The tooth and its supporting periodontium are essential structures of the oral cavity, frequently compromised by conditions such as dental defects, aries, and periodontal diseases, which, if poorly treated, often lead to tooth loss. These conditions, affecting billions of people worldwide, remain significant healthcare and socio-economic challenges. Regenerative dentistry has emerged as a possible therapeutic option, leveraging advances in tissue engineering (TE), stem cell biology, and biophysical stimulation. Oral tissue-derived mesenchymal stem/stromal cells (OMSCs) hold great potential for dental and periodontal regeneration. Electrical stimulation (ES), a biophysical cue known to regulate key cellular behaviors such as migration, proliferation, and differentiation, has gained increasing attention for enhancing the therapeutic capacities of OMSCs. This review explores the biological properties of OMSCs under ES, its role in regenerative dentistry, and recent breakthroughs in ES-based dental and periodontal TE strategies. Furthermore, the current challenges and future directions for translating these innovative approaches into clinical practice are discussed.

This study aimed to investigate autophagy and its associated mechanisms in periodontal ligament stem cells (PDLSCs) within the inflammatory microenvironment of type 2 diabetes mellitus (T2DM) and periodontitis. Periodontal ligament tissues were obtained from healthy individuals, individuals with T2DM, individuals with chronic periodontitis, and individuals with both T2DM and periodontitis. PDLSCs were isolated, cultured, and treated with the autophagy inhibitor 3-methyladenine (3-MA) and the autophagy activator rapamycin (Rapa). Cell proliferative capacity was evaluated, autophagic activity and organelle damage were assessed using transmission electron microscopy, and the relative expression levels of autophagy-related genes (Beclin-1, LC3 II, P62) were measured using real-time quantitative PCR. Compared to PDLSCs derived from healthy individuals, those from individuals with chronic periodontitis or T2DM exhibited no significant morphological differences but demonstrated reduced proliferative capacity. Treatment with 3-MA and Rapa did not significantly alter proliferative capacity across groups. PDLSCs from individuals with chronic periodontitis and T2DM displayed increased autophagosome formation, more severe organelle damage, and upregulated expression of autophagy-related genes Beclin-1 and LC3 II, while P62 expression was downregulated, compared to PDLSCs from healthy individuals. PDLSCs from individuals with T2DM and periodontitis exhibit excessive autophagy and organelle damage. Autophagy dysregulation in PDLSCs within a diabetic and inflammatory microenvironment may contribute to the severity of periodontal destruction observed in individuals with T2DM.

Advanced bioengineering, popularly known as regenerative dentistry, has emerged and is steadily developing with the aim of replacement of lost or injured tissues in the mouth using stem cells and other biomaterials. Conventional therapies for reparative dentistry, for instance fillings or crowns, mainly entail the replenishment of affected tissues without much concern given to the regeneration of tissues. However, these methods do not enable the natural function and aesthetics of the teeth to be maintained in the long term. There are several regenerative strategies that offer the potential to address these limitations to the extent of biologically restoring the function of teeth and their components, like pulp, dentin, bone, and periodontal tissues. Hence, stem cells, especially dental tissue derived stem cells, such as dental pulp stem cells, periodontal ligament stem cells, or apical papilla stem cells, are quite promising in this regard. These stem cells have the potentiality of generating precise dental cell lineages and thus are vital for tissue healing and renewal. Further, hydrogels, growth factors, and synthetic scaffolds help in supporting the stem cells for growth, proliferation, and differentiation into functional tissues. This review aims at describing the process of stem cell-based tissue repair biomaterials in dental regeneration, and also looks into the practice and prospects of regenerative dentistry, analysing several case reports and clinical investigations that demonstrate the efficacy and limitations of the technique. Nonetheless, the tremendous potential for regenerative dentistry is a reality that is currently challenged by biological and technical constraints, such as scarcity of stem cell sources, inadequate vascularization, and the integration of the materials used in the procedure. As we move forward, the prospects for regenerative dentistry are in subsequent developments of stem cell technology, biomaterial optimization, and individualized treatment methods, which might become increasingly integrated in dental practices globally. However, there are regulatory, ethical and economic issues that may pose a hurdle in the further advancement of this discipline.

Mesenchymal stromal cells (MSCs) with high expression of CD146 have superior properties for tissue regeneration. However, high variability in the rate of CD146+ cells among donors is observed. In this study, the possible reasons behind this variability in human periodontal ligament MSCs (hPDL-MSCs) were explored.

hPDL-MSCs were isolated from 22 different donors, and rates of CD146+ cells were analyzed by flow cytometry. Furthermore, populations with various rates of CD146+ cells were isolated with magnetic separation. The dependency of cell proliferation, viability, cell cycle, and osteogenic differentiation on the rates of CD146+ cells was investigated. Besides, the effects of various factors, like cell density, confluence, and inflammatory environment on the CD146+ rate and expression were analyzed.

The rate of CD146+ cells exhibited high variability between donors, with the percentage of CD146+ cells ranging from 3% to 67%. Higher percentage of CD146+ cells was associated with higher proliferation, presumably due to the higher percentage of cells in the S-phase, and higher osteogenic differentiation potential. Prolonged cell confluence and higher cell seeding density led to the decline in the rate of CD146+ cells. The surface rate of CD146 in hPDL-MSCs was stimulated by the treatment with interleukin-1β and tumor necrosis factor-α, and inhibited by the treatment with interferon-γ.

These results suggest that hPDL-MSCs with high rate of CD146+ cells are a promising subpopulation for enhancing the effectiveness of MSC-based regenerative therapies, however the rate of CD146 is affected by various factors, which must be considered for cell propagation and their potential application in vivo.

