Effects of surface nanomorphology on the senescence of periodontal ligament stem cells.

OBJECTIVES: The effect of TiO2 nanotube morphology on the differentiation potency of senescent periodontal ligament stem cells was investigated. METHODS: Two types of titanium sheets with TiO2 nanotube morphology (20V-NT and 70V-NT) were prepared via anodic oxidation at 20 and 70 V separately, and their surface morphology was observed. Young periodontal ligament stem cells were cultivated in an osteogenic induction medium, and the most effective surface morphology in promoting osteogenic differentiation was selected. RO3306 and Nutlin-3a were used to induce the aging of young periodontal ligament stem cells, and senescent periodontal ligament stem cells were obtained. The osteogenic differentiation of senescent periodontal ligament stem cells was induced, and the effect of surface morphology on osteogenic differentiation was observed. RESULTS: Nanotube morphology was achieved on the surfaces of titanium sheets through anodic oxidation, and the diameters of the nanotubes increased with voltage. A significant difference in the effect of nanotube morphology was found among nanotubes with different diameters in the young periodontal ligament stem cells. The surface nanotube morphology of 20V-NT had a more significant effect that promoted osteogenic differentiation. Compared with a smooth titanium sheet, the surface nanotube morphology of 20V-NT increased the number of alkaline phosphatase-positive senescent periodontal ligament stem cells and promoted calcium deposition and the expression of osteogenic marker genes Runt-related transcription factor 2, osteopontin, and osteocalcin. CONCLUSIONS: A special nanotube morphology enhances the differentiation ability of senescent periodontal ligament stem cells, provides an effective method for periodontal regeneration, and further improves the performance of implants.

LepR-expressing cells are a critical population in periodontal healing post periodontitis.

Identification of promising seed cells plays a pivotal role in achieving tissue regeneration. This study demonstrated that LepR-expressing cells (LepR+ cells) are required for maintaining periodontal homeostasis at the adult stage. We further investigated how LepR+ cells behave in periodontal healing using a ligature-induced periodontitis (PD) and a self-healing murine model with LepRCre/+; R26RtdTomato/+ mice. Lineage tracing experiments revealed that the largely suppressed osteogenic ability of LepR+ cells results from periodontal inflammation. Periodontal defects were partially recovered when the ligature was removed, in which the osteogenic differentiation of LepR+ cell lineage was promoted and contributed to the newly formed alveolar bone. A cell ablation model established with LepRCre/+; R26RtdTomato/+; R26RDTA/+ mice further proved that LepR+ cells are an important cell source of newly formed alveolar bone. Expressions of ß-catenin and LEF1 in LepR+ cells were upregulated when the inflammatory stimuli were removed, which are consistent with the functional changes observed during periodontal healing. Furthermore, the conditional upregulation of WNT signaling or the application of sclerostin neutralized antibody promoted the osteogenic function of LepR+ cells. In contrast, the specific knockdown of ß-catenin in LepR+ human periodontal ligament cells with small interfering RNA caused arrested osteogenic function. Our findings identified the LepR+ cell lineage as a critical cell population for endogenous periodontal healing post PD, which is regulated by the WNT signaling pathway, making it a promising seed cell population in periodontal tissue regeneration.

[The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells].

PURPOSE: To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells (hPDLSCs), as well as the underlying mechanism of electronic cigarette (E-cig)-induced cell apoptosis. METHODS: PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic reasons. Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. Then the cells were exposed to different doses of menthol-favored E-liquid (at 59 mg/L nicotine concentration) in the culture median (the final nicotine concentrations were 0.1 µg/mL, 1.0 µg/mL, 10 µg/mL, 50 µg/mL, 0.1 mg/mL, 0.2 mg/mL and 0.5 mg/mL, respectively) for different period of times (24, 48 and 72 h). The cell viability was analyzed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry (7-AAD and Annexin V staining) and TUNEL assay. Reactive oxygen species (ROS) production was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry. The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK, JNK, c-Jun, p-c-Jun, Bcl-2, Bax and cleaved-caspase 3) were analyzed by Western blot. Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK. After addition of NAC, a ROS scavenger, and MAPK/JNK specific blocker SP600125, their effects on E-cig-induced cell apoptosis were evaluated. Statistical analysis was performed with Graph Pad 5.0 software package. RESULTS: Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. CCK8 assay indicated cell viability was significantly(P＜0.001) different among all concentration groups at various time points (24, 48 or 72 h), and the difference in apoptosis rate among all concentration groups was also statistically significant (P＜0.001). After exposure to E-liquid with nicotine concentration ≥50 µg/mL, cell viability was significantly reduced, and the proportion of apoptotic cells and the cellular ROS level was significantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL). Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner. Either NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3. CONCLUSIONS: ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apoptosis of hPDLSCs.

