Periodontal Ligament Reactions to Orthodontic Force: A Transcriptomic Study on Maxillary and Mandibular Human Premolars.

AIMS: Orthodontic force (OF) induces a variety of reactions in the periodontal ligament (PDL) that could potentially account for individual variability regarding orthodontic tooth movement (OTM). This study investigates the transcriptomic profile of human PDL tissue subjected to OF in vivo for 7 and 28 days, additionally comparing the differences between maxillary and mandibular PDL. METHODS: Healthy patients requiring orthodontic premolar extractions were randomly assigned to one of three groups: control (CG) where no OF was applied, 7 days and 28 days, where premolars were extracted either 7 or 28 days after the application of a 50-100 g OF. Total RNA was extracted from the PDL tissue and analyzed via RNA-seq. Differentially expressed genes (DEGs) were identified using a false discovery rate and fold change threshold of < 0.05 and ≥ 1.5 respectively. Functional and Protein-Protein Interaction analysis were performed. RESULTS: After 7 days of OF, the reaction of PDL to OF is characterized by cell responses to stress, increased bone resorption, inflammation and immune response, and decreased bone formation. In contrast, after 28 days, bone regeneration is more prominent, and processes of bone homeostasis, immune response, and cell migration are present. The response of maxillary and mandibular PDL was different. Bone resorption was observed in the maxilla at 7 and 28 days, while in the mandible expression of cell proliferation and transcriptional activity were predominant after 28 days of OF. CONCLUSIONS: The early reaction of the PDL to OF corresponds with increased bone resorption and decreased bone formation. After 28 days, bone formation became more prominent. The maxillary and mandibular PDL present asynchronous responses during OTM. These findings enhance our comprehension of the mechanisms underlying the origin-specific responses of PDL to different lengths of OF, which is potentially relevant in the development of personalized therapeutic strategies.

Biomechanical effects of a new crimpable gate spring combined with conventional rectangular archwires for torque adjustment of individual anterior teeth : A comparative finite element study.

OBJECTIVE: Precise root torque adjustment of anterior teeth is indispensable for optimizing dental esthetics and occlusal stability in orthodontics. The efficiency of traditional rectangular archwire manipulation within bracket slots seems to be limited. The crimpable gate spring, a novel device, has emerged as a promising alternative. Yet, there is a paucity of guidelines for its optimal clinical application. This study used finite element analysis (FEA) to investigate the biomechanical impact of the gate spring on torque adjustment of individual anterior teeth and to elucidate the most effective application strategy. METHODS: A FEA model was constructed by a maxillary central incisor affixed with an edgewise bracket featuring a 0.022â€¯× 0.028 inch (in) slot. A range of stainless steel rectangular archwires, in conjunction with a gate spring, were modeled and simulated within the bracket slots. A control group utilized a conventional rectangular wire devoid of a gate spring. Palatal root moments were standardized to 9, 18, and 36 Nmm for both experimental and control groups. RESULTS: The gate spring significantly amplified palatal root movement, notably with the 0.019â€¯× 0.025 in archwire. However, this was accompanied by an increase in stress on the tooth and periodontal ligament, particularly in the cervical regions. The synergistic use of a 0.019â€¯× 0.025 in rectangular archwire with a gate spring in a 0.022â€¯× 0.028 in bracket slot was identified as most efficacious for torque control of individual anterior teeth. CONCLUSIONS: The gate spring is a viable auxiliary device for enhancing torque adjustment on individual teeth. However, caution is advised as excessive initial stress may concentrate in the cervical and apical regions of the periodontal ligament and tooth.

1,25-dihydroxyvitamin-D3 distinctly impacts the paracrine and cell-to-cell contact interactions between hPDL-MSCs and CD4+ T lymphocytes.

