Periodontal Ligament Cell Apoptosis Activates Lepr+ Osteoprogenitors in Orthodontics

Abstract

Alveolar bone (AB) remodeling, including formation and absorption, is the foundation of orthodontic tooth movement (OTM). However, the sources and mechanisms underlying new bone formation remain unclear. Therefore, we aimed to understand the potential mechanism of bone formation during OTM, focusing on the leptin receptor+ (Lepr+) osteogenitors and periodontal ligament cells (PDLCs). We demonstrated that Lepr+ cells activated by force-induced PDLC apoptosis served as distinct osteoprogenitors during orthodontic bone regeneration. We investigated bone formation both in vivo and in vitro. Single-cell RNA sequencing analysis and lineage tracing demonstrated that Lepr represents a subcluster of stem cells that are activated and differentiate into osteoblasts during OTM. Targeted ablation of Lepr+ cells in a mouse model disrupted orthodontic force–guided bone regeneration. Furthermore, apoptosis and sequential fluorescent labeling assays revealed that the apoptosis of PDLCs preceded new bone deposition. We found that PDL stem cell–derived apoptotic vesicles activated Lepr+ cells in vitro. Following apoptosis inhibition, orthodontic force–activated osteoprogenitors and osteogenesis were significantly downregulated. Notably, we found that bone formation occurred on the compression side during OTM; this has been first reported here. To conclude, we found a potential mechanism of bone formation during OTM that may provide new insights into AB regeneration.

Keywords

bone remodeling/regeneration osteoblast(s)periodontal ligament (PDL)orthodontic tooth movement extracellular vesicles stem cell(s)

Introduction

Orthodontic treatment is the most commonly used method for improving tooth alignment and occlusion. Its biological basis includes effective bone remodeling and tooth movement mediated by mechanical force (Li et al. 2021). Orthodontic bone remodeling requires the synergistic action of different cell types, including osteoclasts, osteoblasts, and immune cells (Li et al. 2021; Alghamdi et al. 2023). Osteoclasts create a movable space for the tooth root, while osteoblasts maintain tooth stability upon reaching the targeted position. Insufficient osteogenic activity may lead to severe complications, such as a reduction in alveolar bone (AB) height and even tooth loss (Guo et al. 2021). However, the source of osteoblasts and their activation mechanism during bone remodeling remains unclear.

With the development of lineage-tracing techniques, it is now possible to dynamically track the fate of individual cell populations in vivo. Multiple cell markers, including Lepr, Osx, Gli1, and Cxcl12, have been identified, possessing distinct functions in bone formation (Li, Xu, et al. 2022). Among these, leptin receptor+ (Lepr+) bone marrow stem cells, which are an important osteogenic stem cell population in the long bones of adults, are the main source of osteoblasts during injury repair (Shu et al. 2021; Mo et al. 2022). Notably, Lepr+ cells in the AB exhibit similar characteristics (Zhang et al. 2020). Recently, Lepr+ cells in the periodontal ligament (PDL) were identified as mechanoresponsive cells that maintain periodontal homeostasis (Zhang et al. 2023). Developmentally, both AB and PDL originate from neural crest cells. Moreover, orthodontic bone remodeling is regulated by mechanical forces. However, the role of Lepr+ cells in the AB and PDL during orthodontic force–induced bone remodeling has yet to be revealed. Therefore, this study aimed to determine the potential mechanism of AB remodeling in orthodontics by exploring the role of Lepr+ cells and PDLCs.

Herein, we identified Lepr+ cells, activated by force-induced PDLCs apoptosis, as distinct osteoprogenitors during orthodontic bone regeneration. These findings demonstrate the potential mechanism of bone formation during orthodontic tooth movement (OTM) and provide new insights into AB regeneration.

