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.

UNASSIGNED: Periodontal disease is a common oral infection which affects the tooth-supportive tissues directly. Considering the limitation of present regenerative treatments for severe periodontal cases, cytotherapies have been gradually introduced. Human periodontal ligament-derived mesenchymal stromal cells (hPDLMSCs), while identified as one of the promising cell sources for periodontal regenerative therapy, still hold some problems in the clinical application especially their limited life span. To solve the problems, human induced pluripotent stem cells (hiPSCs) are taken into consideration as a robust supply for hPDLMSCs.
UNASSIGNED: The induction of hPDLMSCs was performed based on the generation of neural crest-like cells (NCLCs) from hiPSCs. Fibronectin and laminin were tested as coating materials for NCLCs differentiation when following previous protocol, and the characteristics of induced cells were identified by flow cytometry and RT-qPCR for evaluating the induction efficiency. Subsequently, selected dental ectoderm signaling-related cytokines were applied for hPDLMSCs induction for 14 days, and dental mesenchyme-related genes, dental follicle-related genes and hPDL-related genes were tested by RT-qPCR for the evaluation of differentiation.
UNASSIGNED: Compared to the 58% in laminin-coated condition, fibronectin-coated condition had a higher induction efficiency of CD271high cells as 86% after 8-day induction, while the mesenchymal potential of induced NCLCs was similar between two coating materials.It was shown that the gene expressions of dental mesenchyme, dental follicles and hPDL cells were significantly enhanced with the stimulation of the combination with fibroblast growth factor 8b (FGF8b), FGF2, and bone morphogenetic protein 4 (BMP4).
UNASSIGNED: FN coating was more effective in NCLCs induction, and the FGF8b+FGF2+BMP4 growth factor cocktail was effective in hPDLMSC-like cell generation. These findings underscored the likely regenerative potential of hiPSCs as an applicable and promising curative strategy for periodontal diseases.

Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research. Key features • This protocol builds upon a method described by Cunningham et al. [1] for selective release of Treponema outer membrane proteins. • We adapted the protocol for the purification of biologically active, detergent-stable outer membrane protein complexes from large batch cultures of T. denticola. • The protocol involves large-scale preparative electrophoresis using a Model 491 Prep Cell. • We then use gelatin zymography to demonstrate the activity of the purified dentilisin complex by its ability to activate matrix metalloproteinase 2 (MMP-2).

UNASSIGNED: The three-dimensional morphological structures of periodontal ligaments (PDLs) are important data for periodontal, orthodontic, prosthodontic, and implant interventions. This study aimed to employ a deep learning (DL) algorithm to segment the PDL automatically in cone-beam computed tomography (CBCT).
UNASSIGNED: This was a retrospective study. We randomly selected 389 patients and 1734 axial CBCT images from the CBCT database, and designed a fully automatic PDL segmentation computer-aided model based on instance segmentation Mask R-CNN network. The labels of the model training were \'teeth\' and \'alveolar bone\', and the \'PDL\' is defined as the region where the \'teeth\' and \'alveolar bone\' overlap. The model\'s segmentation performance was evaluated using CBCT data from eight patients outside the database.
UNASSIGNED: Qualitative evaluation indicates that the PDL segmentation accuracy of incisors, canines, premolars, wisdom teeth, and implants reached 100%. The segmentation accuracy of molars was 96.4%. Quantitative evaluation indicates that the mIoU and mDSC of PDL segmentation were 0.667 ± 0.015 (>0.6) and 0.799 ± 0.015 (>0.7) respectively.
UNASSIGNED: This study analysed a unique approach to AI-driven automatic segmentation of PDLs on CBCT imaging, possibly enabling chair-side measurements of PDLs to facilitate periodontists, orthodontists, prosthodontists, and implantologists in more efficient and accurate diagnosis and treatment planning.

