incubated with primary antibodies at 4°C overnight, followed by washing with PBS. Subsequently, the cells were stained with fluorophore-conjugated secondary antibodies for 1 h at room temperature and 4′, 6-diamidino-2-phenylindole (DAPI, Solarbio) for 5 min. The results were visualized using an IX73 inverted microscope (Olympus). Fluorescence intensity was quantified using ImageJ.

All antibodies used for immunofluorescence characterizations in this study are listed in Table 2.

2.8 Flow cytometric analysis

The cell surface markers of macrophages in mouse dental pulp were analysed by flow cytometry (BD Biosciences) according to the manufacturer's instructions. Briefly, after washing with PBS containing 3% FBS, the cells were incubated with monoclonal antibodies against mouse F4/80 (Biolegend, Cat. No. 123107) in the dark at 4°C for 1h; cells without pretreatment by any antibody were used as blank control. Subsequently, the cells were washed with PBS and analysed by flow cytometry.

2.9 ELISA

ELISA was conducted to quantitatively estimate cytokines, including IL-1b, IL-10, VEGF and TNF-α, in the culture supernatants of MDPCs. Commercially available kits (from Elabscience) were employed as per the manufacturer's instructions. Supernatants were collected and stored at −80°C until analysis.

2.10 Statistical analysis

Data with a normal distribution were presented as mean ± standard deviation for each group, consisting of three or more independent samples. GraphPad Prism 9.0 software (GraphPad Software Inc.) was used for statistical analysis. Differences between two experimental groups were assessed using the Student's t-test, while differences between three groups were analysed using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

3.1 Inflammation and hypoxia occur in pulp tissue during orthodontic tooth movement

During the application of orthodontic force, the pulpal tissue underwent significant changes over time. In the pulp cavity region, we observed a gradual disorganization of the odontoblasts arrangement (indicated by white arrows) from the 1 day group (Figure 1A[a]) to the 14 days group (Figure 1A[d]). At the same time, the pulpal characteristics changed, with an increase in inflammatory cell infiltration and showed significant pulp vacuolation and vascular dilatation (indicated by *). However, in the 28 days group, the disorganization of dentin cell arrangement and pulpal vacuoles subsided (Figure 1A[e]). Changes were more pronounced in the root canal region. We observed disorganized odontoblasts arrangement in the 3 days group (Figure 1B[b], indicated by white arrows). The root canals showed more pronounced vascularization, beginning with the 7 days group (Figure 1B[c]). A substance with light purple staining, suggesting the presence of plasma proteins (indicated by black arrows), indicated hypoxia and the onset of vascularization. This was accompanied by marked vasodilatation (indicated by *). The above indicates an inflammatory response of the pulp to mechanical force and vascularization is a compensatory change in the pulp in response to hypoxia. This is consistent with our previous study, which found elevated HIF-1α and VEGF expression in the dental pulp.¹²

3.2 Macrophages polarize to the M2 type in pulp during orthodontic tooth movement

To clarify the polarization of the macrophages under mechanical force, we analysed the expression of iNOS, a marker of M1 polarization, and Arg-1, a marker of M2 polarization in the pulp. Immunohistochemical results indicated that there was no significant difference in the expression of iNOS and Arg-1 on the first day when compared to the control group. However, iNOS expression significantly increased at 3 days, followed by a declining trend after 7 days (Figure 2A,C). On the other hand, Arg-1 expression displayed an upward trend after 3 days of force application and did not exhibit a decrease until the 28th day (Figure 2B,D). Furthermore, we observed that as the appearance of vascularization and inflammation resolution, iNOS expression decreased, and Arg-1 expression correspondingly increased. This suggests a gradual polarization of macrophages towards M2 type, which may be involved in pulpal inflammation resolution and pulpal vascularization.

3.3 Hypoxia promotes the polarization of macrophages to the M2 type

To further verify the effect of hypoxia on macrophage polarization, we, respectively, induced macrophage polarization towards pro-inflammatory (M1) using 100 ng/mL LPS (24 h) and anti-inflammatory (M2) using 10 ng/mL IL-4 (24 h). We observed that LPS culture significantly increased the expression levels of iNOS and TNF-α, indicating the classical activation of pro-inflammatory macrophages (M1). Conversely, IL-4 culture significantly increased the expression of Arg-1 and decreased the expression of iNOS, signifying the selective activation of anti-inflammatory macrophages (M2) (Figure 3B).

The normal cultured RAW264.7 cells exhibited round, rod-shaped or irregular shapes with inconspicuous pseudopods. M1 direction-induced RAW264.7 cells displayed a distinct ‘omelette’ morphology with more pseudopods and fork-like changes. In contrast, M2 direction-induced RAW264.7 cells adopted a stretched ‘spindle-shaped’ appearance with fewer pseudopods (Figure 3A). The point is, RAW264.7 cells cultured with hypoxia for 24 h exhibited morphological similarities to anti-inflammatory cells, appearing as pike-shaped cells with fewer protruding pseudopods. In addition, the results of qRT-PCR indicated that iNOS and inflammatory factor TNF-α expression was decreased and Arg-1 was increased in hypoxic cultured RAW264.7 cells (Figure 3C). Western Bolt also reflected the characteristics of anti-inflammatory macrophages (M2) with decreased iNOS and increased Arg-1 (Figure 3D). This is consistent with the macrophage polarization results we have observed in vivo. The above results further validate that mechanical force may promote macrophage M2 polarization through hypoxia in the pulp.

3.4 Inhibition of macrophages M2 polarization is detrimental to pulpal vascularization and inflammation resolution

We isolated MDPCs by enzyme digestion and microscopic techniques. Microscopically, MDPCs primarily comprised fibroblasts with elongated spindle-like or stellate short protrusions, alongside a variety of pulp defence cells including macrophages, dendritic cells and lymphocytes (Figure 4A). Flow cytometric analysis of the cell surface marker F4/80 indicated that macrophages constituted approximately 15%–20% of the total cell quantity (Figure 4B). Subsequently, we subjected the extracted MDPCs to 24 h of hypoxic culture. The analysis of F4/80 immunofluorescence results suggested that the macrophage percentage remained relatively stable (Figure 4C). To examine the role of macrophages in pulp during orthodontic tooth movement, we cultured MDPCs with hypoxia after adding a selectively activated (M2) polarization inhibitor. By analysing the mRNA expression of IL-1β, IL-10, TNF-α and VEGF, it revealed that inflammatory factors such as IL-1β and TNF-α exhibited an increase and the secretion of anti-inflammatory factor IL-10 and the VEGF decreased (Figure 4D). ELISA was also performed for the expression of the above factors in the culture supernatants. The results also showed an anti-inflammatory trend (Figure 4E). These suggest that, for the entire pulp tissue, inhibition of macrophage M2 polarization would be detrimental to inflammatory regression and vascularization. Hypoxia induces macrophages to participate in pulpal vascularization and promoting inflammatory regression during orthodontic tooth movement.