Effect of Mitophagy on the Tension-Driven Osteogenic Differentiation of Periodontal Ligament Stem Cells During Ageing

Abstract

Objectives

Age-related differences in orthodontic tooth movement (OTM) and mechanical force-induced osteogenesis have been reported. Mitophagy plays a crucial role in bone metabolism and various age-related diseases, and BCL2-interacting protein 3 (BNIP3) is a mitophagy-related receptor. This study aimed to elucidate the role of mitophagy associated with BNIP3 on age-related changes in the orthodontic tension-driven osteogenic differentiation of periodontal ligament stem cells.

Materials and methods

Periodontal ligament stem cells (PDLSCs) from adolescent (6-week-old) and adult (8-month-old) rats were cultured and stretched using a Flexcell system. The effects of mitophagy associated with BNIP3 were assessed via real-time quantitative PCR and western blot analyses. Moreover, a rat model of OTM across different ages was established for in vivo analyses. The function of mitophagy in age-related osteogenic differentiation induced by orthodontic force on the tension side was evaluated via microcomputed tomography and immunohistochemistry analyses.

Results

Under tension, the expression of the mitophagy factor BNIP3, the autophagy factor microtubule-associated protein light chain 3 (LC3), and the osteogenic factors Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) significantly increased in rPDLSCs over time. The expression of these factors was also upregulated in the rat OTM model under orthodontic force. Compared with the adolescent group, the adult group exhibited lower levels of mitophagy and osteogenic differentiation after tension both in vivo and in vitro. Enhanced mitophagy induced by carbonyl cyanide m-chlorophenyl hydrazone upregulated the expression of the aforementioned factors in an adult rat OTM model.

Conclusions

Mitophagy is associated with osteogenic activity induced by tension force in PDLSCs and may play a substantial role in regulating age-related changes in the OTM process.

Graphical abstract

Under the action of orthodontic tension force, mitophagy is activated, the periodontal ligament stem cells undergo osteogenic differentiation in vivo and vitro, and this process exhibits age-related changes. Compared with adolescent rats, adult rats have decreased osteogenic differentiation ability and reduced tooth movement distance, which is associated with mitophagy suppression. Upregulating the level of mitophagy in adult rats can improve these conditions.

Introduction

The number of adult orthodontic patients is increasing, and orthodontic treatment in adult patients is often associated with relatively greater challenges and limitations.¹ Adults exhibit slower tissue responses to orthodontic forces and decreased bone remodelling activity compared with younger individuals, which results in a prolonged treatment duration and possibly increase the incidence of complications.² A deeper understanding of age-related differences and exploration of potential regulatory mechanisms are crucial for enhancing orthodontic treatment.

Periodontal ligament stem cells (PDLSCs), mechanosensitive mesenchymal stem cells, play a crucial role in bone remodelling during orthodontic tooth movement (OTM).^3,4 Zhang et al⁵ reported that as age increased, the proliferation, migration potential, and differentiation ability of human PDLSCs decreased, demonstrating age-related changes in human. PDLSCs may play a key role in the age-related decline in orthodontic responsiveness.⁶

Mitophagy, a type of selective autophagy, eliminates and recycles damaged or dysfunctional mitochondria to maintain mitochondrial homeostasis and functional integrity.⁷ Mitophagy occurs through two main regulatory pathways: the ubiquitin-dependent pathway (the PTEN-induced putative kinase 1 [PINK1]/Parkin pathway) and the ubiquitin-independent pathway (also known as the receptor-mediated pathway).⁸ BCL2-interacting protein 3 (BNIP3) is a mitophagy-related receptor associated with the ubiquitin-independent pathway that can interact directly with microtubule-associated protein light chain 3 (LC3) and GABAA-receptor-associated protein through typical or atypical LC3-interacting regions, thereby facilitating autophagic degradation, especially under hypoxic conditions.⁹

