Periodontal Ligament Tissue Research Articles

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

ILC1s and ILC3s Exhibit Inflammatory Phenotype in Periodontal Ligament of Periodontitis Patients.

Innate lymphoid cells (ILCs) are emerging as important players in inflammatory diseases. The oral mucosal barrier harbors all ILC subsets, but how these cells regulate the immune responses in periodontal ligament tissue during periodontitis remains undefined. Here, we show that total ILCs are markedly increased in periodontal ligament of periodontitis patients compared with healthy controls. Among them, ILC1s and ILC3s, particularly NKp44+ILC3 subset, are the predominant subsets accumulated in the periodontal ligament. Remarkably, ILC1s and ILC3s from periodontitis patients produce more IL-17A and IFN-γ than that from healthy controls. Collectively, our results highlight the role of ILCs in regulating oral immunity and periodontal ligament inflammation and provide insights into targeting ILCs for the treatment of periodontitis.

Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

Role and mechanism of low-dose lipopolysaccharide-treated human periodontal ligament stem cells on the expression of macrophage pro-inflammatory factors

Objective: To investigate the effect of low dose lipopolysaccharide (LPS)-treated human periodontal ligament stem cells (HPDLSC) on the expression of macrophage pro-inflammatory factors and the mechanism involved. Methods: The primary HPDLSCs were obtained from healthy third molar periodontal ligament tissue. Phosphate buffer saline (PBS), 100 μg/L or 10 mg/L of LPS were used to treat HPDLSCs for 48 h, and their conditioned media were respectively co-cultured with THP-1-derived macrophages for 48 h. The corresponding experimental groups were PBS-treated HPDLSC-derived conditioned medium (CM-C) group, low dose LPS-treated HPDLSC-derived conditioned medium (CM-L) group, and high dose LPS-treated HPDLSC-derived conditioned medium (CM-H) group. Quantitative real-time PCR was performed to explore the mRNA expressions of macrophage interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor-α (TNF-α) in the CM-C, CM-L and CM-H groups, and the expressions of nuclear factor (erythroid-derived 2)-like 2 (NRF2), glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1) in the CM-C and CM-L groups. Meanwhile, Western blotting was used to detect the change of nuclear and cytoplasmic NRF2 and the levels of GCLC and HO-1 in the CM-C and CM-L groups. The 2', 7'-dichlorofluorescein probe was adopted to detect the reactive oxygen species (ROS) levels of macrophages in the CM-C and CM-L groups and the data were characterized by the mean fluorescent intensity (MFI). Results: The mRNA expressions of macrophage pro-inflammatory factors IL-6, IL-8, IL-12 and TNF-α in the CM-H group (2.332±0.594, 3.601±0.639, 2.120±0.677 and 2.468±0.236) were significantly upregulated compared with those in the CM-C group (1.000±0.321, 1.000±0.151, 1.000±0.059 and 1.000±0.095) (P<0.05); while the relative mRNA levels of IL-6, IL-12 and TNF-α in the CM-L group (0.056±0.002, 0.215±0.024 and 0.567±0.071) were much lower than those in the CM-C group (1.000±0.209, 1.000±0.220 and 1.000±0.220) (P<0.05). At the mRNA level, the expression of NRF2 was significantly increased in the CM-L group (1.864±0.198) compared with that in the CM-C group (1.000±0.094) (P<0.05). At the protein level, the cytoplasmic NRF2 and nuclear NRF2 were increased in CM-L group (1.175±0.104 and 1.308±0.082) compared with those in the CM-C group (1.000±0.025 and 1.000±0.049) (P<0.05). Furthermore, the antioxidative genes, i.e. GCLC and NQO1, localized in NRF2 downstream, were significantly upregulated in the CM-L group (1.786±0.278 and 1.444±0.078) compared with the CM-C group (1.000±0.139 and 1.000±0.226) (P<0.05). The protein levels of GCLC and HO-1 were augmented in the CM-L group (1.159±0.036 and 1.412±0.075) in contrast with those in the CM-C group (1.000±0.050 and 1.000±0.013) (P<0.05). In addition, the MFI in the CM-L group (123 419±1 302) was significantly lower than that in the CM-C group (139 193±1 241) (P<0.05). Conclusions: Low-dose LPS-treated HPDLSCs could regulate oxidative stress response through activating the NRF2 signaling pathway of macrophages and further downregulating the expressions of macrophage pro-inflammatory factors.