BackgroundMesenchymal stem cells-derived exosomes, crucial in regenerative medicine, have been explored for their potential for the functional modification of bone scaffolds.ObjectiveTo design a functionally modified biomimetic nanohydroxyapatite using exosomes and explore its effects on bone regeneration.MethodsA biomimetic nanohydroxyapatite (named as tHA) was fabricated as previous methods using a polydopamine (pDA) structure as a template, and exosomes (Exo) derived from periodontal ligament stem cells (PDLSCs) were used to functionally modify the tHA scaffold material through pDA. The effects of functional composite scaffold (tHA-Exo) on cells proliferation and osteogenic differentiation were investigated. Furthermore, their effect on bone regeneration was also evaluated in vivo.ResultsExosomes can be loaded onto the tHA via pDA and the tHA-Exo releases exosomes in a sustained and stable manner. tHA-Exo showed improved cytocompatibility compared to controls. Additionally, tHA-Exo significantly enhanced the proliferation and osteogenic differentiation of PDLSCs. More importantly, animal experiments have shown that tHA-Exo could dramatically promote bone regeneration.ConclusionThe tHA nanoparticles, functionally modified by the PDLSCs-Exo through pDA, significantly promoted bone regeneration by improving its cytocompatibility and osteogenic potential, which could serve as a promising material for promoting bone regeneration.

Cell homing, a process that leverages the body's natural ability to recruit cells and repair damaged tissues, presents a promising alternative to cell transplantation methods. Central to this approach is the recruitment of endogenous stem/progenitor cells-such as those from the apical papilla, bone marrow, and periapical tissues-facilitated by chemotactic biological cues. Moreover, biomaterial scaffolds embedded with signaling molecules create supportive environments, promoting cell migration, adhesion, and differentiation for the regeneration of the pulp-dentin complex. By analyzing in vivo animal studies using cell homing strategies, this review explores how biomolecules and scaffold materials enhance the recruitment of endogenous stem cells to the site of damaged dental pulp tissue, thereby promoting repair and regeneration. It also examines the key principles, recent advancements, and current limitations linked to cell homing-based regenerative endodontic therapy, highlighting the interplay of biomaterials, signaling molecules, and their broader clinical implications.

Static magnetic field (SMF) as an effective physical stimulus is capable of osteogenic differentiation for multiple mesenchymal stem cells, including human periodontal ligament stem cells (hPDLSCs). However, the exact molecular mechanism is still unknown. Therefore, this study intends to excavate molecular mechanisms related to SMF in hPDLSCs using functional experiments.

hPDLSCs were treated with different intensities of SMF, H19 lentivirus, and Wnt/β-catenin pathway inhibitor (XAV939). Changes in osteogenic markers (Runx2, Col Ⅰ, and BMP2), Wnt/β-catenin markers (β-catenin and GSK-3β), and calcified nodules were examined using RT-qPCR, western blotting, and alizarin red staining in hPDLSCs.

SMF upregulated the expression of H19, and SMF and overexpressing H19 facilitated the expression of osteogenic markers (Runx2, Col Ⅰ, and BMP2), activation of the Wnt/β-catenin pathway, and mineralized sediment in hPDLSCs. Knockdown of H19 alleviated SMF function, and treatment with XAV939 limited SMF- and H19-mediated osteogenic differentiation of hPDLSCs. Notably, the expression of hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p in hPDLSCs was regulated by SMF, and may form an endogenous competitive RNA mechanism with H19 and β-catenin.

SMF contributes to the osteogenic differentiation of hPDLSCs by mediating the H19/Wnt/β-catenin pathway, and hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p may be the key factors in it.

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

This study assessed the impact of low-level laser irradiation on the viability and proliferation of human periodontal ligament stem cells (hPDLSCs) cultivated on polylactic acid (PLA) scaffolds. hPDLSCs were obtained, characterized, and grown on the surface of PLA films produced via the solvent casting technique. The study involved two groups: the control group, which was not exposed to radiation, and the laser group, which was irradiated with a diode laser (InGaAIP) with a power of 30 mW, a wavelength of 660 nm, and a single dose of 1 J/cm² emitted continuously. Cell viability was assessed 24 and 48 hours after irradiation using the Alamar blue and Live/Dead assays. Flow cytometry was used to assess cell cycle events, and scanning electron microscopy (SEM) was used to evaluate the interaction between cells and the biomaterial. The results revealed a statistically significant increase in cell metabolic activity in the laser group compared with the control group at 24 hours (p <0.05) and 48 hours (p <0.001), as indicated by the Alamar blue assay. The Live/Dead assay also revealed a greater density of viable cells in the laser group. The cell cycle analysis revealed a significant increase in the number of cells in the proliferative phase (G2/M) in the laser group compared with the control group (p <0.001). The SEM images demonstrated that the irradiated group had a greater concentration of cells while still maintaining their cell shape and projections. This study demonstrated that photobiomodulation can increase the viability and proliferation of periodontal stem cells cultured on PLA scaffolds, suggesting the potential of this protocol for future studies on periodontal tissue engineering.

Periodontitis can lead to the destruction of periodontal tissues and potentially tooth loss. Numerous periodontal tissue-derived cells display osteogenic differentiation potential. The presence of differentially expressed long non-coding RNAs (lncRNAs) in these cells indicate their ability to regulate the process of osteogenic differentiation. We aim to elucidate the various lncRNA-mediated regulatory mechanisms in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis at epigenetic modification, transcriptional, and post-transcriptional levels.

We systematically searched the PubMed, Web of Science, and ScienceDirect databases to identify relevant literature in the field of periodontitis discussing the role of lncRNAs in regulating osteogenic differentiation of periodontal tissue-derived cells. The identified literature was subsequently summarized for comprehensive review.

In this review, we have comprehensively summarized the regulatory mechanisms of lncRNAs in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis and discussed how these lncRNAs provide novel perspectives for understanding the pathogenesis and progression of periodontitis.

These results indicate the pivotal role of lncRNAs as regulators in the osteogenic differentiation of periodontal tissue-derived cells, providing a solid basis for future investigations on the role of lncRNAs in the periodontitis field.

