Periodontal Ligament Tissue Research Articles

Periodontal ligament tissues support neutrophil differentiation and maturation processes

IntroductionPeriodontal ligament is the soft connective tissue joining the roots of teeth with alveolar bone. The periodontal ligament presents significant cellular heterogeneity, including fibroblasts, endothelial cells, cementoblasts, osteoblasts, osteoclasts, and immune cells such as macrophages and neutrophils. These cells have crucial roles for periodontium homeostasis and function. However, certain cell types, such as neutrophils, remain poorly characterized in this tissue, despite their natural abundance and relevance in processes and diseases affecting the periodontal ligament.MethodsIn order to characterize neutrophils present in periodontal ligament, and get some insight into their functions, single-cell RNA sequencing data from published reports was analyzed to integrate and create a comprehensive map of neutrophil heterogeneity within the murine periodontal ligament under steady-state conditions.ResultsFour distinct neutrophil populations were identified based on their unique transcriptional signatures. Comparison and trajectory analysis revealed that these populations represent discrete stages of neutrophils undergoing maturation. These neutrophil populations were also classified, based on their granule content-associated signatures, as azurophil, specific, a transitional stage between specific and gelatinase (specific/gelatinase), and gelatinase. This reflects the sequential order of granule formation during neutrophil development (granulopoiesis) in the bone marrow.DiscussionTogether, our findings indicate that the periodontal ligament may serve as a microenvironment where the ordered and sequential maturation of neutrophils takes place. This suggests that similarly to other niches, the murine periodontal ligament can support, to some extent, hematopoietic processes such as granulopoiesis.

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

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

Engineered 3D Periodontal Ligament Model with Magnetic Tensile Loading

In vitro models are invaluable tools for deconstructing the biological complexity of the periodontal ligament (PDL). Model systems that closely reproduce the 3-dimensional (3D) configuration of cell–cell and cell–matrix interactions in native tissue can deliver physiologically relevant insights. However, 3D models of the PDL that incorporate mechanical loading are currently lacking. Hence, we developed a model where periodontal tissue constructs (PTCs) are made by casting PDL cells in a collagen gel suspended between a pair of slender, silicone posts for magnetic tensile loading. Specifically, one of the posts was rigid and the other was flexible with a magnet embedded in its tip so that PTCs could be subjected to tensile loading with an external magnet. Additionally, the deflection of the flexible post could be used to measure the contractile force of PDL cells in the PTCs. Prior to tensile loading, second harmonics generation analysis of collagen fibers in PTCs revealed that incorporation of PDL cells resulted in collagen remodeling. Biomechanical testing of PTCs by tensile loading revealed an elastic response at 4 h, permanent deformation by 1 d, and creep elongation by 1 wk. Subsequently, contractile forces of PDL cells were substantially lower for PTCs under tensile loading. Immunofluorescence analysis revealed that tensile loading caused PDL cells to increase in number, express higher levels of F-actin and α–smooth muscle actin, and become aligned to the tensile axis. Second harmonics generation analysis indicated that collagen fibers in PTCs progressively remodeled over time with tensile loading. Gene expression analysis also confirmed tension-mediated upregulation of the F-actin/Rho pathway and osteogenic genes. Our model is novel in demonstrating the mechanobiological behavior that results in cell-mediated remodeling of the PDL tissue in a 3D context. Hence, it can be a valuable tool to develop therapeutics for periodontitis, periodontal regeneration, and orthodontics.

Biomaterial Scaffolds for Periodontal Tissue Engineering.