Materials and Methods

Animals

All procedures were approved by the Animal Care and Use Committee of Peking University (approval number: PUIRB-LA2023099). This study conformed with the ARRIVE 2.0 guidelines. Lepr-CreER and ROSA-tdTomato mice were purchased from the Shanghai Model Organisms Center (Shanghai, China). The ROSA-iDTR mice were purchased from Cyagen Biosciences (Suzhou, China). Caspase3 -/- mice were kindly provided by Professor Zhengfan Jiang (School of Life Sciences, Peking University). The wild-type mice were from Vital River Laboratory Animal Technology Co. (Beijing, China). All mice were housed in a pathogen-free environment. For all in vivo studies, sex-matched littermate mice were used in the control and experimental groups. We made every effort to minimize the pain and stress that animals suffered in this study. To induce Cre recombinase, tamoxifen (Sigma-Aldrich) dissolved in corn oil (25 mg/mL) was injected intraperitoneally (125 mg/kg body weight) for 3 consecutive days.

To trace the fate of Lepr+ cells during OTM, tamoxifen induction was performed on Lepr-CreER;tdTomato mice before OTM. The mice were harvested at 3, 7, 14, and 28 d after OTM.

To study the impact of ablation of Lepr+ cells, Lepr-CreER;tdTomato;iDTR mice were generated by mating Lepr-CreER;tdTomato mice and iDTR mice. After tamoxifen induction and OTM, the mice were intraperitoneally administered with either 100 µL of physiological saline or 100 µL of diphtheria toxin (DT; 2 μg/mL, Sigma-Aldrich) dissolved in physiological saline solution for 7 consecutive days to ablate Lepr+ cells. The mice were sacrificed at 14 d of OTM.

To understand the process of destruction and reconstruction of the PDL during OTM, wild-type mice were used. Sirius red staining was performed at 0, 3, 14, and 28 d after OTM. At 3 d after OTM, TUNEL assay and immunofluorescence staining of Caspase3 were performed.

To investigate the onset of osteogenesis during OTM, triple bone labeling analysis was performed on wild-type mice.

To investigate the role of PDLC apoptosis in orthodontic osteogenesis, we used Caspase3 -/- mice as the experimental group and wild-type mice as the control group. After 7 d of OTM, the samples were harvested.

Details of the methods are included in the appendix materials and methods.

Animal Experiments for OTM

OTM was conducted on 8-week-old mice. The procedure was modified on the basis of a previously described method (Liu et al. 2022). Following general anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg), nickel-titanium coil springs were used to connect the maxillary right first molar and incisors with a continuous force of 20 g. The contralateral maxilla was set as the control. All force application procedures were conducted by the same user to ensure consistency of the force magnitudes. Subsequently, the mice were placed on a heating pad for recovery, and they were fed with a soft-food diet.

Statistical Analyses

All data are presented as mean ± standard deviation. Statistical differences between 2 independent groups were analyzed using an unpaired 2-tailed Student’s test for comparison. One-way analysis of variance was performed for multiple comparisons. Statistical significance at a P value < 0.05 was set.

Supplemental Materials and Methods

The appendix file contains the supplemental materials and methods.

Results

Tracing Lepr+ Cells in Periodontal Tissue

To understand the fate of Lepr+ cells in the periodontium, published scRNA-seq data from human periodontal tissues, including from the AB, PDL, and gingival tissues, were analyzed (Chen et al. 2022). After the cell subclusters were divided, we found that LEPR was highly enriched in cluster 12, showing an expression pattern similar to that of mesenchymal stem cell (MSC) markers (CXCL12 and THY1) and osteoblast markers (SP7 and POSTN) (Fig. 1A –C). However, scRNA-seq data from only the PDL showed that LEPR was highly expressed in fibroblasts and endothelial cells (Appendix Fig. 1A–C) (Pagella et al. 2021). Fluorescent imaging consistently demonstrated significant co-localization of Lepr with the endothelial cell markers Pecam1 in the PDL in the quiescent state (Fig. 1D, E and Appendix Fig. 1D, E). However, this co-localization was lower in AB (Fig. 1E and Appendix Fig. 1E).

