Periodontal Ligament Cells Research Articles

Investigating the Cytotoxic Effects of Artemisia absinthium Extract on Oral Carcinoma Cell Line

Background: Artemisia absinthium (A. absinthium), commonly known as absinthe, is a perennial plant with distinctive broad ovate pointed leaves of a silvery-gray color, reaching a height of 1.5 m. The utilization of this herb as a source of natural compounds and as the primary ingredient in the alcoholic beverage absinthe has recently seen a resurgence following a period of prohibition. This study investigates the biological effects of A. absinthium extract on healthy human periodontal ligament stem cells (hPDLSCs) and the human tongue squamous carcinoma cell line (HSC-3). Methods: A. absinthium element characterization was performed using High-Performance Liquid Chromatography (HPLC) and the Folin method. Alizarin assays evaluated the osteogenic capacity of human periodontal ligament cells (hPDLSCs) while CCK-8 and MTT determined the cytotoxicity of the extract against HSC-3 and hPDLSCs. Results: High artemisinin levels were detected, revealing a concentration of 89 μM (25 μg/mL). The total phenolic concentration of the extract was 1.07 mM +/− 0.11. The in vitro cytotoxicity assays revealed the biocompatible profile of the Artemisia extract in hPDLSCs without exhibiting any osteogenic potential. After 24 h of incubation with HSC-3, Artemisia extract (10 µM) decreased cancer cell viability by 99% and artemisinin by 64%, and increased the expression of Caspase 3 and 9 almost six and two times, respectively. Conclusions: In summary, our preliminary findings suggest that A. absinthium extract exhibits a toxic effect against carcinoma cell lines without affecting healthy human periodontal ligament stem cells.

Beneficial effects of non-invasive physical plasma on human periodontal ligament cells in vitro.

Periodontitis is a chronic inflammatory disease of the periodontium that can lead to the loss of affected teeth if left untreated. It is induced by a multifactorial process centered on microbial pathogens such as Fusobacterium nucleatum (F.n.). Non-invasive physical plasma (NIPP), a highly reactive gas, has become a focus of research, not only for its hemostatic, proliferation-enhancing and apoptotic properties, but also for its antimicrobial potential. The objective of this study was to examine the impact of NIPP on human periodontal ligament (PDL) cells that had been induced into a state of periodontal infection in vitro. Initially, the solitary effect of NIPP was evaluated by measuring temperature and pH and analyzing reactive oxygen species (ROS). Additionally, DAPI and phalloidin staining were employed to investigate possible cytotoxic effects. The cells were pre-incubated with F.n. and treated with NIPP after 24 hours. Interleukin (IL)-6 and IL-8 were analyzed at mRNA and protein levels, respectively, by real-time PCR and ELISA. NIPP alone had no significant effect on PDL cells. However, the F.n.-induced upregulation of IL-6 and IL-8 was counteracted by NIPP. Thus, the utilization of NIPP may be regarded as a promising therapeutic strategy for the treatment of periodontal diseases.

Biomimetic In Vitro Model of Canine Periodontal Ligament.

Periodontal disease affects about 80% of dogs, highlighting the importance of addressing periodontitis in veterinary dental care. The periodontal ligament (PDL) is a key structure holding the potential to regenerate the entire periodontal complex. This work presents an in vitro model of canine PDL-derived cell cultures that mimic the PDL's regenerative capacity for both mineralised and soft tissues. Explant outgrowth-derived PDL cells were cultured under standard conditions in osteoinductive medium and with hydroxyapatite nanoparticles (Hap NPs). Cell behaviour was assessed for viability/proliferation, morphology, growth patterns, and the expression of osteogenic and periodontal markers. Osteogenic conditions, either achieved with osteoinducers or an osteoconductive biomaterial, strongly promoted PDL-derived cells' commitment towards the osteogenic phenotype and significantly increased the expression of periodontal markers. These findings suggest that cultured PDL cells replicate the biological function of the PDL, supporting the regeneration of both soft and hard periodontal tissues under normal and demanding healing conditions. This in vitro model will offer a platform for testing new regenerative treatments and materials, ultimately contributing to canine dental care and better outcomes.