Mitophagy is closely related to bone metabolism-related activities and ensures the proliferation and differentiation of osteogenesis-related cells by maintaining the number and function of mitochondria. Abnormal levels of mitophagy may trigger bone metabolism disorders, leading to the occurrence of diseases such as osteoporosis, osteoarthritis and rheumatoid arthritis.¹⁰ Mitophagy has become a potential target for treating bone-related diseases.¹¹ In recent years, the role of mitophagy in dental-derived mesenchymal stem cells has drawn widespread attention.¹² Mitophagy is involved in the proliferation, osteogenic differentiation, and anaerobic oxidative regulation of PDLSCs.^13,14 Mechanical force can induce PINK1/Parkin-mediated mitophagy in PDLSCs, and regulating mitophagy levels can affect the osteogenic differentiation of PDLSCs induced by tension stimulation.^15,16

Moreover, mitophagy is closely related to age-related changes. The age-dependent decrease in mitophagy contributes to mitochondrial dysfunction, which may result in various age-related diseases.^17,18 A moderate upregulation of mitophagy could alleviate the negative effects of ageing and is considered a potential therapeutic target for the ageing population.¹⁹

Mitophagy plays a crucial role in bone metabolism and age-related diseases, and recent research has focused mainly on PINK1/Parkin-mediated mitophagy. However, the effect of mitophagy on osteogenic differentiation during age-related OTM remains unclear. Therefore, this study aimed to investigate the role of mitophagy associated with BNIP3 in the osteogenic differentiation of PDLSCs induced by tension force at different ages.

Materials and methods

Cell culture

Primary PDLSCs of Sprague-Dawley rats were obtained from Cellverse Bioscience. rPDLSCs were acquired from 6-week-old to 8-month-old rats in the adolescent and adult groups, respectively, and maintained in alpha-modified Eagle’s medium (α-MEM; Gibco) supplemented with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). For subsequent experiments, rPDLSCs were used after three to five passages.

Characterization

After the cell density was adjusted to 1 to 5 × 10⁶ cells/mL, the cells were incubated with primary antibodies conjugated to CD29, CD44, CD34, and CD45 for 30 min in the dark at room temperature. Flow cytometry analysis was performed to determine the phenotype of the rPDLSCs.

Upon reaching 80% to 100% confluency, the cells were cultured in preprepared osteogenic or adipogenic differentiation medium (Cyagen Biosciences) for 2 weeks according to the manufacturer’s protocol. The cells were fixed with 4% formalin, stained with alizarin red or oil red O, and observed through microscopy.

Samples of rPDLSCs were incubated with the Cell Counting Kit-8 (CCK-8) (Dojindo) reagent in an incubator for 2 hours in the dark. The absorbances were measured at 450 nm by using a microplate reader.

After fixation, the cells were incubated with a senescence-associated β-galactosidase (SA-β-Gal) staining kit (Beyotime) at 37°C overnight in the absence of CO₂ and then observed through microscopy.

Mechanical stimulation

Three to five passages of rPDLSCs were seeded on BioFlex culture plates (Flexcell Corporation) at a density of 2 × 10⁵ cells per well. Using a Flexcell FX-5000 Strain Unit, the cells were subjected to half-sine periodic tensile stress (0.1 Hz, 12% elongation) for 0, 1, 3, 6, and 12 hours.

Real-time quantitative polymerase chain reaction

RNAiso Plus reagent (Takara) was used to extract total RNA from the rPDLSCs. A PrimeScript RT reagent kit (Takara) was used for cDNA synthesis, and SYBR Premix Ex Taq (Takara) was used for Real-time quantitative polymerase chain reaction following the manufacturer’s protocol. All genes were normalized to the expression of GAPDH, and the results were analysed using the 2^–△△Ct method.

