Nocardamine mitigates cellular dysfunction induced by oxidative stress in periodontal ligament stem cells.

BACKGROUND: The role of periodontal ligament stem cells (PDLSCs) in repairing periodontal destruction is crucial, but their functions can be impaired by excessive oxidative stress (OS). Nocardamine (NOCA), a cyclic siderophore, has been shown to possess anti-cancer and anti-bacterial properties. This study aimed to investigate the protective mechanisms of NOCA against OS-induced cellular dysfunction in PDLSCs. METHODS: The cytotoxicity of NOCA on PDLSCs was assessed using a CCK-8 assay. PDLSCs were then treated with hydrogen peroxide (H2O2) to induce OS. ROS levels, cell viability, and antioxidant factor expression were analyzed using relevant kits after treatment. Small molecule inhibitors U0126 and XAV-939 were employed to block ERK signaling and Wnt pathways respectively. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining of mineralized nodules. Expression levels of osteogenic gene markers and ERK pathway were determined via real-time quantitative polymerase chain reaction (RT-qPCR) or western blot (WB) analysis. ß-catenin nuclear localization was examined by western blotting and confocal microscopy. RESULTS: NOCA exhibited no significant cytotoxicity at concentrations below 20 µM and effectively inhibited H2O2-induced OS in PDLSCs. NOCA also restored ALP activity, mineralized nodule formation, and the expression of osteogenic markers in H2O2-stimulated PDLSCs. Mechanistically, NOCA increased p-ERK level and promoted ß-catenin translocation into the nucleus; however, blocking ERK pathway disrupted the osteogenic protection provided by NOCA and impaired its ability to induce ß-catenin nuclear translocation under OS conditions in PDLSCs. CONCLUSIONS: NOCA protected PDLSCs against H2O2-induced OS and effectively restored impaired osteogenic differentiation in PDLSCs by modulating the ERK/Wnt signaling pathway.

NOR1 promotes the osteoblastic differentiation of human periodontal ligament stem cells via TGF-ß signaling pathway.

Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.

KAT6A/YAP/TEAD4 pathway modulates osteoclastogenesis by regulating the RANKL/OPG ratio on the compression side during orthodontic tooth movement.

BACKGROUND: Orthodontic tooth movement (OTM) is a dynamic equilibrium of bone remodeling, involving the osteogenesis of new bone and the osteoclastogenesis of old bone, which is mediated by mechanical force. Periodontal ligament stem cells (PDLCSs) in the periodontal ligament (PDL) space can transmit mechanical signals and regulate osteoclastogenesis during OTM. KAT6A is a histone acetyltransferase that plays a part in the differentiation of stem cells. However, whether KAT6A is involved in the regulation of osteoclastogenesis by PDLSCs remains unclear. RESULTS: In this study, we used the force-induced OTM model and observed that KAT6A was increased on the compression side of PDL during OTM, and also increased in PDLSCs under compression force in vitro. Repression of KAT6A by WM1119, a KAT6A inhibitor, markedly decreased the distance of OTM. Knockdown of KAT6A in PDLSCs decreased the RANKL/OPG ratio and osteoclastogenesis of THP-1. Mechanistically, KAT6A promoted osteoclastogenesis by binding and acetylating YAP, simultaneously regulating the YAP/TEAD axis and increasing the RANKL/OPG ratio in PDLSCs. TED-347, a YAP-TEAD4 interaction inhibitor, partly attenuated the elevation of the RANKL/OPG ratio induced by mechanical force. CONCLUSION: Our study showed that the PDLSCs modulated osteoclastogenesis and increased the RANKL/OPG ratio under mechanical force through the KAT6A/YAP/TEAD4 pathway. KAT6A might be a novel target to accelerate OTM.

Biocompatibility, bioactivity and immunomodulatory properties of three calcium silicate-based sealers: an in vitro study on hPDLSCs.