Advanced periodontitis poses a significant threat to oral health, causing extensive damage and loss of both hard and soft periodontal tissues. While traditional therapies such as scaling and root planing can effectively halt the disease's progression, they often fail to fully restore the original architecture and function of periodontal tissues due to the limited capacity for spontaneous regeneration. To address this challenge, periodontal tissue engineering has emerged as a promising approach. This technology centers on the utilization of biomaterial scaffolds, which function as three-dimensional (3D) templates or frameworks, supporting and guiding the regeneration of periodontal tissues, including the periodontal ligament, cementum, alveolar bone, and gingival tissue. These scaffolds mimic the extracellular matrix (ECM) of native periodontal tissues, aiming to foster cell attachment, proliferation, differentiation, and, ultimately, the formation of new, functional periodontal structures. Despite the inherent challenges associated with preclinical testing, the intensification of research on biomaterial scaffolds, coupled with the continuous advancement of fabrication technology, leads us to anticipate a significant expansion in their application for periodontal tissue regeneration. This review comprehensively covers the recent advancements in biomaterial scaffolds engineered specifically for periodontal tissue regeneration, aiming to provide insights into the current state of the field and potential directions for future research.

IRF4 Suppresses Osteogenic Differentiation of Periodontal Ligament Stem Cells by Activating IL-18 Signaling Pathway in Periodontitis.

The present study aims to investigate the role of interferon regulatory factor 4 (IRF4) in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and analyze the underlying signaling of these processes. In this study, IRF4 is upregulated in periodontitis periodontal ligament tissues, as compared to healthy periodontal ligament tissues. IRF4 knockdown increases cell proliferation, decreases levels of tumor necrosis factor-alpha, interleukin-6, and interleukin-8, enhances osteogenic activity, and increases the expression of RUNX family transcription factor 2, Collagen I, and Osteocalcin in PDLSCs. The opposite results are observed in IRF4 overexpressed PDLSCs. Additionally, GSEA shows that IRF4 activates the interleukin-18 (IL-18) signaling pathway. The expressions of IL-18, B-cell translocation gene 2, interleukin-1beta, and caspase-3 are decreased by IRF4 knockdown, while increased by IRF4 overexpression. IL-18 overexpression eliminates the promoting effect of IRF4 knockdown on osteogenic differentiation of PDLSCs. In conclusion, IRF4 suppresses osteogenic differentiation of PDLSCs by activating the IL-18 signaling pathway.

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

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. 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). 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. 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.

Weighted Gene Co-expression Network Analysis (WGCNA) of Wnt Signaling Related to Periodontal Ligament Formation: A Bioinformatics-Based Analysis.

Introduction The Wnt signaling pathway is crucial for tooth development, odontoblast differentiation, and dentin formation. It interacts with epithelial cadherin (E-cadherin) and beta-catenin in tooth development and periodontal ligament (PDL) formation. Dysregulation of Wnt signaling is linked to periodontal diseases, requiring an understanding of therapeutic interventions. Weighted gene co-expression network analysis (WGCNA) can identify co-expressed gene modules. Our study aims to identify hub genes in WGCNA analysis of Wnt signaling-based PDL formation. Methods The study used a microarray datasetGSE201313 from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus to analyze the impact of DMP1 expression on XLH dental pulp cell differentiation and PDL formation. The standardized dataset was used for WGCNA analysis, which generated a co-expression network by calculating pairwise correlations between genes and constructing an adjacency matrix. The topological overlap matrix (TOM) was transformed into a hierarchical clustering tree and then cut into modules or clusters of highly interconnected genes. The module eigengene (ME) was calculated for each module, and the genes within this module were identified as hub genes. Gene ontology (GO) and KEGG pathway enrichment analysis were performed to gain insights into the biological functions of the hub genes. The integrated Differential Expression and Pathway analysis (iDEP) tool (http://bioinformatics.sdstate.edu/idep/; South Dakota State University, Brookings, USA) was used for WGCNA analysis. Results The study used the WGCNA package to analyze 1,000 differentially expressed genes, constructing a gene co-expression network and generating a hierarchical clustering tree and TOM. The analysis reveals a scale-free topology fitting index R2 and mean connectivity for various soft threshold powers, with an R2 value of 5. COL6A1, MMP3, BGN, COL1A2, and FBN2 are hub genes implicated in PDL development. Conclusion The study identified key hub genes, including COL6A1, MMP3, BGN, and FBN2, crucial for PDL formation, tissue remodeling, and cell-matrix interactions, guiding future therapeutic strategies.

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.

Role of Oxidative Stress in Periodontal Diseases.