The sequences of primers used were as follows: BNIP3 forward primer: 5′-TTAAACACCCGAAGCGCACA-3′, reverse primer: 5′-ACTGTGTGAGCAGAAGGCAG-3′; LC3B forward primer: 5′-AAAGAGTGGAAGATGTCCGGC-3′, reverse primer: 5′-GGCTTGGTTAGCATTGAGCTG-3′; TOMM20 forward primer: 5′-AAATGCAATCGCTGTGTGTGG-3′, reverse primer: 5′-ATGTTGGTGTCTGGCTCATTCC-3′; RUNX2 forward primer: 5′-GTGCCTCCAACCTGTGTTTT-3′, reverse primer: 5′-TTTGCTACTGGGTTTC-3′; OSX forward primer: 5′-GGTCCTGGCAACACTCCTAC-3′, reverse primer: 5′-AAGAGGTGGGGTGCTGGATA-3′; p53 forward primer: 5′-GGAGGATTCACAGTCGGATATG-3′, reverse primer: 5′-CTGTGGTGGGCAGAATATCA-3′; p21 forward primer: 5′-TTGCCACTTCTTACCTGGGG-3′, reverse primer: 5′-GTGACAAGGAGACCCCGAAG-3′; p16 forward primer: 5′-GATGGGCAACGTCAAAGTGG-3′, reverse primer: 5′-TCGTGATGTCCCCGCTCTA-3′; and GAPDH forward primer: 5′-CAAGTTCAACGGCACAGTCA-3′, reverse primer: 5′-CCCCATTTGATGTTAGCGGG-3′.

Western blotting

Total protein was extracted using radioimmunoprecipitation lysis buffer (Solarbio) and quantified via a bicinchoninic acid protein assay kit (Thermo Fisher). The protein samples were separated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After 1 hour of blocking in fat-free milk, the membranes were incubated with a specific primary antibody overnight at 4°C. After the membranes were incubated with a secondary antibody at room temperature for 1 hour, the bands were detected using an enhanced chemiluminescence detection system. ImageJ was used to assess protein expression, which was normalized to that of HSP90.

Transmission electron microscopy

After mechanical force was applied for 6 hours, the rPDLSCs were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer. Then the rPDLSCs were dehydrated, embedded, sectioned, and stained for observation via transmission electron microscope (HT-7700).

Establishment of rat OTM model and injection of drugs

All animal experiments were approved by the Animal Ethics and Welfare Committee of the School of Stomatology, Capital Medical University, and complied with the ARRIVE guidelines.

Thirty-five male Sprague-Dawley rats (SPF Biotechnology) were used. Fifteen 6-week-old rats and 20 8-month-old rats were used to represent the adolescent and adult groups, respectively.

After general anaesthesia with 1.25% tribromoethanol (Avertin, 10 ml/kg; M2960; AibeiBio) was administered, a nickel-titanium (Ni-Ti) coil spring was inserted between the maxillary incisors and the maxillary left first molar, providing a force of 25 g for mesial movement of the maxillary left first molar for 7 days (Figure 4A). A soft diet was provided to avoid damage to the orthodontic device.

Fig. 4.

Establishment and micro-CT results of the rat OTM model. (A) Diagram of orthodontic tooth movement model establishment. (B) Diagram of the zone of interest for histopathological analysis on the tension side of the periodontal ligament of the left maxillary first molar. (C and D) Comparison of the orthodontic tooth movement between 6w and 8m rats. (E-H) The statistics results of BV/TV, Tb.N, Tb.Sp, and Tb.Th on tension side in Days 0 and 7 between 6w and 8m rats. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001.

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which disrupts the mitochondrial membrane, is a common mitophagy inducer.²⁰ In adult rats, CCCP (MCE) was locally injected into the distobuccal and distopalatal alveolar bone of the left maxillary first molar at a dose of 200 µL at a concentration of 10 µM/L on Days 0 and 4. Normal saline (SJZ NO.4 Pharmaceutical) was injected into the same areas and in the same amount and frequency as those used in the experimental animals in both adult and adolescent control rats (Figure 6A).

Fig. 6.

Orthodontic tooth movement and micro-CT results in 6w-NS, 8m-NS, and 8m-CCCP rats. (A) Flowchart of drug injection and specimen collection. (B and C) Comparison of the orthodontic tooth movement among 6w-NS, 8m-NS, and 8m-CCCP rats. (D-G) The statistics results of BV/TV, Tb.N, Tb.Sp, and Tb.Th on tension side among 6w-NS, 8m-NS, and 8m-CCCP rats. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001.

Before force was applied, 5 adolescent and 5 adult rats were randomly selected and sacrificed to establish a baseline (6w-0d and 8m-0d). The other rats were divided into different groups after orthodontic force loading: 5 adult rats and 5 adolescent rats without any injections (6w-7d and 8 m-7d), 5 adult rats and 5 adolescent rats injected with normal saline (6w-NS and 8m-NS), and 5 adult rats injected with CCCP (8m-CCCP). They were sacrificed on Day 7.