OBJECTIVES: To assess the biocompatibility, bioactivity, and immunomodulatory properties of three new calcium silicate cement-based sealers: Ceraseal (CS), Totalfill BC Sealer (TFbc) and WellRoot ST (WR-ST) on human periodontal ligament stem cells (hPDLSCs). MATERIALS AND METHODS: HPDLSCs were isolated from extracted third molars from healthy patients. Eluates (1:1, 1:2, and 1:4 ratio) and sample discs of CS, TFbc and WR-ST after setting were prepared. A series of assays were performed: cell characterization, cell metabolic activity (MTT assay) cell attachment and morphology (SEM assay), cell migration (wound-healing assay), cytoskeleton organization (phaloidin-based assay); IL-6 and IL-8 release (ELISA); differentiation marker expression (RT-qPCR assay), and cell mineralization (Alizarin Red S staining). HPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media were used as a comparison. Statistical significance was established at p < 0.05. RESULTS: All the tested sealers exhibited similar results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group. CS and TFbc exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. CS and TFbc also showed a significantly higher calcified nodule formation than the negative and positive control groups. Both the marker expression and calcified nodule formation were significantly higher in CS-treated cells than TFbc treated cells. WR-ST exhibited similar results to the control group. CS and TFbc-treated cells exhibited a significant downregulation of IL-6 after 72 h of culture compared to the negative control group (p < 0.05). CONCLUSION: All the tested sealers exhibited an adequate cytocompatibility. CS significantly enhances cell differentiation by upregulating the expression of key genes associated with bone and cementum formation. Additionally, CS was observed to facilitate the mineralization of the extracellular matrix effectively. In contrast, the effects of TFbc and WR-ST on these processes were less pronounced compared to CS. Furthermore, both CS and TFbc exhibited an anti-inflammatory potential, contributing to their potential therapeutic benefits in regenerative endodontics. CLINICAL RELEVANCE: This is the first study to compare the biological properties and immunomodulatory potential of Ceraseal, Totalfill BC Sealer, and WellRoot ST. The results act as supporting evidence for their use in root canal treatment.

[Effects of Morinda officinalis polysaccharides on FN and FN-EDA of periodontal ligament fibroblasts in inflammatory microenvironment].

PURPOSE: To investigate the effect of Morinda officinalis polysaccharides(MOP) on the expression of fibronectin(FN) and fibronectin containing extra domain A(FN-EDA) in inflammatory periodontal ligament fibroblasts. METHODS: Thirty six rats were randomly divided into a control group(n=12) and a model group (n=24). The model group used orthodontic wire ligation to establish periodontitis. After three weeks, 6 rats from each group were selected and confirmed by Micro-CT to complete the modeling. The remaining rats in the model group were randomly divided into periodontitis group, normal saline(NS) group, and MOP group. In the MOP group, MOP (200 mg/kg for 3 d, 50 µL for 4 weeks) was injected into the palatal side of the left maxillary first molar of the rats. In the NS group, same volume of NS was injected, and no treatment was performed in the periodontitis group. The left maxillary tissue of rats were taken and the pathological changes of periodontal tissue were observed by H-E staining. The expression of FN and FN-EDA was detected by immunohistochemistry. Periodontal ligament fibroblasts were cultured in vitro, the effect of MOP on cell activity detected by CCK-8. The fourth generation cells were divided into control group, inflammation group (10 mg/mL lipopolysaccharide), and experimental group (12.5 µg/mL MOP, 12.5 µg/mL MOP+10 mg/mL lipopolysaccharide). The expression of FN and FN-EDA was detected by qRT-PCR and Western blot. The data were statistically analyzed using Prism 8.0 software package. RESULTS: In vivo experiments, the expression of FN-EDA in the MOP group was significantly lower than that in the periodontitis group and NS group(P＜0.05), and the infiltration of inflammatory cells was reduced. However, there was no significant difference in the expression of FN in each group. In vitro experiments, compared with the control group, the expression of FN-EDA mRNA and protein in the inflammation group was significantly increased(P＜0.000 1). MOP significantly reduced the expression of FN-EDA in inflammatory cells, but had no significant effect on FN expression. CONCLUSIONS: With increased expression of FN-EDA in inflammatory periodontal ligament tissues and cells, MOP may play a role in inhibiting inflammation by down-regulating FN-EDA.