Evaluation of Propolis, Aloe Vera Gel, Noni, Pomegranate Juice Natural Storage Medium for Management of Avulsed Teeth

ABSTRACT Objectives: To ascertain the effectiveness of propolis, aloe vera gel, noni, and pomegranate juice natural storage solution in preserving the vitality of avulsed teeth PDL cells. Materials and Methods: Forty recently extracted human premolars that were recommended for orthodontic purposes were used in the current investigation. Each of the four storage medium groups (N = 10)—Group A: propolis, Group B: pomegranate juice, Group C: aloe vera, and Group D: noni—was assigned to them at random. The teeth were immersed in one of the three experimental media for forty minutes after being dried for twenty-five minutes at room temperature. Each tooth’s root surface was treated with an enzymatic solution containing 1 mL of Collagenase Type II and Dispase Type I in phosphate buffer saline following drying and soaking. Following the application of 0.4% Trypan blue labeling, the cells were counted using a hemocytometer under a 10x light microscope magnification. The obtained data was statistically analyzed. Results: The samples placed in propolis indicated the maximum number of viable PDL cells subsequently pomegranate juice, aloe vera gel, and noni. There was considerable disparity in the amount of viable PDL cells among the groups. Conclusion: The current research findings suggest that propolis, pomegranate juice, aloe vera gel, and non-natural media can be employed to preserve the viability of PDL cells in avulsed teeth.

Role of DDR1 in Regulating MMPs in External Root Resorption.

Human periodontal ligament cells (hPDLCs) express matrix metalloproteinases (MMPs), a group of enzymes responsible for the destruction of most extracellular matrix proteins in dental tissues, especially MMP-1, MMP-2, and MMP-13. Exploring the regulatory mechanism of MMPs is crucial for understanding external root resorption (ERR), one of the most severe complications, along with substantial loss of dental tissue, induced by trauma, pulpal infection, tooth bleaching, and orthodontic treatment, etc. Discoidin domain receptor 1 (DDR1), a cell surface receptor binding to collagen, has the potential to regulate the expression of MMP-1, MMP-2, and MMP-13, but the mechanism remains unclear. Thus, the present study aimed to investigate the connection and underlying mechanism between MMP-1, MMP-2, MMP-13, and DDR1 in hPDLCs. Our post-replantation ERR model revealed that Mmp-1, Mmp-2, Mmp-13, and Ddr1 all increased in the sites of ERR. hPDLCs with DDR1 knockdown exhibited a substantial reduction in MMP-1, MMP-2, and MMP-13 expression. To further confirm the underlying mechanism, we conducted further in vitro experiments, including RNA sequencing, RNA interference, RT-qPCR, Western blotting, and ELISA. Based on our results, MMP-1 was positively regulated by the Smad2/3 and MEK-ERK1/2 pathways and negatively regulated by the PI3K-Akt pathway through CCN2. MMP-2 and MMP-13 were positively regulated by the Smad2/3 pathway. MMP-13 was positively regulated by the MEK-ERK1/2 and PI3K/Akt signaling pathways. Collectively, DDR1 is a potent regulator of MMP-1, MMP-2, and MMP-13 expression through the Smad2/3, MEK-ERK1/2, and PI3K/Akt signaling pathways. Clarifying the significance and underlying mechanism by which DDR1 is involved in ERR might bring the chances to hinder the pathogenic process of ERR, hence reducing its incidence rate.

Tensile stress induced osteogenesis of periodontal ligament cells via Piezo1 mediated TAZ-Cbfα1 signaling