Microcomputed tomography (micro-CT) scanning and analysis

All the specimens were fixed in 4% paraformaldehyde for 24 hours and then scanned using SkyScan1276 (SkyScan). DataViewer was used to correct the direction on each axis and measure the OTM distance, which was defined as the distance between the distal convex point of the left maxillary first molar crown and the mesial convex point of the second molar crown. CTAn (CT-analysis software) was used to calculate trabecular bone parameters in regions of interest. Bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) were measured. The measurement method was described in our previous study.²¹

Immunohistochemical (IHC) staining

IHC staining was performed using an IHC kit (ZSBio). The primary antibodies used included monoclonal rabbit antirat BNIP3 (ab109362; Abcam), polyclonal rabbit antirat LC3 (ab48394; Abcam), monoclonal rabbit antirat TOMM20 (ab186735; Abcam), monoclonal rabbit antirat Runt-related transcription factor 2 (RUNX2; ab236639; Abcam), and monoclonal rabbit antirat Osterix (ab209484; Abcam). Three random fields were selected from the periodontal membrane of the first molar on the tension side for each sample (Figure 4B). The average optical density and percentage of positive cells were calculated via Image-Pro Plus.²²

Statistical analysis

All the data were processed using GraphPad Prism 8.0 and are presented as the means ± standard deviations from three independent experiments. The Shapiro–Wilk test was used to evaluate the normality of all datasets. All groups subjected to parameter analysis were assumed to be normal. Student’s t test or one-way analysis of variance (ANOVA) was employed to evaluate the differences. The Tukey’s HSD test was used as posthoc multiple comparison tests applying after ANOVA. Statistical significance was set at P < .05.

Results

Culture and characterization rat PDLSCs

The rPDLSCs were spindle-shaped in the 6w group, while the rPDLSCs exhibited flattened structures and increased branching in the 8 m group (Figure 1A). Alizarin red and oil red O staining revealed calcified nodules and reddish colouration after osteogenic and adipogenic induction, indicating the osteogenic and adipogenic differentiation potential of the rPDLSCs (Figure 1B, C). rPDLSCs from the 8m group had relatively weak osteogenic differentiation ability and tended to undergo adipogenic differentiation. SA-β-gal staining was more strongly blue in the 8m group than in the 6w group (Figure 1D). Flow cytometry results demonstrated high expression of the positive markers CD29 and CD44 and low expression of the negative markers CD34 and CD45 in both the 6w and 8m groups (Figure 1E, F). The overall growth curve of the third-generation cultured rPDLSCs was ‘S shaped’, with slower growth in the 8m group (Figure 1G). The expression levels of age-related factors p53, p21, and p16 in the 8m group were significantly higher than those in the 6w group (Figure 1H-J, P < .01).

Fig. 1.

Characterization of PDLSCs from 6-week-old to 8-month-old rats. (A) Morphology of the third-generation rat PDLSCs. (B) After 2 weeks of osteogenic induction, rPDLSCs formed red-stained mineralized nodules. (C) After 2 weeks of adipogenic induction, rPDLSCs formed red-stained spherical lipid. (D) SA-β-Gal staining of rPDLSCs. Original magnification × 100. Scale bar for (A-D) = 100 μm. (E) For rPDLSCs from 6-week-old rats, the positive expression rates of positive markers CD29 and CD44 were 98.0% and 99.0%, respectively. The positive expression of negative markers CD34 and CD45 were 0.50% and 0.65%, respectively. (F) For rPDLSCs from 8-month-old rats, the positive expression rates of positive markers CD29 and CD44 were 96.5% and 97.7%, respectively. The positive expression of negative markers CD34 and CD45 were 0.71% and 0.97%, respectively. (G) The growth curve was represented as an ‘S’ shape. (H-J) The western blot images and relative expression of p53, p21, and p16 protein and mRNA. n = 5, *P < .05, **P < .01, ***P < .001.

