A Comparative Evaluation of the Periodontal Ligament Cell Viability Using Placentrex, Propolis, and Hanks' Balanced Salt Solution: An In-Vitro Study.

BACKGROUND AND OBJECTIVES: Most of the dento-alveolar trauma that occurs frequently in childhood is often linked to avulsion injuries. Two considerable elements influencing the prognosis of tooth avulsion after replantation are extraoral dry duration and the characteristics of the storage media to support the viability of cells. The study aimed to compare and assess the effectiveness of Hanks' balanced salt solution (HBSS) (HiMedia Laboratories, Thane, India) and Placentrex (Albert David Limited, Kolkata, India) against propolis (5% and 10%) as storage media in preserving the vitality of periodontal ligament (PDL) cells. MATERIALS AND METHODS: Four groups with 15 samples each were selected for the study. Sixty recently extracted premolars were left to incubate for 30 minutes in 15-ml falcon tubes containing 2.5 ml of collagenase 0.2 mg/ml in phosphate-buffered saline and 2.4 mg/ml of dispase in HBSS, Placentex, propolis 5%, and propolis 10%. After the addition of bovine serum, it was centrifuged for 4 minutes. Trypan blue 0.4% was utilized to recognize the cells, and a hemocytometer was employed for counting the live PDL cells under a light microscope. RESULTS: Propolis 5% and 10%, HBSS, and Placentrex all showed statistically significant differences in PDL cell viability; however, HBSS was significantly outperformed by Placentrex and propolis. CONCLUSION: Placentrex is a superior substitute storage medium in cases of tooth avulsion as opposed to alternatives such as propolis 5% and 10%.

Cyclic stretch induced epigenetic activation of periodontal ligament cells.

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.

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1.

OBJECTIVES: Oculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. METHODS: Periodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. RESULTS: RNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. CONCLUSIONS: Our findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

A comparison of osteogenic effect of newly manufactured calcium silicate-based sealers in vitro.

This study aimed to assess the effect of different calcium silicate-based root canal sealers (CSRS) on osteogenic effect in human periodontal ligament cells (hPDLCs). hPDLCs were cultured in a medium containing extract of 5 types of CSRS. The specimens were assessed by the cell cytotoxicity test, alkaline phosphatase staining, alizarin red S staining, quantitative real-time PCR, Western blot analysis, and enzyme-linked immunosorbent assay. The diluted concentrations of extracted solutions had no significant effect on the viability of hPDLCs. There was a statistically significant difference in the mRNA expression level of bone sialoprotein (BSP), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) among some groups. The protein expressions of BSP, OCN, and RUNX2 were significantly higher in some groups compared to the control group. The CSRS did not interfere with the osteogenic differentiation of hPDLCs, compared to the control group. CSRS are shown to have biocompatibility and osteogenic differentiation effect on hPDLCs.

Irisin: A Myokine With Therapeutic And Diagnostic Potential In Clinical Dentistry.

Irisin is a novel adipomyokine which has extensive systemic and local effects in different tissues of the body. The scientific interest in understanding the physiological roles of irisin in the body has increased tremendously in the past few years due to its vast therapeutic potential in different fields of medicine. The current narrative review was planned to describe the molecular mechanisms by which irisin regulates oral hard and soft tissues. The information gleaned provided useful insights for future researchers to investigate newly discovered roles of irisin in craniofacial health and disease, and to explore the potential of irisin as a promising therapeutic and diagnostic agent in clinical dentistry.

The Comparative Analysis of the Effectiveness of Four Different Storage Media (Placentrex, Propolis 10%, Pomegranate Juice 5%, and Hank's Balanced Salt Solution) in Preserving the Viability of Periodontal Ligament Cells: An In Vitro Study.