Introduction: Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) possess a strong ability to modulate the immune response, executed via cytokine-boosted paracrine and direct cell-to-cell contact mechanisms. This reciprocal interaction between immune cells and hPDL-MSCs is influenced by 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). In this study, the participation of different immunomodulatory mechanisms on the hPDL-MSCs-based effects of 1,25(OH)2D3 on CD4+ T lymphocytes will be elucidated using different co-culture models with various cytokine milieus. Material and methods: hPDL-MSCs and CD4+ T lymphocytes were co-cultured indirectly and directly with inserts (paracrine interaction only) or directly without inserts (paracrine and direct cell-to-cell contact interaction). They were stimulated with TNF-α or IL-1ß in the absence/presence of 1,25(OH)2D3. After five days of co-cultivation, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the gene expression of soluble and membrane-bound immunomediators was determined in hPDL-MSCs. Results: In the indirect and direct co-culture model with inserts, 1,25(OH)2D3 decreased CD4+ T lymphocyte proliferation and viability. The direct co-culture model without inserts caused the opposite effect. 1,25(OH)2D3 mainly decreased the CD4+ T lymphocyte-associated secretion of cytokines via hPDL-MSCs. The degree of these inhibitions varied between the different co-culture setups. 1,25(OH)2D3 predominantly decreased the expression of the soluble and membrane-bound immunomediators in hPDL-MSCs to a different extent, depending on the co-culture models. The degree of all these effects depended on the absence and presence of exogenous TNF-α and IL-1ß. Conclusion: These data assume that 1,25(OH)2D3 differently affects CD4+ T lymphocytes via the paracrine and direct cell-to-cell contact mechanisms of hPDL-MSCs, showing anti- or pro-inflammatory effects depending on the co-culture model type. The local cytokine microenvironment seems to be involved in fine-tuning these effects. Future studies should consider this double-edged observation by executing different co-culture models in parallel.

Effects of Stress on Biological Characteristics and Metabolism of Periodontal Ligament Stem Cells of Deciduous Teeth.

INTRODUCTION AND AIMS: Periodontal ligament stem cells (PDLSCs) from deciduous teeth (DePDLSCs) can perceive and respond to mechanical signals upon exposure to various environments. The effects of mechanical stress on the biological characteristics and metabolism of DePDLSCs were investigated using in vitro stress loading. METHODS: DePDLSCs were subjected to mechanical stresses of different strengths. Cell proliferation, expression of osteogenic/osteoclastic factors, apoptosis, and oxidative stress levels were evaluated using CCK-8 assays, alkaline phosphatase staining, real-time PCR, flow cytometry, and malondialdehyde and superoxide dismutase assays. Liquid chromatography-mass spectrometry was used to perform nontargeted metabolomic detection and analysis. RESULTS: Under stresses of 75 and 150 kPa, the expression of osteogenesis-related factors OPG, ALP, and RUNX2 decreased, and the ratio of RANKL/OPG significantly increased. A pressure of 150 kPa induced oxidative stress and caused a significant increase in cell apoptosis. Among the differential metabolites screened from the 150 kPa group, spermine, spermidine, ceramide, phosphatidylethanolamine, lysophosphatidylethanolamine, linoleic acid, and docosatrienoic acid were the most significantly upregulated. The metabolites screened from the 75 kPa group were mainly related to glycerophospholipid and sphingolipid metabolism, oxidative phosphorylation, and mineral absorption, which were common pathways affected in both experimental groups. CONCLUSION: A certain degree of mechanical stress can inhibit the proliferative activity and osteogenic differentiation of DePDLSCs, enhance their osteoclast-inducing ability, and cause elevated levels of cell apoptosis and oxidative stress. The metabolic expression profile of DePDLSCs changed significantly under stress. Understanding changes in cellular activity and metabolic reactions may provide an experimental basis for elucidating the role of mechanical stress in root resorption and periodontal tissue remodelling of deciduous teeth. CLINICAL RELEVANCE: Mechanical stress may affect periodontal tissue remodeling and root resorption of DePDLSc.

A hierarchical Bilayered scaffold for periodontal complex structure regeneration.

