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Low-level laser irradiation can promote the healing process in soft and hard tissue but the precise mechanisms are unclear. In this study, we examined the effect of LLLT (low-level laser therapy) on the healing of extraction sockets in diabetic and healthy rats. Forty-eight Sprague-Dawley rats were divided into normal (n = 24) and diabetic (n = 24) rats, and streptozotocin (STZ) injection was used to induce diabetes in the latter. The left and right maxillary first molars of all the rats were extracted. In the non-diabetic rats, the left extraction sockets were not irradiated (group 1) and the right ones were irradiated daily for 3, 5, 7, and 14 days after extraction with a galium-aluminum-arsenide (GaAlAs) diode laser (group 2), and in the diabetic rats, similarly the left ones were not irradiated (group 3) and the right ones were irradiated (group 4). Specimens acquired at these intervals were examined by hematoxylin and eosin (H&E) staining and reverse transcription polymerase chain reaction (RT-PCR). Histological observations and gene expression analyses revealed that groups 2 (normal rats with LLLT) and 4 (diabetic rats with LLLT) showed faster initial healing and more new alveolar bone formation than group 1 (normal rats without LLLT) and group 3 (diabetic rats without LLLT), respectively. We conclude that 980-nm GaAlAs low-intensity diode laser irradiation is beneficial for the initial stages of alveolar bone healing and for further calcification in both diabetic and normal rats when applied every day at a dose of 13.95 J/cm(2) for 60 s.

Hesperetin (3′,5,7-trihydroxy-4-methoxyflavanone) is a metabolite of hesperidin (hesperetin-7-O-rutinoside), which belongs to the flavanone subgroup and is found mainly in citrus fruits. Hesperetin has been reported to be an effective osteoinductive compound in various in vivo and in vitro models. However, how hesperetin effects osteogenic differentiation is not fully understood. In this study, we investigated the capacity of hesperetin to stimulate the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to relieve the anti-osteogenic effect of high glucose. Osteogenesis of PDLSCs was assessed by measurement of alkaline phosphatase (ALP) activity, and evaluation of the mRNA expression of ALP, runt-related gene 2 (Runx2), osterix (OSX), and FRA1 as osteogenic transcription factors, as well as assessment of protein expression of osteopontin (OPN) and collagen type IA (COLIA). When PDLSCs were exposed to a high concentration (30 mM) of glucose, osteogenic activity decreased compared to control cells. Hesperetin significantly increased ALP activity at doses of 1, 10, and 100 µM. Pretreatment of cells with hesperetin alleviated the high-glucose-induced suppression of the osteogenic activity of PDLSCs. Hesperetin scavenged intracellular reactive oxygen species (ROS) produced under high glucose condition. Furthermore, hesperetin increased the activity of the PI3K/Akt and β-catenin pathways. Consistent with this, blockage of Akt or β-catenin diminished the protective effect of hesperetin against high glucose-inhibited osteogenic differentiation. Collectively, our results suggest that hesperetin alleviates the high glucose-mediated suppression of osteogenic differentiation in PDLSCs by regulating ROS levels and the PI3K/Akt and β-catenin signaling pathways.

Objectives:To evaluate the long-term influence of residual bone height (RBH), sinus membrane perforation and the presence of voids on the survival of implants placed simultaneously with lateral maxillary sinus floor augmentation (MSFA). Materials and Methods:A total of 207 patients (613 implants) who underwent MSFA from 1999 to 2003 and followed up until 2018 were included in this study. RBH and marginal bone loss were assessed using a series of panoramic and periapical radiographs. Cumulative survival rates (CSR) with log-rank tests and hazard ratios of implant failure according to RBH, membrane perforation, and the presence of voids were assessed. Results:The overall 10-year and 20-year CSRs were 95% (95% CI: 84.95%-85.05%) and 85% (95% CI: 84.95%-85.05%), respectively. CSR was significantly higher for implants with ≥3 mm RBH (n = 260, 92.4%) than those with <3 mm RBH (n = 353, 78.8%) (p = .002). CSR of non-smoking group (n = 312, 90.0%) was higher than that of smoking group (n = 301, 77.1%) (p = .009). There was no significant difference in the hazard ratio of implant failure between the perforated membrane group (n = 245) and the non-perforated group (n = 368). In two out of 11 patients with voids, the inflammation of peri-implantitis progressed into voids, resulting in severe bone resorption. Conclusions:The survival of implants placed in <3 mm of RBH, even though it showed a lower CSR, could be considered acceptable. Neither sinus membrane perforation nor the presence of voids appeared to affect implant survival as long as supportive maintenance therapy with proper oral hygiene was provided. K E Y W O R D Sbone regeneration, bone substitutes, guided tissue regeneration, sinus floor elevation, smoking

Lee S‐I, Kang K‐L, Shin S‐I, Herr Y, Lee Y‐M, Kim E‐C. Endoplasmic reticulum stress modulates nicotine‐induced extracellular matrix degradation in human periodontal ligament cells. J Periodont Res 2012; 47: 299–308. © 2012 John Wiley & Sons A/S Background and Objective:  Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine‐induced cytotoxicity and ECM degradation. Material and Methods:  Cytotoxicity and cell death were measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT‐PCR and western blot analysis. Results:  Treatment with nicotine reduced cell viability and increased the proportion of annexin V‐negative, propidium iodide‐positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase‐like ER‐resident kinase, phosphorylated eukaryotic initiation factor‐2α and glucose‐regulated protein‐78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP‐1, MMP‐2, MMP‐8 and MMP‐9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine‐induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor‐κB. Conclusion:  These results indicate that nicotine‐induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.