Non-coding RNA has many types which has rich functions and plays an important role in the study of basic molecular mechanisms. Many non-coding RNA have important implications for pluripotent stem cells and embryonic stem cells. It has been found to affect the self-renewal and osteogenesis of many types of stem cells. They have also been found to regulate stem cell proliferation and induct bone differentiation. Periodontal ligament stem cells are essential for the regeneration of periodontal tissue. In recent years, in the field of stomatology, studies have found that many non-coding RNA also have significant regulatory effects on the proliferation and differentiation of periodontal stem cells and may become potential therapeutic targets for many common periodontal diseases such as periodontitis, bone/tooth/soft tissue loss and orthodontic treatment. Therefore, we summarized the current research status of non-coding RNA in the field of molecular mechanism of periodontal ligament stem cells and prospected its future progress.

Type 2 diabetes (T2DM), a recognized risk factor for periodontitis, is characterized by insulin resistance. However, the molecular mechanisms concerning the role of insulin resistance in linking T2DM and periodontitis remain poorly elucidated due to the absence of an appropriate T2DM cell model. We aimed to explore an appropriate model of T2DM in human periodontal ligament stem cells (hPDLSCs) and uncover the involved mechanisms.

hPDLSCs were incubated with common reagents for recapitulating insulin resistance state including high glucose (HG) (15, 25, 35, 45 mM), glucosamine (0.8, 8, 18, 28, 38 mM), or palmitic acid (PA; 100, 200, 400, 800 μM), combined with LPS for 48 h. The insulin signaling pathway, inflammation, and pyroptosis were detected by western blots and quantitative real-time polymerase chain reaction (RT-qPCR). The effects on osteogenesis were evaluated by alkaline phosphatase staining, alizarin red S staining, RT-qPCR, and western blots.

HG failed to recapitulate insulin resistance. Glucosamine was sufficient to induce insulin resistance but failed to trigger inflammation. In total, 100 and 200 μM PA exhibited the most proinflammatory, insulin resistance, and pyroptosis induced role, and inhibited the osteogenic differentiation of hPDLSCs.

Palmitic acid is a promising candidate for developing T2DM model in hPDLSCs.

Oral diseases, including periodontal disorders, oral cancer, periodontitis, and mucositis are the major challenges for both patients and healthcare providers. These conditions often involve inflammation, oxidative stress, and impaired cellular processes, leading to symptoms ranging from discomfort to severe debilitation. Conventional treatments for such oral diseases exhibit constraints, prompting the investigation of innovative therapeutic approaches. Considering the anti-inflammatory, anti-oxidant, and anti-cancer effects of melatonin, this study was carried out to investigate the potential protective effects of melatonin in mitigating the severity of oral diseases. Studies indicate that melatonin influences the differentiation of periodontal stem cells, inhibits oral cancer progression, reduces inflammation associated with periodontitis, and alleviates the severity of oral mucositis. Melatonin has demonstrated potential efficacy in both preclinical and clinical investigations; however, findings are frequently heterogeneous and contingent upon contextual factors. This review provides a comprehensiveoverview of current state of knowledge in this domain, elucidating the multifaceted role that melatonin may assume in combatingoral diseases. Further research should be directed toward determining the most effective dosing, timing, and administration methods for melatonin-based therapies for oral diseases.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

We aimed to evaluate the materials based on 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane (Super-bond [SB]) and nano hydroxyapatite (naHAp) for the repair of perforation at pulp chamber floor (PPF) in vitro and in vivo models. SB and naHAp were mixed in the mass ratio of 10% or 30% to produce naHAp/SB. Human periodontal ligament stem cells (HPDLSCs) were cultured on resin discs of SB or naHAp/SB to analyze the effects of naHAp/SB on cell adhesion, proliferation, and cementoblastic differentiation. A rat PPF model was treated with SB or naHAp/SB to examine the effects of naHAp/SB on the healing of defected cementum and periodontal ligament (PDL) at the site of PPF. HPDLSCs were spindle-shaped and adhered to all resin discs. Changing the resin from SB to naHAp/SB did not significantly alter cell proliferation. Both 10% and 30% naHAp/SB were more effective than SB in promoting cementoblastic differentiation of HPDLSCs. In the rat PPF model, 30% naHAp/SB was more effective than SB in promoting the formation Sharpey's fiber-like structures with expression of the PDL-related marker and cementum-like structures with expression of cementum-related markers. In conclusion, 30% naHAp/SB can be the new restorative material for PPF because it exhibited the abilities of adhering to dentin and healing of defected periodontal tissue.

The present systematic review was performed to assess the application of orally derived stem cells in periodontal regenerative therapy, and because of this, the following PICO question was proposed: "In patients with periodontitis, can the adjunctive use of orally derived stem cells provide additional clinical and radiographic benefits for periodontal regeneration?". Randomized clinical studies were electronically and manually searched up until December 2023. Quantitative analyses were performed with the aim of evaluating the mean differences (MDs) between the treatment and control groups in terms of clinical attachment level (CAL) gain, probing pocket depth (PPD) reduction, gingival recession (GR), and radiographic bone gain (RBG) using random effect models. A total of seven studies were selected for the systematic review. Meta-analyses excluding studies with a high risk of bias highlighted a non-statistically significant result for the use of stem cells when compared to the control groups in terms of CAL gain [MD = 1.05; 95% CI (-0.88, 2.97) p = 0.29] and PPD reduction [MD = 1.32; 95% CI (-0.25, 2.88) p = 0.10]. The same also applied to GR [MD = -0.08; 95% CI (-0.79, 0.63) p = 0.83] and RBG [MD = 0.50; 95% CI (-0.88, 1.88) p = 0.48]. Based on the high heterogeneity, there is not enough evidence to consider the adjunctive application of orally derived mesenchymal stem cells as a preferential approach for periodontal regenerative treatment, as compared to standard procedures.

