Effects of the multi-channeled oral irrigation (MCOI) unit in preventing dental plaque formation and gingivitis: A randomized controlled trial.

PURPOSE: To evaluate the efficacy of COMORAL a new multi-channeled oral irrigation (MCOI) unit with pulsating water jet, in plaque score reduction and gingivitis. METHODS: This was a single-blinded clinical randomized controlled trial (NCT05031260). Forty-two healthy subjects between 18 to 35 years old were initially recruited, and the control group (n = 20) and the intervention group (n = 17) were randomly assigned. Both groups were asked to brush their teeth one or two times a day without any supplementary oral hygiene products while the intervention group used COMORAL 3 times a day, 5 days a week. Clinical indices including gingival index (GI), plaque index (PI), bleeding on probing (BOP), pocket depth (PD), gingival recession (GR), and clinical attachment loss (CAL) were obtained at the baseline (D0), day 14 (D14), and day 28 (D28). Saliva was collected to examine the presence of periodontal pathogens. The repeated measures analysis of variance or generalized estimating equation was used to compare the interaction between groups and time points. The independent t-test or Mann-Whitney test were used for intergroup differences at each time point. RESULTS: At V0, PI, GI, BOP, and PD scores showed no differences between the two groups. At V1 and V2, these scores showed significant difference between two groups (P < 0.05) such that the intervention group showed gradual decreases while the control group showed no change. There were no differences in GR, CAL, and periodontal pathogens between the two groups. COMORAL showed improvement in reducing gingival inflammation and dental plaque formation adjuvant to routine toothbrushing in healthy adults. CLINICAL SIGNIFICANCE: The results of this study can be useful to clinicians when selecting oral hygiene devices that can help improve patients' routine oral hygiene practice and their overall oral health.

GV1001 Inhibits the Severity of the Ligature-Induced Periodontitis and the Vascular Lipid Deposition Associated with the Periodontitis in Mice.

GV1001, a 16 amino acid peptide derived from the catalytic segment of human telomerase reverse transcriptase, was developed as an anti-cancer vaccine. Subsequently, it was found to exhibit anti-inflammatory and anti-Alzheimer's disease properties. Periodontitis is a risk factor for a variety of systemic diseases, including atherosclerosis, a process in which chronic systemic and vascular inflammation results in the formation of plaques containing lipids, macrophages, foam cells, and tissue debris on the vascular intima. Thus, we investigated the effect of GV1001 on the severity of ligature-induced periodontitis, vascular inflammation, and arterial lipid deposition in mice. GV1001 notably reduced the severity of ligature-induced periodontitis by inhibiting gingival and systemic inflammation, alveolar bone loss, and vascular inflammation in wild-type mice. It also significantly lowered the amount of lipid deposition in the arterial wall in ApoE-deficient mice receiving ligature placement without changing the serum lipid profile. In vitro, we found that GV1001 inhibited the Receptor Activator of NF-κB ligand (RANKL)-induced osteoclast formation and tumor necrosis factor-α (TNF-α)-induced phenotypic changes in endothelial cells. In conclusion, our study suggests that GV1001 prevents the exacerbation of periodontitis and atherosclerosis associated with periodontitis partly by inhibiting local, systemic, and vascular inflammation and phenotypic changes of vascular endothelial cells.

Epithelial cells release IL-36α in extracellular vesicles following mechanical damage.

The epithelium is an integral part of barrier tissues, and plays a critical role in the initiation of the innate immune responses. The pro-inflammatory cytokine IL-36α has been previously reported to be strongly expressed during oral mucosal wound healing, but regulation of IL-36α expression and secretion in the oral mucosa are not well known. The objective of this study was to determine the types of stimuli that lead to expression and secretion of IL-36α in epithelial cells. Maxillary tissues from C57BL/6J mice during wound healing were utilized to identify endogenous expression of IL-36α, ß, and γ in oral epithelial tissue. Immortalized HaCaT cells and primary normal human oral keratinocytes were subjected to Escherichia coli derived lipopolysaccharide (LPS), Poly(I:C), heat killed Candida albicans (HKCa), and mechanical damage. IL-36α and IL-1ß levels in supernatant were assessed by sandwich ELISA, and expression of pro-inflammatory cytokines and IL-36 family genes were assessed by quantitative real-time PCR in HaCaT cells. Migration ability of keratinocytes was assessed with or without functional IL-36 signaling. IL-36α but not IL-36ß or γ levels in the oral epithelium were elevated during wound healing. Treatment of epithelial cells with LPS, Poly(I:C), HKCa and mechanical damage revealed little to no soluble IL-36α in the media supernatant. However, sonication of the supernatant to disrupt the membranes of extracellular vesicles revealed a dose-dependent increase in IL-36α for each of the tested conditions. IL-1 superfamily genes were upregulated following mechanical damage in keratinocytes. Abrogation of IL-36 signaling led to severe inhibition of migration. Our data show for the first time that IL-36α is released primarily in extracellular vesicles by oral keratinocytes. Additionally, we show that IL-36α - but not IL-36ß or γ - is upregulated in keratinocytes following mechanical damage, and that IL-36 signaling is important for keratinocyte migration.

DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

DYRK1A, one of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), plays an important role in various biological processes by regulating downstream targets via kinase-dependent and independent mechanisms. Here, we report a novel role of DYRK1A in maintaining tumor growth and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. Deletion of DYRK1A from OSCC cells abrogated their in vivo tumorigenicity and self-renewal capacity, the key features of cancer stem-like cells (CSCs; also referred to as tumor-initiating cells). The DYRK1A deletion also induced the suppression of CSC populations and properties, such as migration ability and chemoresistance. Conversely, ectopic expression of DYRK1A in OSCC cells augmented their CSC phenotype. Among five DYRK members (DYRK1A, 1B, 2, 3, and 4), DYRK1A is the most dominantly expressed kinase, and its expression is upregulated in OSCC compared to normal oral epithelial cells. More importantly, DYRK1A was highly enriched in various CSC-enriched OSCC populations compared to their corresponding non-CSC populations, indicating its pivotal role in cancer progression and stemness. Further, our study revealed that fibroblast growth factor 2 (FGF2) is a key regulator in the DYRK1A-mediated CSC regulation. Functional studies demonstrated that the loss of DYRK1A inhibits CSC phenotype via reduction of FGF2. Overexpression of DYRK1A promotes CSC phenotype via upregulation of FGF2. Our study delineates a novel mechanism of cancer stemness regulation by DYRK1A-FGF2 axis in OSCC. Thus, inhibition of DYRK1A would lead to a potential novel therapeutic option for targeting CSCs in OSCC.

Chronic Alcohol Exposure Promotes Cancer Stemness and Glycolysis in Oral/Oropharyngeal Squamous Cell Carcinoma Cell Lines by Activating NFAT Signaling.

Alcohol consumption is associated with an increased risk of several cancers, including oral/oropharyngeal squamous cell carcinoma (OSCC). Alcohol also enhances the progression and aggressiveness of existing cancers; however, its underlying molecular mechanism remains elusive. Especially, the local carcinogenic effects of alcohol on OSCC in closest contact with ingestion of alcohol are poorly understood. We demonstrated that chronic ethanol exposure to OSCC increased cancer stem cell (CSC) populations and their stemness features, including self-renewal capacity, expression of stem cell markers, ALDH activity, and migration ability. The ethanol exposure also led to a significant increase in aerobic glycolysis. Moreover, increased aerobic glycolytic activity was required to support the stemness phenotype of ethanol-exposed OSCC, suggesting a molecular coupling between cancer stemness and metabolic reprogramming. We further demonstrated that chronic ethanol exposure activated NFAT (nuclear factor of activated T cells) signaling in OSCC. Functional studies revealed that pharmacological and genetic inhibition of NFAT suppressed CSC phenotype and aerobic glycolysis in ethanol-exposed OSCC. Collectively, chronic ethanol exposure promotes cancer stemness and aerobic glycolysis via activation of NFAT signaling. Our study provides a novel insight into the roles of cancer stemness and metabolic reprogramming in the molecular mechanism of alcohol-mediated carcinogenesis.

Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Removal of Pre-Existing Periodontal Inflammatory Condition before Tooth Extraction Ameliorates Medication-Related Osteonecrosis of the Jaw-Like Lesion in Mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but detrimental intraoral lesion that predominantly occurs in patients with long-term use of antiresorptive agents, such as bisphosphonate and denosumab, a human anti-receptor activator of NF-κB ligand (RANKL) monoclonal antibody (Ab). Surgical intervention, such as tooth extraction, is a known risk factor for MRONJ, which is often performed to eliminate preexiting pathologic inflammatory conditions, such as periodontal diseases. Nonetheless, it remains unknown whether pre-existing periodontal disease condition exacerbates, or removal of such condition ameliorates, MRONJ development after tooth extraction. In this study, we combined the ligature-induced periodontitis and the tooth extraction mouse models under the administration of zoledronic acid (ZOL) or anti-RANKL Ab, and provide experimental evidence that a pre-existing pathologic inflammatory condition exacerbates MRONJ development after tooth extraction in mice. Under ZOL administration, tooth extraction alone induced ONJ lesions; however, extraction of a ligature-placed tooth further exacerbated ONJ development. When the ligature was removed and the inflammatory condition was deescalated, ONJ development was ameliorated. Anti-RANKL Ab administration resulted in similar outcomes. Interestingly, unlike ZOL-administered mice, anti-RANKL Ab-administered mice exhibited complete absence of osteoclasts, suggesting that physical presence of osteoclasts is not directly involved in ONJ development. Collectively, our study demonstrated that periodontal disease is a functionally linked risk factor that predisposes ONJ development after tooth extraction in the presence of bisphosphonate and denosumab.