Periodontal disease, a significant worldwide health burden, is characterized by chronic inflammation and destruction of periodontal tissues, including the cementum, periodontal ligament(PDL), alveolar bone, and gingival tissue. Recent research has linked the development and progression of periodontal disease to oxidative stress. This study provides comprehensive explanations of the mechanisms behind oxidative stress in periodontal disease, with a focus on the generation of reactive oxygen species (ROS) and their effects on periodontal tissues. Oxidative stress triggers a number of detrimental reactions, including lipid peroxidation, protein oxidation, and damage to deoxyribonucleic acid (DNA). Alveolar bone resorption, connective tissue degradation, and periodontal inflammation are further conditions exacerbated by these processes. In addition, the delicate balance between antioxidants and oxidants is upset by oxidative stress, which impairs antioxidant defense systems and exacerbates periodontal tissue damage. This review highlights the negative effects of oxidative stress and enhancesperiodontal health outcomes.

Revealing Genetic Dynamics: scRNA-seq Unravels Modifications in Human PDL Cells across In Vivo and In Vitro Environments.

The periodontal ligament (PDL) is a highly specialized fibrous tissue comprising heterogeneous cell populations of an intricate nature. These complexities, along with challenges due to cell culture, impede a comprehensive understanding of periodontal pathophysiology. This study aims to address this gap, employing single-cell RNA sequencing (scRNA-seq) technology to analyze the genetic intricacies of PDL both in vivo and in vitro. Primary human PDL samples (n = 7) were split for direct in vivo analysis and cell culture under serum-containing and serum-free conditions. Cell hashing and sorting, scRNA-seq library preparation using the 10x Genomics protocol, and Illumina sequencing were conducted. Primary analysis was performed using Cellranger, with downstream analysis via the R packages Seurat and SCORPIUS. Seven distinct PDL cell clusters were identified comprising different cellular subsets, each characterized by unique genetic profiles, with some showing donor-specific patterns in representation and distribution. Formation of these cellular clusters was influenced by culture conditions, particularly serum presence. Furthermore, certain cell populations were found to be inherent to the PDL tissue, while others exhibited variability across donors. This study elucidates specific genes and cell clusters within the PDL, revealing both inherent and context-driven subpopulations. The impact of culture conditions-notably the presence of serum-on cell cluster formation highlights the critical need for refining culture protocols, as comprehending these influences can drive the creation of superior culture systems vital for advancing research in PDL biology and regenerative therapies. These discoveries not only deepen our comprehension of PDL biology but also open avenues for future investigations into uncovering underlying mechanisms.

GroEL triggers NLRP3 inflammasome activation through the TLR/NF-κB p-p65 axis in human periodontal ligament stem cells.

The interaction between bacteria and the host plays a vital role in the initiation and progression of systemic diseases, including gastrointestinal and oral diseases, due to the secretion of various virulence factors from these pathogens. GroEL, a potent virulence factor secreted by multiple oral pathogenic bacteria, is implicated in the damage of gingival epithelium, periodontal ligament, alveolar bone and other peripheral tissues. However, the underlying biomechanism is still largely unknown. In the present study, we verify that GroEL can trigger the activation of NLRP3 inflammasome and its downstream effector molecules, IL-1β and IL-18, in human periodontal ligament stem cells (hPDLSCs) and resultantly induce high activation of gelatinases (MMP-2 and MMP-9) to promote the degradation of extracellular matrix (ECM). GroEL-mediated activation of the NLRP3 inflammasome requires the participation of Toll-like receptors (TLR2 and TLR4). High upregulation of TLR2 and TLR4 induces the enhancement of NF-κB (p-p65) signaling and promotes its nuclear accumulation, thus activating the NLRP3 inflammasome. These results are verified in a rat model with direct injection of GroEL. Collectively, this study provides insight into the role of virulence factors in bacteria-induced host immune response and may also provide a new clue for the prevention of periodontitis.