Improper mechanical stress may induce side effects during orthodontic treatment. If the roots and alveolar bones are extensively resorbed following excess mechanical stress, unplanned tooth mobility and inflammation can occur. Although multiple factors are believed to contribute to the development of side effects, the cause is still unknown. Sonic hedgehog (Shh), one of the hedgehog signals significantly associated with cell growth and cancer development, promotes osteoclast formation in the jawbone. Shh may be associated with root and bone resorptions during orthodontic treatment. In this study, we investigated the relationships between Shh, RANKL, and IL-6 in human periodontal ligament (hPDL) cells exposed to improper mechanical force. Weights were placed on hPDL cells and human gingival fibroblasts (HGFs) for an optimal orthodontic force group (1.0 g/cm2) and a heavy orthodontic force group (4.0 g/cm2). A group with no orthodontic force was used as a control group. Real-time PCR, SDS-PAGE, and Western blotting were performed to examine the effects of orthodontic forces on the expression of Shh, RANKL, and IL-6 at 2, 4, 6, 8, 12, and 24 h after the addition of pressure. The protein expression of Shh was not clearly induced by orthodontic forces of 1.0 and 4.0 g/cm2 compared with the control in HGFs and hPDL cells. In contrast, RANKL and IL-6 gene and protein expression was significantly induced by 1.0 and 4.0 g/cm2 in hPDL cells for forces lasting 6~24 h. However, neither protein was expressed in HGFs. RANKL and IL-6 expressions in response to orthodontic forces and in the control were clearly inhibited by Shh inhibitor RU-SKI 43. Shh did not directly link to RANKL and IL-6 for root and bone resorptions by orthodontic force but was associated with cell activities to be finally guided by the production of cytokines in hPDL cells.

Novel functional biomaterials are expected to bring about breakthroughs in developing immunotherapy and regenerative medicine through their application as drug delivery systems and scaffolds. Nanogels are defined as nanoparticles with a particle size of 100 nm or less and as having a gel structure. Nanogels have a three-dimensional network structure of cross-linked polymer chains, which have a high water content, a volume phase transition much faster than that of a macrogel, and a quick response to external stimuli. As it is possible to transmit substances according to the three-dimensional mesh size of the gel, a major feature is that relatively large substances, such as proteins and nucleic acids, can be taken into the gel. Furthermore, by organizing nanogels as a building block, they can be applied as a scaffold material for tissue regeneration. This review provides a brief overview of the current developments in nanogels in general, especially drug delivery, therapeutic applications, and tissue engineering. In particular, polysaccharide-based nanogels are interesting because they have excellent complexation properties and are highly biocompatible.

Piezo1 (2010) was identified as a mechanically activated cation channel capable of sensing various physical forces, such as tension, osmotic pressure, and shear force. Piezo1 mediates mechanosensory transduction in different organs and tissues, including its role in maintaining bone homeostasis. This review aimed to summarize the function and possible mechanism of Piezo1 in the mechanical receptor cells in bone tissue. We found that it is a potential therapeutic target for the treatment of bone diseases.

Periodontal disease, characterized by inflammation and infection of the supporting structures of teeth, presents a significant challenge in dentistry and public health. Current treatment modalities, while effective to some extent, have limitations in achieving comprehensive periodontal tissue regeneration. This comprehensive review explores the potential of stem cell therapy in advancing the field of periodontal regeneration. Stem cells, including mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), hold promise due to their immunomodulatory effects, differentiation potential into periodontal tissues, and paracrine actions. Preclinical studies using various animal models have revealed encouraging outcomes, though standardization and long-term assessment remain challenges. Clinical trials and case studies demonstrate the safety and efficacy of stem cell therapy in real-world applications, especially in personalized regenerative medicine. Patient selection criteria, ethical considerations, and standardized treatment protocols are vital for successful clinical implementation. Stem cell therapy is poised to revolutionize periodontal regeneration, offering more effective, patient-tailored treatments while addressing the systemic health implications of periodontal disease. This transformative approach holds the potential to significantly impact clinical practice and improve the overall well-being of individuals affected by this prevalent oral health concern. Responsible regulatory compliance and a focus on ethical considerations will be essential as stem cell therapy evolves in periodontal regeneration.

An overview of various cell engineering techniques being developed for modern conservative and reconstructive periodontology is presented. The accelerated development of cellular engineering technologies poses to medicine and, in particular, periodontics, the task of early implementation of the results of such experiments into patient management protocols. The main groups of promising techniques that are closest to practical healthcare are: isolation and use of stem cells; synthesis of biologically active (inductive) signaling molecules; development of scaffolds that ensure three-dimensional tissue growth.

Stem cell therapy has been considered to play a paramount role in the treatment modalities available for regenerative dentistry. The established beneficial effects of photobiomodulation (PBM) at the cellular level have led to the combined use of these two factors (PBM and stem cells). The main goal of this study was firstly to critically appraise the effects of PBM on periodontal ligament stem cells (PDLSCs), and secondly to explore the most effective PBM protocols applied.

Pubmed, Cochrane, Scopus, Science Direct, and Google Scholar search engines were used to identify experimental in vitro studies in which PBM was applied to cultured PDLSCs. After applying specific keywords, additional filters, and inclusion/exclusion criteria, a preliminary number of 245 articles were narrowed down to 11 in which lasers and LEDs were used within the 630 - 1064 nm wavelength range. Selected articles were further assessed by three independent reviewers for strict compliance with PRISMA guidelines, and a modified Cochrane risk of bias to determine eligibility.

The dataset analysed was extracted from the studies with sufficient and clearly presented PBM protocols. Simple univariate regression analysis was performed to explore the significance of contributions of potential quantitative predictor variables toward study outcomes, and a one-way ANOVA model was employed for testing differences between the laser or LED sources of the treatments. The significance level for testing was set at α = 0.05.

