TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Tumor gene signatures that correlate with release of extracellular vesicles shape the immune landscape in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) evade immune responses through multiple resistance mechanisms. Extracellular vesicles (EVs) released by the tumor and interacting with immune cells induce immune dysfunction and contribute to tumor progression. This study evaluates the clinical relevance and impact on anti-tumor immune responses of gene signatures expressed in HNSCC and associated with EV production/release. Expression levels of two recently described gene sets were determined in The Cancer Genome Atlas Head and Neck Cancer cohort (n = 522) and validated in the GSE65858 dataset (n = 250) as well as a recently published single-cell RNA sequencing dataset (n = 18). Clustering into HPV(+) and HPV(-) patients was performed in all cohorts for further analysis. Potential associations between gene expression levels, immune cell infiltration, and patient overall survival were analyzed using GEPIA2, TISIDB, TIMER, and the UCSC Xena browser. Compared to normal control tissues, vesiculation-related genes were upregulated in HNSCC cells. Elevated gene expression levels positively correlated (P < 0.01) with increased abundance of CD4(+) T cells, macrophages, neutrophils, and dendritic cells infiltrating tumor tissues but were negatively associated (P < 0.01) with the presence of B cells and CD8(+) T cells in the tumor. Expression levels of immunosuppressive factors NT5E and TGFB1 correlated with the vesiculation-related genes and might explain the alterations of the anti-tumor immune response. Enhanced expression levels of vesiculation-related genes in tumor tissues associates with the immunosuppressive tumor milieu and the reduced infiltration of B cells and CD8(+) T cells into the tumor.

Impact of PIEZO1-channel on inflammation and osteoclastogenesis mediated via periodontal ligament fibroblasts during mechanical loading.

The identification of mechanosensitive ion channels and their importance in innate immunity provides new starting points to elucidate the molecular mechanisms of orthodontic tooth movement. The mechanosensitive electron channel PIEZO1 (Piezo Type Mechanosensitive Ion Channel Component 1) may play a crucial role in orthodontic tooth movement. To investigate the role of the PIEZO1 channel, periodontal ligament fibroblasts (PDLF) were subsequently treated with a PIEZO1 inhibitor (GsMTx) with simultaneous pressure application or with an activator (JEDI2) without mechanical strain. The expression of genes and proteins involved in orthodontic tooth movement was examined by RT-qPCR, Western blot and ELISA. In addition, the effect on PDLF-mediated osteoclastogenesis was investigated in a coculture model using human monocytes. Inhibition of PIEZO1 under pressure application caused a reduction in RANKL (receptor activator of NF-kB ligand) expression, resulting in decreased osteoclastogenesis. On the other hand, activation of PIEZO1 without mechanical strain downregulated OPG (osteoprotegerin), resulting in increased osteoclastogenesis. PIEZO1 appears to play a role in the induction of inflammatory genes. It was also shown to influence osteoclastogenesis.

An Evaluation of Different 3D Cultivation Models on Expression Profiles of Human Periodontal Ligament Fibroblasts with Compressive Strain.

The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT-qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.

Influence of Single-Nucleotide Polymorphisms on Vitamin D Receptor Expression in Periodontal Ligament Fibroblasts as a Response to Orthodontic Compression.

This study aimed to evaluate if single-nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene are associated with gene expression in human periodontal ligament (hPDL) fibroblasts under simulated orthodontic compressive force. hPDL samples from 57 patients were used. A physiological compressive strain was performed to simulate orthodontic tooth movement in pressure areas under cell culture conditions. The RNA from hPDL fibroblasts was isolated to determine the relative gene expression (mRNA) of the VDR. The DNA was also isolated for the genotyping analysis of five SNPs in the VDR gene: BglI (rs739837, G/T), BsmI (rs1544410, T/C), ApaI (rs7975232, A/C), FokI (rs2228570, A/G), and TaqI (rs731236, A/G). Real-time polymerase chain reaction was used for both analyses. Kruskal−Wallis tests were used to compare VDR expression among genotypes of each SNP. A linear regression analysis was performed to evaluate SNP−SNP interaction. An established alpha of 5% was used. The relative mRNA VDR expression according to the genotypes in the SNPs BglI, BsmI, ApaI, FokI, and TaqI was not statistically significantly different (p > 0.05). The SNP−SNP interaction evaluated by regression analysis did not demonstrate any statistically significant association. No association was observed (p > 0.05). In conclusion, the SNPs BglI (rs739837), BsmI (rs1544410), ApaI (rs7975232), FokI (rs2228570), and TaqI (rs731236) did not show an impact on VDR gene expression in hPDL fibroblasts under simulated orthodontic compressive force.

Impact of melatonin on periodontal ligament fibroblasts during mechanical strain.

