Differentiation Of Periodontal Ligament Cells Research Articles

The Expression and Regulatory Roles of Long Non-Coding RNAs in Periodontal Ligament Cells: A Systematic Review.

Periodontal ligament (PDL) cells play a pivotal role in periodontal and bone homeostasis and have promising potential for regenerative medicine and tissue engineering. There is compelling evidence that long non-coding RNAs (lncRNAs) are differentially expressed in PDL cells compared to other cell types and that these lncRNAs are involved in a variety of biological processes. This study systematically reviews the current evidence regarding the expression and regulatory functions of lncRNAs in PDL cells during various biological processes. A systematic search was conducted on PubMed, the Web of Science, Embase, and Google Scholar to include articles published up to 1 July 2021. Original research articles that investigated the expression or regulation of lncRNAs in PDL cells were selected and evaluated for a systematic review. Fifty studies were ultimately included, based on our eligibility criteria. Thirteen of these studies broadly explored the expression profiles of lncRNAs in PDL cells using microarray or RNA sequencing. Nineteen studies investigated the mechanisms by which lncRNAs regulate osteogenic differentiation in PDL cells. The remaining 18 studies investigated the mechanism by which lncRNAs regulate the responses of PDL cells to various stimuli, namely, lipopolysaccharide-induced inflammation, tumor necrosis factor alpha-induced inflammation, mechanical stress, oxidative stress, or hypoxia. We systematically reviewed studies on the expression and regulatory roles of lncRNAs in diverse biological processes in PDL cells, including osteogenic differentiation and cellular responses to inflammation, mechanical stress, and other stimuli. These results provide new insights that may guide the development of lncRNA-based therapeutics for periodontal and bone regeneration.

Energy Metabolism in Osteogenic Differentiation and Reprogramming: A Possible Future Strategy for Periodontal Regeneration

Energy metabolism is crucial in stem cells as they harbor various metabolic pathways depending on their developmental stages. Moreover, understanding the control of their self-renewal or differentiation via manipulation of their metabolic state may yield novel regenerative therapies. Periodontal ligament (PDL) cells existing between the tooth and alveolar bone are crucial for maintaining homeostasis in the periodontal tissue. In addition, they play a pivotal role in periodontal regeneration, as they possess the properties of mesenchymal stem cells and are capable of differentiating into osteogenic cells. Despite these abilities, the treatment outcome of periodontal regenerative therapy remains unpredictable because the biological aspects of PDL cells and the mechanisms of their differentiation remain unclear. Recent studies have revealed that metabolism and factors affecting metabolic pathways are involved in the differentiation of PDL cells. Furthermore, understanding the metabolic profile of PDL cells could be crucial in manipulating the differentiation of PDL cells. In this review, first, we discuss the energy metabolism in osteoblasts and stem cells to understand the metabolism of PDL cells. Next, we summarize the metabolic preferences of PDL cells during their maintenance and cytodifferentiation. The perspectives discussed have potential applicability for creating a platform for reliable regenerative therapies for periodontal tissue.

LncPVT1 regulates osteogenic differentiation of human periodontal ligament cells via miR-10a-5p/brain-derived neurotrophic factor.