Upregulated BNIP3 expression and mitophagy in rat PDLSCs under cyclic tension

The expression of BNIP3, LC3, and TOMM20 in the 8m group was lower than that in the 6w group before cyclic tension loading. The expression of BNIP3 and LC3 was upregulated by tension force and was significantly lower in the 8m group than in the 6w group. BNIP3 protein and mRNA expression were significantly lower in the 8m group than in the 6w group at 6 and 12 hours (Figure 2A, B, C, P < .05). The increase in the LC3-II/I ratio in the 8m group was lower than that in the 6w group at 1, 3 and 12 hours (Figure 2D, E, P < .05). LC3 mRNA expression was lower in the 8m group than in the 6w group at all time points (Figure 2F, P < .05). With the application of tension force, the TOMM20 protein expression in the 6w group decreased slightly, whereas that in the 8m group decreased significantly at 3 and 6 hours and were lower than those in the 6w group at all time points (Figure 2G, H, P < .01). TOMM20 mRNA expression decreased at 1 and 3 hours, and increased at 6 and 12 hours. And it was lower in the 8m group than in the 6w group at 3, 6, and 12 hours (Figure 2I, P < .01).

Fig. 2.

Upregulated mitophagy level in response to tension force. (A-C) The western blot images and relative expression of BNIP3 protein and mRNA at 0, 1, 3, 6, and 12 hours. (D-F) The western blot images and relative expression of LC3 protein and mRNA at 0, 1, 3, 6, and 12 hours. (G-I) The western blot images and relative expression of TOMM20 protein and mRNA at 0, 1, 3, 6, and 12 hours. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001. (J) The transmission electron microscopy image of rPDLSCs after 0 and 6 hours of stretching. Red arrow: autophagosome/autophagosome. Yellow arrow: mitochondria. Scale bar = 500 nm.

Transmission electron microscopy revealed mitochondria and autophagic vesicles in rPDLSCs before force loading. Six-hour cyclic tension resulted in a decrease in mitochondrial quantity and size but an increase in the number of autophagic vesicles. The numbers of mitochondria and autophagic vesicles were lower in the 8m group than in the 6w group (Figure 2J).

Increased osteogenesis-related factors expression in rat PDLSCs under cyclic tension

The levels of osteogenic-related factors in the 8m group were essentially equivalent to those in the 6w group before tension stimulation and increased markedly under tension stimulation. However, this increase was slower and lower in the 8m group than in the 6w group. Compared with that in the 6w group, RUNX2 protein expression in the 8m group was significantly lower at 6 hours (Figure 3A, B, P < .01), with lower expression of RUNX2 mRNA at 1, 3, 6 and 12 hours (Figure 3C, P < .05); OSX protein expression in the 8m group was notably lower at 1, 3 and 6 hours (Figure 3D, E, P < .05), with lower expression of OSX mRNA at 1, 3 and 12 hours (Figure 3F, P < .01).

Fig. 3.

The osteogenesis ability of rPDLSCs in response to tension force. (A-C) The western blot images and relative expression of RUNX2 protein and mRNA at 0, 1, 3, 6, and 12 hours. (D-F) The western blot images and relative expression of OSX protein and mRNA at 0, 1, 3, 6, and 12 hours. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001.

Establishment of the rat OTM model

The micro-CT results revealed significant OTM after 7 days of orthodontic force application, with 8m rats exhibiting less movement than 6w rats did (Figure 4C, D, P < .001). Initially, the BV/TV and Tb.N were greater (P < .05), whereas the Tb.Sp and Tb.Th were lower in 8m-0d rats than in 6w-0d rats. Under orthodontic force, BV/TV and Tb.N decreased significantly in both groups (P < .05); Tb.Sp increased notably (P < .01), whereas Tb.N decreased slightly (Figure 4E-H).

Orthodontic tension force upregulated expression of BNIP3 and mitophagy- and osteogenesis-related factors

IHC staining revealed that in the initial state, BNIP3 and LC3 expression was significantly lower in 8m-0d rats than in 6w-0d rats (P < .05). BNIP3 and LC3 expression was upregulated in the periodontal ligament on the tension side after orthodontic force loading in both groups (P < .01), but these increases were lower in the 8m-7d group than in the 6w-7d group (Figure 5A-D, P < .01).

Fig. 5.