Upregulated ATF1 Promotes Lipopolysaccharide Induced Inflammatory Response and Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Regulating NF-κB Pathway.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis. METHODS: Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 µg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability. RESULTS: LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway.

The heightened prevalence and accelerated progression of periodontitis in individuals with diabetes is primarily attributed to inflammatory responses in human periodontal ligament cells (HPDLCs). This study is aimed at delineating the regulatory mechanism of nucleotide-binding oligomerization domain-like receptors (NLRs) in mediating inflammation incited by muramyl dipeptide (MDP) in HPDLCs, under the influence of advanced glycation end products (AGEs), metabolic by-products associated with diabetes. We performed RNA-seq in HPDLCs induced by AGEs treatment and delineated activation markers for the receptor of AGEs (RAGE). It showed that advanced glycation end products modulate inflammatory responses in HPDLCs by activating NLRP1 and NLRP3 inflammasomes, which are further regulated through the NF-κB signaling pathway. Furthermore, AGEs synergize with NOD2, NLRP1, and NLRP3 inflammasomes to augment MDP-induced inflammation significantly.

Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

A comprehensive understanding of the extracellular matrix (ECM) is essential for developing biomimetic ECM scaffolds for tissue regeneration. As the periodontal ligament cell (PDLC)-derived ECM has shown potential for periodontal tissue regeneration, it is vital to gain a deeper understanding of its comprehensive profile. Although the PDLC-derived ECM exhibits extracellular environment similar to that of periodontal ligament (PDL) tissue, details of its molecular composition are lacking. Thus, using a multiomics approach, we systematically analyzed cultured mouse PDLC-derived ECM and compared it to mouse PDL tissue as a reference. Proteomic analysis revealed that, compared to PDL tissue, the cultured PDLC-derived ECM had a lower proportion of fibrillar collagens with increased levels of glycoprotein, corresponding to an immature ECM status. The gene expression signature was maintained in cultured PDLCs and was similar to that in cells from PDL tissues, with additional characteristics representative of naturally occurring progenitor cells. A combination of proteomic and transcriptomic analyses revealed that the cultured mouse PDLC-derived ECM has multiple advantages in tissue regeneration, providing an extracellular environment that closely mimics the environment in the native PDL tissue. These findings provide valuable insights for understanding PDLC-derived ECM and should contribute to the development of biomimetic ECM scaffolds for reliable periodontal tissue regeneration.

Calcitonin gene-related peptide regulates periodontal tissue regeneration.

Calcitonin gene-related peptide (CGRP), a neuropeptide composed of 37 amino acids secreted from the sensory nerve endings, reportedly possesses various physiological effects, such as vasodilation and neurotransmission. Recently, there have been increasing reports of the involvement of CGRP in bone metabolism; however, its specific role in the pathogenesis of periodontitis, particularly in the repair and healing processes, remains to be elucidated. Therefore, this study aimed to investigate dynamic expression patterns of CGRP during the destruction and regeneration processes of periodontal tissues in a mouse model of experimental periodontitis. We also explored the effects of CGRP on periodontal ligament cells, which can differentiate to hard tissue-forming cells (cementoblasts or osteoblasts). Our findings demonstrated that CGRP stimulation promotes the differentiation of periodontal ligament cells into hard tissue-forming cells. Experimental results using a ligature-induced periodontitis mouse model also suggested fluctuations in CGRP expression during periodontal tissue healing, underscoring the vital role of CGRP signaling in alveolar bone recovery. The study results highlight the important role of nerves in the periodontal ligament not only in sensory reception in the periphery, as previously known, but also in periodontal tissue homeostasis and tissue repair processes.