[Preparation and biological characteristics of extracellular matrix vesicle mimetics].

Objective: To investigate the characteristics of extracellular matrix vesicle mimetics prepared by mechanical extrusion and their effects on the cell viability and osteogenic differentiation potential of human periodontal ligament stem cells (PDLSC). Methods: PDLSC derived extracellular matrix vesicles were prepared by collagenase digestion, while the cell derived vesicle mimetics were simulated by mechanical extrusion. The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. MVs, O-MVs, CVMs and O-CVMs do not affect the cell viability of PDLSC, and can promote the osteogenic differentiation potential of PDLSC to a certain extent.

Anti-inflammatory effects and related mechanisms of naringenin in human periodontal ligament stem cells under lipopolysaccharide stimulation based on RNA sequencing.

OBJECTIVES: RNA sequencing (RNA-seq) and bioinformatic analysis were combined and used to explore the anti-inflammatory effects and mechanisms of naringenin (Nar) in lipopolysaccharide (LPS)-stimulated human periodontal ligament stem cells (hPDLSCs). METHODS: Cell counting kit-8, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA) were adopted to detect the effects of Nar on the proliferation and expression of inflammatory factors in LPS-stimulated hPDLSCs, screening for the optimal anti-inflammatory concentration of Nar. Differentially expressed genes (DEGs) were screened using |log2FC|≥1 and P≤0.05 as criteria. Volcano plot analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the String database, and the MCODE module of Cytoscape were utilized to select core genes and enriched pathways. The effects on the nuclear factor κB (NF-κB) signaling pathway were verified using ELISA, qRT-PCR, and Western blot. RESULTS: Appropriate concentrations of Nar could alleviate the expression of inflammatory factors and promote the proliferation of hPDLSCs stimulated by LPS. The best anti-inflammatory effect was achieved with 20 µmol/L Nar. RNA-seq showed significant enrichment of inflammation-related signaling pathways. The anti-inflammatory effect of Nar was mediated by inhibiting the NF-κB signaling pathway, similar to the effect of the NF-κB inhibitor BAY 11-7802. CONCLUSIONS: Nar could exert its anti-inflammatory effects by inhibiting the NF-κB signaling pathway, making it a potential therapeutic option for the adjuvant treatment of periodontitis.

Fabricating Multiphasic Angiogenic Scaffolds Using Amyloid/Roxadustat-Assisted High-Temperature Protein Printing.

Repairing multiphasic defects is cumbersome. This study presents new soft and hard scaffold designs aimed at facilitating the regeneration of multiphasic defects by enhancing angiogenesis and improving cell attachment. Here, the nonimmunogenic, nontoxic, and cost-effective human serum albumin (HSA) fibril (HSA-F) was used to fabricate thermostable (up to 90 °C) and hard printable polymers. Additionally, using a 10.0 mg/mL HSA-F, an innovative hydrogel was synthesized in a mixture with 2.0% chitosan-conjugated arginine, which can gel in a cell-friendly and pH physiological environment (pH 7.4). The presence of HSA-F in both hard and soft scaffolds led to an increase in significant attachment of the scaffolds to the human periodontal ligament fibroblast (PDLF), human umbilical vein endothelial cell (HUVEC), and human osteoblast. Further studies showed that migration (up to 157%), proliferation (up to 400%), and metabolism (up to 210%) of these cells have also improved in the direction of tissue repair. By examining different in vitro and ex ovo experiments, we observed that the final multiphasic scaffold can increase blood vessel density in the process of per-vascularization as well as angiogenesis. By providing a coculture environment including PDLF and HUVEC, important cross-talk between these two cells prevails in the presence of roxadustat drug, a proangiogenic in this study. In vitro and ex ovo results demonstrated significant enhancements in the angiogenic response and cell attachment, indicating the effectiveness of the proposed design. This approach holds promise for the regeneration of complex tissue defects by providing a conducive environment for vascularization and cellular integration, thus promoting tissue healing.

Extracellular Matrix Remodelling of the Periodontium under Orthodontic Force.