ObjectiveCyclic tensile stress (CTS) is known to induce osteogenesis of periodontal ligament cells (PDLCs), in which the molecular mechanism remains to be elucidated.This study aimed to investigate the role of the mechanosensitive calcium channel Piezo1 in the osteogenesis of PDLCs under tensile strain, as well as the signal regulation of the TAZ-Cbfα1 pathway in this process. DesignPDLCs were isolated from periodontal ligament tissues and subjected to CTS. Alizarin red staining (ARS) and alkaline phosphatase (ALP) assay were used to detect the osteogenesis of PDLCs. RT-qPCR and Western blot were used to detect the transcripts and protein expression levels of Piezo1, Transcriptional co-activator with PDZ binding motif (TAZ), and Core-binding factor α1 (Cbfα1) respectively. Immunofluorescence staining was used to detect the nuclear aggregation of TAZ. Small interfering RNA (siRNA) targeting Piezo1 (Piezo1-siRNA) was adopted to inhibit Piezo1 mRNA expression. ResultsThe results showed that the osteogenic differentiation capacity of PDLCs was significantly enhanced under CTS, along with elevated mRNA and protein expression levels of Piezo1, TAZ, and Cbfα1. Moreover, the ALP activity and the formation of calcium nodules by ARS staining were significantly increased. In addition, Piezo1 siRNA infection significantly inhibited the CTS-induced TAZ-Cbfα1 pathway and the osteogenesis of PDLCs, suggesting the regulatory role of Piezo1. ConclusionsWe provided evidence that the application of CTS encourages the osteogenic differentiation of PDLCs, which could be mediated by the Piezo1 targeted TAZ-Cbfα1 signaling.

Development of a Custom Fluid Flow Chamber for Investigating the Effects of Shear Stress on Periodontal Ligament Cells.

The periodontal ligament (PDL) is crucial for maintaining the integrity and functionality of tooth-supporting structures. Mechanical forces applied to the tooth during orthodontic tooth movement generate pore pressure gradients, leading to interstitial fluid movement within the PDL. The generated fluid shear stress (FSS) stimulates the remodeling of PDL and alveolar bone. Herein, we present the construction of a parallel fluid-flow apparatus to determine the effect of FSS on PDL cells. The chamber was designed and optimized using computer-aided and computational fluid dynamics software. The chamber was formed by PDMS using a negative molding technique. hPDLCs from two donors were seeded on microscopic slides and exposed to FSS of 6 dyn/cm2 for 1 h. The effect of FSS on gene and protein expression was determined using RT-qPCR and Western blot. FSS upregulated genes responsible for mechanosensing (FOS), tissue formation (RUNX2, VEGFA), and inflammation (PTGS2/COX2, CXCL8/IL8, IL6) in both donors, with donor 2 showing higher gene upregulation. Protein expression of PTGS2/COX2 was higher in donor 2 but not in donor 1. RUNX2 protein was not expressed in either donor after FSS. In summary, FSS is crucial in regulating gene expression linked to PDL remodeling and inflammation, with donor variability potentially affecting outcomes.

Apelin Counteracts the Effects of Fusobacterium nucleatum on the Migration of Periodontal Ligament Cells In Vitro

To better understand the link between periodontitis and metabolic diseases, our in vitro study aimed to assess the influence of the adipokine apelin and/or the periodontal pathogen Fusobacterium nucleatum on periodontal cells. Periodontal ligament (PDL) cells were exposed to F. nucleatum in the presence and absence of apelin. Scratch assays were used to analyze the in vitro wound healing and velocity of cell migration. To investigate if F. nucleatum and/or apelin have a regulatory effect on cell proliferation and apoptosis, proliferation and viability assays were performed as well as an analysis of caspase 9 expression. Both the in vitro wound closure and the cell migration rate were significantly reduced by F. nucleatum. Simultaneous incubation with apelin counteracted the adverse effects of F. nucleatum. The proliferation assay demonstrated that neither apelin nor F. nucleatum significantly affected PDL cell proliferation. Furthermore, neither apelin nor F. nucleatum was cytotoxic or affected apoptosis after 48 h. Apelin could play a modulatory role in the pathogenesis of periodontitis, as it was able to compensate for the inhibitory effects of the periodontal pathogen F. nucleatum on PDL cell migration in vitro.

MiR-146a Is Mutually Regulated by High Glucose-Induced Oxidative Stress in Human Periodontal Ligament Cells.