IHC staining images of the rat OTM model. (A and B) IHC staining for BNIP3 and expression level of BNIP3 on tension side in 6w and 8m rats. (C and D) IHC staining for LC3 and expression level of LC3 on tension side in 6w and 8m rats. (E and F) IHC staining for TOMM20 and expression level of TOMM20 on tension side in 6w and 8m rats. (G and H) IHC staining for RUNX2 and percentage of RUNX2-positive cells on tension side in 6w and 8m rats. (I and J) IHC staining for Osterix and percentage of Osterix-positive cells on tension side in 6w and 8m rats. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001.R, root; P, PDL; B, alveolar bone. Scale bar = 50 μm.

The expression of TOMM20 in 8m-0d rats was lower than that in 6w-0d rats, but the difference was not significant. After force was applied, the change in TOMM20 expression was not notable in either the 8m-7d or the 6w-7d groups (Figure 5E, F).

Before force loading, the difference in the expression of osteogenesis-related factors between 8m-0d rats and 6w-0d rats was not significant. Orthodontic tension force promoted the percentage of RUNX2- and Osterix-positive cells in both groups (P < .05). However, RUNX2 and Osterix expression was lower in 8m-7d rats than in 6w-7d rats (Figure 5G-J, P < .05).

Mitophagy affected osteogenic differentiation in adult rats during OTM

Micro-CT revealed that the amount of OTM in 8m-CCCP rats was greater than that in 8m-NS rats (P < .05), but was still substantially lower than that in 6w-NS rats (Figure 6B, C, P < .001). The BV/TV in 8m-CCCP rats was greater than that in 8m-NS rats and significantly greater than that in 6w-NS rats (Figure 6D, P < .001). Tb.N in 8m-CCCP rats was significantly greater than that in 8m-NS and 6w-NS rats (Figure 6E, P < .001). Tb.Sp in 8m-CCCP rats was significantly lower than that in 8m-NS rats (P < .001) and lower than that in 6w-NS rats (Figure 6F, P < .05). The difference in Tb.Th among the three groups was not remarkable (Figure 6G).

Compared with that in 8m-NS rats, the expression of BNIP3 and LC3 on the tension side in 8m-CCCP rats increased markedly (P < .05), indicating that mitophagy was upregulated and was comparable to that in 6w-NS rats (Figure 7A-D). The change in the expression level of TOMM20 was not significant (Figure 7E, F). The number of RUNX2- and Osterix-positive cells increased in 8m-CCCP rats compared with 8m-NS rats (P < .05) and was comparable to that in 6w-NS rats (Figure 7G-J).

Fig. 7.

CCCP upregulated osteogenic differentiation during OTM in adult rats. (A and B) IHC staining for BNIP3 and expression level of BNIP3 on tension side. (C and D) IHC staining for LC3 and expression level of LC3 on tension side. (E and F) IHC staining for TOMM20 and expression level of TOMM20 on tension side. (G and H) IHC staining for RUNX2 and percentage of RUNX2-positive cells on tension side. (I and J) IHC staining for Osterix and percentage of Osterix-positive cells on tension side. Data are presented as ‘mean ± standard deviation’. n = 5, *P < .05, **P < .01, ***P < .001. R, root; P, PDL; B, alveolar bone. Scale bar = 50 μm.

Discussion

There are significant biological and therapeutic differences between adults and adolescents during orthodontic treatment.²³ The mobilization and remodelling of periodontal tissue are slower in adults than in adolescents,²⁴ which may affect OTM. OTM is a complex biological process that requires osteogenic differentiation of PDLSCs, which is a critical step for bone remodelling, and its mechanism has not been fully elucidated. In our study, we selected 6-week-old and 8-month-old rats, corresponding to humans aged 11 to 13 years and 25 to 30 years, respectively,²⁵ to investigate the potential effect of age on osteogenic differentiation in PDLSCs subjected to tension force.