Identifying the factors affecting osteoblast differentiation of periodontalligament cells(PDLCs) can help enhance the regeneration of periodontal tissue. LncRNA plasmacytoma variant translocation 1 (lncPVT1) is an important regulatory factor involved in many biological processes, but its role in osteogenesis remains unclear. Expressions of osteogenic markers were detected by quantitative reverse transcription polymerase chain reaction and Western blot analysis. Alkaline phosphatase staining was conducted for early osteoblast differentiation and alizarin red S staining was used for mineral deposition. RNA sequencing was used to identify the miRNAs regulated by lncPVT1 during osteogenesis. Cell transfection was used to overexpress or knockdown lncPVT1 and miR-10a-5p. Dual luciferase reporter assays were conducted to analyze the binding of miR-10a-5p to brain-derived neurotrophic factor (BDNF). LncPVT1 was significantly increased during osteogenic induction of PDLCs. Overexpression of lncPVT1 promoted osteogenesis, whereas lncPVT1 knockdown inhibited this process. RNA sequencing showed that miR-10a-5p expression was significantly increased after lncPVT1 knockdown. RNA immunoprecipitation assay further demonstrated the binding potential of lncPVT1 and miR-10a-5p. MiR-10a-5p inhibited the osteogenesis of PDLCs, and partially reversed the stimulatory effects of lncPVT1. Subsequently, we identified a predicted binding site for miR-10a-5p on BDNF and confirmed it using dual luciferase reporter assays. Moreover, lncPVT1 upregulated the expression of BDNF, whereas miR-10a-5p downregulated BDNF expression. BDNF promoted osteogenesis and partially rescued the si-lncPVT1-mediated inhibition of PDLCs osteogenic differentiation. LncPVT1 positively regulated the osteogenic differentiation of PDLCs via miR-10a-5p and BDNF. Our results provide a promising target for enhancing the osteogenic potential of PDLCs.

Enhancement of Osteoblast Differentiation Using No-Ozone Cold Plasma on Human Periodontal Ligament Cells.

Periodontitis is an inflammatory disease that leads to periodontal tissue destruction and bone resorption. Proliferation and differentiation of cells capable of differentiating into osteoblasts is important for reconstructing periodontal tissues destroyed by periodontitis. In this study, the effects of the nozone (no-ozone) cold plasma (NCP) treatment on osteoblastic differentiation in periodontal ligament (PDL) cells were investigated. To test the toxicity of NCP on PDL cells, various NCP treatment methods and durations were tested, and time-dependent cell proliferation was analyzed using a water-soluble tetrazolium salts-1 assay. To determine the effect of NCP on PDL cell differentiation, the cells were provided with osteogenic media immediately after an NCP treatment to induce differentiation; the cells were then analyzed using alkaline phosphatase (ALP) staining, an ALP activity assay, real time PCR, and Alizarin Red S staining. The NCP treatment without toxicity on PDL cells was the condition of 1-min NCP treatment immediately followed by the replacement with fresh media. NCP increased ALP, osteocalcin, osteonectin, and osteopontin expression, as well as mineralization nodule formation. NCP treatment promotes osteoblastic differentiation of PDL cells; therefore, it may be beneficial for treating periodontitis.

Role of transient receptor potential channel 6 in the osteogenesis of periodontal ligament cells

Transient receptor potential channel 6 (TRPC6) is a receptor-operated Ca2+ channel that plays an important role in Ca2+ influx in the majority of non–excitable cells and influences calcium signalling and cellular responses. Therefore, the purpose of the present study was to gain insight into the role of TRPC6 in the osteogenesis of periodontal ligament cells (PDLCs). By western blot and immunohistochemical staining, the protein level of TRPC6 was found to be increased in a time-dependent manner during osteoblastic differentiation of PDLCs. In addition, the TRPC6 inhibitor SKF96365 was used to block the function of TRPC6 and inhibit osteoblastic differentiation of PDLCs. The TRPC6 activator hyperforin dicyclohexylammonium salt (hyperforin DCHA) was used to activate TRPC6 and promote osteoblastic differentiation of PDLCs. In vivo, wild-type mice showed better bone regeneration than TRPC6−/− mice, suggesting that TRPC6 has notable osteogenic induction properties and is important for bone defect repair. In conclusion, the current data demonstrated that TRPC6 plays a significant role in osteoblastic differentiation of PDLCs, suggesting that it may be a promising therapeutic target in osteogenesis.

The Effect of Menopause Hypoestrogenism on Osteogenic Differentiation of Periodontal Ligament Cells (PDLC) and Stem Cells (PDLCs): A Systematic Review.