To analyze the differentiation process of Lepr+ cells in the quiescent state, colocalization of Lepr/tomato with the MSC markers Thy1 and Mcam was detected at various time points after tamoxifen induction (Fig. 1F). This co-localization demonstrated a significant increase in the percentage of MSCs derived from Lepr+ cells over time (Fig. 1G –I). Three months after induction, this percentage was approximately 60% in the bone marrow and 25% in the periodontium. These findings suggest that Lepr+ cells are progenitors of MSCs in both the AB and PDL tissues, and Lepr+ cells in AB may have greater proliferative capacity than that possessed by those in PDL.

Lepr+ Cells Participate in Orthodontic Force–Guided Bone Regeneration

Lepr-CreER;tdTomato mice were generated for lineage tracing during OTM. We found that Lepr+ cells were activated by orthodontic forces and differentiated into osteoblasts (Fig. 2A). Orthodontic force was applied after tamoxifen induction, and osteoblasts in the AB and PDL tissues were detected at various stages of OTM (Appendix Fig. 2A, B). Osteoblasts are stained by Osx; we found that Lepr+ cell–derived osteoblasts were stained by both Osx and tdTomato fluorescence. Orthodontic force significantly increased the proportion of Lepr+ osteoblasts in AB from the third day of OTM (Fig. 2B, C). The progeny of Lepr+ cells contributed to more than 70% of the osteoblasts in the AB at the 28th day of OTM (Fig. 2C, Appendix Fig. 2C–F). Orthodontic force, comprising both the compression and tensile forces, increased the number of Lepr+ cell–derived osteoblasts in the PDL (Fig. 2D, E). Notably, we found that the proportion of Osx+&Tomato+ cells increased significantly to nearly 40% by the 28th day, following exposure to tensile force (Fig. 2E, Appendix Fig. 2G–I). These results indicate that Lepr+ cells participate in orthodontic tension–guided bone regeneration.

Furthermore, parabiosis experiments showed that Lepr+ cells did not originate from the peripheral blood (Appendix Fig. 2J, K). To investigate the process by which Lepr+ cells differentiated into osteoblasts in periodontal tissue, an EdU proliferation assay was performed (Fig. 2F). In the Lepr-CreER;tdTomato mouse OTM model, the Lepr+ cell progeny, which were segregated after EdU injection, were labeled with both Tomato and EdU. These results demonstrate that orthodontic force markedly promoted the proliferation of Lepr+ cells in both AB and PDL (Fig. 2G–M). Notably, in the quiescent state, Lepr+ cells exhibited greater proliferation (Fig. 2H, K) and were activated earlier in the AB than that in the PDL during OTM (third day vs. the seventh day, Fig. 2I, M). This difference may be attributed to variations in the cell location or gene expression patterns.

Ablation of Lepr+ Cells Disrupts Orthodontic Force–Guided Bone Regeneration

To further explore the role of Lepr+ cells in orthodontic force–guided bone regeneration, Lepr-CreER;tdTomato;iDTR mice were generated to selectively ablate Lepr+ cells during OTM (Fig. 3A). Most Lepr+ cells were successfully ablated after tamoxifen and DT treatment (Fig. 3B–D). Activation of Lepr+ cells by orthodontic force was significantly impeded in both the AB and PDL (Fig. 3B–D). Consistently, the proportion of osteoblasts marked by Osx decreased substantially in both the control and OTM groups (Fig. 3E–G, Appendix Fig. 3). Furthermore, micro–computed tomography (micro-CT) analysis demonstrated that OTM guided AB regeneration. However, in Lepr+ cell–ablated mice, the AB was degenerated (Fig. 3H). Quantitative analysis showed that bone mineralization density and bone volume in both the control and OTM groups significantly decreased after Lepr+ cell ablation (Fig. 3I–L). Furthermore, double bone labeling analysis demonstrated that the promoting effect of orthodontic tension on AB deposition was significantly inhibited after depletion of Lepr+ cells (Fig. 3M, N). These results further confirm the essential role of Lepr+ cells in orthodontic force–guided bone regeneration.