BACKGROUND: Out of all traumas, dentoalveolar trauma occurs quite frequently and is commonly linked to avulsion injuries. Of all severe lesions to the permanent dentition, tooth avulsion accounts for 1%-16% of cases. The extraoral dry time and the storage medium in which the tooth is stored and its capacity to promote cell viability are two important variables influencing the prognosis of an avulsed tooth following replantation. AIM: The aim of this paper is to assess and contrast the effectiveness of placentrex, propolis 10%, pomegranate juice 5%, and Hank's balanced salt solution (HBSS) as a storage medium in preserving the periodontal ligament (PDL) cells' viability. METHODS: Sixty freshly extracted premolars were put in four various media for 45 minutes each, including HBSS, placentrex, propolis 10%, and pomegranate juice 5%, and placed in an incubator for 30 minutes in 15 mL falcon tubes with 2.5 mL solutions of collagenase 0.2 mg/mL and dispase 2.4 mg/mL in phosphate-buffered saline. Bovine serum was added after incubation, and the mixture was centrifuged for four minutes. Trypan blue 0.4% was used to identify the cells. Under a light microscope, a hemocytometer was utilized to quantify the number of periodontal ligament cells that were still alive. RESULTS: Propolis, pomegranate, and placentrex are all significantly inferior to HBSS in the context of the sustainability of the cells. It was discovered that HBSS with placentrex and pomegranate juice 5% was statistically insignificant. The difference in periodontal ligament cell viability between propolis 10%, HBSS, placentrex, and pomegranate juice 5% was shown to be statistically significant. CONCLUSION: When compared to other media such as propolis 10% and pomegranate juice 5%, placentrex is a better alternative storage medium for avulsed teeth.

YAP/WNT5A/FZD4 axis regulates osteogenic differentiation of human periodontal ligament cells under cyclic stretch.

OBJECTIVE: To verify the role of YAP/WNT5A/FZD4 axis in stretch-induced osteogenic differentiation of hPDLCs. BACKGROUND: During orthodontic tooth movement, differentiation of human periodontal ligament cells (hPDLCs) at the tension side of the periodontal ligament mediates new bone formation. WNT5A promotes osteogenesis and its regulator Yes-associated protein (YAP) is responsive to mechanical stimulation in hPDLCs. However, the mechanisms of YAP and WNT5A in alveolar bone remodeling remain unclear. METHODS: Cyclic stretch was applied to hPDLCs to mimic the orthodontic stretching force. Osteogenic differentiation was determined by alkaline phosphatase (ALP) activity, Alizarin Red staining, qRT-PCR and western blotting. To detect activation of YAP and expression of WNT5A and its receptor Frizzled-4 (FZD4), western blotting, immunofluorescence, qRT-PCR and ELISA were performed. Verteporfin, Lats-IN-1, small interfering RNAs and recombinant protein were used to explore the relationship of YAP, WNT5A and FZD4, and the effect of their relationship on stretch-induced osteogenesis of hPDLCs. RESULTS: WNT5A, FZD4 and nuclear localization of YAP were upregulated by cyclic stretch. YAP positively regulated WNT5A and FZD4 expression and osteogenic differentiation of hPDLCs under cyclic stretch by YAP inhibition or activation assay. Knockdown of WNT5A and FZD4 attenuated YAP-induced and stretch-induced osteogenic differentiation. Recombinant WNT5A rescued the suppressed osteogenic differentiation by YAP inhibitor in hPDLCs, whereas knockdown of FZD4 weakened the effect of WNT5A and amplified the suppression. CONCLUSIONS: WNT5A/FZD4 could be positively regulated by YAP and the YAP/WNT5A/FZD4 axis mediated osteogenic differentiation of hPDLCs under cyclic stretch. This study provided further insight into the biological mechanism of orthodontic tooth movement.

Evaluation of delphinidin as a storage medium for avulsed teeth.

BACKGROUND: Delphinidin (DP), an anthocyanidin found in blueberries, has antioxidant and anti-inflammatory effects. This study aimed to investigate the efficacy of DP as a storage medium for avulsed teeth. METHODS: Human periodontal ligament cells were cultured and exposed to DP solution (10, 50, and 100 µM), Dulbecco's modified Eagle's medium, Hank's balanced salt solution and tap water. Cell counting kit-8 assays were performed after 0.5, 1, 6, and 24 h to measure the cell viability. Nitric oxide assays and gelatin zymography were performed to evaluate the anti-inflammatory effects of DP. Reverse transcription-polymerase chain reaction was used to determine the expression levels of inflammatory cytokines. RESULTS: The viability of periodontal ligament cells was greatest at 100 µM DP. At 1 h, 100 µM DP decreased nitric oxide synthesis (p < .0167). Matrix metallopeptidase-9 activity was inhibited by DP in a dose-dependent manner (p < .0167). Moreover, treatment with 100 µM DP decreased the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 in periodontal ligament cells (p < .0167). CONCLUSIONS: Within the limits of this study, DP preserved the viability and suppressed the inflammatory response of periodontal ligament cells. These findings suggest that DP could be promising for preservation of avulsed teeth.