Estimation of the maximum permissible intrusive force for intrusion of a canine tooth: one-dimensional finite element study

The aim of the present research study is to estimate the maximum permissible intrusive force mathematically with one-dimensional finite element method and to achieve the safe intrusion of a canine tooth without any damage to periodontal ligament (PDL). For this purpose, one-dimensional finite element (FE) model of a canine tooth was developed consisting of tooth (crown and root) and PDL structure. The maximum permissible intrusive force was computed mathematically. For this purpose, the maximum permissible stress was considered as ‘EPDL/1.5’, where ‘EPDL’ is the Young’s Modulus of PDL and factor of safety (FOS) is 1.5. The maximum permissible intrusive force was computed to be approximately 10 g-force (gf). For verification of this computed force value, finite element analysis was carried out on 3-dimensional canine tooth, PDL and bone structures for better verification and it is proved that 10 g-force (gf) value is safe without any damage to PDL tissues.

Matrix metalloproteinases and Th17 cytokines in the gingival crevicular fluid during orthodontic tooth movement.

Matrix metalloproteinases (MMPs) contribute to remodeling in orthodontic tooth movement (OTM). Moreover, IL-17 can promote the production of MMPs. This study aimed to investigate the regulation of Th17 on MMPs expression during OTM. Eighteen children undergoing orthodontic treatment were recruited. The gingival crevicular fluid (GCF) was collected at different time points: the day of application (T0), one hour (T1), 24 hours (T2), one week (T3), 4 weeks (T4) and 12 weeks (T5) after the application of orthodontic force. Th17 cell-related cytokines and MMPs expression were measured in GCF by Multiplex Luminex analyser. Human periodontal ligament (hPDL) tissues were stimulated by IL-17. The levels of IL-17 and MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13) of the study teeth at T2-T4 were significantly up-regulated compared with that of T0 and T1 and decreased to baseline level at T5. We found that the expression of IL-17 was correlated with MMPs. After rhIL-17 treatment, the expression of MMP-1, MMP-2, and MMP-9 were up-regulated significantly. The IL-17 expression was positively correlated with MMPs. IL-17 promotes the expression of MMP-1, MMP-2, and MMP-9 by hPDL cells, suggesting that IL-17 plays a crucial role in the remodeling during OTM.

Store in nature - A medium for avulsed tooth

Avulsion of tooth is complete displacement of tooth out of its socket that leads to mutilation of periodontal ligaments. The desirable treatment option is replantation of the tooth. However, unsuccessful replantation is a matter of great discontentment. Protection of teeth from desiccation (drying of the periodontal ligament tissue), by keeping it in storage media can improve the result of the treatment. Ability to maintain periodontal cell viability for a longer time is warranted in cases of major accidents, where teeth can be replanted only after other major surgeries. This paper reviews the different storage media that have been used for avulsed teeth based on full-length research papers retrieved from GOOGLE search using the key words ‘storage medium’, ‘avulsion’, ‘tooth avulsion’, ‘replantation’, ‘tooth replantation’, ‘milk’ , ‘propolis’, ‘coconut water’, ‘pomegranate’ and natural products. Natural products have easy availability, greater efficacy and longer storage time as compared to Hank’s balanced salt solution which has been recommended by the International Association of Dental Traumatology as standard solution for storage of avulsed teeth. In case of emergency, it is important for dentists to consider the circumstances of the accident, the location and suggest an appropriate Natural transport/storage medium for the avulsed tooth which score better over the Hank’s Balance Salt Solution, based on their storage time, ease of availability and economical price. Keywords: Avulsion, Storage media, Natural products, Propolis, Coconut water, Pomegranate.

Wnt5a up-regulates Periostin through CaMKII pathway to influence periodontal tissue destruction in early periodontitis.

Periostin is essential for periodontal tissue integrity and homeostasis and also associated with periodontitis and periodontitis healing. This study aims to investigate the temporal and spatial expression of Periostin and Wnt5a/CaMKII in periodontitis and how the Wnt5a regulates Periostin through CaMKII signaling pathway in PDLCs in inflammatory environment. The experimental periodontitis mice were adopted to clarify the temporal and spatial expression of Wnt5a, CaMKII and Periostin during early periodontitis. And the Wnt5a, CaMKII and Periostin expression pattern and regulation mechanism in PDLCs were clarified in Porphyromonas gingivalis Lipopolysaccharide (P.g. LPS) induced inflammatory condition. Along with the periodontitis development, Wnt5a, CaMKII and Periostin significantly increased in periodontal ligament and partially increased in gingiva during 0 to 6day (P < 0.05). They were involved in early periodontitis homeostasis especially in periodontal ligament tissue. Meanwhile, Wnt5a, CaMKII and Periostin were significantly decreased at 12h (P < 0.05) and increased at 48h (P < 0.05) in PDLCs after induced by P.g. LPS. Besides, Wnt5a significantly enhanced total CaMKII protein (P < 0.05), pCaMKII (P < 0.001) and Periostin (P < 0.001), and this could be blocked by CaMKII inhibitor KN93 (P < 0.05). In conclusions, in early periodontitis, Wnt5a/CaMKII and Periostin should be involved in maintaining periodontal homeostasis and Wnt5a could up-regulate Periostin via CaMKII pathway in inflammation, which would provide new clues for us to understand the pathogenesis of periodontitis and develop better therapeutic strategies.