The periodontal tissue comprises alveolar bone, cementum, and periodontal ligament (PDL), forming a highly hierarchical architecture. Although current therapies could regenerate the hard tissue well, the simultaneous reconstruction of hard and soft tissue remains a great clinical challenge with the major difficulty in highly orientated PDL regeneration. Using the unidirectional freeze-casting method and biomimetic mineralization technique, we construct a hierarchical bilayer scaffold with the aligned chitosan scaffold with ZIF-8 resembling PDL, and intrafibrillarly mineralized collagen resembling alveolar bone. The hierarchical bilayer scaffold exhibits different geomorphic clues and chemical microenvironments to realize a perfect simulation of the natural periodontal hierarchical architecture. The aligned scaffold with ZIF-8 could induce the fibrogenic differentiation of bone mesenchymal stromal cells (BMSCs), and the mineralized scaffold could induce osteogenic differentiation of BMSCs. The hierarchical bilayer scaffold could simulate periodontal complex tissue, exhibiting great promise for synchronized multi-tissue regeneration of periodontal tissue.

NIR Laser Irradiation Promotes Osteogenic Differentiation of PDLSCs Through the Activation of TRPV1 Channels and Subsequent Calcium Signaling.

Near-infrared (NIR) irradiation has shown potential to stimulate osteogenic differentiation, but the mechanisms are not fully understood. The study is to investigate the effects of NIR laser irradiation on osteoblastic differentiation. Human periodontal ligament stem cells (hPDLSCs) were cultured in osteogenic medium and exposed to 810 nm NIR laser at 0.5 J/cm2 every 48 h. The transient receptor potential vanilloid (TRPV1) channel inhibitor capsazepine (CPZ) was used to evaluate the role of calcium influx. Osteogenic differentiation was assessed by proliferation (CCK-8), alkaline phosphatase (ALP) activity, mineralization (Alizarin Red), and expression of bone markers by PCR and Western blot over 2 weeks. Intracellular calcium was measured by Fluo-4M dye and flow cytometry. Results showed that NIR irradiation enhanced hPDLSC proliferation, ALP activity, mineralization, and bone marker expression, indicating increased osteogenic differentiation. These effects were inhibited by CPZ. NIR induced a transient rise in intracellular calcium peaking at 3 min, which was blocked by CPZ. In conclusion, this study demonstrates that NIR laser irradiation promotes osteogenic differentiation of PDLSCs through the activation of TRPV1 channels and subsequent calcium signaling. Further research is warranted to optimize the treatment parameters and elucidate the detailed signaling pathways involved, paving the way for the clinical application of NIR therapy in the treatment of bone disorders and periodontal disease.

ANXA2 promotes osteogenic differentiation and inhibits cellular senescence of periodontal ligament cells (PDLCs) in high glucose conditions.

Background: Periodontal ligament cells (PDLCs) are a major component of the periodontal ligament and have an important role in the regeneration of periodontal tissue and maintenance of homeostasis. High glucose can affect the activity and function of PDLCs in a variety of ways; therefore, it is particularly important to find ways to alleviate the effects of high glucose on PDLCs. Annexin A2 (ANXA2) is a calcium- and phospholipid-binding protein involved in a variety of cellular functions and processes, including cellular cytokinesis, cytophagy, migration, and proliferation. Aim: The aim of this study was to exploring whether ANXA2 attenuates the deleterious effects of high glucose on PDLCs and promotes osteogenic differentiation capacity. Methods and results: Osteogenic differentiation potential, cellular senescence, oxidative stress, and cellular autophagy were detected. Culturing PDLCs with medium containing different glucose concentrations (CTRL, 8 mM, 10 mM, 25 mM, and 40 mM) revealed that high glucose decreased the protein expression of ANXA2 (p < 0.0001). In addition, high glucose decreased the osteogenic differentiation potential of PDLCs as evidenced by decreased calcium deposition (p = 0.0003), lowered ALP activity (p = 0.0010), and a decline in the expression of osteogenesis-related genes (p = 0.0008). Moreover, ß-Galactosidase staining and expression of p16, p21 and p53 genes showed that it increased cellular senescence in PDLCs (p < 0.0001). Meanwhile high glucose increased oxidative stress in PDLCs as shown by ROS (p < 0.0001). However, these damages caused by high glucose were inhibited after the addition of 1 µM recombinant ANXA2 (rANXA2), and we found that rANXA2 enhanced autophagy in PDLCs under high glucose conditions. Conclusions and discussion: Therefore, our present study demonstrates that alterations in ANXA2 under high glucose conditions may be a factor in the decreased osteogenic differentiation potential of PDLCs. Meanwhile, ANXA2 is associated with autophagy, oxidative stress, and cellular senescence under high glucose conditions.