Circ_0036490 and DKK1 competitively bind miR-29a to promote lipopolysaccharides-induced human gingival fibroblasts injury

MicroRNA (miRNA) plays a regulatory role in periodontitis. This study aimed to explore whether miR-29a could affect lipopolysaccharides (LPSs)-induced injury in human gingival fibroblasts (HGFs) through the competitive endogenous RNAs (ceRNA) mechanism. Periodontal ligament (PDL) tissues and HGFs were derived from patients with periodontitis and healthy volunteers. Periodontitis cell model was established by treating HGFs with LPS. Expression levels of circ_0036490, miR-29a, and DKK1 were evaluated by the reverse transcription quantitative real-time PCR (RT-qPCR) method. Western blotting assay was performed to assess protein expression levels of pyroptosis-related proteins and Wnt signalling related proteins. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. Concentration of lactate dehydrogenase (LDH), interleukin (IL)-1β, and IL-18 were determined by Enzyme-linked immunosorbent assay (ELISA). Pyroptosis rate were determined by flow cytometry assay to evaluate pyroptosis. The interaction between miR-29a and circ_0036490 or DKK1 was verified by dual-luciferase reporter and RNA pull-down assays. MiR-29a expression was lower in PDL tissues of patients with periodontitis than that in healthy group; likewise, miR-29a was also downregulated in LPS-treated HGFs. Overexpression of miR-29a increased cell viability and decreased pyroptosis of HGFs induced by LPS while inhibition of miR-29a exerted the opposite role. MiR-29a binds to circ_0036490 and elevation of circ_0036490 contributed to dysfuntion of LPS-treated HGFs and reversed the protection function of elevated miR-29a. In addition, miR-29a targets DKK1. Overexpression of DKK1 abrogated the effects of overexpressed miR-29a on cell vaibility, pyroptosis, and protein levels of Wnt signalling pathway of LPS-treated HGFs. Circ_0036490 and DKK1 competitively bind miR-29a to promote LPS-induced HGF injury in vitro. Wnt pathway inactivated by LPS was activated by miR-29a. Thence, miR-29a may be a promising target for periodontitis.

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.

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

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. 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. 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. PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

Circ_0087199 depletion attenuates lipopolysaccharides-induced human periodontal ligament cell injury through the miR-527/TLR4 axis.

Circular RNAs participate in the development of periodontitis. The present work aims to reveal the role and mechanism of circ_0087199 in human periodontal ligament cell (PDLC) injury during periodontitis. PDLCs were treated with lipopolysaccharides (LPS) to establish a periodontitis cell model. Quantitative real-time polymerase chain reaction was used to detect the expression of circ_0087199, miR-527, toll-like receptor 4 (TLR4). Western blot analysis assay was performed to assess protein expression. Cell viability, proliferation, apoptosis and inflammation were investigated by cell counting kit-8, EdU assay, flow cytometry and enzyme-linked immunosorbent assay, respectively. Oxidative stress was evaluated by malondialdehyde assay kit and superoxide dismutase activity assay kit. The interaction between miR-527 and circ_0087199 or TLR4 was confirmed by a dual-luciferase reporter assay. Circ_0087199 and TLR4 expression levels were significantly increased, while miR-527 was decreased in the periodontal ligament tissues of periodontitis patients and LPS-stimulated PDLCs when compared with controls. LPS treatment inhibited cell viability and proliferation but induced cell apoptosis, inflammation and oxidative stress, whereas these effects were attenuated after circ_0087199 knockdown. Circ_0087199 bound to miR-527 and regulated LPS-caused PDLC damage by targeting miR-527. Additionally, the overexpression of TLR4, a target gene of miR-527, rescued miR-527 mimic-mediated effects on LPS-treated PDLCs. Further, the regulation of circ_0087199 toward TLR4 involved miR-527. Circ_0087199 knockdown attenuated LPS-induced apoptosis, inflammation and oxidative stress of PDLCs by regulating the miR-527/TLR4 pathway.

Evaluation of Random and Aligned Polycaprolactone Nanofibrous Electrospun Scaffold for Human Periodontal Ligament Engineering in Biohybrid Titanium Implants.