The high-glucose conditions caused by diabetes mellitus (DM) exert several effects on cells, including inflammation. miR-146a, a kind of miRNA, is involved in inflammation and may be regulated mutually with reactive oxygen species (ROS), which are produced under high-glucose conditions. In the present study, we used human periodontal ligament cells (hPDLCs) to determine the effects of the high-glucose conditions of miR-146a and their involvement in the regulation of oxidative stress and inflammatory cytokines using Western blotting, PCR, ELISA and other methods. When hPDLCs were subjected to high glucose (24 mM), cell proliferation was not affected; inflammatory cytokine expression, ROS induction, interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) expression increased, but miR-146a expression decreased. Inhibition of ROS induction with the antioxidant N-acetyl-L-cysteine restored miR-146a expression and decreased inflammatory cytokine expression compared to those under high-glucose conditions. In addition, overexpression of miR-146a significantly suppressed the expression of the inflammatory cytokines IRAK1 and TRAF6, regardless of the glucose condition. Our findings suggest that oxidative stress and miR-146a expression are mutually regulated in hPDLCs under high-glucose conditions.

Mesenchymal stem cell-derived protein extract induces periodontal regeneration

BackgroundPeriodontal disease is characterized by chronic inflammation and destruction of supporting periodontal tissues, ultimately leading to tooth loss. In recent years, “cell-free treatment” without stem cell transplantation has attracted considerable attention for tissue regeneration. This study investigated the effects of extracts of mesenchymal stem cells (MSC-extract) and their protein components (MSC-protein) on the proliferation and migration of periodontal ligament (PDL) cells and whether MSC-protein can induce periodontal regeneration. MethodsMSC-extract and MSC-protein were obtained by subjecting mesenchymal stem cells (MSCs) to freeze–thaw cycles and acetone precipitation. Cell proliferation was examined using a WST-8 assay and Ki67 immunostaining, and cell migration was examined using Boyden chambers. The MSC-protein content was analyzed using liquid chromatography-mass spectrometry, protein arrays, and enzyme-linked immunosorbent assays (ELISAs). Gene expression in MSC-protein-treated PDL cells was examined using RNA-sequencing and Gene Ontology analyses. The regenerative potential of MSC-protein was examined using micro-computer tomography (CT) and histological analyses after transplantation into a rat periodontal defect model. ResultsMSC-extract and MSC-protein promoted the proliferation and migration of PDL cells. Protein array and ELISA revealed that MSC-protein contained high concentrations of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). Exogenous bFGF promoted the proliferation and migration of PDL cells. Furthermore, the transplantation of MSC-protein enhanced periodontal tissue regeneration with the formation of new alveolar bone and PDLs. ConclusionsThese results indicate that the MSC-protein promotes the proliferation and migration of PDL cells and induces significant periodontal tissue regeneration, suggesting that the MSC-protein could be used as a new cell-free treatment for periodontal disease.

Fabrication of 3D bioactive melt electrowriting composite scaffold with high osteogenic potential

A key challenge in using melt electrowriting (MEW) technology is incorporating large amounts of bioactive inorganic materials, such as hydroxyapatite (HA). In the present study, following optimization of the fabrication parameters, 40 %-HA (HA40) nanoparticles were pre-mixed into medical-grade polycaprolactone (PCL) and processed using the MEW (MEW) technique to mimic the structure and function of the natural extracellular matrix (ECM) for bone regeneration. The HA40 fibrous composite scaffolds showed continuous writing and obtained a well-connected and orderly stacked fibre with a small diameter size (67 ± 8.5 µm). A major result of the present study was the successful enrichment and accumulation of the HA particles, which mostly occurred on the MEW fibre external surfaces. This design allows for direct interfacial interaction with human periodontal ligament cells (hPDLCs). We systematically investigated the behaviour and function of hPDLCs on the HA40 composite scaffold, alongside parameters related to mineralization. The HA40 scaffold demonstrated significantly higher metabolic activity and enhanced expression of osteopontin compared to PCL-only scaffolds, as well as increased levels of ALP and COL1. The study’s findings demonstrate that bioactive composite scaffolds, incorporating 40 % HA into m-PCL via MEW, effectively enhance the biological response of the ECM and are promising for potential applications in bone regeneration.

Annexin A1 protects periodontal ligament cells against lipopolysaccharide-induced inflammatory response and cellular senescence: An implication in periodontitis.