The proliferation rate, osteogenic differentiation, and expression of different indicative genes for osteogenesis and inflammation suppression were found to be positively affected by the application of various types of lasers and LEDs. With regard to the PBM protocol, only the wavelength variable appeared to affect the treatment outcome; indeed, the 940 nm wavelength parameter was found not to exert a favourable effect.

Photobiomodulation can enhance the stemness and differentiation capacities of periodontal ligament stem cells. Therefore, for PBM protocols, there remains no consensus amongst the scientific community. Statistical analyses performed here indicated that the employment of a near-infrared (NIR) wavelength of 940 nm may not yield a significant favourable outcome, although those within the 630 - 830 nm range did so. Concerning the fluence, it should not exceed 8 J/cm2 when therapy is applied by LED devices, and 4 J/cm2 when applied by lasers, respectively.

This study aimed to investigate the functions of 19 types of Wnt ligands during the process of osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4.

The expression levels of 19 types of Wnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14 days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability.

During hPDLSCs osteogenic differentiation, the expression of 19 types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly.

Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis.

Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

The aim of the present study is to determine the possibility of isolation and characterization of the human periodontal ligament stem cells (hPDLSCs) using limited harvested periodontal ligament (PDL) tissue of only one patient's wisdom teeth (2-4 teeth) under the more compatible terms of use in clinical application without using the fetal bovine serum (FBS).

In this pilot study, hPDLSCs were isolated from the impacted third molar, and tissue was scraped from the roots of the impacted third molar of 10 volunteers to enzymatically digest using collagenase. The cells were sub-cultured. The samples of the first seven patients and half of the eighth patient's sample were cultured in alpha modified of Eagle's medium (α-MEM) (-FBS) medium and the other part of the eighth patient's sample was cultured with prior medium supplemented with +FBS 15% as a control of the cultivation protocol. While for the past two patients (9th and 10th the α-MEM medium was supplemented with L-Glutamine, anti/anti 2X, and 20% knock-out serum replacement (KSR). Two more nutritious supplements (N2 and B27) were added to the medium of the tenth sample. Flow-cytometric analysis for the mesenchymal stem cell surface markers CD105, CD45, CD90, and CD73 was performed. Subsequent polymerase chain reaction was undertaken on three samples cultured with two growth media.

Cultivation failed in some of the samples because of the lack of cell adhesion to the culturing dish bottom (floating cells), but it was successful for the 9th and 10th patients, which were cultured in the α-MEM serum supplemented with KSR 20%. Flow cytometry analysis was positive for CD105, CD90, and CD73 and negative for CD45. The PDL stem cells (PDLSCs) expressed CD105, CD45, and CD90 but were poor for CD73.

According to the limited number of sample tests in this study, isolation and characterization of PDLSCs from collected PDL tissue of one patient's wisdom teeth (2-4) may be possible by the proper setup in synthetic FBS-free serum.

Periodontitis is an inflammatory disease characterized by alveolar bone loss. Periodontal ligament stem cells (PDLSCs) have osteogenic differentiation potential, which can be influenced by epigenetics regulation in periodontitis. Therefore, this review aimed to shed light on the role of different epigenetic mechanisms in the osteogenic differentiation of PDLSCs and to consider the prospects of their possible therapeutic applications in periodontitis. Databases MEDLINE (through PubMed) and Web of Science were searched for the current knowledge of epigenetics in osteogenic differentiation of PDLSCs using the keywords "periodontal ligament stem cells", "epigenetic regulation", "epigenetics", "osteogenic differentiation", and "osteogenesis". All studies introducing epigenetic regulation and PDLSCs were retrieved. This review shows that epigenetic factors like DNMT, KDM6A, HDACi, some miRNAs, and lncRNAs can induce the osteogenic differentiation of PDLSCs in the noninflammatory microenvironment. However, the osteogenic differentiation of PDLSCs is inhibited in the inflammatory microenvironment through the upregulated DNA methylation of osteogenesis-related genes and specific changes in histone modification and noncoding RNA. Epigenetics of osteogenic differentiation of PDLSCs in inflammation exhibits the contrary effect compared with a noninflammatory environment. The application of epigenetic drugs to regulate the abnormal epigenetic status in periodontitis and focus on alveolar bone regeneration is promising.

Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration.

To assess the cytocompatibility and bioactive potential of the new calcium silicate-based cement Ceraputty on human periodontal ligament stem cells (hPDLSCs) compared to Biodentine and Endosequence BC root repair material (ERRM).

hPDLSCs were isolated from extracted third molars from healthy donors. Standardized sample discs and 1:1, 1:2, and 1:4 eluates of the tested materials were prepared. The following assays were performed: surface element distribution via SEM-EDX, cell attachment and morphology via SEM, cell viability via a MTT assay, osteo/cemento/odontogenic marker expression via RT-qPCR, and cell calcified nodule formation via Alizarin Red S staining. hPDLSCs cultured in unconditioned or osteogenic media were used as negative and positive control groups, respectively. Statistical analysis was performed using one-way ANOVA or two-way ANOVA and Tukey's post hoc test. Statistical significance was established at p < 0.05.

The highest Ca2+ peak was detected from Biodentine samples, followed by ERRM and Ceraputty. hPDLSC viability was significantly reduced in Ceraputty samples (p < 0.001), while 1:2 and 1:4 Biodentine and ERRM samples similar results to that of the negative control (p > 0.05). Biodentine and ERRM exhibited an upregulation of at least one cemento/odonto/osteogenic marker compared to the negative and positive control groups. Cells cultured with Biodentine produced a significantly higher calcified nodule formation than ERRM and Ceraputty (p < 0.001), which were also higher than the control groups (p < 0.001).