Background: Stem cells are introduced to regenerate some living tissue to restore function and longevity. The study aims to isolate in vitro human periodontal ligament stem cells (hPDLSCs) and investigate their proliferation rate on plasma-treated aligned and random polycaprolactone (PCL) nanofibrous scaffolds made via an electrospinning technique to attempt periodontal-like tissue in dental implants. Materials and Methods: hPDLSCs were isolated from extracted human premolars and cultured on plasma-treated or untreated PCL-aligned and random scaffolds to enhance adhesion of periodontal ligament (PDL) cells as well as interaction and proliferation. Cell morphology, adhesion, and proliferation rate were evaluated using field emission scanning electron microscopy (FESEM) and the methyl tetrazolium (MTT) assay. The wettability of PCL scaffolds was tested using a goniometer. Results: The hydrophilicity of plasma-treated scaffolds was significantly increased (p ≤ 0.05) in both aligned and random nanofibers compared to the nontreated nanofibrous scaffold. Cells arranged in different directions on the random nanofiber scaffold, while for aligned scaffold nanofibers, the cells were arranged in a pattern that followed the direction of the aligned electrospun nanofibres. The rate of hPDLSC proliferation on an aligned PCL nanofiber scaffold was significantly higher than on a random PCL nanofibrous scaffold with a continuous, well-arranged monolayer of cells, as shown in FESEM. Conclusion: The aligned PCL nanofiber scaffold is superior to random PCL when used as an artificial scaffold for hPDLSC regeneration in PDL tissue engineering applications.

Sustained delivery of chemically modified mRNA encoding amelogenin from self-assembling hydrogels for periodontal regeneration

Periodontitis is a growing clinical problem worldwide. Despite various approaches that have been devoted to repairing periodontal defects, the clinical outcomes are still limited due to the decline in tissue regenerative potential after periodontitis. Fortunately, chemically modified mRNA (modRNA)-based therapeutics have emerged as a promising strategy to combat incurable diseases. However, the use of modRNA for treating periodontitis is rare and challenging. Herein, a novel therapeutic mRNA platform that allows for sustained delivery and targeted osteogenesis and cementogenesis was developed for periodontal regeneration by combining a self-assembling PLA-PEG-PLA hydrogel and newly synthesized modRNA encoding amelogenin (AMELX) via in vitro transcription. The thermosensitive PLA-PEG-PLA hydrogel significantly prevented modRNA degradation and prolonged the release of encapsulated modRNA by forming an in situ hydrogel depot at 37 °C, resulting in sustained and strong expression of AMELX in human periodontal ligament stem cells (PDLSCs). Moreover, the AMELX modRNA exhibited a great capacity for promoting osteogenesis and cementogenesis of PDLSCs, as evidenced by elevated alkaline phosphatase (ALP) activity, calcium nodule formation, and osteogenesis- and cementogenesis-related gene expression (e.g., ALP, BMP2, CAP, BSP, COL1A1). As a result, after a single local injection, the PLA-PEG-PLA hydrogel enabled AMELX modRNA to persist and release at the defect site, thus successfully regenerating bone and periodontal ligament tissues in periodontal defects of rats. These findings provide a new approach for treating periodontal defects using self-assembling hydrogels to enable safe and effective delivery of AMELX modRNA, thereby offering various possibilities for functional tissue reconstruction with mRNA therapeutics.

Developmental endothelial locus-1 promotes osteogenic differentiation and alveolar bone regeneration in experimental periodontitis with type 2 diabetes mellitus.