Periodontitis is an inflammatory condition that affects the tooth-supporting structures, triggered by the host's immune response toward the bacterial deposits around the teeth. Annexin A1 (AnxA1), a vital member of the annexin superfamily, is known for its diverse physiological functions, particularly its anti-inflammatory and anti-senescence properties. We hypothesized that AnxA1 has a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses and cellular damage in periodontal ligament cells (PDLCs). In this study, we demonstrate that LPS stimulation significantly reduced telomerase activity in PDLCs, a decline that was dose-dependently reversed by AnxA1. Importantly, AnxA1 protected the cells from LPS-induced cellular senescence and the downregulation of human telomerase reverse transcriptase (hTERT) expression. In line with this, AnxA1 suppressed the LPS-induced expression of p21 and p16 at both the mRNA and protein levels. Furthermore, AnxA1 demonstrated potent anti-inflammatory effects by inhibiting the secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). It also mitigated LPS-induced oxidative stress by reducing the levels of phosphorylated Foxo3a (Ser253) and restored sirtuin 1 (SIRT1) expression. Notably, SIRT1 silencing abolished AnxA1's protective effects on Foxo3a phosphorylation and cellular senescence, suggesting that SIRT1 mediates AnxA1's actions. In conclusion, AnxA1 protected PDLCs against LPS-triggered inflammation and cell senescence by activating SIRT1 signal pathway. These findings indicate that AnxA1 could serve as a promising therapeutic strategy for the treatment of periodontitis.

Single nucleotide polymorphism rs854560 in paraoxonase-1 regulates the cytodifferentiation of human periodontal ligament cells

Single nucleotide polymorphism rs854560 in paraoxonase-1 regulates the cytodifferentiation of human periodontal ligament cells

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

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. The aim of this study was to exploring whether ANXA2 attenuates the deleterious effects of high glucose on PDLCs and promotes osteogenic differentiation capacity. 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 40mM) 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, β-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. 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.

Cryopreservation of human teeth using vitrification method with cryoprotectant cocktails and N-acetylcysteine for banking and clinical applications

Preserving freshly-extracted healthy human teeth offers an optional resource for potential tooth transplantation and cell therapy. This study aimed to assess the impact of vitrification, utilizing a blend of cryoprotectant agents and N-acetylcysteine (NAC), on the cryopreservation of periodontal ligament tissues, and investigate the underlying mechanisms of NAC on the tooth cryopreservation. Periodontal ligament cells were isolated from freshly-extracted healthy human permanent teeth, and cell sheets of PDLCs were fabricated. The samples including cell sheets, freshly-extracted human and rat teeth were cryopreserved with or without NAC for three months. The viability, ROS level, gene expressions and microstructure of PDLCs within cell sheets were assessed. The expression of SOD-2, Caspase3, LC3A/B and Catalase were evaluated through western blotting. Histological assessments of cryopreserved cell sheets and teeth were conducted. PDLCs were isolated from cryopreserved teeth, and their immunophenotype and differentiation ability were evaluated. The data was analyzed using one-way analysis of variance. The vitrification method showed good performance in preserving the viability and differentiation potential of PDLCs. Cryopreservation supplemented with NAC improved the survival rate of PDLCs, enhanced osteogenic differentiation ability, upregulated the expression of SOD-2 and Catalase, and inhibited cell apoptosis. Additionally, mRNA sequencing analysis revealed a significant activation of the PI3K-AKT pathway following cryopreservation via vitrification. Adding a PI3K-AKT activator improved the survival rates of PDLCs post-cryopreservation. The vitrification strategy combining various CPAs and NAC proved to be feasible for tooth cryopreservation. Targeting the PI3K-AKT pathway may improve the efficacy of tooth cryopreservation.

The expression of signal regulatory protein alpha (SIRPα) in periodontal cells and tissue.