The role of noncoding RNAs in the osteogenic differentiation of human periodontal ligament-derived cells.

Human periodontal ligament-derived cells are important seed cells for periodontal regeneration, and their osteogenic potential closely affects alveolar bone repair and periodontal regeneration. Human periodontal ligament stem cells are pluripotent stem cells of mesenchymal origin, which can differentiate in osteoblasts and cementoblasts. However, the molecular mechanism of this differentiation activity is poorly studied. Noncoding RNAs (ncRNAs) belong to RNAs, which do not encode proteins and represent a large segment of the human transcriptome, mainly including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs). It was shown that ncRNAs is involved in the proliferation and differentiation of cells, epigenetic modifications, apoptosis, as well as in complex control and pathogenesis of various diseases. NcRNAs are actively involved in the regulation of osteogenic genes in human periodontal ligament-derived cells. This article reviews the research progress of ncRNAs in the regulatory targets, pathways and functions of ncRNAs in the osteogenic differentiation of human periodontal ligament-derived cells.

Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation.

OBJECTIVES: This study aimed to explore the specific mechanism, mediated by the reactive oxygen species (ROS) and PINK1/Parkin pathway, of the mitochondrial autophagy of human periodontal ligament cells (hPDLCs) under starvation conditions. METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. Earle's balanced salt solution (EBSS) was used to simulated a starvation environment and thus stimulate hPDLCs mitochondrial autophagy. N-Acetyl-L-cysteine (NAC) was used to inhibit ROS production to explore the role of ROS in hPDLC mitochondrial autophagy. Cyclosporin A was used to inhibit the PINK1/Parkin pathway to study the role of ROS and the PINK1/Parkin pathway in hPDLCs activation under starvation. The mitochondrial membrane potential was detected by flow cytometry with a JC-1 mitochondrial membrane potential detection kit. The morphological structure of mitochondria and the formation of mitochondrial autophagosome were observed by transmission electron microscopy. Mito tracker red cmxros and lyso tracker green staining were used to observe the localization of mitochondria and lysosomes. The formation intensity of ROS was detected with a DCFH-DA ROS fluorescent probe. The expression levels of mitochondrial autophagy genes (Tomm20 and Timm23) and the PINK1/Parkin pathway were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of mitochondrial autophagy proteins (Tomm20 and Timm23) and PINK1/Parkin protein were detected by Western blot. RESULTS: EBSS starvation for 30 min induced the strongest activation of hPDLCs mitochondrial autophagy, increased the expression of ROS, downregulated the expression of mitochondrial autophagy-related genes (Tomm20 and Timm23) (P<0.001), and upregulated the PINK1/Parkin pathway (P<0.001). After NACinhibited ROS production, mitochondrial autophagy was also inhibited. Meanwhile, the expression of Tomm20 and Timm23 was upregulated (P<0.001 and P<0.05), and the expression of the PINK1/parkin pathway (P<0.001 and P<0.05) was down regulated. When cyclosporin A inhibited the expression of the PINK1/Parkin pathway (P<0.05 and P<0.05), it reversed the mitochondrial autophagy of hPDLCs (P<0.001 and P<0.01) and also upregulated the expression of Tomm20 and Timm23 (P<0.001 and P<0.01). CONCLUSIONS: ROS enhanced the mitochondrial autophagy of hPDLCs primarily through the PINK1/Parkin pathway under starvation conditions.

The Role of Inflammasome NLPR3 in the Development and Therapy of Periodontitis.