In line with previous research, we found that PDLSCs from adult rats exhibited age-related changes, such as reduced proliferation and osteogenic differentiation abilities, increased SA-β-gal positivity, and expression of age-related factors.^26,27 Our results revealed increased protein and mRNA expression of RUNX2 and OSX, demonstrating that tension force promoted osteogenic differentiation of PDLSCs, and the same results were obtained in vivo.²⁸ We compared alveolar bone changes in adolescent and adult rats. The baseline BV/TV and Tb.N in adult rats was greater than that in adolescent rats, indicating the alveolar bone from the tension side becomes denser in adult rats.²⁹ With respect to orthodontic force, the alveolar bone tends to be looser in adults than in adolescents, and the number of RUNX2- and Osterix-positive cells is lower. Similar to that in previous studies, the osteogenic activity in adult rats was lower.³⁰

So far, many theories have been proposed regarding the mechanism of reduced reactivity in adults, including oxidative stress, DNA damage, genomic instability, epigenetic and metabolic disarray, inflammation, apoptosis, and mitochondrial injury.³¹ In the selective elimination of damaged, aged, and redundant mitochondria, mitophagy maintains cellular and organismal homeostasis through mitochondrial quality control. An age-dependent decrease in mitophagy leads to the accumulation of dysfunctional mitochondria and, consequently, the deterioration of cell function.³² BNIP3, a mitochondrial protein anchored in the outer mitochondrial membrane, can exert multiple cellular effects on mitochondria. BNIP3 homodimerizes and binds to LC3 to dock mitochondria to autophagosomes, thereby ensuring their removal through mitophagy.³³ Our results demonstrated that the expression of BNIP3 and LC3 was lower in adult rat samples in vivo and in vitro, indicating an age-dependent decrease in BNIP3-related mitophagy, which was similar to the findings of previous studies.^34,35

Previous studies have shown that mechanical stimuli impair mitochondrial function while activating mitophagy to maintain mitochondrial homeostasis.^36,37 Zhang et al¹⁵ reported that 10% elongation and 0.5 Hz mechanical tension stimulated mitochondrial fission and altered oxidative patterns in PDLSCs, which induced mitophagy. In our study, the average size of the mitochondria decreased after tension. Mitophagy was induced, as evidenced by the upregulated protein and mRNA expression of BNIP3 and LC3. TOMM20 is an indicator of mitochondrial number.³⁸ An intriguing observation was the opposite changes in the expression of TOMM20 in vitro versus in vivo. This divergence likely mirrors the distinct biological contexts: acute mechanical stress in isolation versus chronic adaptation within a complex tissue.³⁹ In vitro, mitochondrial damage occurs in isolated cells under tension force, mitophagy is activated rapidly to clear damaged mitochondria and prevent injury propagation, leading to a net reduction in mitochondrial content, so the expression of TOMM20 decreased. However, in vivo, tension force initiates a high-energy-demand process involving inflammation, angiogenesis, and active bone remodelling. While mitophagy associated with BNIP3 is activated, mitochondrial biogenesis program is simultaneously and cooperatively activated to meet the energetic demand. The increased TOMM20 expression in vivo may represent the net outcome of this dynamic turnover, supporting the elevated energy requirements of successful OTM. In addition, adult rats presented lower levels of BNIP3, LC3, and TOMM20, indicating an age-related decline in the response of mitophagy to mechanical tension.

The facilitation of mitophagy can prolong lifespan and improve tissue homeostasis.⁴⁰ Mitophagy is emerging as a potential target for therapeutic interventions against age-related diseases, such as neurodegeneration,⁴¹ cardiovascular diseases,⁴² osteoporosis¹⁹ and muscle atrophy.⁴³ The functional upregulation of BNIP3-mediated mitophagy could improve organismal homeostasis and health with age.⁴⁴

Mitophagy plays a crucial role in the stem cell fate, such as promoting survival, maintenance, and differentiation of stem cells. Mitophagy participates in the processes like erythrocyte maturation, myogenesis, and neurogenesis.⁴⁵ The reduction of mitophagy could induce stem cell senescence and loss of stemness and pluripotency.⁴⁶ The appropriate role of mitophagy is vital for successful osteogenic differentiation. Defects in mitophagy could influence osteogenic differentiation by failing to maintain healthy mitochondria. In Pink1-knockout mice with impaired mitophagy, the differentiation of osteoblasts was inhibited, the bone mass and collagen deposition were decreased.⁴⁷ Some biomaterials could enhance osteogenic differentiation and bone regeneration through the upregulation mitophagy.^48,49 Enhanced mitophagy in bone marrow mesenchymal stem cells and osteoblasts can increase the expression levels of osteogenic markers such as ALP, OCN, OPN, RUNX2 and COL1A to address bone formation dysfunction in osteoporosis.^50,51 Mitophagy can promote the osteogenic differentiation of dental pulp stem cells to accelerate dentin restoration.⁵² Activation of mitophagy reverses age-associated decreases in osteogenic activity in senescent PDLSCs and restored bone regeneration in aged mice.⁵³