In our previous studies, we confirmed that low-level laser therapy (LLLT) with a 980-nm gallium-aluminum-arsenide diode laser was beneficial for the healing of the alveolar bone in rats with systemic disease. However, many factors can affect the biostimulatory effects of LLLT. Thus, we attempted to investigate the effects of irradiation time on the healing of extraction sockets by evaluating the expressions of genes and proteins related to bone healing. The left and right first maxillary molars of 24 rats were extracted. Rats were randomly divided into four groups in which extraction sockets were irradiated for 0, 1, 2, or 5 min each day for 3 or 7 days. Specimens containing the sockets were examined using quantitative real-time reverse transcription polymerase chain reaction and western blotting. LLLT increased the expressions of all tested genes, Runx2, collagen type 1, osteocalcin, platelet-derived growth factor-B, and vascular endothelial growth factor, in a time-dependent manner. The highest levels of gene expressions were in the 5-min group after 7 days. Five minutes of irradiation caused prominent increases of the expression of all tested proteins after both 3 and 7 days. The expression level of each protein in group 4 was higher by almost twofold compared with group 1 after 7 days. Laser irradiation for 5 min caused the highest expressions of genes and proteins related to bone healing. In conclusion, LLLT had positive effects on the early stages of bone healing of extraction sockets in rats, which were irradiation time-dependent.

BackgroundOxysterols, oxygenated by-products of cholesterol biosynthesis, play roles in various physiological and pathological systems. However, the effects of oxysterols on periodontal regeneration are unknown. This study investigated the effects of the specific oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on the regeneration of periodontal tissues using in-vitro periodontal ligament stem cells (PDLSCs) and in-vivo models of alveolar bone defect.MethodsTo evaluate the effects of the combined oxysterols on PDLSC biology, we studied the SS-induced osteogenic differentiation of PDLSCs by assessing alkaline phosphatase activity, intracellular calcium levels [Ca2+]i, matrix mineralization, and osteogenic marker mRNA expression and protein levels. To verify the effect of oxysterols on alveolar bone regeneration, we employed tooth extraction bone defect models.ResultsOxysterols increased the osteogenic activity of PDLSCs compared with the control group. The expression of liver X receptor (LXR) α and β, the nuclear receptors for oxysterols, and their target gene, ATP-binding cassette transporter A1 (ABCA1), increased significantly during osteogenesis. Oxysterols also increased protein levels of the hedgehog (Hh) receptor Smo and the transcription factor Gli1. We further confirmed the reciprocal reaction between the LXRs and Hh signaling. Transfection of both LXRα and LXRβ siRNAs decreased Smo and Gli1 protein levels. In contrast, the inhibition of Hh signaling attenuated the LXRα and LXRβ protein levels. Subsequently, SS-induced osteogenic activity of PDLSCs was suppressed by the inhibition of LXRs or Hh signaling. The application of SS also enhanced bone formation in the defect sites of in-vivo models, showing equivalent efficacy to recombinant human bone morphogenetic protein-2.ConclusionsThese findings suggest that a specific combination of oxysterols promoted periodontal regeneration by regulating PDLSC activity and alveolar bone regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0725-9) contains supplementary material, which is available to authorized users.

Grainyhead-Like 2 (GRHL2) is an epithelial-specific transcription factor that regulates epithelial morphogenesis and differentiation. Prior studies suggested inverse regulation between GRHL2 and TGF-β in epithelial plasticity and potential carcinogenesis. Here, we report the role of GRHL2 in oral carcinogenesis in vivo using a novel Grhl2 knockout (KO) mouse model and the underlying mechanism involving its functional interaction with TGF-β signaling. We developed epithelial-specific Grhl2 conditional KO mice by crossing Grhl2 floxed mice with those expressing CreER driven by the K14 promoter. After induction of Grhl2 KO, we confirmed the loss of GRHL2 and its target proteins, while Grhl2 KO strongly induced TGF-β signaling molecules. When exposed to 4-nitroquinoline 1-oxide (4-NQO), a strong chemical carcinogen, Grhl2 wild-type (WT) mice developed rampant oral tongue tumors, while Grhl2 KO mice completely abolished tumor development. In cultured oral squamous cell carcinoma (OSCC) cell lines, TGF-β signaling was notably induced by GRHL2 knockdown while being suppressed by GRHL2 overexpression. GRHL2 knockdown or KO in vitro and in vivo, respectively, led to loss of active p-Erk1/2 and p-JNK MAP kinase levels; moreover, ectopic overexpression of GRHL2 strongly induced the MAP kinase activation. Furthermore, the suppressive effect of GRHL2 on TGF-β signaling was diminished in cells exposed to Erk and JNK inhibitors. These data indicate that GRHL2 activates the Erk and JNK MAP kinases, which in turn suppresses the TGF -β signaling. This novel signaling represents an alternative pathway by which GRHL2 regulates carcinogenesis, and is distinct from the direct transcriptional regulation by GRHL2 binding at its target gene promoters, e.g., E-cadherin, hTERT, p63, and miR-200 family genes. Taken together, the current study provides the first genetic evidence to support the role of GRHL2 in carcinogenesis and the underlying novel mechanism that involves the functional interaction between GRHL2 and TGF-β signaling through the MAPK pathways.