The age-related effects on orthodontic tooth movement and the surrounding periodontal environment.

Orthodontic treatment in adults is often related to longer treatment time as well as higher periodontal risks compared to adolescents. The aim of this review is to explore the influence of age-related chages on orthodontic tooth movement (OTM) from macro and micro perspectives. Adults tend to show slower tooth movement speed compared to adolescence, especially during the early phase. Under orthodontic forces, the biological responses of the periodontal ligament (PDL) and alveolar bone is different between adult and adolescents. The adult PDL shows extended disorganization time, increased cell senescence, less cell signaling and a more inflammatory microenvironment than the adolescent PDL. In addition, the blood vessel surface area is reduced during the late movement phase, and fiber elasticity decreases. At the same time, adult alveolar bone shows a higher density, as well as a reduced osteoblast and osteoclast activation, under orthodontic forces. The local cytokine expression also differs between adults and adolescents. Side-effects, such as excessive root resorption, greater orthodontic pain, and reduced pulpal blood flow, also occur more frequently in adults than in adolescents.

Regulation of TGF-ß1-induced fibroblast differentiation of human periodontal ligament stem cells through the mutually antagonistic action of ectonucleotide pyrophosphatase/phosphodiesterase 1 and 2.

Human periodontal ligament stem cells (hPDLSCs) differentiate into periodontal ligament (PDL) fibroblasts, osteoblasts, and cementoblasts. To identify inducers of PDL fibroblastic differentiation, monoclonal antibody series were developed a series of against membrane/extracellular matrix (ECM) molecules through decoy immunization. The anti-PDL13 antibody targets ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), renowned for regulating skeletal and soft tissue mineralization. ENPP1 accumulates in the periodontal ligament region of tooth roots, and specifically localizes to the cell boundaries and elongated processes of the fibroblastic cells. As ENPP1 expression increases during fibroblastic differentiation, mineralization induced by tissue-nonspecific alkaline phosphatase (TNAP), a pyrophosphate-degrading enzyme, is completely inhibited. This is consistent with ENPP1 and TNAP acting in opposition, and TGF-ß1-induced ENPP1 expression creates an essential environment for PDL fibroblast differentiation. Representative fibroblastic differentiation markers decrease with endogenous ENPP1 inhibition by siRNA and antibody blocking. ENPP2 generates lipid signaling molecules. In contrast to ENPP1, ENPP2 disappears in TGF-ß1-induced PDL fibroblasts. Ectopic expression of ENPP2 hinders TGF-ß1-induced PDL fibroblastic differentiation. Suppression of ENPP1 and ENPP2 leads to severe defects in undifferentiated and differentiated cells, demonstrating that these two factors play opposing roles in soft and hard tissue differentiation but can complement each other for cell survival. In conclusion, increased ENPP1 is crucial for TGF-ß1-induced PDL differentiation, while ENPP2 and TNAP can inhibit ENPP1. ENPP1 and ENPP2 exhibit complementary functions in the cell survival.

"Periodontal ligament-on-chip" as a Novel Tool for Studies on the Physiology and Pathology of Periodontal Tissues.