The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.

Carbon Monoxide Releasing Molecule-3 Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Carbon Monoxide Release.

PurposeLimited intrinsic regeneration capacity following bone destruction remains a significant medical problem. Multiple regulatory effects of carbon monoxide releasing molecule-3 (CORM-3) have been reported. The aim of this study was to investigate the effect of CORM-3 on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) during osteogenesis.Patients and MethodshPDLSCs obtained from healthy periodontal ligament tissues were cultured and identified with specific surface antigens by flow cytometry. Effect of CORM-3 on the proliferation of hPDLSCs was determined by CCK-8 assay. Alizarin red staining and alkaline phosphatase (ALP) activity were used to assess the osteogenic differentiation of hPDLSCs. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect the expression of the indicated genes. Critical-sized skull defect was made in Balb/c-nude mice, microcomputed tomography (Micro-CT) and Masson trichrome staining were used to assess the new bone regeneration in mice.ResultsCORM-3 (400 μmol/l) significantly promoted the proliferation of hPDLSCs. CORM-3 pretreatment not only notably enhanced the mRNA and protein expression of osteo-specific marker OPN, Runx2 and ALP, but also increased mineral deposition and ALP activity by the release of CO on day 3, 7 and 14 (P<0.05). Degassed CORM-3 did not show the same effect as CORM-3. In animal model, application of CORM-3 with hPDLSCs transplantation highly increased new bone formation in skull defect region.ConclusionCORM-3 promoted osteogenic differentiation of hPDLSCs, and increased hPDLSCs-induced new bone formation in mice with critical-sized skull defect, which suggests an efficient and promising strategy in the treatment of disease with bone defect.

Cell-Based Therapy for Tooth Replantation Following Avulsion: A Systematic Review.

The management of avulsed teeth undergoing delayed replantation remains a clinical challenge as there are currently no effective interventions that can improve periodontal healing and prevent replacement root resorption. While several preclinical studies have reported varied success using cell-based tissue engineering to improve periodontal healing, a consensus is required before further clinical translation. Therefore, this systematic review seeks to evaluate the efficacy of cell-based therapy in promoting periodontal healing following delayed replantation in animal models. MEDLINE (PubMed) and Embase were searched on September 27, 2020. Ten studies involving rodent and dog models met the inclusion criteria. Cell sources included gingiva, periodontal ligament (PDL), bone marrow, and adipose tissues. Generally, cell-based therapy had increased the proportion of root surfaces displaying periodontal healing and concomitantly reduced the proportion presenting with replacement root resorption and ankylosis. The best outcomes were observed following treatment with PDL-derived cells of various potency. Future preclinical studies will benefit from adopting measures to minimize bias during the conduct of animal experiments and the standardization of the outcome measures reporting. This will facilitate future reviews with possible pooling of results in the form of meta-analyses, allowing a consensus to be obtained from the literature. In addition, further research will be required to shed light on the implications of using allogeneic cells as well as the optimization of cell delivery protocols. The findings of this systematic review demonstrated the therapeutic potential of certain cell-based therapies in promoting periodontal healing following delayed replantation, thus highlighting their prospective clinical benefits and translational value. Impact statement Current therapies cannot predictably promote periodontal healing following delayed replantation of an avulsed tooth, especially when there is already significant root surface damage. This review systematically assessed the literature for preclinical studies employing cell-based therapies to promote periodontal healing following delayed replantation. The results showed that certain cell-based therapies significantly increased the formation of new periodontal ligament and reduced adverse healing outcomes of replacement root resorption and ankylosis. This highlights the potential clinical benefits and translational value of cell-based therapy for the replantation of avulsed teeth.