Ceraputty evidenced a reduced cytocompatibility towards hPDLSCs on its lowest dilutions compared to the other tested cements and the control group. Biodentine and ERRM promoted a significantly higher mineralization and osteo/cementogenic marker expression on hPDLSCs compared with Ceraputty. Further studies are necessary to verify the biological properties of this new material and its adequacy as a retrograde filling material.

This is the first study to elucidate the adequate biological properties of Ceraputty for its use as a retrograde filling material.

The osteogenic differentiation capacity of periodontal mesenchymal stem cells (PDLSCs) can be influenced by different levels of static mechanical strain (SMS) in an inflammatory microenvironment. Long non-coding RNAs (lncRNAs) are involved in various physiological processes. However, the mechanisms by which lncRNAs regulate the osteogenic differentiation of PDLSCs remain unclear.

We investigated the responses of PDLSCs obtained from periodontitis patients and healthy people to 8% and 12%SMS. Gene microarray and bioinformatics analyses were implemented and identified lncRNA00638 as a target gene for the osteogenesis of PDLSCs from periodontitis patients under SMS. Competing endogenous RNA (ceRNA) network analysis was applied and predicted interactions among lncRNA00638, miRNA-424-5p, and fibroblast growth factor receptor 1 (FGFR1). Gene expression levels were regulated by lentiviral vectors. Cell Counting Kit-8 assays, alkaline phosphatase assays, and Alizarin Red S staining were used to examine the osteogenic potential. RT-qPCR and Western blot were performed to detect the expression levels of related genes and proteins.

We found that 8% and 12% SMS exerted distinct effects on HPDLSCs and PPDLSCs, with 12% SMS having the most significant effect. By microarray analysis, we detected differentially expressed lncRNAs/mRNAs between 12% SMS strained and static PPDLSCs, among which lncRNA00638 was detected as a positive target gene to promote the osteogenic differentiation of PPDLSCs under SMS loading. Mechanistically, lncRNA00638 may act as a ceRNA for miR-424-5p to compete with FGFR1. In this process, lncRNA00638 and miR-424-5p suppress each other and form a network to regulate FGFR1.

Our findings demonstrate that the lncRNA00638/miRNA-424-5p/FGFR1 regulatory network is actively involved in the regulation of PDLSC osteogenic differentiation from periodontitis patients under SMS loading, which may provide evidence for optimizing orthodontic treatments in patients with periodontitis.

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Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines' or growth factors' mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes.

Tumor necrosis factor-alpha (TNF-α) is one of the crucial inflammatory factors in alveolar bone metabolism during the process of periodontitis. Autophagy is indispensable for proper osteoblast function. However, the effects of autophagy on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory microenvironment and the underlying mechanisms remain to be clarified. The aim of the present study was to investigate whether autophagy participates in hPDLSCs differentiation after treated with TNF-α and explore the underlying mechanisms.

Characterizations of hPDLSCs were evaluated by Alizarin-red S staining, Oil red staining and flow cytometry. hPDLSCs were treated with various concentrations of TNF-α. Rapamycin or 3MA was used to achieve or inhibit autophagy activation. AKT signaling was inhibited using ARQ092. Cell proliferation was evaluated using Cell Counting Kit-8 (CCK8) assay. Real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR), western blot, alkaline phosphatase (ALP) staining and Alizarin Red S staining were applied to evaluate levels of osteogenic differentiation and autophagy. CCK8 showed that low concentrations of TNF-α had no influence on cell proliferation, while high concentrations of TNF-α inhibited proliferation. Low concentrations of TNF-α promoted osteogenic differentiation and autophagy, while high concentrations of TNF-α inhibited osteogenic differentiation and autophagy in hPDLSCs. The levels of osteogenic differentiation in hPDLSCs were partly effected after co-incubation with 0.1 ng/mL TNF-α with 3MA or Rapamycin. ARQ092 enhanced 0.1 ng/mL TNF-α-induced ALP expression and mineral nodule formation.

Low concentrations of TNF-α promote hPDLSCs osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR signaling.

Periodontal ligament progenitor cells (PDL cells) isolated from patients with inflammatory periodontitis have impaired regenerative capacity, but it is unknown whether this capacity can be recovered upon treatment and stabilization of the periodontal condition. The study aimed to investigate the expression of surface markers and the proliferation and osteogenic potential of PDL cells isolated from patients with treated stable periodontitis (S-PDL cells), periodontally healthy individuals (H-PDL cells), and patients with inflammatory periodontitis (I-PDL cells).

H-PDL, I-PDL, and S-PDL cells were isolated from the extracted teeth of individuals who (1) were periodontally healthy, (2) had inflammatory periodontitis, and (3) had treated stable periodontitis, respectively. The expression levels of surface markers and the proliferative and osteogenic capacities of the PDL cells were assessed.

PDL cells derived from all three sources exhibited mesenchymal stem cell (MSC) characteristics. They were positive for MSC-related markers and negative for a hematopoiesis-related marker. However, S-PDL cells had higher proliferation rates, higher expression levels of osteogenic markers, higher alkaline phosphatase activity, and more calcium nodules than I-PDL cells. But all of these parameters remained lower in S-PDL cells than in H-PDL cells.

S-PDL cells proliferated faster and had greater osteogenic potential than I-PDL cells, although these values remained lower than those in H-PDL cells.

To review the role of the immune cells and their interaction with cells found in gingiva, periodontal ligament, and bone that leads to net bone loss in periodontitis or bone remodeling in orthodontic tooth movement.