Periodontitis is a chronic inflammatory disease that affects tooth-supporting tissues and even leads to tooth loss. NLRP3 inflammasomes play a critical role in periodontitis pathogenesis. Aberrant activation or overexpression of NLRP3 inflammasomes in cellular players, including osteoclasts, osteoblasts, periodontal ligament fibroblasts, and leukocytes often contributes to cellular dysfunction and environment abnormality, thus resulting in the disorganization of ligament and alveolar bone. In this review, we mainly focus on the negative regulation of NLRP3 inflammasome in periodontitis and highlight the importance of NLRP3 inflammasome as a candidate therapeutic target in periodontitis treatment. Then we elucidate the development status of NLRP3 inflammasome inhibitors and show their application potential for treating periodontitis. In summary, this review reveals the recent progress and perspectives of NLRP3 inflammasome and the therapeutic potential of NLRP3 inflammasome inhibitors in periodontitis.

Histone Demethylase Jmjd3 Regulates the Osteogenic Differentiation and Cytokine Expressions of Periodontal Ligament Cells.

Periodontal ligament (PDL) cells are critical for the bone remodeling process in periapical lesions since they can differentiate into osteoblasts and secrete osteoclastogenesis-promoting cytokines. Post-translational histone modifications including alterations of the methylation status of H3K27 are involved in cell differentiation and inflammatory reaction. The histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes methylation of H3K27. We investigated whether Jmjd3 is involved in the osteogenic differentiation and secretion of PDL cells' inflammatory factors. Jmjd3 expression in periapical lesions was examined by immunostaining. Using siRNA specific for Jmjd3 or the specific Jmjd3 inhibitor GSK-J4, we determined Jmjd3's roles in osteogenic differentiation and cytokine production by real-time RT-PCR. The locations of Jmjd3 and NF-κB were analyzed by immunocytochemistry. Compared to healthy PDLs, the periapical lesion samples showed higher Jmjd3 expression. Treatment with GSK-J4 or Jmjd3 siRNA suppressed PDL cells' osteogenic differentiation by suppressing the expressions of bone-related genes (Runx2, Osterix, and osteocalcin) and mineralization. Jmjd3 knockdown decreased the expressions of cytokines (TNF-α, IL-1ß, and IL-6) induced by lipopolysaccharide extracted from Porphyromonas endodontalis (Pe-LPS). Pe-LPS induced the nuclear translocations of Jmjd3 and NF-κB; the latter was inhibited by GSK-J4 treatment. Jmjd3 appears to regulate PDL cells' osteogenic differentiation and proinflammatory cytokine expressions.

Peroxisome proliferator-activated receptor gamma preserves intracellular homeostasis of insulin-resistant periodontal ligament stem cells.

Background: Diabetes and periodontitis develop and influence each other. The peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone (RSG) controls blood glucose and hence the systemic diseases associated with diabetes by increasing the sensitivity of tissues to insulin. However, whether and how RSG can treat diabetic periodontitis is poorly understood. Methods: Insulin-resistant periodontal ligament stem cells (IR-PDLSCs) were induced by glucosamine (18 mM, 24 h) in the presence or absence of RSG or GW9662 (a PPARγ antagonist). The glucose uptake rate was tested to evaluate insulin sensitivity. A scratch test was carried out to measure cell proliferation and motility. We used 2,7-dichlorodihydrofluorescein diacetate (DFCH-DA) and JC-1 kits to detect oxidative stress (OS), and cytoskeleton staining and Calcein-AM/PI kits were used to determine cell viability. Interferon-gamma (IFN-γ) and interleukin-10 (IL-10) ELISA kits were used to evaluate inflammation levels. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) analysis were used to assess the expression of osteogenic/odontogenic differentiation-related genes or proteins. Results: Our results showed that RSG exhibited a protective effect on IR-PDLSCs, with increased insulin sensitivity and migration efficiency, an alleviation of glucosamine-induced OS, and a downregulated pro-inflammatory cytokine secretion through activation of PPARγ receptors. Moreover, RSG alleviated the suppressed odontogenic differentiation ability of IR-PDLSCs. Conclusions: RSG preserves the biological functions of IR-PDLSCs in maintaining intracellular homeostasis by increasing insulin sensitivity, reducing OS, and suppressing inflammation.

Irisin attenuates P. gingivalis-suppressed osteogenic/cementogenic differentiation of periodontal ligament cells via p38 signaling pathway.