Irradiation with red light-emitting diode enhances proliferation and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to evaluate the effects of low-energy red light-emitting diode (LED) irradiation on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). PDLSCs were derived from human periodontal ligament tissues of premolars and were irradiated with 0 (control group), 1, 3, or 5 J/cm2 red LED in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) results. Osteoblast-associated proteins (Runx2, OCN, OPN, and BSP) were detected using western blotting. The results of the MTT assay indicated that PDLSCs in the irradiation groups exhibited a higher proliferation rate than those in the control group (P < 0.05). ALP results showed that after 7 days of illumination, only 5 J/cm2 promoted the expression of ALP of PDLSCs. However, after 14 days of illumination, the irradiation treatments did not increase ALP activity. The results of alizarin red staining showed that red LED promoted osteogenic differentiation of the PDLSCs. The real-time polymerase chain reaction (RT-PCR) results demonstrated that red LED upregulated the expression levels of osteogenic genes. Expression of the proteins BSP, OPN, OCN, and Runx2 in the irradiation groups was higher than that in the control group. Our results confirmed that low-energy red LED at 1, 3, and 5 J/cm2 promotes proliferation and osteogenic differentiation of PDLSCs.

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

BackgroundLong non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).MethodsHealthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-β-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).ResultsHPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3′-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.ConclusionThis study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

Role and application of stem cells in dental regeneration: A comprehensive overview

Recently, a growing attention has been observed toward potential advantages of stem cell (SC)-based therapies in regenerative treatments. Mesenchymal stem/stromal cells (MSCs) are now considered excellent candidates for tissue replacement therapies and tissue engineering. Autologous MSCs importantly contribute to the state-of-the-art clinical strategies for SC-based alveolar bone regeneration. The donor cells and immune cells play a prominent role in determining the clinical success of MSCs therapy. In line with the promising future that stem cell therapy has shown for tissue engineering applications, dental stem cells have also attracted the attention of the relevant researchers in recent years. The current literature review aims to survey the variety and extension of SC-application in tissue-regenerative dentistry. In this regard, the relevant English written literature was searched using keywords: “tissue engineering”, “stem cells”, “dental stem cells”, and “dentistry strategies”. According to the available database, SCs application has become increasingly widespread because of its accessibility, plasticity, and high proliferative ability. Among the growing recognized niches and tissues containing higher SCs, dental tissues are evidenced to be rich sources of MSCs. According to the literature, dental SCs are mostly present in the dental pulp, periodontal ligament, and dental follicle tissues. In this regard, the present review has described the recent findings on the potential of dental stem cells to be used in tissue regeneration.

High Hydrostatic Pressure Induces Nucleus Translocation of FOXO3

Occlusion force pressurizes the periodontal ligament tissue. However, the effect of pressure on cells at a molecular level is poorly understood due to the lack of methods that observe cell motility under high pressure conditions. Here we used high hydrostatic pressure system to apply mechanical stress to cells in vitro and investigated cell morphology, molecules, and gene expression levels under mechanical stress conditions. We observe forkhead box O 3 (FOXO3) proteins, one of the transcription factors, translocate to nucleus from cytosol in a short time (∼ 5min) under the high pressure condition.

Curcumin reduces apoptosis and promotes osteogenesis of human periodontal ligament stem cells under oxidative stress in vitro and in vivo

AimsHuman periodontal ligament stem cells (hPDLSCs) tether the teeth to the surrounding bone and are considered as major functional stem cells responsible for regeneration of the alveolar bone and periodontal ligament tissue. However, the outcome of stem cell regenerative therapy is affected by the survival rate and their differentiation potential of transplanted cells. This is primarily because of local oxidative stress and chronic inflammation at the transplantation site. Therefore, our study aimed to explore whether a natural antioxidant, curcumin could increase the tissue regeneration ability of transplanted hPDLSCs. Main methodsA hydrogen peroxide environment and a rat cranial bone defect model were built to mimic the oxidative stress conditions in vitro and in vivo, respectively. We evaluated the effect of curcumin on oxidative status, apoptosis, mitochondrial function and osteogenic differentiation of H2O2-stimulated hPDLSCs in vitro. We also measured the effect of curcumin on cell viability and bone repair ability of transplanted hPDLSCs in vivo. Key findingsOur data showed that curcumin enhanced cell proliferation, reduced the reactive oxygen species (ROS) levels and apoptosis, maintained the standard mitochondrial structure and function, and promoted osteogenic differentiation of H2O2-stimulated hPDLSCs. The extracellular regulated protein kinases 1/2 (Erk1/2) signaling pathway was determined to be involved in the osteogenic differentiation of the H2O2-stimulated hPDLSCs. Moreover, curcumin enhanced the viability and the bone repair ability of hPDLSCs in vivo. SignificanceCurcumin reduced apoptosis and promoted osteogenesis of the hPDLSCs under oxidative stress, and might therefore have a potential clinical use with respect to tissue regeneration.