As the biological mechanisms of orthodontic tooth movement have been explored further, scholars have gradually focused on the remodelling mechanism of the extracellular matrix (ECM) in the periodontal ligament (PDL). The ECM of the PDL consists of various types of collagens and other glycoproteins. The specific process and mechanism of ECM remodelling during orthodontic tooth movement remains unclear. Collagen I and III, which constitute major components of the PDL, are upregulated under orthodontic force. The changes in the contents of ECM proteins also depend on the expression of ECM-related enzymes, which organise new collagen fibre networks to adapt to changes in tooth position. The matrix metalloproteinase family is the main enzyme that participates in collagen hydrolysis and renewal and changes its expression under orthodontic force. Moreover, ECM adhesion molecules, such as integrins, are also regulated by orthodontic force and participate in the dynamic reaction of cell adhesion and separation with the ECM. This article reviews the changes in ECM components, related enzymes and adhesion molecules in the PDL under orthodontic force to lay the foundation for the exploration of the regulatory mechanism of ECM remodelling during orthodontic tooth movement.

Role of the GRP78-c-Src signaling pathway on osteoblast differentiation of periodontal ligament fibroblasts induced by cyclic mechanical stretch.

OBJECTIVES: This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism. METHODS: FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining. RESULTS: GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch. CONCLUSIONS: GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.

Biomimetic Mineralized Hydroxyapatite-Fish-Scale Collagen/Chitosan Nanofibrous Membranes Promote Osteogenesis for Periodontal Tissue Regeneration.

Commercial mammalian collagen-based membranes used for guided tissue regeneration (GTR) in periodontal defect repair still face significant challenges, including ethical concerns, cost-effectiveness, and limited capacity for periodontal bone regeneration. Herein, an enhanced biomimetic mineralized hydroxyapatite (HAp)-fish-scale collagen (FCOL)/chitosan (CS) nanofibrous membrane was developed. Specifically, eco-friendly and biocompatible collagen extracted from grass carp fish scales was co-electrospun with CS to produce a biomimetic extracellular matrix membrane. An enhanced biomimetic mineralized HAp coating provided abundant active calcium and phosphate sites, which promoted cell osteogenic differentiation, and showed greater in vivo absorption. In vitro experiments demonstrated that the HAp-FCOL/CS membranes exhibited desirable properties with no cytotoxicity, provided a mimetic microenvironment for stem cell recruitment, and induced periodontal ligament cell osteogenic differentiation. In rat periodontal defects, HAp-FCOL/CS membranes significantly promoted new periodontal bone formation and regeneration. The results of this study indicate that low-cost, eco-friendly, and biomimetic HAp-FCOL/CS membranes could be promising alternatives to GTR membranes for periodontal regeneration in the clinic.

Role of AOX1 on RXR signaling regulates osteoblastogenesis in hPDLMSCs.

Alveolar bone loss resulting from periodontal disease ultimately leads to tooth loss. Periodontal ligament mesenchymal stem cells (PDLMSCs) are the tissue-specific cells responsible for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. In this study, we explored the role of aldehyde oxidase 1 (AOX1) in regulating the osteoinduction of human periodontal ligament stem cells (hPDLMSCs). hPDLMSCs were isolated from clinically healthy donors, and AOX1 expression was assessed by comparing inducted and non-inducted hPDLMSCs. Remarkably, we observed a significant upregulation of AOX1 expression during osteoinduction, while AOX1 silencing resulted in the enhanced osteogenic potential of hPDLMSCs. Subsequent experiments and analysis unveiled the involvement of retinoid X receptor (RXR) signaling in the inhibition of osteogenesis in hPDLMSCs. Ligands targeting the RXR receptor mirrored the effects of AOX1 on osteogenesis, as evidenced by alterations in alkaline phosphatase (ALP) activity and bone formation levels. Collectively, these findings underscore the potential regulatory role of AOX1 via RXR signaling in the osteogenesis of hPDLMSCs. This elucidation is pivotal for advancing hPDLMSC-based periodontal regeneration strategies and lays the groundwork for the development of targeted therapeutic interventions aimed at enhancing bone formation in the context of periodontal disease.

Exploring the role of innate lymphoid cells in the periodontium: insights into immunological dynamics during orthodontic tooth movement.