(1) Background: Menopause is a physiological condition typified by drastic hormonal changes, and the effects of this transition have long-term significant clinical implications on the general health, including symptoms or physical changes. In menopausal women, the periodontium can be affected directly or through neural mechanism by oestrogen (E2) deficiency. The majority of the biological effects of E2 are modulated via both oestrogen receptor-α (ERα) and oestrogen receptor- β (ERβ). There is evidence that hypoestrogenism has a substantial impact on the aetiology, manifestation and severity of periodontitis, via the regulation of the expression of osteoprogesterin and RANKL in human periodontal ligament cells through ERβ. However, the mechanistic understanding of oestrogen in periodontal status has been partially clarified. The aim of this paper was to synopsize the recent scientific evidence concerning the link between the menopause and periodontitis, through the investigation of physio-pathological impact of the oestrogen deficiency on osteogenic differentiation of PDLSCs and PDLSC, as well as the dynamic change of ERα and ERβ. (2) Methods: Search was conducted for significant studies by exploring electronic PubMed and EMBASE databases, and it was independently performed by two researchers. All studies on the impact of oestrogen level on alveolar bone resorption were searched from 2005 to July 2020. Data selection was in concordance with PRISMA guidelines. (3) Results: Eight studies met the criteria and were included in this systematic review. All studies reported that oestrogen deficiency impairs the osteogenic and osteoblastic differentiation of PDL cells and oestrogen affects the bone formation capacity of cells. Seven studies were conducted on animal samples, divided into two groups: the OVX animals and animals who received the sham operation. (4) Conclusions: There is a multitude of data available showing the influence of menopause on periodontal status. However, the evidence of this line to investigation needs more research and could help explain the physiological linkage between menopause state and periodontal disease.

In vitro comparison of nanofibrillar and macroporous-spongious composite tissue scaffolds for periodontal tissue engineering

ABSTRACT Purpose/Aim of the study The ultimate goal of periodontal treatment is to regenerate the lost periodontal tissues. The interest in nanomaterials in dentistry is growing rapidly and has focused on improvements in various biomedical applications, such as periodontal regeneration and periodontal tissue engineering. To enhance periodontal tissue regeneration, hydroxyapatite (HA) was used in conjunction with other scaffold materials, such as Poly lactic-co-glycolic-acid (PLGA) and collagen (C). The main target of this study was to compare the effects of nano and macrostructures of the tissue scaffolds on cell behavior in vitro for periodontal tissue engineering. Materials and Methods Nanofibrillar and macroporous-spongious composite tissue scaffolds were produced using PLGA/C/HA. Subgroups with BMP-2 signal molecule and without HA were also created. The scaffolds were characterized by FTIR, SEM/EDX techniques, and mechanical tests. The scaffolds were compared in the periodontal ligament (PDL) and MCT3-E1 cell cultures. The cell behaviors; adhesions by SEM, proliferation by WST-1, differentiation by ALP and mineralization with Alizarin Red Tests were determined. Results Cell adhesion and mineralization were higher in the nanofibrillar scaffolds compared to the macroporous-spongious scaffolds. Macroporous-spongious scaffolds seemed better for the proliferation of PDL cells and differentiation of MC3T3-E1-preosteoblastic cells, while nanofibrillar scaffolds were more convenient for the differentiation of PDL cells and proliferation of MC3T3-E1-preosteoblastic cells. Conclusions In general, nanofibrillar scaffolds showed more favorable results in cell behaviors, compared to the macroporous-spongious scaffolds, and mostly, BMP-2 and HA promoted the activities of the cells.

Scleraxis upregulated by transforming growth factor-β1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-β1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-β receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-β1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

Simultaneous Quantitative Analysis of Ginsenosides Isolated from the Fruit of Panax ginseng C.A. Meyer and Regulation of HO-1 Expression through EGFR Signaling Has Anti-Inflammatory and Osteogenic Induction Effects in HPDL Cells.