GDF15 Modulates the Zoledronic-Acid-Induced Hyperinflammatory Mechanoresponse of Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) is thought to be impeded by bisphosphonate (BP) therapy, mainly due to increased osteoclast apoptosis and changes in the periodontal ligament (PdL), a connecting tissue between the alveolar bone and teeth. PdL cells, mainly fibroblasts (PdLFs), are crucial regulators in OTM by modulating force-induced local inflammatory processes. Recently, we identified the TGF-ß/BMP superfamily member GDF15 as an important modulator in OTM, promoting the pro-inflammatory mechanoresponses of PdLFs. The precise impact of the highly potent BP zoledronate (ZOL) on the mechanofunctionality of PdLFs is still under-investigated. Therefore, the aim of this study was to further characterize the ZOL-induced changes in the initial inflammatory mechanoresponse of human PdLFs (hPdLFs) and to further clarify a potential interrelationship with GDF15 signaling. Thus, two-day in vitro treatment with 0.5 µM, 5 µM and 50 µM of ZOL altered the cellular properties of hPdLFs partially in a concentration-dependent manner. In particular, exposure to ZOL decreased their metabolic activity, the proliferation rate, detected using Ki-67 immunofluorescent staining, and survival, analyzed using trypan blue. An increasing occurrence of DNA strand breaks was observed using TUNEL and an activated DNA damage response was demonstrated using H2A.X (phosphoS139) staining. While the osteogenic differentiation of hPdLFs was unaffected by ZOL, increased cellular senescence was observed using enhanced p21Waf1/Cip1/Sdi1 and ß-galactosidase staining. In addition, cytokine-encoding genes such as IL6, IL8, COX2 and GDF15, which are associated with a senescence-associated secretory phenotype, were up-regulated by ZOL. Subsequently, this change in the hPdLF phenotype promoted a hyperinflammatory response to applied compressive forces with an increased expression of the pro-inflammatory markers IL1ß, IL6 and GDF15, as well as the activation of monocytic THP1 cells. GDF15 appeared to be particularly relevant to these changes, as siRNA-mediated down-regulation balanced these hyperinflammatory responses by reducing IL-1ß and IL-6 expression (IL1B p-value < 0.0001; IL6 p-value < 0.001) and secretion (IL-1ß p-value < 0.05; IL-6 p-value < 0.001), as well as immune cell activation (p-value < 0.0001). In addition, ZOL-related reduced RANKL/OPG values and inhibited osteoclast activation were enhanced in GDF15-deficient hPdLFs (both p-values < 0.0001; all statistical tests: one-way ANOVA, Tukey's post hoc test). Thus, GDF15 may become a promising new target in the personalized orthodontic treatment of bisphosphonatepatients.

3D-printed bioink loading with stem cells and cellular vesicles for periodontitis-derived bone defect repair.

The suitable microenvironment of bone regeneration is critically important for periodontitis-derived bone defect repair. Three major challenges in achieving a robust osteogenic reaction are the exist of oral inflammation, pathogenic bacteria invasion and unaffluent seed cells. Herein, a customizable and multifunctional 3D-printing module was designed with glycidyl methacrylate (GMA) modified epsilon-poly-L-lysine (EPLGMA) loading periodontal ligament stem cells (PDLSCs) and myeloid-derived suppressive cells membrane vesicles (MDSCs-MV) bioink (EPLGMA/PDLSCs/MDSCs-MVs, abbreviated as EPM) for periodontitis-derived bone defect repair. The EPM showed excellent mechanical properties and physicochemical characteristics, providing a suitable microenvironment for bone regeneration.In vitro, EPMs presented effectively kill the periodontopathic bacteria depend on the natural antibacterial properties of the EPL. Meanwhile, MDSCs-MV was confirmed to inhibit T cells through CD73/CD39/adenosine signal pathway, exerting an anti-inflammatory role. Additionally, seed cells of PDLSCs provide an adequate supply for osteoblasts. Moreover, MDSCs-MV could significantly enhance the mineralizing capacity of PDLSCs-derived osteoblast. In the periodontal bone defect rat model, the results of micro-CT and histological staining demonstrated that the EPM scaffold similarly had an excellent anti-inflammatory and bone regeneration efficacyin vivo. This biomimetic and multifunctional 3D-printing bioink opens new avenues for periodontitis-derived bone defect repair and future clinical application.

Cardamonin decreases inflammatory mediator expression in IL-1ß-stimulated human periodontal ligament cells.