Background: The periodontal ligament (PDL) experiences considerable mechanical stresses between teeth and bone, vital for tissue adaptation, especially in orthodontic tooth movement (OTM). While recent research emphasizes the role of innate lymphoid cells (ILCs) in regulating sterile inflammation, their involvement in periodontal tissues during OTM remains largely unexplored. Methods: In this study, PDL tissues from orthodontic patients (n = 8) were examined using flow cytometry to detect ILC subtypes. Transwell co-culture systems were used to expose PDL cells to mechanical strain, followed by measuring migration and ratios of sorted ILC subtypes. Statistical analyses were conducted using paired Student's t-test, Kruskal-Wallis test, Dunn's post-test and one-way/two-way ANOVA with Tukey's post-test (p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001). Results: Our findings demonstrate a significant increase in CD127+ CD161+ ILC frequencies in PDL tissues during OTM, indicating ILC involvement in sterile inflammation induced by orthodontic forces. Co-culture assays show directed migration of ILC subsets towards PDL cells and substantial proliferation and expansion of ILCs. Conclusions: This study is the first to comprehensively investigate the role of ILCs in sterile inflammation during OTM, revealing their presence and distribution within PDL tissues' innate immune response in vivo, and exploring their migratory and proliferative behavior in vitro. The results suggest a crosstalk between ILCs and PDL cells, potentially influencing the inflammatory response and tissue remodeling processes associated with OTM.

Unraveling Divergent Transcriptomic Profiles: A Comparative Single-Cell RNA Sequencing Study of Epithelium, Gingiva, and Periodontal Ligament Tissues.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

Adipogenic differentiation effect of human periodontal ligament stem cell initial cell density on autologous cells and human bone marrow stromal cells.

Stem cells demonstrate differentiation and regulatory functions. In this discussion, we will explore the impacts of cell culture density on stem cell proliferation, adipogenesis, and regulatory abilities. This study aimed to investigate the impact of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the adipogenic differentiation of autologous cells. Our findings indicate that the proliferation rate of hPDLSCs increased with increasing initial cell density (0.5-8 × 104 cells/cm2). After adipogenic differentiation induced by different initial cell densities of hPDLSC, we found that the mean adipose concentration and the expression levels of lipoprotein lipase (LPL), CCAAT/enhancer binding protein α (CEBPα), and peroxisome proliferator-activated receptor γ (PPAR-γ) genes all increased with increasing cell density. To investigate the regulatory role of hPDLSCs in the adipogenic differentiation of other cells, we used secreted exocrine vesicles derived from hPDLSCs cultivated at different initial cell densities of 50 µg/mL to induce the adipogenic differentiation of human bone marrow stromal cells. We also found that the mean adipose concentration and expression of LPL, CEBPα, and PPARγ genes increased with increasing cell density, with an optimal culture density of 8 × 104 cells/cm2. This study provides a foundation for the application of adipogenic differentiation in stem cells.

Effect of Cells Derived from Periodontal Ligament Tissue on Bone Formation.

BACKGROUND/AIM: Recent reports indicate that sclerostin is secreted by periodontal ligament tissue-derived (PDL) cells during orthodontic force loading and that the secreted sclerostin contributes to bone metabolism. However, the detailed mechanism is poorly understood. The aim of this study was to determine how PDL cells affect bone formation. MATERIALS AND METHODS: Rat periodontal ligament tissue was immunohistochemically stained for sclerostin. Cultured primary PDL cells, osteoblasts, and skin fibroblasts (Sfbs) isolated from rat periodontal ligament tissue, calvaria, and skin, respectively, were examined. Osteoblasts were cultured with control conditioned medium (Cont-CDM) and PDL cell culture conditioned medium (PDL-CDM) for up to 21 days. Cultured osteoblasts were then stained with alkaline phosphatase and von Kossa stain. Osteoblasts cultured in each conditioned medium were analyzed by real-time quantitative PCR for bone Gla protein (Bgp), Axin2, and Ki67 expression. PDL cells used to obtain conditioned medium were analyzed for Sost, Ectodin and Wnt1 expression and compared with expression in Sfbs. RESULTS: Expression of sclerostin was observed in periodontal ligament tissue by immunohistochemical staining. The formation of mineralization nodules was inhibited in PDL-CDM compared with Cont-CDM in osteoblast culture. In PDL-CDM, the expression levels of Bgp and Axin2 in osteoblasts were decreased compared with Cont-CDM. In PDL cells, expression levels of Sost and Ectodin were much higher than in Sfbs; however, expression of Wnt1 was lower in PDL cells compared with Sfbs. CONCLUSION: PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