Teeth exert fundamental physiological functions, such as mastication and speech, and are a key feature of oral health that affects life quality. Teeth are anchored to the alveolar bone via the periodontal ligament, which provides stability to the teeth and absorbs mechanical stresses during mastication. Periodontal infection leads to periodontitis, a severe inflammation of the supporting soft tissues that ultimately cause tooth loss. Despite the pressing need of periodontal regeneration for improved oral care, efficient in vitro models of the periodontal tissues are still missing, thus hampering the development of novel, faster, and more effective therapy modalities. Herein, a novel "periodontal ligament (PDL)-on-chip" model that integrates patient-derived periodontal ligament cells (PDLCs) and endothelial cells is introduced. This microfluidic platform provides optimal conditions for the formation of extensive and perfusable vascular networks. Furthermore, PDLCs elicit blood vessels' development and maturation while establishing close contacts with the endothelial cells. Potential applications for inflammatory periodontal diseases are also successfully displayed in the "PDL-on-chip" by stimulating inflammation and detecting inflammatory cytokines. This work offers a cornerstone for more complex and specialized microfluidic dental models, which are necessary to unravel complex oral diseases that affect individuals' general health that go beyond the field of dentistry.

FoxO1-Overexpressed Small Extracellular Vesicles Derived from hPDLSCs Promote Periodontal Tissue Regeneration by Reducing Mitochondrial Dysfunction to Regulate Osteogenesis and Inflammation.

Purpose: Periodontitis is a chronic infectious disease characterized by progressive inflammation and alveolar bone loss. Forkhead box O1 (FoxO1), an important regulator, plays a crucial role in maintaining bone homeostasis and regulating macrophage energy metabolism and osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, FoxO1 was overexpressed into small extracellular vesicles (sEV) using engineering technology, and effects of FoxO1-overexpressed sEV on periodontal tissue regeneration as well as the underlying mechanisms were investigated. Methods: Human periodontal ligament stem cell (hPDLSCs)-derived sEV (hPDLSCs-sEV) were isolated using ultracentrifugation. They were then characterized using transmission electron microscopy, Nanosight, and Western blotting analyses. hPDLSCs were treated with hPDLSCs-sEV in vitro after stimulation with lipopolysaccharide, and osteogenesis was evaluated. The effect of hPDLSCs-sEV on the polarization phenotype of THP-1 macrophages was also evaluated. In addition, we measured the reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells. Experimental periodontitis was established in vivo in mice. HPDLSCs-sEV or phosphate-buffered saline (PBS) were injected into periodontal tissues for four weeks, and the maxillae were collected and assessed by micro-computed tomography, histological staining, and small animal in vivo imaging. Results: In vitro, FoxO1-overexpressed sEV promoted osteogenic differentiation of hPDLSCs in the inflammatory environment and polarized THP-1 cells from the M1 phenotype to the M2 phenotype. Furthermore, FoxO1-overexpressed sEV regulated the ROS level, ATP production, mitochondrial characteristics, and metabolism of hPDLSCs and THP-1 cells in the inflammatory environment. In the in vivo analyses, FoxO1-overexpressed sEV effectively promoted bone formation and inhibited inflammation. Conclusion: FoxO1-overexpressed sEV can regulate osteogenesis and immunomodulation. The ability of FoxO1-overexpressed sEV to regulate inflammation and osteogenesis can pave the way for the establishment of a therapeutic approach for periodontitis.

Nanosilicates facilitate periodontal regeneration potential by activating the PI3K-AKT signaling pathway in periodontal ligament cells.

The recent development of nanobiomaterials has shed some light on the field of periodontal tissue regeneration. Laponite (LAP), an artificially synthesized two-dimensional (2D) disk-shaped nanosilicate, has garnered substantial attention in regenerative biomedical applications owing to its distinctive structure, exceptional biocompatibility and bioactivity. This study endeavors to comprehensively evaluate the influence of LAP on periodontal regeneration. The effects of LAP on periodontal ligament cells (PDLCs) on osteogenesis, cementogenesis and angiogenesis were systematically assessed, and the potential mechanism was explored through RNA sequencing. The results indicated that LAP improved osteogenic and cementogenic differentiation of PDLCs, the regulatory effects of LAP on PDLCs were closely correlated with activation of PI3K-AKT signaling pathway. Moreover, LAP enhanced angiogenesis indirectly via manipulating paracrine of PDLCs. Then, LAP was implanted into rat periodontal defect to confirm its regenerative potential. Both micro-CT and histological analysis indicated that LAP could facilitate periodontal tissue regeneration in vivo. These findings provide insights into the bioactivity and underlying mechanism of LAP on PDLCs, highlighting it might be a potential therapeutic option in periodontal therapy.