Signal regulatory protein alpha (SIRPα) is mainly expressed by cells of myeloid origin. This membrane glycoprotein is shown to be involved in regulation of different inflammatory conditions, such as colitis and arthritis. However, SIRPα has not been investigated in relationship to periodontitis, an inflammatory condition affecting the tooth supporting tissues. We aim to investigate if resident cells in the periodontium express SIRPα and whether a possible expression is affected by inflammatory conditions. Primary human keratinocytes, fibroblasts, periodontal ligament cells, and osteoblasts were cultured with or without the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) or interleukin-1-beta (IL-1β). All different periodontal cell types showed a basal mRNA expression of SIRPα. Pro-inflammatory cytokines induced a 2-3-fold significant increase in SIRPα expression in both cultured human gingival fibroblasts and osteoblasts but neither in keratinocytes nor in periodontal ligament cells. Tissue sections from human gingival tissue biopsies were histochemically stained for SIRPα. Epithelial keratinocytes and gingival fibroblasts stained positive in sections from periodontally healthy as well as in sections from periodontitis. In periodontitis sections, infiltrating leukocytes stained positive for SIRPα. We highlight our finding that oral keratinocytes, gingival fibroblasts, and periodontal ligament cells do express SIRPα, as this has not been presented before. The fact that inflammatory stimulation of gingival fibroblasts increased the expression of SIRPα, while an increased expression by gingival fibroblasts in periodontitis tissue in situ could not be detected, is indeed contradictory.

Single-cell RNA-seq analysis of rat molars reveals cell identity and driver genes associated with dental mesenchymal cell differentiation

BackgroundThe molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge.ResultsHere, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar.ConclusionsOur results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.

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.

Ubiquitin C-terminal hydrolase L1 activation in periodontal ligament cells mediates orthodontic tooth movement via the MAPK signaling pathway

ABSTRACT Purpose Periodontal ligament cells (PDLCs) play a significant role in orthodontic force induced bone remodeling. However, the molecular mechanisms by which PDLCs respond to mechanical stimuli and influence osteoclastic activities remain unclear. This study aims to investigate the role of UCHL1, a key deubiquitinating enzyme involved in protein degradation and cellular responses, in force-treated PDLCs during orthodontic tooth movement (OTM). Materials and Methods In this study, we conducted in vivo and in vitro experiments using human PDLCs and a rat model of OTM. Mechanical stress was applied to PDLCs, and UCHL1 expression was analyzed through quantitative real-time polymerase chain reaction (qPCR), Western blot, and immunofluorescence staining. UCHL1 knockdown was achieved using siRNA, and its effects on osteoclast differentiation were assessed. The role of the MAPK/ERK pathway was investigated using the MEK-specific inhibitor U0126. An animal model of OTM was established, and the impact of UCHL1 inhibitor-LDN57444 on OTM and osteoclastic activity was evaluated through micro-CT analysis, histological staining, and immunohistochemistry. Results Mechanical force induced UCHL1 expression in PDLCs during OTM. UCHL1 knockdown downregulated the RANKL/OPG ratio in PDLCs, affecting osteoclast differentiation. LDN57444 inhibited OTM and osteoclastic activity. UCHL1 activation correlated with ERK1/2 phosphorylation in force-treated PDLCs. Conclusions Mechanical force mediated UCHL1 activation in PDLCs promotes osteoclast differentiation via the ERK1/2 signaling pathway during OTM.

Heavy mechanical force decelerates orthodontic tooth movement via Piezo1-induced mitochondrial calcium down-regulation

Orthodontic tooth movement (OTM) depends on periodontal ligament cells (PDLCs), which sense biomechanical stimuli and initiate alveolar bone remodeling. Light (optimal) forces accelerate OTM, whereas heavy forces decelerate it. However, the mechanisms by which PDLCs sense biomechanical stimuli and affect osteoclastic activities under different mechanical forces (MFs) remain unclear. This study demonstrates that mechanosensitive ion channel Piezo1-mediated Ca2+ signal conversion is crucial for sensing and delivering biomechanical signals in PDLCs under heavy-force conditions. Heavy MF up-regulated Piezo1 in PDLCs, reducing mitochondrial Ca2+ influx by inhibiting ITPR3 expression in mitochondria-associated membranes. Decreased mitochondrial calcium uptake led to reduced cytoplasmic release of mitochondrial DNA and inhibited the activation of the cGAS‒STING signaling cascade, subsequently inhibiting monocyte-to-osteoclast differentiation. Inhibition of Piezo1 or up-regulation of STING expression under heavy MF conditions significantly increased osteoclast activity and accelerated OTM. These findings suggest that heavy MF-induced Piezo1 expression in PDLCs is closely related to the control of osteoclast activity during OTM and plays an essential role in alveolar bone remodeling. This mechanism may be a potential therapeutic target for accelerating OTM.