Periodontitis is a set of chronic inflammatory diseases caused by the accumulation of Gram-negative bacteria on teeth, resulting in gingivitis, pocket formation, alveolar bone loss, tissue destruction, and tooth loss. In this study, the contents of ginsenosides isolated from Panax ginseng fruit extract were quantitatively analyzed, and the anti-inflammatory effects were evaluated in human periodontal ligament cells. The major ginsenosides, Re, Ra8, and Rf, present in ginseng fruit were simultaneously analyzed by a validated method using high-performance liquid chromatography with a diode-array detector; Re, Ra8, and Rf content per 1 g of P. ginseng fruit extract was 1.01 ± 0.03, 0.33 ± 0.01, and 0.55 ± 0.04 mg, respectively. Ginsenosides-Re, -Ra8, and -Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 (HO-1), promoting osteoblast differentiation of periodontal ligament cells, suppressing alveolar bone loss, and promoting the expression of osteoblast-specific genes, such as alp, opn, and runx2. An inhibitory effect of these ginsenosides on periodontitis and alveolar bone loss was observed via the regulation of HO-1 and subsequent epidermal growth factor receptor (EGFR) signaling. Silencing EGFR with EGFR siRNA confirmed that the effect of ginsenosides on HO-1 is mediated by EGFR. In conclusion, this study evaluated the contents of ginsenosides-Re, -Ra8, and -Rf isolated from P. ginseng fruit extract. Therefore, these results provide important basic data for future P. ginseng fruit component studies and suggest that ginsenosides Re, Ra8, and Rf have potential as future treatment options for periodontitis.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

LncRNA DCST1-AS1 inhibits PDLCs' proliferation in periodontitis and may bind with miR-21 precursor to upregulate PLAP-1.

This study aimed to investigate the potential interactions among long noncoding RNA domain containing 1-antisense (lncRNA DCST1-AS1), miR-21, and periodontal ligament-associated protein-1 (PLAP-1) in periodontitis. It has been verified that miR-21 can target PLAP-1 to regulate the osteogenic differentiation of periodontal ligament cells (PDLCs). Differential expression of DCST1-AS1 and miR-21 in PDLCs derived from periodontitis patients and healthy controls was determined by qPCR and unpaired t test. QPCR and Western blots were conducted to evaluate the effects of overexpression of DCST1-AS1 and miR-21 on the expression of PLAP-1. CCK-8 assay was applied to evaluate the effect of DCST1-ASI, miR-21, or PLAP-1 on PDLCs' proliferation. Western blotting was conducted to detect the expression levels of CKD family (CDK4, CDK6, and CCND1). DCST1-AS1 was downregulated in PDLCs derived from periodontitis patients, and its expression was inversely correlated with the expression of miR-2 but positively correlated with PLAP-1. Bioinformatics analysis showed that DCST1-AS1 might bind with miR-21 precursor but not mature miR-21. Transfection experiments showed that overexpression of DCST1-AS1 led to decreased expression levels of miR-21 and significantly increased the expression levels of PLAP-1 at both mRNA and protein levels, while overexpression of miR-21 resulted in a dramatic lower level of PLAP-1. CCK-8 assay indicated that overexpression of DCST1-AS1 or PLAP-1 prohibited PDLCs' proliferation. However, elevation of miR-21 had a contrary effect on the proliferation of PDLCs. And increased expression levels of DCST1-AS1 could significantly inhibit the expression of CDK4, CDK6, and CCND-1, while overexpression of miR-21 inversed the effects of DCST1-AS1. Therefore, the expression levels of DCST1-AS1 are much lower in periodontitis patients compared to that in healthy controls, and overexpression of DCST1-AS1 can significantly elevate the expression of PLAP-1 by inhibiting miR-21 in PDLCs.

Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides

BackgroundPeriodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS).MethodsHuman PDLCs were incubated with baicalein (0–100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot.ResultsMTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs.ConclusionsWith the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

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.

Wnt antagonist secreted frizzled-related protein I (sFRP1) may be involved in the osteogenic differentiation of periodontal ligament cells in chronic apical periodontitis.