This study sought to explore the role of developmental endothelial locus-1 (DEL-1) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and investigate the therapeutic effect of DEL-1 in ligature-induced experimental periodontitis with type 2 diabetes mellitus (T2DM). T2DM is a significant risk factor for periodontitis. Treatment modalities for periodontitis with T2DM are being explored. DEL-1 is a versatile protein that can modulate the different stages of inflammatory diseases including periodontitis. The direct effect of DEL-1 on osteogenic differentiation of PDLSCs in periodontitis with T2DM is poorly understood. Primary hPDLSCs were isolated from periodontal ligament tissue and identified by flow cytometry. In osteogenesis experiments, alkaline phosphatase (ALP), Alizarin Red staining and western blot were used to assess the osteogenic effect of DEL-1 on hPDLSCs in high glucose and inflammation environments. The mouse model of ligature-induced experimental periodontitis was established. H&E and Masson's trichrome staining were used to assess the change of periodontal tissue after local periodontal injection of DEL-1. Immunohistochemical staining was used to evaluate osteogenic-related protein expression. hPDLSCs expressed mesenchymal stem cell (MSC)-specific surface markers and were negative for hematopoietic cell surface markers. hPDLSCs had the potential for multidirectional differentiation. DEL-1 could enhance the osteogenic differentiation of hPDLSCs in high glucose and inflammation environments, although it did not return to the control level. Histological staining showed that DEL-1 contributed to alveolar bone regeneration and osteogenic-related protein expression, but the degree of improvement in T2DM mice was lower than in non-T2DM mice. In summary, we demonstrated that DEL-1 could promote osteogenic differentiation of hPDLSCs in high glucose and inflammation environment and rescue alveolar bone loss in experimental periodontitis with T2DM, which could provide a novel therapeutic target for periodontitis with T2DM.

Leucine-Rich Repeat Containing 15-Mediated Cell Adhesion Is Essential for Integrin Signaling in TGF-β1-Induced PDL Fibroblastic Differentiation.

Human periodontal ligament cells (hPDLCs) cultured from periodontal ligament (PDL) tissue contain postnatal stem cells that can be differentiated into PDL fibroblasts. We obtained PDL fibroblasts from hPDLCs by treatment with low concentrations of TGF-β1. Since the extracellular matrix and cell surface molecules play an important role in differentiation, we had previously developed a series of monoclonal antibodies against PDL fibroblast-specific cell surface molecules. One of these, the anti-PDL51 antibody, recognized a protein that was significantly upregulated in TGF-β1-induced PDL fibroblasts and highly accumulated in the PDL region of the tooth root. Mass spectrometry revealed that the antigen recognized by the anti-PDL51 antibody was leucine-rich repeat containing 15 (LRRC15), and this antibody specifically recognized the extracellular glycosylated moiety of LRRC15. Experiments presented here show that as fibroblastic differentiation progresses, increased amounts of LRRC15 localized at the cell surface and membrane. Inhibition of LRRC15 by siRNA-mediated depletion and by antibody blocking resulted in downregulation of the representative PDL fibroblastic markers. Moreover, following LRRC15 inhibition, the directed and elongated cell phenotypes disappeared, and the long processes of the end of the cell body were no longer found. Through a specific interaction between integrin β1 and LRRC15, the focal adhesion kinase signaling pathway was activated in PDL fibroblasts. Furthermore, it was shown that increased LRRC15 was important for the activation of the integrin-mediated cell adhesion signal pathway for regulation of cellular functions, including fibroblastic differentiation, proliferation, and cell migration arising from the expression of PDL-related genes in TGF-β1-induced PDL fibroblastic differentiation.

Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells.

Periodontal ligament (PDL) stem-like cells (PDLSCs) are promising for regeneration of the periodontium because they demonstrate multipotency, high proliferative capacity, and the potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability. Since PDLSCs are derived from neural crest cells (NCs) and NCs persist in adult PDL tissue, we devised to promote the regeneration of the periodontium by activating NCs to differentiate into PDLSCs. SK-N-SH cells, a neuroblastoma cell line that reportedly has NC-like features, seeded on the extracellular matrix of PDL cells for 2 weeks, resulted in the significant upregulation of PDL marker expression. SK-N-SH cell-derived PDLSCs (SK-PDLSCs) presented phenotypic characteristics comparable to induced pluripotent stem cell (iPSC)-derived PDLSCs (iPDLSCs). The expression levels of various hyaluronic acid (HA)-related genes were upregulated in iPDLSCs and SK-PDLSCs compared with iPSC-derived NCs and SK-N-SH cells, respectively. The knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while low-molecular HA (LMWHA) induction enhanced SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells could be applied as a new model to induce the differentiation of NCs into PDLSCs and that the LMWHA-CD44 relationship is important for the differentiation of NCs into PDLSCs.