Regeneration of periodontal hard tissues damaged by Porphyromonas gingivalis (P. gingivalis) is essential for tooth stability and dental health. Irisin, a myokine secreted by skeletal muscle, is involved in different biological processes, such as myogenesis, adipogenesis, neurogenesis and osteogenesis. However, whether irisin regulates the osteogenic/cementogenic differentiation of human periodontal ligament cell (hPDLCs), especially under P. gingivalis-triggered inflammation, remains unknown. In this study, we verified the suppression role of P. gingivalis in the osteogenic/cementogenic differentiation of hPDLCs. Also, compared with the control cells, hPDLCs with irisin stimulation showed higher expression of osteogenic-/cementogenic-related markers, ALP activity and mineralization ability, as measured by RT-qPCR, western blotting, ALP staining and Alizarin red staining, respectively. Moreover, the osteogenic/cementogenic differentiation-facilitating role of irisin was also demonstrated under P. gingivalis-elicited inflammation, which implied a rescue function of irisin in P. gingivalis-suppressed hPDLC differentiation. Finally, the underlying mechanism involved in the process was explored. We observed that the p38 signaling pathway was activated during irisin-accelerated hPDLC differentiation. Furthermore, hPDLC differentiation was weakened after the p38 inhibitor was applied. In summary, we found that irisin can facilitate the osteogenic/cementogenic differentiation of hPDLCs partially through the p38 signaling pathway, which may provide evidence for the regeneration of P. gingivalis-destroyed periodontal hard tissues.

Effects of DDR1 on migration and adhesion of periodontal ligament cells and the underlying mechanism.

BACKGROUND AND OBJECTIVE: As one of the widely expressed cell surface receptors binding to collagen, the most abundant component of the extracellular matrix (ECM), knowledge of the expression, functions, and mechanisms underlying the role of discoidin domain receptor 1 (DDR1) in human periodontal ligament cells (hPDLCs) is incomplete. This study determined the expression of DDR1 in hPDLCs and the effect of DDR1 upon migration and adhesion to hPDLCs, as well as the related regulatory mechanisms. MATERIALS AND METHODS: The expression of DDR1 and the DDR1 isoforms in hPDLCs from six donors were tested. The migratory ability (horizontal and vertical) and adhesive capacity of hPDLCs with or without specific knockdown of DDR1 were evaluated. After treatment with MEK-ERK1/2 inhibitors (PD98059 and U0126) with or without RNAi, the migratory and adhesive capacity of hPDLCs were re-tested. Western blotting was performed to verify p-MEK1/2 and p-ERK1/2, the key factors of the MEK-ERK1/2 signaling pathways. RESULTS: DDR1 was detected in hPDLCs in the mRNA and protein level; DDR1b was the dominant isoform. Knockdown of DDR1 almost halved the migratory capacity and significantly downregulated the adhesive capacity of hPDLCs. The use of MEK-ERK1/2 inhibitors caused declined migratory and adhesive capacity of hPDLCs as well. After DDR1 was knocked down, the expression of p-MEK and p-ERK protein declined significantly while total MEK and ERK showed no obvious change, which means the ratio of p-MEK/MEK and p-ERK/ERK was markedly reduced. CONCLUSIONS: DDR1 plays an important role in the migration and adhesion of hPDLCs and might be regulated via the MEK-ERK1/2 signaling pathway.

FNDC5/irisin is expressed and regulated differently in human periodontal ligament cells, dental pulp stem cells and osteoblasts.

OBJECTIVE: To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs). METHODS: FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs. RESULTS: FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs. CONCLUSIONS: FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs.

[Functions of non-coding RNAs in the osteogenic differentiation of human periodontal ligament-derived cells].

Human periodontal ligament-derived cells serve as an important source of seeding cells in periodontal regenerative medicine, and their osteogenic potential is closely related to alveolar bone repair and periodontal regeneration. Non-coding RNA (ncRNA), such as microRNA, long non-coding RNA, and circular RNA, play important roles in the regu-lation of osteogenic genes in human periodontal ligament-derived cells. In this review, we summarize the target genes, path-ways, and functions of the ncRNA network during osteogenic differentiation of periodontal ligament-derived cells.

[Preliminary study on the expression and distribution of S100A8 and S100A9 in healthy and experimental periodontitis tissues].