BACKGROUND: Cardamonin is classified as a natural chalcone, and has been reported to possess various bioactive effects. However, there have been limited attempts to utilize cardamonin in the treatment of periodontitis. This study aimed to investigate whether cardamonin has anti-inflammatory effects on human periodontal ligament cells (HPDLCs), which are a component cell of periodontal tissue. Specifically, the study seeks to determine whether cardamonin affects the expression of inflammatory mediators, such as cytokines and adhesion molecules, induced by interleukin-1ß (IL-1ß) in HPDLCs, as well as the signaling pathways activated by IL-1ß. METHODS: Cytokine and chemokine levels in supernatants of HPDLCs were measured by ELISA. Western blot analysis was used to measure protein expression and signal transduction pathway activation in HPDLCs. RESULTS: We found that IL-1ß-induced CC chemokine ligand (CCL)2, CCL5, CCL20, CXC-chemokine ligand (CXCL)10, and interleukin (IL)-6 production and intercellular adhesion molecule (ICAM)-1 and cyclooxygenase (COX)-2 expression in HPDLCs were suppressed by cardamonin treatment. We also found that cardamonin suppressed IL-1ß-activated nuclear factor (NF)-κB pathway, and the phosphorylation of signal transducer and activator of transcription (STAT)3. Furthermore, cardamonin treatment enhanced the expression of the antioxidant enzymes, heme oxygenase (HO)-1 and NAD(P)H dehydrogenase [quinone] 1 (NQO1), in HPDLCs. CONCLUSION: In this study, we found that cardamonin could suppress the production of inflammatory mediators in HPDLCs as well as the activation of several signaling pathways induced by IL-1ß treatment.

Substance P/calcitonin gene-related peptide and their receptors expression in human periodontal ligament after root canal preparation with five different systems.

AIM: The purpose of this study was to quantify the effect of five different root canal preparation instruments on Substance P (SP), Calcitonin gene-related peptide (CGRP) and their receptors expression in healthy human periodontal ligament. METHODOLOGY: STROBE guidelines were used to design a study using 60 periodontal ligament samples obtained from healthy lower premolars where extraction was indicated for orthodontic reasons. Prior to extraction 40 of these premolars were equally divided into four groups and root canals were prepared using different systems: Mtwo, Reciproc Blue, HyFlex EDM and Plex-V. Ten premolars were prepared with hand files and served as a positive control group. The remaining 10 premolars where extracted without treatment and served as a negative control group. All periodontal ligament samples were processed to measure the expression of SP, CGRP and their receptors by radioimmunoassay. Kruskal-Wallis and Duncan tests were performed to determine statistically significant differences between the groups for each variable. RESULTS: Greater expression of all the peptides measured were found in the hand-file preparation group, followed by the Reciproc Blue, Mtwo, HyFlex EDM and Plex-V groups. The lower SP, CGRP and their receptors values were for the intact teeth control group. Kruskal-Wallis test showed statistically significant differences amongst groups (p < .001). Dunn post-hoc tests showed statistically significant differences in SP, CGRP and their receptors expression between the intact teeth and the hand-file and Reciproc Blue groups. Hand-file group showed significant differences with the other groups, except with Reciproc Blue, where no differences were observed in any of the peptides measured. Finally, no differences were observed between Plex-V and HyFlex in any of the peptides measured. CONCLUSIONS: Root canal preparation with hand files and Reciproc Blue generates the highest expression of SP, CGRP, NK1 and CGRP1R in human periodontal ligament, whilst Plex-V and HyFlex maintain the basal expression of neuropeptides and their receptors. Mtwo showed intermediate results between Reciproc Blue and HyFlex.

LncRNA ADAMTS9-AS2 regulates periodontal ligament cell migration under mechanical compression via ADAMTS9/fibronectin.

BACKGROUND: Periodontal ligament cells (PDLCs) are key mechanosensory cells involved in extracellular matrix (ECM) remodeling during orthodontic tooth movement (OTM). Mechanical force changes the ECM components, such as collagens and matrix metalloproteinases. However, the associations between the changes in ECM molecules and cellular dynamics during OTM remain largely uncharacterized. OBJECTIVES: To investigate the influence of mechanical force on the morphology and migration of PDLCs and explore the interaction between ECM remodeling and cellular dynamics, including the detailed mechanisms involved. METHODS: Human PDLCs (hPDLCs) were subjected to a static mechanical compression to mimic the compression state of OTM in vitro. A mouse OTM model was used to mimic the OTM procedure in vivo. The migration of hPDLCs was compared by wound healing and transwell migration assays. Moreover, expression levels of ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) and fibronectin (FN) in hPDLCs were determined via western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assays. Expression levels of ADAMTS9 and FN in mice were assessed via immunohistochemical staining. Additionally, the relative expression of long non-coding RNA (lncRNA) ADAMTS9-antisense RNA 2 (ADAMTS9-AS2) was assessed via quantitative real-time polymerase chain reaction. ADAMTS9-AS2 knockdown was performed to confirm its function in hPDLCs. RESULTS: Mechanical compression induced changes in the morphology of hPDLCs. It also promoted migration and simultaneous upregulation of FN and downregulation of ADAMTS9, a fibronectinase. The mouse OTM model showed the same expression patterns of the two proteins on the compression side of the periodontium of the moved teeth. RNA sequencing revealed that lncRNA ADAMTS9-AS2 expression was significantly upregulated in hPDLCs under mechanical compression. After knocking down ADAMTS9-AS2, hPDLCs migration was significantly inhibited. ADAMTS9 expression was increased as FN expression decreased compared to that in the control group. Moreover, knockdown of ADAMTS9-AS2 reduced the effect of mechanical compression on hPDLCs migration and reversed the expression change of ADAMTS9 and FN. RNA immunoprecipitation revealed direct binding between ADAMTS9-AS2 and ADAMTS9 protein. CONCLUSION: Our study suggests that mechanical compression induces the expression of ADAMTS9-AS2, which directly binds to ADAMTS9 and inhibits its function, leading to the promotion of downstream FN expression and ECM remodeling to facilitate hPDLCs migration and maintain the stability of the periodontium.