New Bone Formation Occurred following Orthodontic Force–Induced PDLC Apoptosis

Considering that the PDL is the prime location whereby a mechanical force is converted into a biological signal, changes in PDLCs were analyzed to understand the mechanism by which Lepr+ progenitors were activated. Sirius red staining was used to understand the process of “destruction and reconstruction” of PDL during OTM (Fig. 4A–D). Destruction of the PDL began at an early stage (3 d) of OTM (Fig. 4C, D). Furthermore, TUNEL staining and Caspase3 immunofluorescence staining confirmed that the orthodontic force comprising both compression and tensile forces induced PDLC apoptosis from the third day of OTM (Fig. 4E–H, Appendix Fig. 4A). Western blotting conducted on the cells cultured in vitro showed a notable increase in the levels of apoptosis-related markers and a decrease in the expression levels of apoptosis-inhibiting proteins 24 h after applying tension or compression forces to PDLCs (Fig. 4I, J and Appendix Fig. 4B).

Following confirmation of PDLC apoptosis during the early stage of OTM, the time at which orthodontic force–guided bone regeneration began was investigated. New bone formation during OTM was recorded using sequential fluorescent labeling at different time points (Appendix Fig. 4C). Orthodontic tension force promoted new bone formation 5 d after OTM (Appendix Fig. 4D). Interestingly, new bone formation was observed on the compression side (Appendix Fig. 4D), consistent with the osteoblasts observed on the compression side (Fig. 2D, Appendix Fig. 2G). To clarify the precise time point at which orthodontic force–guided bone regeneration began, more detailed sequential fluorescent labeling was conducted (Fig. 4K). The amount of new bone deposited on the tension side before the fifth day was much lower than that deposited after the fifth day (Fig. 4K). These results indicate that orthodontic force induces PDLC apoptosis before guiding new bone deposition.

Defective Apoptosis Process Inhibits Orthodontic Force–Guided Bone Regeneration

Apoptotic vesicles are derived from programmed cell death and play an essential role in maintaining MSCs and bone homeostasis (Liu et al. 2018). To investigate whether PDLC-derived apoptotic vesicles activated Lepr+ osteoprogenitors, tomato+ cells were sorted from Lepr-CreER;tdTomato mice femurs after tamoxifen induction for 5 consecutive days. Further experiments revealed that apoptotic vesicles from PDL stem cells (PDLSCs) significantly enhanced the proliferative and osteogenic capacities of Lepr+ cells derived from the maxillary bone marrow (Fig. 5A –D, Appendix Fig. 5A and B). Caspase3 is critical for the final phase of apoptosis. To investigate the effect of cell apoptosis on osteogenesis during OTM, Caspase3 knockout (Caspase3 -/-) mice were used as apoptosis-deficient models. Micro-CT analysis demonstrated that impaired apoptosis dysregulated bone homeostasis and hindered new bone formation during OTM (Fig. 5E–I). Immunohistochemical staining demonstrated that osteoblasts in the PDL were significantly inhibited in the apoptosis-deficient OTM models (Fig. 5J). Furthermore, our findings showed that orthodontic force promoted the expression of apoptotic markers (cleaved Caspase3) and osteogenic markers (Osx and Runx2); these markers were significantly inhibited in the AB of apoptosis-deficient mice (Fig. 5K and Appendix Fig. 5C). In conclusion, we propose that orthodontic force–induced PDLC apoptosis activates Lepr+ osteoprogenitor cells (Fig. 5L).