Effects of Scutellaria baicalensis Extract-Induced Exosomes on the Periodontal Stem Cells and Immune Cells under Fine Dust.

In adverse environments, fine dust is linked to a variety of health disorders, including cancers, cardiovascular, neurological, renal, reproductive, motor, systemic, and respiratory diseases. Although PM10 is associated with oral inflammation and cancer, there is limited research on biomaterials that prevent damage caused by fine dust. In this study, we evaluated the effects of biomaterials using microRNA profiling, flow cytometry, conventional PCR, immunocytochemistry, Alizarin O staining, and ELISA. Compared to SBE (Scutellaria baicalensis extract), the preventive effectiveness of SBEIEs (SBE-induced exosomes) against fine dust was approximately two times higher. Furthermore, SBEIEs promoted cellular differentiation of periodontal ligament stem cells (PDLSCs) into osteoblasts, periodontal ligament cells (PDLCs), and pulp progenitor cells (PPCs), enhancing immune modulation for oral health against fine dust. In terms of immune modulation, SBEIEs activated the secretion of cytokines such as IL-10, LL-37, and TGF-ß in T cells, B cells, and macrophages, while attenuating the secretion of MCP-1 in macrophages. MicroRNA profiling revealed that significantly modulated miRNAs in SBEIEs influenced four biochemical categories: apoptosis, cellular differentiation, immune activation, and anti-inflammation. These findings suggest that SBEIEs are an optimal biomaterial for developing oral health care products. Additionally, this study proposes functional microRNA candidates for the development of pharmaceutical liposomes.

Mechanisms of mechanical force in periodontal homeostasis: a review.

Mechanical forces affect periodontal health through multiple mechanisms. Normally, mechanical forces can boost soft and hard tissue metabolism. However, excessive forces may damage the periodontium or result in irreversible inflammation, whereas absence of occlusion forces also leads to tissue atrophy and bone resorption. We systemically searched the PubMed and Web of Science databases and found certain mechanisms of mechanical forces on immune defence, extracellular matrix (ECM) metabolism, specific proteins, bone metabolism, characteristic periodontal ligament stem cells (PDLSCs) and non-coding RNAs (ncRNAs) as these factors contribute to periodontal homeostasis. The immune defence functions change under forces; genes, signalling pathways and proteinases are altered under forces to regulate ECM metabolism; several specific proteins are separately discussed due to their important functions in mechanotransduction and tissue metabolism. Functions of osteocytes, osteoblasts, and osteoclasts are activated to maintain bone homeostasis. Additionally, ncRNAs have the potential to influence gene expression and thereby, modify tissue metabolism. This review summarizes all these mechanisms of mechanical forces on periodontal homeostasis. Identifying the underlying causes, this review provides a new perspective of the mechanisms of force on periodontal health and guides for some new research directions of periodontal homeostasis.

Role of sodium-dependent vitamin C transporter 2 in human periodontal ligament fibroblasts.