Modeling the Etiology of p53-mutated Cancer Cells.

p53 gene mutations are among the most common alterations in cancer. In most cases, missense mutations in one TP53 allele are followed by loss-of-heterozygosity (LOH), so tumors express only mutant p53. TP53 mutations and LOH have been linked, in many cases, with poor therapy response and worse outcome. Despite this, remarkably little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. Nutlin-3a occupies the p53-binding site in MDM2 and blocks p53-MDM2 interaction, resulting in the stabilization and activation of p53 and subsequent growth arrest or apoptosis. We leveraged the powerful growth inhibitory activity of Nutlin-3a to select p53-mutated cells and examined how TP53 mutations arise and how the remaining wild-type allele is lost or inactivated. Mismatch repair (MMR)-deficient colorectal cancer cells formed heterozygote (p53 wild-type/mutant) colonies when cultured in low doses of Nutlin-3a, whereas MMR-corrected counterparts did not. Placing these heterozygotes in higher Nutlin-3a doses selected clones in which the remaining wild-type TP53 was silenced. Our data suggest silencing occurred through a novel mechanism that does not involve DNA methylation, histone methylation, or histone deacetylation. These data indicate MMR deficiency in colorectal cancer can give rise to initiating TP53 mutations and that TP53 silencing occurs via a copy-neutral mechanism. Moreover, the data highlight the use of MDM2 antagonists as tools to study mechanisms of TP53 mutation acquisition and wild-type allele loss or silencing in cells with defined genetic backgrounds.

Elevated expression of JMJD6 is associated with oral carcinogenesis and maintains cancer stemness properties.

Cancer stem cells (CSCs) are defined as a small subpopulation of cancer cells within a tumor and responsible for initiation and maintenance of tumor growth. Thus, understanding of molecular regulators of CSCs is of paramount importance for the development of effective cancer therapies. Here, we identified jumonji domain-containing protein 6 (JMJD6) as a novel molecular regulator of oral CSCs. JMJD6 is highly expressed in CSC-enriched populations of human oral squamous cell carcinoma (OSCC) cell lines. Moreover, immunohistochemical staining revealed significantly high level of JMJD6 in OSCC tissues compared to normal human oral epithelia, suggesting that expression of JMJD6 positively correlates with oral carcinogenesis. Subsequent functional analysis showed that knockdown of endogenous JMJD6 in OSCC strongly suppressed self-renewal capacity, a key characteristic of CSCs, and anchorage-independent growth. Conversely, ectopic expression of JMJD6 enhanced CSC characteristics including self-renewal, ALDH1 activity, migration/invasion and drug resistance. Expression of CSC-related genes was also markedly affected by modulating JMJD6 expression. Mechanistically, JMJD6 induces interleukin 4 (IL4) transcription by binding to its promoter region. IL4 rescues self-renewal capacity in JMJD6- knocked down OSCC cells, suggesting the importance of JMJD6-IL4 axis in oral CSCs. Our studies identify JMJD6 as a molecular determinant of CSC phenotype, suggesting that inhibition of JMJD6 may offer an effective therapeutic modality against oral cancer.

Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells.

Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs.

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

In this study, we investigated the effects of p63 modulation in epithelial plasticity in human keratinocytes. The p63 isoforms ΔNp63α, ΔNp63ß, 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 ΔNp63ß 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.

Orai1 mediates osteogenic differentiation via BMP signaling pathway in bone marrow mesenchymal stem cells.

Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1(-/-)) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1(-/-) BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1(-/-) BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1(-/-) BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.

Impaired bone resorption and woven bone formation are associated with development of osteonecrosis of the jaw-like lesions by bisphosphonate and anti-receptor activator of NF-κB ligand antibody in mice.