Discussion

Effective bone remodeling forms the basis of OTM. In this study, we investigated the sources of osteoblasts and their activation mechanisms during orthodontic force–induced osteogenesis using a mouse model. Our findings demonstrated that during OTM, the PDL is subjected to compressive and tensile forces, leading to apoptosis of PDLCs. This process triggers the recruitment of Lepr+ cells to the stress region, where they become activated and participate in osteogenesis. Furthermore, we discovered that orthodontic force–induced apoptosis of PDLCs activates Lepr+ osteoprogenitors. During the OTM process, Lepr+ cells acted as a prominent source of osteoblasts, actively contributing to osteogenic activities. Numerous studies have investigated the role of PDLSCs in osteogenesis (Huang et al. 2019; Dieterle et al. 2021; Lin et al. 2021). However, other potential sources of osteoblasts, particularly stem cell populations in the AB, have long been overlooked. Developmentally, both the PDL and AB originate from neural crest cells. This suggests that AB stem cells may share a biological response pattern similar to that of PDLSCs. Previous studies have reported that Lepr+ osteoprogenitor cells account for approximately 94% of the stem cell population in adult long bones (Zhou et al. 2014), serving as an important source of osteoblasts that rapidly respond to stress conditions, such as fractures, radiation damage, and aging (Shu et al. 2021; Mo et al. 2022). In the quiescent state, the progeny of Lepr+ cells make up 2% of osteoblasts and 7% of PDLSCs in the AB (Men et al. 2020), contributing to 50% of osteoblasts in the AB during injury repair (Zhang et al. 2020). Recently, Zhang et al. (2023) identified Lepr+ cells as a developmental origin for other cell populations in the periodontium through pseudotime analysis and cell tracing. Consistent with previous research, this study revealed that Lepr+ cells within the AB act as reserve cells in the quiescent state, swiftly responding to orthodontic force stimulation and differentiating into osteoblasts.

In addition, Gli1+ stem cell populations were found participate in osteogenesis during OTM (Liu et al. 2020; Seki et al. 2023). The Gli1+ stem cell population not only develops into osteoblasts but also massively differentiates into fibroblasts in the PDL (Men et al. 2020). However, Gli1+ cells are more abundant in the adolescent population and are distinctly decreased in the adult population. In contrast, Lepr+ cells are highly enriched with osteoprogenitors throughout postnatal life (Shi et al. 2017; Men et al. 2020; Mo et al. 2022). As AB regeneration in response to orthodontic treatment can occur throughout an individual’s life after tooth eruption, this study focused on Lepr+ cells to accurately identify the source of osteoblasts during OTM.

Lepr+ cells in the AB exhibited higher proliferative activity than those in the PDL. ScRNA-seq analysis revealed that Lepr expression in the AB overlapped with the expression of osteogenic genes, while in the PDL, Lepr overlapped with both osteogenic and endothelial cell markers, demonstrating greater heterogeneity. This is consistent with our observation that Lepr+ cells in the AB maintained a higher proliferation rate than those in the PDL in the quiescent state. In addition, this suggests that stem cells derived from the AB possess a stronger ability to respond to external stimuli. These differences may be attributed to the distinct tissue microenvironments of the PDL and the AB. Given that the differences between Lepr+ cells in the AB and PDL do not constitute the primary focus of this study, an in-depth investigation into the underlying causes was not pursued. In the scRNA-seq analysis by Zhang et al. (2023), the overlap of Lepr+ with other stem cell markers might be due to the exclusion of other cell populations from their analysis; thus, the expression of Lepr in these excluded cell populations was not analyzed.

The question remains: How does the PDL serve as a crucial connection between the tooth and the AB, converting mechanical signals into biological signals? In this study, we found that apoptosis of PDLCs precedes osteogenesis during OTM and that osteogenesis during OTM was significantly inhibited in mice with apoptosis defects. Therefore, we proposed that apoptosis of PDLCs induced by orthodontic forces promotes the osteogenic differentiation of Lepr+ osteoprogenitors. This finding of orthodontic force–induced apoptosis in PDLCs is consistent with that from previous research (Hao et al. 2009). Accumulating evidence suggests that apoptotic vesicles play a notable role in maintaining and promoting the osteogenesis of MSCs (Liu et al. 2018; Ma et al. 2020). Our study introduces another novel piece of evidence demonstrating bone deposition on the compression side at the micro level. This is contrary to the classic OTM theory, which typically suggests bone resorption on the compression side at the macro level. Previous studies have reported that mechanical compression inhibits the osteogenic ability of PDLCs (Huang et al. 2019) and promotes osteoclast differentiation (Huang et al. 2022). Our results appear to challenge these claims. This contradiction may be explained by the activation of Lepr+ precursor cells by PDLC-derived apoptotic vesicles. This newly proposed theory also aligns with recent clinical findings, in which significant bone formation was observed in the retraction direction of the upper anterior teeth once retraction had ceased (Guo et al. 2023).