To elucidate the role of mitophagy in age-related osteogenic differentiation induced by orthodontic tension force, CCCP, a pharmacological promoter of mitophagy, was applied to adult rats. Fan et al⁵⁴ reported that the osteogenic differentiation level of MSCs was restored by CCCP. In our study, the change in mitophagy levels after CCCP injection was consistent with the change in osteogenic activity, suggesting that upregulation of mitophagy could promote osteogenesis on the tension side, which is similar to the findings of a previous study.⁵⁵ We also compared the data between adult rats injected with CCCP and adolescent rats injected with saline to confirm the positive role of mitophagy in reversing the adulthood state. Numbers of RUNX2- and Osterix-positive cells changed significantly in response to CCCP-mediated regulation and even reached the levels in adolescent rats. Compared with adult rats injected with saline, adult rats injected with CCCP exhibited accelerated tooth movement and denser bone on the tension side.

This study has several limitations. In this study, mitophagy level in adult rats was only upregulated via CCCP injection. Future research should use mitophagy promoters and inhibitors in both adolescent and adult rats to conduct an in-depth investigation of the role of mitophagy in age-related osteogenesis induced by orthodontic force. Although studies have confirmed that CCCP can promote the expression of BNIP3,56, 57, 58 CCCP can affect multiple pathways (like activating PINK1/Parkin pathway, enhancing activation of AMPK and inhibition of mTORC1) to induce mitophagy, elicit mitochondrial-derived signaling, alter global cellular energy metabolism, and even trigger proapoptotic signals.^59,60 Future research should combine pharmacological or genetic approaches to better verify the role of BNIP3-mediated mitophagy in the osteogenic differentiation of PDLSCs under tension. Meanwhile, in the compression side, mitophagy is also activated. The environment of ischemia and hypoxia leads to an increase in ROS and hypoxia-inducible factor-1α expression.⁶¹ And BNIP3 is itself a known hypoxia-inducible factor-1α target gene. So, research regarding the compression side should be included to better elucidate the role of mitophagy in OTM. Additional in-depth investigations are needed to identify potential therapeutic targets for modulating bone remodelling during OTM, especially in adult patients.

Conclusions

In this study, we revealed that mitophagy responded to tension force. There were age-related changes in osteogenic differentiation and decrease in the mitophagy level with age under orthodontic tension force. When the mitophagy level in adult rats was upregulated, osteogenesis increased, and the amount of OTM increased to some extent. These findings demonstrate that mitophagy may play a substantial role in regulating age-related changes during OTM, and the BNIP3 pathway may be involved. This study provides novel insights for improving orthodontic therapeutic approaches, especially for adults, although further research is needed.

Funding

This study was supported by the National Nature Science Foundation of China (Grant Number 82471009), the Natural Science Foundation of Beijing Municipality (Grant Number L232106) to Kai Yang, Young Scientist Program of Beijing Stomatological Hospital, Capital Medical University (No. YSP202313) to Bowen Xu.

Ethics statement

This project was approved by Animal Experimentation Ethics Committee of School of Stomatology, Capital Medical University, Beijing, China (Approval No. KQYY-202207-005).

Author contributions

Chuhan Peng: Experiment operation, specimen collection, data collection and analysis, illustration drawing, and writing of the original draft. Yidan Zhang, Linna Bai, and Xinyu Zhang: Experiment operation, specimen collection, data collection, and analysis. Bowen Xu: Experiment operation, writing instruction, draft outline organization, and funding acquisition. Kai Yang: Conceptualization, experimental instruction, supervision, project administration, and funding acquisition. All authors reviewed and approved the final manuscript.

Conflict of 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 article.