Objective: To investigate the systemic expression profile of S100A8 and S100A9 in healthy and inflamed periodontal tissues. Methods: Experimental periodontitis models were established by ligations around the mandibular second molars of six beagle dogs for 12 weeks (ligation group). The mandibular second molars on the opposite side were kept clean (healthy control group). The expressions of S100A8 and S100A9 in healthy and inflamed periodontal tissues of six beagle dogs were examined by immunohistochemistry. The expressions of S100A8 and S100A9 in primary human gingival fibroblasts (hGF) from 3 subjects and human periodontal ligament cells (hPDLC) from 3 other subjects were detected by immunocytochemistry. Results: After the ligation for 12 weeks, the mean probing depth of ligation group [(3.86±0.14) mm] was significantly higher than that of healthy control group [(2.11±0.28) mm] (P<0.01). Results of immunohistochemistry analysis indicated that S100A8 and S100A9 could be expressed in gingival epithelial cells and might infiltrated neutrophils in the healthy periodontium. Except for the gingival epithelial cells and neutrophils, both proteins were induced and expressed in gingival fibroblasts, periodontal ligament cells, microvascular endothelial cells and bone marrow fibroblasts under inflammatory conditions. The distribution of S100A8 and S100A9 differed in the healthy oral gingival epithelium (OGE), which becomes consistent in inflamed OGE. Additionally, the expressions of S100A8 and S100A9 were confirmed in primary hGF and hPDLC. Conclusions: Periodontal inflammation might enlarge the expression scope of S100A8 and S100A9 and enrich multiple cells with expressions of S100A8 and S100A9.

Periodontal ligament cells regulate osteogenesis via miR-299-5p in mesenchymal stem cells.

BACKGROUND: The periodontal ligament contains periodontal ligament cells, which is a heterogeneous cell population, and includes progenitor cells that can differentiate into osteoblasts/cementoblasts. Mesenchymal stem cells (MSCs) can differentiate into various cells and can be used for periodontal regenerative therapy. Therefore, transplanted MSCs can be affected by humoral factors from periodontal ligament cells via the transcription factors or microRNAs (miRNAs) of MSCs. In addition, periostin (POSTN) is secreted from HPL cells and can regulate periodontal regeneration and homeostasis. To clarify the regulatory mechanism of humoral factors from periodontal ligament cells, we attempted to identify key genes, specifically microRNAs, involved in this process. METHODS: Human MSCs (hMSCs) were indirectly co-cultured with human periodontal ligament cells (HPL cells) and then evaluated for osteogenesis, undifferentiated MSCs markers, and miRNA profiles. Furthermore, hMSCs were indirectly co-cultured with HPL cells in the presence of anti-POSTN monoclonal antibody (anti-POSTN Ab) to block the effect of POSTN from HPL cells, and then evaluated for osteogenesis or undifferentiated MSC markers. Moreover, hMSCs showed alterations in miRNA expression or cultured with HPL were challenged with POSTN during osteogenesis, and cells were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: hMSCs co-cultured with HPL cells showed suppressed osteogenesis and characteristic expression of SOX11, an undifferentiated MSC marker, as well as miR-299-5p. Overexpression of miR-299-5p regulated osteogenesis and SOX11 expression as observed with indirect co-culture with HPL cells. Furthermore, MSCs co-cultured with HPL cells were recovered from the suppression of osteogenesis and SOX11 mRNA expression by anti-POSTN Ab. However, POSTN induced miR-299-5p and SOX11 expression, and enhanced osteogenesis. CONCLUSION: Humoral factors from HPL cells suppressed osteogenesis in hMSCs. The suppressive effect was mediated by miR-299-5p and SOX11 in hMSCs.

Functions of non-coding RNAs in the osteogenic differentiation of human periodontal ligament-derived cells / 华西口腔医学杂志

Human periodontal ligament-derived cells serve as an important source of seeding cells in periodontal regenerative medicine, and their osteogenic potential is closely related to alveolar bone repair and periodontal regeneration. Non-coding RNA (ncRNA), such as microRNA, long non-coding RNA, and circular RNA, play important roles in the regu-lation of osteogenic genes in human periodontal ligament-derived cells. In this review, we summarize the target genes, path-ways, and functions of the ncRNA network during osteogenic differentiation of periodontal ligament-derived cells.