MicroRNA-125a-5p modulates macrophage polarization by targeting E26 transformation-specific variant 6 gene during orthodontic tooth movement

ObjectiveThe aim of this study was to investigate the role of microRNA-125a-5p (miR-125a-5p) in macrophages during orthodontic tooth movement (OTM). DesignPeriodontal ligament tissues were collected from patients underwent OTM. Periodontal ligament cells were isolated from periodontal ligament tissues. Periodontal ligament stem cells were isolated from normal human impacted third molars. The miR-125-5p levels were measured by real-time quantitative polymerase chain reaction. The impact of miR-125-5p on macrophage polarization was tested by alizarin red staining assay. The effects of miR-125-5p and E26 transformation-specific variant 6 gene (ETV6) on M1/M2 macrophages phenotype markers were determined by real-time quantitative polymerase chain reaction, western blot, and flow cytometry analyses. The interaction between miR-125-5p and ETV6 was verified using luciferase reporter and RNA immunoprecipitation assays. ResultsPeriodontal miR-125a-5p was upregulated under the force. Macrophage polarization facilitated osteogenesis by cocultured system. Moreover, miR-125a-5p was upregulated in macrophages polarized with M2 conditions. MiR-125a-5p downregulation promoted the expression of M1 phenotype markers, while suppressed the expression of M2 markers. Mechanistically, ETV6 was confirmed to be a target of miR-125a-5p. ETV6 overexpression increased the expression of M1 polarized markers, while decreased the expression of M2 polarized markers. Furthermore, ETV6 knockdown reversed the effects of miR-125a-5p inhibitor on both M1 macrophages and M2 macrophages. ConclusionsOverall, miR-125a-5p facilitates bone healing by targeting ETV6 to promote macrophage M2 polarization.

Angiogenic Effects of Secreted Factors from Periodontal Ligament Stem Cells.

Periodontal disease is a chronic inflammation of tooth-supporting tissues, and the destruction of these tissues results in tooth loss. Regeneration of periodontal tissues is the ultimate goal of periodontal treatment. We previously reported that transplantation of conditioned medium (CM) of periodontal ligament stem cells (PDLSCs) demonstrated the enhancement of periodontal tissue regeneration, compared to CM from fibroblasts (Fibroblast-CM). We hypothesized that the angiogenic effects of PDLSC-CM might participate in the enhanced wound healing of periodontal tissues. The aim of this study was to investigate the effect of PDLSC-CM on the functions of endothelial cells. PDLSCs were cultured from periodontal ligament tissues obtained from healthy volunteers. Human gingival epithelial cells, dermal fibroblasts, osteoblasts, and umbilical vein endothelial cells (HUVECs) were purchased from commercial sources. The functions of endothelial cells were examined using immunostaining of Ki67, observation of nuclear fragmentation and condensation (apoptosis), and network formation on Matrigel. Vascular endothelial cell growth factor (VEGF) level was measured using an ELISA kit. HUVECs demonstrated higher cell viability in PDLSC-CM when compared with those in Fibroblast-CM. HUVECs demonstrated a higher number of Ki67-positive cells and lower apoptosis cells in PDLSC-CM, compared to Fibroblast-CM. Additionally, HUVECs formed more capillary-like structures in PDLSC-CM than Fibroblast-CM. PDLSC-CM contained higher levels of angiogenic growth factor, VEGF, than Fibroblast-CM. Our results showed that PDLSC-CM increased cell viability, proliferation, and capillary formation of HUVECs compared to Fibroblast-CM, suggesting the angiogenic effects of PDLSC-CM, and the effect is a potential regenerative mechanism of periodontal tissues by PDLSC-CM.

Autophagy facilitates type I collagen synthesis in periodontal ligament cells

Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

The Role of Noncoding RNAs in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

Chronic inflammatory diseases, including periodontitis, are the most common causes of bone tissue destruction. Periodontitis often leads to loss of connective tissue homeostasis and reduced alveolar bone levels. Human periodontal ligament stem cells (PDLSCs), a population of multipotent stem cells derived from periodontal ligament tissues, are considered as candidate cells for the regeneration of alveolar bone and periodontal tissues. Periodontitis impairs the osteogenic differentiation of human PDLSCs. Noncoding RNAs (ncRNAs), including long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA (circRNA), have been proposed as vital regulators influencing several differentiation processes including bone regeneration. Still, the molecular mechanisms of ncRNAs regulating osteogenic differentiation of human PDLSCs remain poorly understood. Exploring the influence of ncRNAs in the process of osteogenic differentiation of human PDLSCs may provide novel therapeutic strategies for tissue regeneration as the regeneration of the lost periodontium is the ultimate goal of periodontal therapy.