Targeting the Neuropilin-1 receptor with Ovatodiolide and progress in using periodontal ligament organoids for COVID-19 research and therapy.

The discovery of SARS-CoV-2 RNA in the periodontal tissues of patients who tested positive for COVID-19, 24 days post the initial symptom onset, indicates the oral cavity could serve as a viral reservoir. This research aims to investigate the antiviral capabilities of Ovatodiolide, introducing a novel periodontal ligament organoid model for the study of SARS-CoV-2. We have successfully established a reliable and expandable organoid culture from the human periodontal ligament, showcasing characteristics typical of epithelial stem cells. This organoid model enables us to delve into the lesser-known aspects of dental epithelial stem cell biology and their interactions with viruses and oral tissues. We conducted a series of in vitro and ex vivo studies to examine the inhibitory impacts of Ova on SARS-CoV-2. Our findings indicate that Ovatodiolide molecules can bind effectively to the NRP1 active domain. Our study identifies potential interaction sites for Ovatodiolide (OVA) within the b1 domain of the NRP1 receptor. We generated point mutations at this site, resulting in three variants: Y25A, T44A, and a double mutation Y25A/T44A. While these mutations did not alter the binding activity of the spike protein, they did impact the concentration of OVA required for inhibition. The inhibitory concentrations for these variants are 15 µM for Y25A, 15.2 µM for T44A, and 25 µM for the double mutant Y25A/T44A. In addition, in vitro inhibition experiments demonstrate that the EC50 of Ova against the main protease (Mpro) of the SARS-CoV-2 virus is 7.316 µM. Our in vitro studies and the use of the periodontal ligament organoid model highlight Ovatodiolide's potential as a small molecule therapeutic agent that impedes the virus's ability to bind to the Neuropilin-1 receptor on host cells. The research uncovers various pathways and biochemical strategies through which Ovatodiolide may function as an effective antiviral small molecule drug.

The role of fibroblast growth factor-2 in modulating the differentiation of periodontal ligament and alveolar bone-derived stem cells.

OBJECTIVE: This study examined how range concentrations of Fibroblast Growth Factor-2 (FGF-2) influence the differentiation and activity of human-derived periodontal ligament (hPDLSCs) and alveolar bone-derived stem cells (haBMSCs). DESIGN: hPDLSCs and haBMSCs were cultured with varying concentrations of FGF-2 (0, 1, 2.5, 5, 10, 20 ng/mL) and monitored for osteogenic differentiation through alkaline phosphatase (ALP) activity and quantification of gene expression (qRT-PCR) for osteogenesis markers. Additionally, alizarin red staining and a hydroxyproline colorimetric assay evaluated and quantified osteogenic matrix mineralization and collagen deposition. Statistical analyses were performed using one-way ANOVA or two-way ANOVA for multiple comparisons between groups. RESULTS: At low FGF-2 concentrations, hPDLSCs differentiated toward an osteogenic lineage, whereas higher concentrations of FGF-2 inhibited osteogenesis and promoted fibroblastic differentiation. The effect of FGF-2 at the lowest concentration tested (1 ng/mL) led to significantly higher ALP activity than osteogenically induced positive controls at early time points and equivalent RUNX2 expression at early and later time points. FGF-2 supplementation of haBMSC cultures was sufficient, at all concentrations, to increase ALP activity at an earlier time point. Mineralization of haBMSC cultures increased significantly within 5-20 ng/mL FGF-2 concentrations under basal growth media conditions (α-minimal essential medium supplemented with 15 % fetal bovine serum and 1 % penicillin/streptomycin). CONCLUSIONS: FGF-2 has a dual capacity in promoting osteogenic and fibroblastic differentiation within hPDLSCs contingent upon the dosage and timing of administration, alongside supporting osteogenic differentiation in haBMSCs. These findings underscore the need for precision growth factors dosing when considering the design of biomaterials for periodontal regeneration.