To explore the mechanism of secreted frizzled-related protein I (sFRP1) involvement in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) under inflammatory conditions. hPDLCs were cultured in an osteogenic differentiation-inducing medium (odi) and subjected to the stimulation ofPorphyromonas gingivalis lipopolysaccharide (P.gingivalisLPS) with or without the inhibition of sFRP1. Quantitative real-time polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay were carried out to evaluate the expression of osteogenic markers as well as the classic Wnt signalling pathway. Periapical periodontitis was induced in Wistar rats to further confirm the effect of sFRP1 inhibitor on bone loss in vivo. After the Shapiro-Wilk normality test, data were analysed by Student's paired t-test or one-way Anova test with a P value less than 0.05 as the level of statistical significance. Significantly decreased mRNA and protein expression of osteogenic markers were detected in hPDLCs treated withP.gingivalisLPS during osteogenic induction (P<0.001). Increased expression of sFRP1 was observed (P<0.01), whilst Wnt/β-catenin signalling pathway was inhibited by the addition of P.gingivalisLPS (P<0.01). After the addition of the sFRP1 inhibitor, the decrease of osteogenic markers (P<0.05) and the inhibition of Wnt/β-catenin signalling pathway (P<0.05) were reversed significantly. The animal experiment further confirmed thatthe sFRP1 inhibitor significantly reduced bone loss of periapical lesions in vivo (P<0.0001). Wnt antagonist sFRP1 was involved in the osteogenic differentiation of hPDLCs under inflammation. Modulation of the Wnt/β-catenin signalling pathway through the inhibition of sFRP1 may offer a new perspective on the treatment of chronic apical periodontitis.

Local delivery of simvastatin maintains tooth anchorage during mechanical tooth moving via anti-inflammation property and AMPK/MAPK/NF-kB inhibition.

Simvastatin (SMV) could increase tooth anchorage during orthodontic tooth movement (OTM). However, previous studies on its bone‐specific anabolic and anti‐inflammation properties were based on static in vitro and in vivo conditions. AMPK is a stress‐activated kinase that protects tissue against serious damage from overloading inflammation. Rat periodontal ligament cells (PDLCs) were subjected to a serial of SMV concentrations to investigate the optimization that promoted osteogenic differentiation. The PDLCs in static and/or tensile culturing conditions then received the proper concentration SMV. Related factors expression was measured by the protein array, real‐time PCR and Western blot. The 0.05UM SMV triggered osteogenic differentiation of PDLCs. The inhibition of AMPK activation through a pharmacological approach (Compound C) caused dramatic decrease in osteogenic/angiogenic gene expression and significant increase in inflammatory NF‐κB phosphorylation. In contrast, pharmacological activation of AMPK by AICAR significantly inhibited inflammatory factors expression and activated ERK1/2, P38 MAPK phosphorylation. Moreover, AMPK activation induced by SMV delivery significantly attenuated the osteoclastogenesis and decreased the expression of pro‐inflammatory TNF‐α and NF‐κB in a rodent model of OTM. The current studies suggested that SMV could intrigue intrinsic activation of AMPK in PDLCs that promote attenuate the inflammation which occurred under tensile irritation through AMPK/MAPK/NF‐kB Inhibition.

Sympathetic nervous system contributes to orthodontic tooth movement by central neural regulation from hypothalamus.

Orthodontic tooth movement (OTM) is a specific treatment of malocclusion, whose regulation mechanism is still not clear. This study aimed to reveal the relationship between the sympathetic nervous system (SNS) and OTM through the construction of an OTM rat model through the utilization of orthodontic nickeltitanium coiled springs. The results indicated that the stimulation of SNS by dopamine significantly promote the OTM process represented by the much larger distance between the first and second molar compared with mere exertion of orthodontic force. Superior cervical ganglionectomy (SCGx) can alleviate this promotion effect, further proving the role of SNS in the process of OTM. Subsequently, the ability of orthodontic force to stimulate the center of the SNS was visualized by the tyrosin hydroxylase (TH) staining of neurons in ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC) of the hypothalamus, as well as the up-regulated expression of norepinephrine in local alveolar bone. Moreover, we also elucidated that the stimulation of SNS can promote osteoclast differentiation in periodontal ligament cells (PDLCs) and bone marrow-derived cells (BMCs) through regulation of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) system, thus promoting the OTM process. In conclusion, this study provided the first evidence for the involvement of the hypothalamus in the promotion effect of SNS on OTM. This work could provide a novel theoretical and experimental basis for further understanding of the molecular mechanism of OTM.

The role of PHF8 and TLR4 in osteogenic differentiation of periodontal ligament cells in inflammatory environment.