Drug-induced osteonecrosis of the jaw (ONJ) is a detrimental intraoral lesion that often occurs after dental-related interventions in patients undergoing treatment with bisphosphonates or denosumab, the neutralizing human anti-receptor activator of NF-κB ligand (RANKL) antibody (Ab). The cause of ONJ by these drugs has been speculated to their direct effects on osteoclasts. However, the extent to which osteoclasts contribute to ONJ pathogenesis remains controversial. Herein, by using a tooth-extraction mouse model with i.v. administration of mouse anti-RANKL Ab or the bisphosphonate zoledronate (ZOL), we show that unresorbed bone due to impaired formation or suppressed functions of osteoclasts, respectively, is associated with ONJ development. After tooth extraction, ONJ-like lesions developed 50% in the anti-RANKL Ab-treated mice and 30% in the ZOL-treated mice. Nonviable and unresorbed bone was found more in anti-RANKL Ab-treated mice compared with mice receiving ZOL. All mice receiving anti-RANKL Ab had an undetectable tartrate-resistant acid phosphatase (TRAP) level in the serum and no TRAP-positive osteoclasts at the extracted sockets, whereas ZOL-treated mice had a decreased TRAP level without altering the numbers of TRAP-positive osteoclasts. Interestingly, the absence of newly formed woven bone in the extracted sockets was evident in ONJ-like lesions from both anti-RANKL Ab- and ZOL-treated mice. Our study suggests that the lack of osteoclasts' bone-resorptive functions by these drugs and suppression of woven bone formation after dental trauma may be associated with ONJ development.

The maternal microbiome promotes placental development in mice.

The maternal microbiome is an important regulator of gestational health, but how it impacts the placenta as the interface between mother and fetus remains unexplored. Here we show that the maternal gut microbiota supports placental development in mice. Depletion of the maternal gut microbiota restricts placental growth and impairs feto-placental vascularization. The maternal gut microbiota modulates metabolites in the maternal and fetal circulation. Short-chain fatty acids (SCFAs) stimulate angiogenesis-related tube formation by endothelial cells and prevent abnormalities in placental vascularization in microbiota-deficient mice. Furthermore, in a model of maternal malnutrition, gestational supplementation with SCFAs prevents placental growth restriction and vascular insufficiency. These findings highlight the importance of host-microbial symbioses during pregnancy and reveal that the maternal gut microbiome promotes placental growth and vascularization in mice.

The maternal microbiome promotes placental development in mice.

The maternal microbiome is an important regulator of gestational health, but how it affects the placenta as the interface between mother and fetus remains unexplored. Here, we show that the maternal gut microbiota supports placental development in mice. Depletion of the maternal gut microbiota restricts placental growth and impairs feto-placental vascularization. The maternal gut microbiota modulates metabolites in the maternal and fetal circulation. Short-chain fatty acids (SCFAs) stimulate cultured endothelial cell tube formation and prevent abnormalities in placental vascularization in microbiota-deficient mice. Furthermore, in a model of maternal malnutrition, gestational supplementation with SCFAs prevents placental growth restriction and vascular insufficiency. These findings highlight the importance of host-microbial symbioses during pregnancy and reveal that the maternal gut microbiome promotes placental growth and vascularization in mice.

Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.

DeltaNp63α protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.

p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ΔNp63α, a p63 isoform lacking the N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in a TGF-ß-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ΔNp63α or empty vector and serially subcultured until replicative senescence. No phenotypic changes were observed until the culture reached senescence. Then the ΔNp63α-transduced cells underwent morphological changes resembling mesenchymal cells and acquired the EMT phenotype. Treatment with exogenous TGF-ß accelerated EMT in presenescent ΔNp63α-transduced cells, whereas the inhibition of TGF-ß signaling reversed the EMT phenotype. TGF-ß treatment alone led to growth arrest in control NHEK with no evidence of EMT, indicating that ΔNp63α altered the cellular response to TGF-ß treatment. ΔNp63α-transduced cells acquiring EMT gained the ability to be differentiated to osteo-/odontogenic and adipogenic pathways, resembling mesenchymal stem cells. Furthermore, these cells expressed enhanced levels of Nanog and Lin28, which are transcription factors associated with pluripotency. These data indicate that EMT required ΔNp63α transduction and intact TGF-ß signaling in NHEK.

TNFα enhances cancer stem cell-like phenotype via Notch-Hes1 activation in oral squamous cell carcinoma cells.