Periodontal disease is one of the most common oral diseases causing inflammation in the soft and hard tissues of the periodontium and is initiated by bacteria that induce a host response. Although the innate and adaptive immune response function cooperatively to prevent bacterial dissemination, they also play a major role in gingival inflammation and destruction of the connective tissue, periodontal ligament, and alveolar bone characteristic of periodontitis. The inflammatory response is triggered by bacteria or their products that bind to pattern recognition receptors that induce transcription factor activity to stimulate cytokine and chemokine expression. Epithelial, fibroblast/stromal, and resident leukocytes play a key role in initiating the host response and contribute to periodontal disease. Single-cell RNA-seq (scRNA-seq) experiments have added new insight into the roles of various cell types in the response to bacterial challenge. This response is modified by systemic conditions such as diabetes and smoking. In contrast to periodontitis, orthodontic tooth movement (OTM) is a sterile inflammatory response induced by mechanical force. Orthodontic force application stimulates acute inflammatory responses in the periodontal ligament and alveolar bone stimulated by cytokines and chemokines that produce bone resorption on the compression side. On the tension side, orthodontic forces induce the production of osteogenic factors, stimulating new bone formation. A number of different cell types, cytokines, and signaling/pathways are involved in this complex process. Inflammatory and mechanical force-induced bone remodeling involves bone resorption and bone formation. The interaction of leukocytes with host stromal cells and osteoblastic cells plays a key role in both initiating the inflammatory events as well as inducing a cellular cascade that results in remodeling in orthodontic tooth movement or in tissue destruction in periodontitis.

While periodontal ligament cells are sensitive to their 3D biomechanical environment, only a few 3D in vitro models have been used to investigate the periodontal cells mechanobiological behavior. The objective of the current study was to assess the capability of a 3D fibrous scaffold to transmit a mechanical loading to the periodontal ligament cells. Three-dimensional fibrous polycaprolactone (PCL) scaffolds were synthetized through electrospinning. Scaffolds seeded with human periodontal cells (10³ mL^-1) were subjected to static (n = 9) or to a sinusoidal axial compressive loading in an in-house bioreactor (n = 9). At the end of the culture, the dynamic loading seemed to have an influence on the cells' morphology, with a lower number of visible cells on the scaffolds surface and a lower expression of actin filament. Furthermore, the dynamic loading presented a tendency to decrease the Alkaline Phosphatase activity and the production of Interleukin-6 while these two biomolecular markers were increased after 21 days of static culture. Together, these results showed that load transmission is occurring in the 3D electrospun PCL fibrous scaffolds, suggesting that it can be used to better understand the periodontal ligament cells mechanobiology. The current study shows a relevant way to investigate periodontal mechanobiology using 3D fibrous scaffolds.

This study aimed to analyze the ability of G-CSF and TGF-β1 to mobilize periodontal ligament stem cells to obtain populations with better potential for proliferation and osteogenic differentiation.

Primary cultures were established from the periodontal ligament of Wistar rats. After a cell migration assay, four experimental groups were obtained: PDLSC, composed of the primary culture, non-mobilized cells; MPDLSC, the spontaneously migrated cells; MPDLSC-GCSF, the cells mobilized with G-CSF; and MPDLSC-TGF-β1, the cells mobilized with TGF-β1. The expression of mesenchymal stem cell markers was assessed by flow cytometry. Clonogenicity, viability, proliferative potential, and osteogenic differentiation capacity were also analyzed.

All the study groups expressed well-known mesenchymal stem cell markers and exhibited clonogenic capacity. The higher proliferation potential was seen in the PDLSC and MPDLSC groups, while the MPDLSC and MPDLSC-TGFβ1 groups showed a higher number of mineralized deposits in vitro and higher ALP activity after osteogenic differentiation induction. Cells of all the groups also expressed mRNA of genes associated with osteogenic differentiation without previous induction.

Both agents were able to mobilize stem cells from the periodontal ligament, but G-CSF did not show an advantage, whereas TGF-β1 appears to direct the cells towards a state of increased osteogenic differentiation. Furthermore, spontaneous cell migration through a membrane was sufficient to enrich the cell population.

Serous Acinar Cells (ACs) are mature and functional secretory epithelial cells that develop and complete through other stem cells at the end of the ductal system. So, the regeneration of the salivary gland damaged by radiation does not occur without cell therapy. Todays, an accessible tissue like the Periodontal Ligament (PDL) of the tooth was considered to easily extract the Mesenchymal Stem Cells (MSCs). In-vitro differentiation of stem cells before transplantation to damaged tissue reduces the risk of tumorigenesis. This study was conducted to evaluate the feasibility of differentiation of PDLSCs into salivary acinar cells by a co-culture system. PDLSCs were isolated from adult human PDL tissue and co-cultured with rat parotid ACs using an indirect co-culture system. The transdifferentiation of PDLSCs was evaluated by PCR of Aquaporin 5 (AQP5) and Carbonic anhydrase 6 (CA6) genes, then quantitative real-time PCR was used to measure the gene expression levels. The data were analyzed by ANOVA. Specific bond with the correct size on 6% acrylamide gel and TBE5X buffer showed the expression of AQP5 and CA6 in PDLSCs co-cultured with acinar cells. RT-PCR revealed co-cultured PDLSCs with or without KGF (Keratinocyte Growth Factor) showed significantly increased expression of AQP5 genes in compared to the initial PDLSCs. Expression of AQP5 and CA6, indicating successful transdifferentiation of PDLSCs into ACs, in co-culture system for 3 weeks.