Histone methylation is considered to play an important role in the occurrence and development of periodontitis. Plant homeodomain finger protein 8 (PHF8), a histone demethylase, has been shown to regulate inflammation and osteogenic differentiation of bone marrow stromal cells (BMSCs). This study aimed to detect the functions of PHF8 and TLR4 in osteogenic differentiation in an inflammatory environment induced by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) METHODS: A periodontitis mouse model was established, and the mice were treated with TAK-242. Immunohistochemical staining was used to detect the expression of PHF8 in periodontal tissue. Periodontal ligament cells (PDLCs) were treated with mineralization induction medium supplemented with Pg-LPS and/or TAK-242, and a Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of PDLCs. Real-time PCR and western blotting were used to detect the mRNA and protein expression levels, respectively, of PHF8, toll-like receptor 4 (TLR4) and the other osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), Special AT-rich sequence-binding protein 2 (Satb2) and Runt-related transcription factor 2 (Runx2) RESULTS: Periodontitis reduced PHF8 expression in periodontal tissue, and TAK-242 partially reversed this downregulation. An in vitro experiment revealed that the mRNA and protein expression levels of PHF8 were significantly upregulated during the osteogenic differentiation of PDLCs. Alizarin red staining showed that the mineralized nodules of PDLCs in osteogenic induction group were more than those in control group. Real-time PCR and western blot results indicated that Pg-LPS inhibited PHF8 expression and upregulated TLR4 expression in PDLCs. TAK-242 inhibited TLR4 and partially reversed the inhibition of PHF8 expression and osteogenic differentiation induced by Pg-LPS in PDLCs CONCLUSION: PHF8 and TLR4 play important roles in periodontitis. Pg-LPS inhibits the expression of PHF8 via upregulation of TLR4 and might further inhibit the osteogenic differentiation of PDLCs. However, the specific mechanisms involved remain to be explored.

LPCGF and EDTA conditioning of the root surface promotes the adhesion, growth, migration and differentiation of periodontal ligament cells.

Liquid-phase concentrated growth factor (LPCGF), a new-generation platelet concentrate, may potently stimulate human periodontal ligament (PDL) cells. This study assessed the effectiveness of ethylenediaminetetraacetic acid (EDTA) and/or LPCGF on periodontally diseased root surfaces through their effects on PDL cells. Dentin blocks prepared from periodontal teeth were divided into four groups and treated as follows: group I, scaling and root planning (SRP); group II, SRP + EDTA; group III, SRP + LPCGF; and group IV, SRP+EDTA + LPCGF. PDL cells were cultured on dentin blocks, and LPCGF-induced biological effects were evaluated by migration and cell adhesion/proliferation assays. Furthermore, PDL cell differentiation was assessed by real-time polymerase chain reaction (PCR). Significantly more adherent cells were observed in the EDTA, LPCGF and combination treatment groups than in the control group. Root conditioning with EDTA and/or LPCGF enhanced cell proliferation and migration more than SRP did. Compared with the control group, the combined treatment group exhibited significant upregulation of cell differentiation-related genes. Electron microscopy of the tooth surface revealed removal of the smear layer and exposed dentin holes in the EDTA-treated group but not in the control group. EDTA and LPCGF application to periodontitis-affected root surfaces forms a surface suitable for cell attachment, growth, migration and differentiation. Thus, LPCGF is promising in clinical periodontics applications. Further studies to support these results are necessary.