In-vitro effects of different hyaluronic acids on periodontal biofilm-immune cell interaction.

Introduction: Recent studies have demonstrated a positive role of hyaluronic acid (HA) on periodontal clinical outcomes. This in-vitro study aimed to investigate the impact of four different HAs on interactions between periodontal biofilm and immune cells. Methods: The four HAs included: high-molecular-weight HA (HHA, non-cross-linked), low-molecular-weight HA (LHA), oligomers HA (OHA), and cross-linked high-molecular-weight HA (CHA). Serial experiments were conducted to verify the influence of HAs on: (i) 12-species periodontal biofilm (formation and pre-existing); (ii) expression of inflammatory cytokines and HA receptors in monocytic (MONO-MAC-6) cells and periodontal ligament fibroblasts (PDLF) with or without exposure to periodontal biofilms; (iii) generation of reactive oxygen species (ROS) in MONO-MAC-6 cells and PDLF with presence of biofilm and HA. Results: The results indicated that HHA and CHA reduced the bacterial counts in a newly formed (4-h) biofilm and in a pre-existing five-day-old biofilm. Without biofilm challenge, OHA triggered inflammatory reaction by increasing IL-1ß and IL-10 levels in MONO-MAC cells and IL-8 in PDLF in a time-dependent manner, whereas CHA suppressed this response by inhibiting the expression of IL-10 in MONO-MAC cells and IL-8 in PDLF. Under biofilm challenge, HA decreased the expression of IL-1ß (most decreasing HHA) and increased IL-10 levels in MONO-MAC-6 cells in a molecular weight dependent manner (most increasing CHA). The interaction between HA and both cells may occur via ICAM-1 receptor. Biofilm stimulus increased ROS levels in MONO-MAC-6 cells and PDLF, but only HHA slightly suppressed the high generation of ROS induced by biofilm stimulation in both cells. Conclusion: Overall, these results indicate that OHA induces inflammation, while HHA and CHA exhibit anti-biofilm, primarily anti-inflammatory, and antioxidant properties in the periodontal environment.

Ginsenoside Rb3 alleviates the formation of osteoclasts induced by periodontal ligament fibroblasts in the periodontitis microenvironment through the STAT3 pathway.

This study explores the potential role and mechanism of Ginsenoside Rb3 (Rb3) in modulating osteoclastogenesis induced by human periodontal ligament fibroblasts (hPLFs) within the periodontitis microenvironment. We investigated the anti-inflammatory effects of Rb3 on hPLFs stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g-LPS) utilizing quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay techniques. Moreover, the functional role of Rb3 in hPLFs-induced osteoclast formation was assessed by treating human bone marrow-derived macrophages (hBMMs) with conditioned medium from hPLFs, followed by analyses through qPCR, western blot analysis, and staining for tartrate-resistant acid phosphatase (TRAP) and phalloidin. The impact of Rb3 on the activation of the STAT3 signaling pathway was determined via western blot analysis. Results indicated that Rb3 treatment significantly suppressed the upregulation of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, MCP-1, and IL-18) at both gene and protein levels in hPLFs induced by P.g-LPS. Furthermore, conditioned medium from Rb3 plus P.g-LPS treated hPLFs notably decreased the number of TRAP-positive cells, actin ring formations, and the expression of osteoclast marker genes (including CTSK, NFATC1, and ACP5). Rb3 also inhibited the P.g-LPS-induced activation of the STAT3 pathway, with the activation of STAT3 partially reversing the effects of Rb3 on inflammation and osteoclast differentiation. Collectively, Rb3 ameliorates inflammation in P.g-LPS-stimulated hPLFs and reduces hPLFs-induced osteoclastogenesis by inhibiting the STAT3 signaling pathway, suggesting its potential as a therapeutic agent for periodontitis.