Mandibular defect becomes a prevalent maxillofacial disease resulting in mandibular dysfunctions and huge psychological burdens to the patients. Considering the routine presence of oral contaminations and aesthetic restoration of facial structures, the current clinical treatments are however limited, incapable to reconstruct the structural integrity and regeneration, spurring the need for cost-effective mandibular tissue engineering. Hydrogel systems possess great merit for mandibular reconstruction with precise involvement of cells and bioactive factors. In this review, current clinical treatments and distinct mode(s) of mandible formation and pathological resorption are summarized, followed by a review of hydrogel-related mandibular tissue engineering, and an update on the advanced fabrication of hydrogels with improved mechanical property, antibacterial ability, injectable form, and 3D bioprinted hydrogel constructs. The exploration of advanced hydrogel systems will lay down a solid foundation for a bright future with more biocompatible, effective, and personalized treatment in mandibular reconstruction.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that can be recognized by infected host cells and activate the immunoinflammatory response. The purpose of this study is to demonstrate the effect of c-di-AMP on the differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mechanisms. In the present study, we find that the gingival crevicular fluid (GCF) of patients with chronic periodontitis has a higher expression level of c-di-AMP than that of healthy people. In vitro, c-di-AMP influences the differentiation of hPDLSCs by upregulating Toll-like receptors (TLRs); specifically, it inhibits osteogenic differentiation by activating NF-κB and ERK/MAPK and promotes adipogenic differentiation through the NF-κB and p38/MAPK signaling pathways. Inhibitors of TLRs or activated pathways reduce the changes induced by c-di-AMP. Our results establish the potential correlation among bacterial c-di-AMP, periodontal tissue homeostasis and chronic periodontitis pathogenesis.

Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria.

The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1 μM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot.

Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1 μM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD⁺ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20.

We found that melatonin promoted osteogenesis of hPDLCs and 1 μM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.

A relevant alternative to enamel matrix derivatives from animal origin could be the use of synthetic amelogenin-derived peptides. This study aimed to assess the effect of a synthetic amelogenin-derived peptide (ADP-5), alone or included in an experimental gellan-xanthan hydrogel, on periodontal cell behavior (gingival fibroblasts, periodontal ligament cells, osteoblasts and cementoblasts). The effect of ADP-5 (50, 100, and 200 µg/mL) on cell metabolic activity was examined using Alamar blue assay, and cell morphology was assessed by confocal imaging. An experimental gellan-xanthan hydrogel was then designed as carrier for ADP-5 and compared to the commercial gel Emdogain^®. Alizarin Red was used to determine the periodontal ligament and cementoblasts cell mineralization. The inflammatory profile of these two cells was also quantified using ELISA (vascular endothelial growth factor A, tumor necrosis factor α, and interleukin 11) mediators. ADP-5 enhanced cell proliferation and remineralization; the 100 µg/mL concentration was more efficient than 50 and 200 µg/mL. The ADP-5 experimental hydrogel exhibited equivalent good biological behavior compared to Emdogain^® in terms of cell colonization, mineralization, and inflammatory profile. These findings revealed relevant insights regarding the ADP-5 biological behavior. From a clinical perspective, these outcomes could instigate the development of novel functionalized scaffold for periodontal regeneration.

Although multiple regenerative strategies are being developed for periodontal reconstruction, guided periodontal ligament (PDL) regeneration is difficult because of its cellular and fibrous complexities. Here, we manufactured four different types of PDL-mimic fibrous scaffolds on a desired single mat. These scaffolds exhibited a structure of PDL matrix and human PDL fibroblasts (PDLFs) cultured on the scaffolds resembling morphological phenotypes present in native PDLF. The scaffold-seeded PDLF exerted proliferative, osteoblastic, and osteoclastogenic potentials depending on the fiber topographical cues. Fiber surface-regulated behaviors of PDLF were correlated with the expression patterns of yes-associated protein (YAP), CD105, periostin, osteopontin, and vinculin. Transfection with si-RNA confirmed that YAP acted as the master mechanosensing regulator. Of the as-spun scaffolds, aligned or grid-patterned microscale scaffold regulated the YAP-associated behavior of PDLF more effectively than nanomicroscale or random-oriented microscale scaffold. Implantation with hydrogel complex conjugated with microscale-patterned or grid-patterned scaffold, but not other types of scaffolds, recovered the defected PDL with native PDL-mimic cellularization and fiber structure in the reformed PDL. Our results demonstrate that PDL-biomimetic scaffolds regulate topography-related and YAP-mediated behaviors of PDLF in relation to their topographies. Overall, this study may support a clinical approach of the fiber-hydrogel complex in guided PDL regenerative engineering.

The ideal outcome of wound healing is the complete restoration of the structure and function of the original tissue. Stem cells are one of the key factors in this process. Currently, the strategy of periodontal regeneration based on mesenchymal stem cells (MSCs) is generally used to expand stem cells in vitro and then transplant them in vivo. However, their clinical application is limited. In fact, the human body has the capacity to regenerate through stem cells residing in different tissues, even without external therapeutic intervention. Stem cell niches are present in many adult tissues, such as the periodontal ligament and gingiva, and stem cells might remain in a quiescent state in their niches until they are activated in response to a regenerative need. Activated stem cells can exit the niche and proliferate, self-renew, and differentiate to regenerate original structures. Thus, harnessing the regenerative potential of endogenous stem cells in situ has gained increasing attention as a simpler, safer, and more applicable alternative to stem cell transplantation. Nevertheless, there are several key problems to be solved in the application of periodontal mesenchymal stem cells. Thus, animal studies will be especially important to deepen our knowledge of the in vivo mechanisms of mesenchymal stem cells. Studies with conditional knockout mice, in which the expression of different proteins can be eliminated in a tissue-specific manner, are especially important. Post-natal cells expressing the paired-related homeobox protein 1 (PRX1 or PRRX1), a transcription factor expressed in the mesenchyme during craniofacial and limb development, have been shown to have characteristics of skeletal stem cells. Additionally, following wounding, dermal Prx1+ cells are found out of their dermal niches and contribute to subcutaneous tissue repair. Postnatal Prx1+ cells are uniquely injury-responsive. Meanwhile, current evidence shows that Prx1+ cells contribute to promote dentin formation, wound healing of alveolar bone and formation of mouse molar and periodontal ligament. Initial result of our research group also indicates Prx1-expressing cells in bone tissue around the punch wound area of gingiva increased gradually. Collectively, this review supports the future use of PRX1 cells to stimulate their potential to play an important role in endogenous regeneration during periodontal therapy.