AIM: Ascorbic acid (AA) is a water-soluble vitamin that has antioxidant properties and regulates homeostasis of connective tissue through controlling various enzymatic activities. Two cell surface glycoproteins, sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2, are known as ascorbate transporters. The purpose of this study was to investigate the expression pattern and functions of SVCTs in periodontal ligament (PDL) and PDL fibroblast (PDLF). METHODS: Gene expression was examined using real-time polymerase chain reaction (PCR) and reverse transcription PCR. SVCT2 expression was determined by immunofluorescence staining, western blot and flow cytometry. ALP activity and collagen production were examined using ALP staining and collagen staining. Short interfering RNA was used to knock down the gene level of SVCT2. Change of comprehensive gene expression under SVCT2 knockdown condition was examined by RNA-sequencing analysis. RESULTS: Real-time PCR, fluorescent immunostaining, western blot and flowy cytometry showed that SVCT2 was expressed in PDLF and PDL. ALP activity, collagen production, and SVCT2 expression were enhanced upon AA stimulation in PDLF. The enhancement of ALP activity, collagen production, and SVCT2 expression by AA was abolished under SVCT2 knockdown condition. RNA-sequencing revealed that gene expression of CLDN4, Cyclin E2, CAMK4, MSH5, DMC1, and Nidgen2 were changed by SVCT2 knockdown. Among them, the expression of MSH5 and DMC1, which are related to DNA damage sensor activity, was enhanced by AA, suggesting the new molecular target of AA in PDLF. CONCLUSION: Our study reveals the SVCT2 expression in PDL and the pivotal role of SVCT2 in mediating AA-induced enhancements of ALP activity and collagen production in PDLF. Additionally, we identify alterations in gene expression profiles, highlighting potential molecular targets influenced by AA through SVCT2. These findings deepen our understanding of periodontal tissue homeostasis mechanisms and suggest promising intervention targeting AA metabolism.

Enzymatic activity of fibroblast activation protein-α is essential for TGF-ß1-induced fibroblastic differentiation of human periodontal ligament cells.

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-ß1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration.

Preliminary investigation on the mechanism of anti-periodontitis effect of Scutellariae Radix based on bioinformatics analysis and in vitro verification.

Objective: To investigate the material basis, targets and molecular mechanism of Scutellariae Radix against periodontitis to provide theoretical basis for clinical applications. Materials and methods: The active compounds and targets of Scutellariae Radix were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, and the periodontitis-related targets were collected by integrating Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), Genecards and Gene Expression Omnibus (GEO) database together. The potential targets of Scutellariae Radix against periodontitis were obtained from the intersection of two target sets. Metascape database was used for Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Discovery Studio software was used for molecular docking between key targets and compounds to evaluate their binding affinity. Western blot was used to check the expression of PTGS2 and MMP9 to verify the regulatory effects of baicalein, the main active compound of Scutellariae Radix, on human periodontal ligament stem cells (hPDLSCs) cultured under inflammatory environment which induced by lipopolysaccharide (LPS). Results: 15 active compounds of Scutellariae Radix and 53 common targets for periodontitis treatment were identified. Among these targets, the 10 core targets were AKT1, IL-6, TNF, VEGFA, TP53, PTGS2, CASP3, JUN, MMP9 and HIF1A. GO and KEGG analysis mainly focused on response to LPS and pathways in cancer. Molecular docking showed that the main active compounds had good binding affinity with key targets. Cell experiments confirmed that baicalein can interfere the expression of pro-inflammatory factors PTGS2 and MMP9 proteins and exert anti-inflammatory effects. Conclusion: Current study preliminarily analyzed the mechanism of Scutellariae Radix against periodontitis, which provide a new idea for the utilization of Scutellariae Radix and the development of novel medicine for the clinical treatment of periodontitis.

The cytotoxicity assessment of different clear aligner materials.

Background/purpose: Invisible orthodontic treatments are becoming increasingly popular, and numerous brands of invisible aligners are now available. However, concerns remain about the safety of the materials used in these products. This study aimed to assess the cytotoxic effects of both original and thermoformed thermoplastic materials used in orthodontic aligners on human periodontal ligament (HPDL) cells in vitro. Materials and methods: The experiment used six different brands, each containing three types of thermoplastic materials, Polyethylene terephthalateco-1, 4-cyclohexylenedimethylene terephthalate (PETG), thermoplastic polyurethane (TPU), and copolyester polyethylene terephthalate (PET). The original sheets and the thermoformed materials were soaked in a culture medium for seven and fourteen days, and then applied to cultured human periodontal ligament cells. Cells were harvested on the first, third, and fifth days after application, and their viability was analyzed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Results: The findings revealed that some thermoformed materials, notably PETG, exhibited lower survival rates compared to their non-thermoformed versions. However, other materials such as TP and PET maintained over 70% cell viability, indicating only minor cytotoxic effects. Conclusion: These findings highlight the need for further research into the long-term biocompatibility of these materials but generally affirm their safety for use in orthodontic aligners under the tested conditions.