Cancer stem-like cell (CSC; also known as tumor initiating cell) is defined as a small subpopulation of cancer cells within a tumor and isolated from various primary tumors and cancer cell lines. CSCs are highly tumorigenic and resistant to anticancer treatments. In this study, we found that prolonged exposure to tumor necrosis factor alpha (TNFα), a major proinflammatory cytokine, enhances CSC phenotype of oral squamous cell carcinoma (OSCC) cells, such as an increase in tumor sphere-forming ability, stem cell-associated genes expression, chemo-radioresistance, and tumorigenicity. Moreover, activation of Notch1 signaling was detected in the TNFα-exposed cells, and suppression of Notch1 signaling inhibited CSC phenotype. Furthermore, we demonstrated that inhibition of a Notch downstream target, Hes1, led to suppression of CSC phenotype in the TNFα-exposed cells. We also found that Hes1 expression is commonly upregulated in OSCC lesions compared to precancerous dysplastic lesions, suggesting the possible involvement of Hes1 in OSCC progression and CSC in vivo. In conclusion, inflammatory cytokine exposure may enhance CSC phenotype of OSCC, in part by activating the Notch-Hes1 pathway.

The Effect of the Amnion-Chorion or Collagen Membrane as a Matrix on the Microenvironment During a Regenerative Endodontic Procedure.

INTRODUCTION: During regenerative endodontic procedures, the microenvironment of the canal is formed by the degree of disinfection and release of ions from the applied materials onto the top surface. This study aimed to characterize the effects of amnion-chorion membrane and collagen membrane on pulp-dentin regeneration compared to calcium silicate cements (CSCs), focusing on cell migration, mineralization potential, anti-inflammation, and angiogenesis. METHODS: Two CSCs and 2 membranes were used: ProRoot MTA (Dentsply, Tulsa, OK, USA), RetroMTA (BioMTA, Seoul, Korea), Collagen Membrane (Genoss, Suwon, Korea), and BioXclude (amnion-chorion membrane; Snoasis Medical, Colorado, USA). Transwell and scratch assays were used to evaluate cell migration and wound healing. Mineralization potential was evaluated using alkaline phosphatase activity, Alizarin red S staining, and quantitative real-time polymerase chain reaction for the expression of marker genes. Quantitative real-time polymerase chain reaction was used to measure the levels of angiogenic genes and inflammatory mediators. An endothelial tube formation assay was used to assess angiogenesis. RESULTS: The membranes showed superior migration and wound healing compared with CSCs. Except for RetroMTA, ProRoot MTA and the 2 membranes showed high alkaline phosphatase activity and mineralization nodule formation and upregulated mRNA expression of markers for mineralization. Membranes upregulated the mRNA of angiogenesis genes and increased the capillary tube formation of endothelial cells compared to CSCs. Furthermore, the membrane matrix decreased the expression of inflammatory genes. CONCLUSIONS: Collagen membrane and Amnion-chorion membrane showed prominent cell migration, angiogenesis, and healing effects against inflammation, as well as comparable mineralization potential compared to CSCs, recommending the use of membrane as a matrix.

Long-Term Ligature-Induced Periodontitis Exacerbates Development of Bisphosphonate-Related Osteonecrosis of the Jaw in Mice.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a detrimental intraoral lesion that occurs in patients with long-term or high-dose use of anti-resorptive agents such as bisphosphonates. Tooth extraction is a known risk factor for BRONJ, and such intervention is often performed to eliminate existing pathological inflammatory conditions. Previously, we determined that ligature-induced periodontitis (LIP) is a risk factor for the development of osteonecrosis in mice, but it remains unclear whether the chronicity of LIP followed by extraction influences osteonecrosis development. In this study, we assess the effect of short-term and long-term LIP (ligature placed for 3 weeks [S-LIP] or 10 weeks [L-LIP], respectively) on osteonecrosis development in mice receiving 250 µg/kg/week zoledronic acid (ZOL). When compared to S-LIP, L-LIP caused 70% (p ≤ 0.0014) more bone loss without altering microbe composition. In the presence of ZOL, bone loss mediated by LIP was prevented and bone necrosis was induced. When the ligated tooth was extracted, histologic hallmarks of osteonecrosis including empty lacunae and necrotic bone were increased by 88% (p = 0.0374) and 114% (p = 0.0457), respectively, in L-LIP compared to S-LIP. We also observed significant increases in serum platelet factor 4 (PF4) and macrophage inflammatory factor 1 γ (MIP1γ) in mice that received ZOL treatment and had tooth extractions compared to controls, which may be systemic markers of inflammation-associated osteonecrosis development. Additionally, CD3+ T cells were identified as the major immune population in both health and disease, and we observed a 116% (p = 0.0402) increase in CD3+IL23R+ T cells in L-LIP compared to S-LIP lesions following extraction. Taken together, our study reveals that extracting a periodontally compromised tooth increases the formation of necrotic bone compared to extracting a periodontally healthy tooth and that osteonecrosis may be associated with the duration of the preexisting pathological inflammatory conditions. © 2022 American Society for Bone and Mineral Research (ASBMR).