Role of PTH1R Signaling in Prx1+ Mesenchymal Progenitors during Eruption

Tooth eruption is a complex process requiring precise interaction between teeth and adjacent tissues. Molecular analysis demonstrates that bone remodeling plays an essential role during eruption, suggesting that a parathyroid hormone 1 receptor (PTH1R) gene mutation is associated with disturbances in bone remodeling and results in primary failure of eruption (PFE). Recent research reveals the function of PTH1R signaling in mesenchymal progenitors, whereas the function of PTH1R in mesenchymal stem cells during tooth eruption remains incompletely understood. We investigated the specific role of PTH1R in Prx1+ progenitor expression during eruption. We found that Prx1+-progenitors occur in mesenchymal stem cells residing in alveolar bone marrow surrounding incisors, at the base of molars and in the dental follicle and pulp of incisors. Mice with conditional deletion of PTH1R using the Prx1 promoter exhibited arrested mandibular incisor eruption and delayed molar eruption. Micro–computed tomography, histomorphometry, and molecular analyses revealed that mutant mice had significantly reduced alveolar bone formation concomitant with downregulated gene expression of key regulators of osteogenesis in PTH1R-deficient cells. Moreover, culturing orofacial bone-marrow-derived mesenchymal stem cells (OMSCs) from Prx1Cre;PTH1Rfl/fl mice or from transfecting Cre recombinase adenovirus in OMSCs from PTH1Rfl/fl mice suggested that lack of Pth1r expression inhibited osteogenic differentiation in vitro. However, bone resorption was not affected by PTH1R ablation, indicating the observed reduced alveolar bone volume was mainly due to impaired bone formation. Furthermore, we found irregular periodontal ligaments and reduced Periostin expression in mutant incisors, implying loss of PTH1R results in aberrant differentiation of periodontal ligament cells. Collectively, these data suggest that PTH1R signaling in Prx1+ progenitors plays a critical role in alveolar bone formation and periodontal ligament development during eruption. These findings have implications for our understanding of the physiologic and pathologic function of PTH1R signaling in tooth eruption and the progression of PFE.

Functional and cell surface characteristics of periodontal ligament cells (PDLCs) on RGD-synthetic polypeptide conjugate coatings.

Periodontal ligament cells (PDLCs) are an important source for periodontal tissue healing and regeneration. Proper cell adhesion is a key for survival of anchorage-dependent cells and also initiates further intracellular signals for essential cellular functions. We aimed to test 3 different synthetic conjugates with integrin-binding RGD sequence (SAK-c[RGDfC], AK-c[RGDfC], and SAK-opn on the adhesion of human PDLCs and subsequent events including proliferation, migration, behavior of cell surface molecules, and osteogenic differentiation. Synthetic peptides were synthesized by solid-phase technique and attached to branched chain polymeric polypeptides via thioether linkage. Simple adsorption method was used to coat tissue culture plastic or electric arrays. PDLCs were isolated from 24 surgically extracted human third molars. Cell adhesion and proliferation were measured with real-time impedimetric xCELLigence SP system. Cell migration assay was performed with Ibidi® Culture inserts. Cell surface antigens were detected using flow cytometry analysis. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red S staining, and real-time qPCR was performed to analyze the osteoblast-related gene expression. Osteogenic differentiation and adipogenic differentiation of PDLCs were monitored by real-time Electrical Cell-Substrate Impedance Spectroscopy (ECIS). Primary outcome of this study relies on that all three synthetic RGD peptides improved PDLC adhesion (P<.05). When animal serum is absent in culture medium, SAK-c[RGDfC] and AK-c[RGDfC] elevated cell adhesion (P<.05). Cell migration was enhanced by SAK-c[RGDfC] and AK-c[RGDfC] (P<.05). After 1-week treatment, all synthetic peptides elevated CD105 (1.7- to 2.2-fold) and CD146 (1.3- to 1.5-fold) markers and caused different integrin patterns. ALP activity (1.4-fold) and ARS (1.8- and 2.0-fold) were increased by SAK-c[RGDfC] and AK-c[RGDfC] in absence of osteogenic supplements, and all the peptides supported the mineralization under osteogenic condition (P<.05). RT-qPCR revealed the upregulation of bone sialoprotein (5.0- to 7.8-fold), osteocalcin (2.3- to 2.7-fold), and ALP (1.9- to 2.3-fold) gene expression in osteogenesis-induced PDLCs. ECIS monitoring showed that higher impedance was generated by the osteogenic induction compared with the adipogenic or the non-induced (P<.05). Our study demonstrates that SAK-c[RGDfC] and AK-c[RGDfC] improved adhesion and migration of PDLCs and supported osteogenic differentiation of PDLCs. These cyclic RGD peptides proved to be applicable biocompatible material in regenerative medicine.