Hypoxia-inducible Factor 1α Contributes to Matrix Metalloproteinases 2/9 and Inflammatory Responses in Periodontitis.

Periodontitis is a prevalent condition characterized by inflammation and tissue destruction within the periodontium, with hypoxia emerging as a contributing factor to its pathogenesis. Hypoxia-inducible factor 1α (HIF-1α) has a crucial role in orchestrating adaptive responses to hypoxic microenvironments and has been implicated in various inflammatory-related diseases. Understanding the interplay between HIF-1α, matrix metalloproteinases (MMPs), and inflammatory responses in periodontitis could provide insights into its molecular mechanisms. We investigated the relationship between HIF-1α, MMP2, and MMP9 in gingival crevicular fluid (GCF) and periodontal ligament stem cells (PDLSCs) from periodontitis patients. The expression levels of HIF-1α, MMP2, MMP9, and inflammatory factors (IL-6, IL-1ß, TNF-α) were assessed using enzyme-linked immunosorbent assay (ELISA) and real-time PCR (RT-PCR). Additionally, osteogenic differentiation of PDLSCs was identified by alkaline phosphatase activity. Significantly elevated levels of HIF-1α, MMP2, and MMP9 were observed in GCF of periodontitis patients compared to controls. Positive correlations were found between HIF-1α and MMP2/MMP9, as well as with IL-6, IL-1ß, and TNF-α. Modulation of HIF-1α expression in PDLSCs revealed its involvement in MMP2/9 secretion and inflammatory responses, with inhibition of HIF-1α mitigating these effects. Furthermore, HIF-1α inhibition alleviated the reduction in osteogenic differentiation induced by inflammatory stimuli. Our findings elucidate the regulatory role of HIF-1α in MMP expression, inflammatory responses, and osteogenic differentiation in periodontitis. In conclusion, targeting HIF-1α signaling pathways may offer therapeutic opportunities for managing periodontitis and promoting periodontal tissue regeneration.

Heterogeneity in Dental Tissue-Derived MSCs Revealed by Single-Cell RNA-seq.

Mesenchymal stromal cells (MSCs) are multipotent, progenitor cells that reside in tissues across the human body, including the periodontal ligament (PDL) and gingiva. They are a promising therapeutic tool for various degenerative and inflammatory diseases. However, different heterogeneity levels caused by tissue-to-tissue and donor-to-donor variability, and even intercellular differences within a given MSCs population, restrict their therapeutic potential. There are considerable efforts to decipher these heterogeneity levels using different "omics" approaches, including single-cell transcriptomics. Previous studies applied this approach to compare MSCs isolated from various tissues of different individuals, but distinguishing between donor-to-donor and tissue-to-tissue variability is still challenging. In this study, MSCs were isolated from the PDL and gingiva of 5 periodontally healthy individuals and cultured in vitro. A total of 3,844 transcriptomes were generated using single-cell mRNA sequencing. Clustering across the 2 different tissues per donor identified PDL- and gingiva-specific and tissue-spanning MSCs subpopulations with unique upregulated gene sets. Gene/pathway enrichment and protein-protein interaction (PPI) network analysis revealed differences restricted to several cellular processes between tissue-specific subpopulations, indicating a limited tissue-of-origin variability in MSCs. Gene expression, pathway enrichment, and PPI network analysis across all donors' PDL- or gingiva-specific subpopulations showed significant but limited donor-to-donor differences. In conclusion, this study demonstrates tissue- and donor-specific variabilities in the transcriptome level of PDL- and gingiva-derived MSCs, which seem restricted to specific cellular processes. Identifying tissue-specific and tissue-spanning subpopulations highlights the intercellular differences in dental tissue-derived MSCs. It could be reasonable to control MSCs at a single-cell level to ensure their properties before transplantation.