Salivary miR-150-5p as an indicator of periodontitis severity and regulator of human periodontal ligament fibroblast behavior by targeting AIFM2.

OBJECTIVE: This study aimed to evaluate the role of miR-150-5p in the onset and progression of periodontitis, and reveal the potential molecular mechanism underlying its function and to explore a novel biomarker for periodontitis. BACKGROUND: Periodontitis is the leading cause of tooth loss in adults, emphasizing the need for a biomarker to improve its early detection and prevention. The association of miR-150-5p with diseases related to Fuscobacterium nucleatum implies its potential involvement in periodontitis. METHODS: The expression of miR-150-5p in the saliva of patients with periodontitis (n = 77) and healthy individuals (n = 43) was assessed by PCR. Human gingival fibroblasts (HGFs) were induced with an osteogenic culture medium. The regulatory effect of miR-150-5p on the proliferation and migration of HGFs was assessed by CCK8 and transwell assays. Osteogenic differentiation was estimated based on the expression of corresponding factors through western blotting, and the inflammatory response was evaluated by measuring the levels of pro-inflammatory cytokines using ELISA. RESULTS: Significant upregulation of miR-150-5p was observed in patients with periodontitis, which sensitively distinguished them and was closely associated with the severity and periodontal index of the condition. In HGFs, osteogenic induction (OI) resulted in increased miR-150-5p levels, which negatively regulated the expression of AIFM2. Silencing miR-150-5p significantly attenuated OI-induced suppression of proliferation and migration of HGFs. The silencing also alleviated inflammation and osteogenic differentiation, which was reversed upon AIFM2 knockdown. CONCLUSION: Upregulated miR-150-5p in periodontitis served as a diagnostic biomarker, indicating the occurrence and aggravation of disease condition. Silencing miR-150-5p inhibited the osteogenic differentiation and inflammation of HGFs by negatively modulating AIFM2.

Bomidin attenuates inflammation of periodontal ligament stem cells and periodontitis in mice via inhibiting ferroptosis.

AIM: Periodontitis is a prevalent oral immunoinflammatory condition that is distinguished by the compromised functionality of periodontal ligament stem cells (PDLSCs). Bomidin, a new recombinant antimicrobial peptide (AMP), exhibits antibacterial properties and modulates immune responses. Nevertheless, the precise anti-inflammatory impact of bomidin in periodontitis has yet to be fully elucidated. Thus, the study aimed to clarified the role of bomidin in modulating inflammation and its underlying mechanisms. METHODS: TNF-α was applied to treating PDLSCs for establishing a cell model of periodontitis. Bomidin, RSL3, ML385 and cycloheximide were also used to treat PDLSCs. Transcriptome sequencing, RT-qPCR, western blot, immunofluorescence, immunohistochemistry, Fe2+ detection probe, molecular docking, Co-IP assay, ubiquitination assay and murine models of periodontitis were used. RESULTS: Our study demonstrated that bomidin effectively suppressed inflammation in PDLSCs stimulated by TNF-α, through down-regulating the MAPK and NF-κB signaling pathways. Furthermore, bomidin exerted inhibitory effects on ferroptosis and activated the Keap1/Nrf2 pathway in the TNF-α group. There is a strong likelihood of bonding bomidin with Keap1 protein, which facilitated the degradation of Keap1 protein via the ubiquitin-proteasome pathway, leading to an enhanced translocation of Nrf2 protein to the nucleus. CONCLUSIONS: Bomidin can directly bond to Keap1 protein, resulting in the degradation of Keap1 through the ubiquitin-proteasome pathway, thereby further activating the Keap1/Nrf2 pathway. The upregulation of the Keap1/Nrf2 signaling pathway was found to contribute to the suppression of ferroptosis, ultimately alleviating inflammation in treatment of periodontitis.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Osteogenic Differentiation of Human Periodontal Ligament Stromal Cells Influences Their Immunosuppressive Potential toward Allogenic CD4+ T Cells.

