Kyung Lhi Kang

Hesperetin Alleviates the Inhibitory Effects of High Glucose on the Osteoblastic Differentiation of Periodontal Ligament Stem Cells

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.

Differences in implant stability associated with various methods of preparation of the implant bed: An in vitro study

STATEMENT OF PROBLEM It is difficult to achieve the primary stability necessary for immediate loading in the posterior maxilla because of thin cortical bone, low density trabecular bone, and inadequate bone height due to the presence of the maxillary sinus. PURPOSE The purpose of this study was to examine the primary stability of dental implants placed by using different methods of preparation for in vitro monocortical and bicortical models of the posterior maxilla. MATERIAL AND METHODS Sixty screw-shaped implants (4.0 × 10 mm) were inserted into solid rigid polyurethane blocks. The implants were divided into 6 groups (n=10) to test 2 variables: 1) location (monocortical or bicortical block) and 2) preparation method (standard preparation, underpreparation, or the osteotome technique). The insertion and removal torques were measured and resonance frequency analysis (RFA) was performed to determine the primary stability of each implant. Insertion and removal torque data were analyzed by 2-way ANOVA, followed by the post hoc Tukey HSD multiple comparison test. RFA data were analyzed by 2-way and 1-way ANOVAs and the Tukey HSD multiple comparison test (α=.05). The Pearson correlation analysis was also performed to examine correlations among the values. RESULTS The preparation method had a significant effect on insertion torque, RFA value, and removal torque; however location had a significant effect only on the removal torque (P<.001). There was a significant interaction between location and preparation method for RFA values (P=.045) and a significant difference in standard preparation method according to the location (P=.039); however, there was no significant difference in underpreparation (P=1.00) and osteotome technique (P=1.00). Statistically significant correlations were found between insertion torque and RFA values (r=0.529, P< .001), insertion torque and removal torque values (r=0.517, P< .001), and removal torque and RFA values (r=0.481, P<.001). CONCLUSIONS Underpreparation and bicortical fixation significantly increased implant stability and the osteotome technique decreased implant stability in synthetic bone models that mimicked the posterior maxillary region. The primary stability values had statistically significant correlations to each other.

Simvastatin promotes osteogenic differentiation of mouse embryonic stem cells via canonical Wnt/β-catenin signaling

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, has been known to reduce cholesterol biosynthesis. However, recent studies demonstrate that simvastatin shows diverse cholesterol-independent functions including cellular differentiation. In this study, we investigated the stimulatory effect of simvastatin on the osteogenic differentiation of mouse embryonic stem cells (ESCs). The osteogenic effect of simvastatin was observed at relatively low doses (ranging from 1 nM to 200 nM). Incubation of ESCs in simvastatin-supplemented osteogenic medium significantly increased alkaline phosphatase (ALP) activity at day 7. The matrix mineralization was also augmented and demonstrated pivotal levels after 14 days incubation of simvastatin. Osteogenic differentiation of ESCs by simvastatin was determined by upregulation of the mRNA expression of runtrelated gene 2 (Runx2), osterix (OSX), and osteocalcin (OCN) as osteogenic transcription factors. Moreover, the increased protein expression of OCN, osteopontin (OPN), and collagen type I (Coll I) was assessed using Western blot analysis and immunocytochemistry. However, the blockage of canonical Wnt signaling by DKK-1 downregulated simvastatin-induced ALP activity and the mRNA expression of each osteogenic transcription factor. Furthermore, the β-catenin specific siRNA transfection decreased the protein levels of OCN, OPN, and Coll I. Collectively, these findings suggest that simvastatin enhances the differentiation of ESCs toward osteogenic lineage through activation of canonical Wnt/β-catenin signaling.

Nicotinic acetylcholine receptor α7 and β4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells

Nicotine, a major component of cigarette smoking, is the important risk factor for the development of periodontal disease. However, the mechanisms that underlie the cytotoxicity of nicotine in human periodontal ligament stem cells (PDLSCs) are largely unknown. Thus, the purpose of this study was to determine the cytotoxic effect of nicotine by means of nicotinic acetylcholine receptor (nAChR) activation in PDLSCs. We first detected α7 and β4 nAChRs in PDLSCs. The gene expressions of α7 and β4 nAChR were increased by nicotine administration. Nicotine significantly decreased cell viability at a concentration higher than 10−5 M. DNA fragmentation was also detected at high doses of nicotine treatment. Moreover, the detection of sub G1 phase and TUNEL assay demonstrated that nicotine significantly induced apoptotic cell death at 10−2 M concentration. Western blot analysis confirmed that p53 proteins were phosphorylated by nicotine. Under various doses of nicotine, a decrease in the anti-apoptotic protein Bcl-2, but an increase in p53 and cleaved caspase-3 protein levels, was detected in a dose-dependent manner. However, the apoptotic effect of nicotine was inhibited by the pretreatment of α-bungarotoxin, a selective α7 nAChR antagonist or mecamylamine, a non-selective nAChR antagonist. Finally, increases in the subG1 phase and DNA fragmentation by nicotine was attenuated by each nAChR antagonist. Collectively, the presence of α7 and β4 nAChRs in PDLSCs supports a key role of nAChRs in the modulation of nicotine-induced apoptosis.

The p63 Gene Is Regulated by Grainyhead-like 2 (GRHL2) through Reciprocal Feedback and Determines the Epithelial Phenotype in Human Keratinocytes

Background: The p63 isoforms ΔNp63α, ΔNp63β, ΔNp63γ, and Grainyhead-like 2 (GRHL2) play distinct roles in regulating the epithelial phenotype. Results: p63 modulation leads to epithelial-mesenchymal transition in human keratinocytes, and GRHL2 binds directly to the p63 promoter. Conclusion: GRHL2/p63 reciprocal regulation maintains the epithelial phenotype and plasticity. Significance: The GHRL2/p63 model is crucial for understanding epithelial plasticity and metastasis, epithelial wound healing, and tissue regeneration. In this study, we investigated the effects of p63 modulation in epithelial plasticity in human keratinocytes. The p63 isoforms ΔNp63α, ΔNp63β, and ΔNp63γ were ectopically expressed in normal human epidermal keratinocytes (NHEKs). The epithelial or mesenchymal state was determined by morphological changes and altered expression of various markers, e.g. fibronectin, E-Cadherin, and keratin 14. Overexpression of ΔNp63α and ΔNp63β but not ΔNp63γ isoforms led to morphological changes consistent with epithelial-mesenchymal transition (EMT). However, only ΔNp63α overexpression was able to maintain the morphological changes and molecular phenotype consistent with EMT. Interestingly, knockdown of all p63 isoforms by transfection of p63 siRNA also led to the EMT phenotype, further confirming the role of p63 in regulating the epithelial phenotype in NHEKs. EMT in NHKs accompanied loss of Grainyhead-Like 2 (GHRL2) and miR-200 family gene expression, both of which play crucial roles in determining the epithelial phenotype. Modulation of GRHL2 in NHKs also led to congruent changes in p63 expression. ChIP revealed direct GRHL2 binding to the p63 promoter. GRHL2 knockdown in NHK led to impaired binding of GRHL2 and changes in the histone marks consistent with p63 gene silencing. These data indicate the presence of a reciprocal feedback regulation between p63 and GRHL2 in NHEKs to regulate epithelial plasticity.

Evaluating the oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol in periodontal regeneration using periodontal ligament stem cells and alveolar bone healing models

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.

Extensive gingival necrosis and sequestration of the alveolar bone caused by methimazole-induced neutropenia and three-year follow-up.

Purpose Methimazole is an anti-thyroid drug that can cause life-threatening neutropenia in rare situations. The aim of this case report is to describe a set of oral complications associated with methimazole-induced neutropenia and the healing of the gingiva after proper treatment. Methods A 31-year-old female patient hospitalized for systemic symptoms of sore throat and fever and showing extensive gingival necrosis with pain was referred to the Department of Periodontics from the Department of Endocrinology. Methimazole-induced neutropenia was diagnosed based on blood test results and her medical history. Methimazole was discontinued and a range of treatments was administered, including the injection of granulocyte colony stimulating factor. Results After systemic treatment, the gingiva began to heal as the neutrophil count increased. Approximately one year later, the gingiva had returned to a normal appearance. Twenty-one months after treatment, sequestra of the alveolar bone that had broken through the gingiva were removed. Periodic supportive periodontal treatment has been continued uneventfully. Conclusions The oral manifestations of gingival necrosis and ulcerations, in combination with systemic symptoms such as fever and sore throat, are the critical signs presented in the early stages of drug-induced neutropenia. Therefore, dentists need to be aware of these oral complications in order to make an accurate diagnosis and to ensure that prompt medical intervention is provided. Graphical Abstract