OTM is a “sterile” inflammatory process mediated by mechanical force, in which force-induced PDLCs apoptosis enhances local tissue regeneration. During the initial stages of the process, a considerable amount of periodontal fibers are disrupted on both the compression and tension sides. This damage is subsequently repaired and reconstructed. The activation of Lepr+ cells in the PDL occurs slightly later than that in the AB but lasts for a longer duration. This delay may be attributed to the initial PDL damage process during the early stages of OTM. This suggests a role for Lepr+ cells in the repair process of the periodontium. During cell apoptosis, the cell membrane envelops the internal contents to form apoptotic vesicles. Previous studies have found that apoptotic vesicles derived from MSCs promote skull injury repair and can be used for the treatment of bone diseases, such as osteoporosis (Li, Xing, et al. 2022). Apoptosis promotes the function of phagocytic cells (Gerlach et al. 2021). Some studies propose that this may be due to the miRNA or proteins contained in apoptotic vesicles, while others suggest that it may occur through interactions involving apoptotic vesicles or specific proteins on the cell membrane (O’Brien et al. 2020; Ma et al. 2021; Wu et al. 2023). These findings underscore the crucial role of apoptotic vesicles in local and systemic cell-cell communication. Although the role of apoptosis in promoting tissue repair has been verified, the specific biological mechanisms warrant further exploration.

Author Contributions

H. Liu, contributed to conception and design, data acquisition, analysis, and interpretation, drafted and critically revised manuscript; Y. Zhang, contributed to design, data acquisition and analysis, drafted and critically revised manuscript; Y. Zhang, contributed to design, data interpretation, critically revised the manuscript; Y. Huang, contributed to conception and design, data analysis, critically revised the manuscript; Y. Yang, Y. Zhao, contributed to design, data acquisition, drafted the manuscript; S. Chen, contributed to data conception and design, critically revised the manuscript; J. Deng, contributed to conception and design, data analysis and interpretation, critically revised the manuscript; W. Li, B. Han, contributed to conception and design, data interpretation, critically revised the manuscript. All authors provided their final approval and agreed to be accountable for all aspects of this study.

Supplemental Material

sj-docx-1-jdr-10.1177_00220345241262706 – Supplemental material for Periodontal Ligament Cell Apoptosis Activates Lepr+ Osteoprogenitors in Orthodontics

Supplemental material, sj-docx-1-jdr-10.1177_00220345241262706 for Periodontal Ligament Cell Apoptosis Activates Lepr+ Osteoprogenitors in Orthodontics by H. Liu, Y. Zhang, Y. Zhang, Y. Huang, Y. Yang, Y. Zhao, S. Chen, J. Deng, W. Li and B. Han in Journal of Dental Research

Footnotes

Acknowledgements

We thank Professor Zhengfan Jiang (School of Life Sciences, Peking University) for providing the Caspase3 -/- mice. We appreciate the ORAL STEM CELL BANK run by Beijing Tason Biotech Co., Ltd. () for kindly providing the human PDLSCs. The authors have no competing financial interests to declare.

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Fundings

The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by grants from the National Key R&D Program of China (2021YFC2400403), Clinical Research Foundation of Peking University School and Hospital of Stomatology (PKUSS-2023CRF301), National Natural Science Foundation of China (51972005, U21A2055, 82370993, 82201017), Research Foundation of Peking University School and Hospital of Stomatology (PKUSS20230118), Peking University Medicine Sailing Program for Young Scholars’ Scientific & Technological Innovation, the Fundamental Research Funds for the Central Universities (BMU2024YFJHPY009), Beijing Municipal Science Technology Commission (Z211100002921066), China Postdoctoral Science Foundation (2022M710257), Young Elite Scientist Sponsorship Program by CAST (2022QNRC001).

ORCID iDs

H. Liu

Y. Zhang

J. Deng

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Supplementary Material

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