The differentiation ability of human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) in vivo is limited; therefore, some studies considered strategies involving their pre-differentiation in vitro. However, it is not known how the differentiation of hPDL-MSCs influences their immunomodulatory properties. This study investigated how osteogenic differentiation of hPDL-MSCs affects their ability to suppress CD4+ T-lymphocyte proliferation. hPDL-MSCs were cultured for 21 days in osteogenic differentiation or standard culture media. Allogeneic CD4+ T lymphocytes were co-cultured with undifferentiated and differentiated cells in the presence or absence of interferon (IFN)-γ, interleukin (IL)-1ß or tumor necrosis factor (TNF)-α, and their proliferation and apoptosis were measured. Additionally, the effects of these cytokines on the expression of immunomodulatory or pro-inflammatory factors were investigated. Our data show that osteogenic differentiation of hPDL-MSCs reduced their ability to suppress the proliferation of CD4+ T lymphocytes in the presence of IFN-γ and enhanced this ability in the presence of IL-1ß. These changes were accompanied by a slightly decreased proportion of apoptotic CD4+ in the presence of IFN-γ. The osteogenic differentiation was accompanied by decreases and increases in the activity of indoleamine-2,3-dioxygenase in the presence of IFN-γ and IL-1ß, respectively. The basal production of interleukin-8 by hPDL-MSCs was substantially increased upon osteogenic differentiation. In conclusion, this study suggests that pre-differentiation strategies in vitro may impact the immunomodulatory properties of hPDL-MSCs and subsequently affect their therapeutic effectiveness in vivo. These findings provide important insights for the development of MSC-based therapies.

Occlusal Force Maintains Alveolar Bone Homeostasis via Type H Angiogenesis.

Physiologically, teeth and periodontal tissues are exposed to occlusal forces throughout their lifetime. Following occlusal unloading, unbalanced bone remodeling manifests as a net alveolar bone (AB) loss. This phenomenon is termed alveolar bone disuse osteoporosis (ABDO), the underlying mechanism of which remains unclear. Type H vessels, a novel capillary subtype tightly coupled with osteogenesis, reportedly have a role in skeletal remodeling; however, their role in ABDO is not well studied. In the present study, we aimed to explore the pathogenesis of and therapies for ABDO. The study revealed that type H endothelium highly positive for CD31 and endomucin was identified in the periodontal ligament (PDL) but rarely in the AB of the mice. In hypofunctional PDL, the density of type H vasculature and coupled osterix+ (OSX+) osteoprogenitors declined significantly. In addition, the angiogenic factor Slit guidance ligand 3 (SLIT3) was downregulated in the disused PDL, and periodontal injection of the recombinant SLIT3 protein partially ameliorated type H vessel dysfunction and AB loss in ABDO mice. With regard to the molecular mechanism, a mechanosensory signaling circuit, PIEZO1/Ca2+/HIF-1α/SLIT3, was validated by applying cyclic compression to 3-dimensional-cultured PDL cells using the Flexcell FX-5000 compression system. In summary, PDL plays a pivotal role in mechanotransduction by translating physical forces into the intracellular signaling axis PIEZO1/Ca2+/HIF-1α/SLIT3, which promotes type H angiogenesis and OSX+ cell-related osteogenensis, thereby contributing to AB homeostasis. Our findings advance the understanding of PDL in AB disorders. Further therapies targeting SLIT3 may provide new insights into preventing bone loss in ABDO.

Sclerostin antibody improves alveolar bone quality in the Hyp mouse model of X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and impaired mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses and periodontal disease, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases long bone mass, strength, and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg·kg-1 of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (nonphosphorylated) ß-catenin, dentin matrix protein 1 (DMP1) and osteopontin stained alveolar osteocytes. Scl-Ab had no effect on the mass and mineralization of dentin, enamel, acellular or cellular cementum. There was a nonsignificant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fiber structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in adult Hyp mice.

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor­α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells.

OBJECTIVES: This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. METHODS: HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. RESULTS: 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). CONCLUSIONS: 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.