UNASSIGNED: The periodontium is a connective tissue which consists of periodontal ligament, alveolar bone, cementum and gingiva. Periodontal ligament (PDL) is a specialized connective tissue that connects the cementum - coating the surface of the tooth - to the alveolar bone. Mohawk homeobox (Mkx) is a transcription factor that is expressed in PDL, that is known to play a vital role in the development and homeostasis of PDL. A detailed functional analysis of Mkx in the periodontal ligament for alveolar bone and cementum metabolism has not yet been conducted.
UNASSIGNED: Alveolar bone height, bone mineral density (BMD) and bone volume fractions (Bone volume/Total volume: BV/TV) were measured and analyzed using micro-computed tomography (Micro-CT) and 3DBon on 7-week-old male wild-type (WT) (Mkx+/+) (n = 10) and Mkx-knockout (Mkx-/-) (n = 6) rats. Hematoxylin and Eosin (H&E), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and Masson Trichrome staining were performed on 5, 6, and 7-week-old Mkx+/+ and Mkx-/- rats. Cementum surface area and the number of TRAP-positive osteoclasts/mm were quantified, measured, and compared for 5,6 and 7-week-old Mkx+/+ and Mkx-/- rats (n = 3 each).
UNASSIGNED: The level of alveolar bone height was significantly higher in Mkx-/- rats than in Mkx+/+ rats. On the other hand, there was significantly less BMD in Mkx-/- alveolar bone. A significant increase in cellular cementum could be observed as early as 5 weeks in Mkx-/- rats when compared with Mkx+/+ rats of the same age. More TRAP-positive osteoclasts were observed in Mkx-/- rats.
UNASSIGNED: Our findings further reveal the essential roles of Mkx in the homeostasis of the periodontal tissue. Mkx was found to contribute to bone and cementum metabolism and may be essential to the prevention of diseases such as periodontitis, and could show potential in regenerative treatments.

UNASSIGNED: Periodontitis is a long-term, multifactorial inflammatory condition that is triggered by bacterial germs and interacts with the host\'s immune system. The unique attachment of fibrous tissue between the cementum and bone presents a challenge for periodontal regeneration.
UNASSIGNED: To achieve the lowest optimum dose of BMP-7 that helps in periodontal regeneration, involving newly formed cementum, PDL and bone.
UNASSIGNED: Five healthy mongrel dogs were used for the study. A critical class III furcation defect was created using rotating burs. The bone defects (ten defects for each group) were allocated to one of the subsequent groups: (Group 1) control with the surgical defect only. (Group 2) Surgical defect implanted with hydrogel only (CS/β-GP). (Group 3) Surgical defect implanted with CS/BMP-7 (50 ng/ml). (Group 4) Surgical defect implanted with CS/BMP-7 (100 ng/ml).
UNASSIGNED: Histomorphometric and H&E analysis revealed a statistically significant difference in bone, PDL, and cementum regeneration defects filled with CS/BMP-7 (100 ng/ml) compared with other groups.
UNASSIGNED: The standard effective dose for BMP-7 use in periodontal regeneration is 100 ng/ml.

OBJECTIVE: Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis.
METHODS: DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro.
RESULTS: The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts.
CONCLUSIONS: Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

Periodontal ligament (PDL) attaches tooth root to the surrounding bone. Its existence between tooth and jaw bone is of utmost importance due to its significant role in absorbing and distributing physiological and para-physiological loading. According to the previous studies, various mechanical tests have been performed to characterize the mechanical properties of the PDL; however, all of them have been done at room temperature. To the best of our knowledge, this is the first study in which the testing was performed at body temperature. The present research was planned to measure the dependency of PDL\'s viscoelastic behavior on temperature and frequency. Three different temperatures, including body and room temperature, were opted to perform the dynamic compressive tests of the bovine PDL. In addition, a Generalized Maxwell model (GMM) was presented based on empirical outcomes. At 37 °C, amounts of loss factor were found to be greater than those in 25 °C, which demonstrates that the viscous phase of the PDL in higher temperatures plays a critical role. Likewise, by raising the temperature from 25 °C to 37 °C, the model parameters show an enlargement in the viscous part and lessening in the elastic part. It was concluded that the PDL\'s viscosity in body temperature is much higher than that in room temperature. This model would be functional for a more accurate computational analysis of the PDL at the body temperature (37 °C) in various loading conditions such as orthodontic simulations, mastication, and impact.

Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to the thin and vulnerable periodontal ligament is the key reason for failure. Cell- or stem cell-based regenerative medicine has emerged in the past two decades as a promising clinical treatment modality to improve treatment outcomes. This concept has also been tested for the management of avulsed teeth in animal models. This review focuses on the discussion of limitation of current management protocols for avulsed teeth, cell-based therapy for periodontal ligament (PDL) regeneration in small and large animals, the challenges of de novo regeneration of PDL on denuded root in the edentulous region using a mini-swine model, and establishing a prospective new clinical protocol to manage avulsed teeth based on the current progress of cell-based PDL regeneration studies.

BACKGROUND: The functional interplay between cementum of the root and alveolar bone of the socket is tuned by a uniquely positioned 70-80 µm wide fibrous and lubricious ligament in a dentoalveolar joint (DAJ). In this study, structural and biomechanical properties of the DAJ, periodontal ligament space (PDL-space also known as the joint space), alveolar bone of the socket, and cementum of the tooth root that govern the biomechanics of a lipopolysaccharide (LPS)-affected DAJ were mapped both in space and time.
METHODS: The hemi-maxillae from 20 rats (4 control at 6 weeks of age, 4 control and 4 LPS-affected at 12 weeks of age, 4 control and 4 LPS-affected at 16 weeks of age) were investigated using a hybrid technique; micro-X-ray computed tomography (5 µm resolution) in combination with biomechanical testing in situ. Temporal variations in bone and cementum volume fractions were evaluated. Trends in mineral apposition rates (MAR) in additional six Sprague Dawley rats (3 controls, 3 LPS-affected) were revealed by transforming spatial fluorochrome signals to functional growth rates (linearity factor - RW) of bone, dentin, and cementum using a fast Fourier transform on fluorochrome signals from 100-µm hemi-maxillae sections.
RESULTS: An overall change in LPS-affected DAJ biomechanics (a 2.5-4.5X increase in tooth displacement and 2X tooth rotation at 6 weeks, no increase in displacement and a 7X increase in rotation at 12 weeks; 27% increase in bone effective strain at 6 weeks and 11% at 12 weeks relative to control) was associated with structural changes in the coronal regions of the DAJ (15% increase in PDL-space from 0 to 6 weeks but only 5% from 6 to 12 weeks compared to control). A significant increase (p < 0.05) in PDL-space between ligated and age-matched control was observed. The bone fraction of ligated at 12 weeks was significantly lower than its age-matched control, and no significant differences (p > 0.05) between groups were observed at 6 weeks. Cementum in the apical regions grew faster but nonlinearly (11% and 20% increase in cementum fraction (CF) at 6 and 12 weeks) compared to control. Alveolar bone revealed site-specific nonlinear growth with an overall increase in MAR (108.5 µm/week to 126.7 µm/week after LPS treatment) compared to dentin (28.3 µm/week in control vs. 26.1 µm/week in LPS-affected) and cementum (126.5 µm/week in control vs. 119.9 µm/week in LPS-affected). A significant increase in CF (p < 0.05) in ligated specimens was observed at 6 weeks of age.
CONCLUSIONS: Anatomy-specific responses of cementum and bone to the mechano-chemo stimuli, and their collective temporal contribution to observed changes in PDL-space were perpetuated by altered tooth movement. Data highlight the \"resilience\" of DAJ function through the predominance of nonlinear growth response of cementum, changes in PDL-space, and bone architecture. Despite the significant differences in bone and cementum architectures, data provided insights into the reactionary effects of cementum as a built-in compensatory mechanism to reestablish functional competence of the DAJ. The spatial shifts in architectures of alveolar bone and cementum, and consequently ligament space, highlight adaptations farther away from the site of insult, which also is another novel insight from this study. These adaptations when correlated within the context of joint function (biomechanics) illustrate that they are indeed necessary to sustain DAJ function albeit being pathological.