BACKGROUND: The endogenous hormone melatonin regulates the circadian rhythm and impacts on bone metabolism. As patient compliance to wear removable orthodontic appliances is generally higher at night, when melatonin release is increased, a boosting effect on tooth movement would be favourable for therapy, whereas an inhibiting effect would indicate daytime wear to be more therapy-effective. We hypothesize that melatonin has either a stimulating or impeding effect on the expression profile of periodontal ligament fibroblasts (PDLF) during simulated orthodontic compressive and tensile strain, which would suggest either an accelerating or inhibiting impact on orthodontic tooth movement in vivo. METHODS: PDLF were preincubated with melatonin for 24 h and then subjected to tensile or compressive strain to mimic tension and pressure sides in PDL. In addition, the selective melatonin MTNR1B-receptor antagonist 4P-PDOT was used. We investigated melatonin effects on collagen synthesis, expression of inflammatory and bone-remodelling genes/proteins by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and total collagen assays. PDLF-induced osteoclastogenesis was analysed in a coculture model by tartrate-resistant acid phosphatise (TRAP) staining. RESULTS: Expression of melatonin receptors in PDLF was not affected by compressive strain. Melatonin increased expression of inflammatory factors and elevated collagen synthesis during mechanical strain. Melatonin showed no effects on OPG or RANKL expression without mechanical strain, but increased RANKL gene expression during compression. CONCLUSIONS: Expression of melatonin receptors by PDLF enable them to detect fluctuating melatonin concentrations in the periodontal ligament. Melatonin increased collagen synthesis and expression of inflammatory mediators, but had no effect on genes involved in bone remodelling. Therefore, we suggest that melatonin has no accelerating effect on PDLF-induced osteoclastogenesis.

The first quantitative histomorphological analyses of bone vitality and inflammation in surgical specimens of patients with medication-related osteonecrosis of the jaw.

BACKGROUND: The purpose of the study was to categorize the vitality and inflammation of resected bone of patients with medication-related osteonecrosis of the jaw (MRONJ) and to correlate the grade of inflammation with the surgical success. METHODS: This prospective study includes 44 patients with stage III MRONJ. Necrotic bone was resected in a block fashioned way. After demineralization and staining, histological analyses were performed by measuring the areas of necrotic, vital, and regenerative bone. Areas of chronic and acute inflammation were categorized as non, mild, moderate, and severe and were correlated with surgical success and parameters of inflammation in blood plasma (C-reactive protein and leukocytes). RESULTS: An average area of 59.0% was necrotic in the examined specimen. Vital bone was measured with an average area of 40.9%. The stage of chronic inflammation correlated with the amount of vital bone (P < .001) and the success of surgery (P = .002). If acute inflammation was dominant, chronic inflammation areas were found less while necrotic areas were observed more (P < .001). Also, the risk of relapses, wound healing disorders, and the level of C-reactive protein were elevated if acute inflammation was severe or moderate (P = .031). Areas of bone regeneration were seen only in 11.3% of vital bone areas and occurred independently of infection stages. CONCLUSION: If possible, surgery should be delayed in patients with signs of severe acute inflammation. Patients may profit from prolonged pre-operative antibiotic therapy to reduce the level of acute inflammation.

Are annoyance scores based on sound pressure levels suitable for snoring assessment in the home environment?

PURPOSE: An objective statement about the annoyance of snoring can be made with the Psychoacoustic Snore Score (PSS). The PSS was developed based on subjective assessments and is strongly influenced by observed sound pressure levels. Robustness against day-to-day interfering noises is a fundamental requirement for use at home. This study investigated whether or not the PSS is suitable for use in the home environment. METHODS: Thirty-six interfering noises, which commonly occur at night, were played in the acoustic laboratory in parallel with 5 snoring sounds. The interfering noises were each presented at sound pressure levels ranging from 25 to 55 dB(A), resulting in 3255 distinct recordings. Annoyance was then assessed using the PSS. RESULTS: In the case of minimally annoying snoring sounds, interfering noises with a sound pressure level of 25 dB(A) caused significant PSS changes from 40 to 55 dB(A) for annoying snoring sounds. If the interfering noise was another snoring sound, the PSS was more robust depending on the sound pressure level of the interfering noise up to 10 dB(A). Steady (no-peak) interfering noises influenced the PSS more strongly than peak noises. CONCLUSIONS: The PSS is significantly distorted by quiet interfering noises. Its meaningfulness therefore depends strongly on the acoustic environment. It may therefore be assumed that scores dependent on sound pressure level are suitable for measurements when there is minimal ambient noise, as in the sleep laboratory. However, for measurements where noise is incalculable, as in the home environment, interfering noises may distort the results.

Staging more important than grading? Evaluation of malignancy grading, depth of invasion, and resection margins in oral squamous cell carcinoma.

OBJECTIVES: The present study evaluated the predictive value of staging and grading parameters concerning the presence of lymph-node metastases, overall survival (OS), and relapse-free survival (RFS) of patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: HE-stains of 135 surgically treated (R0) primary OSCCs were analyzed using a both microscopic and software-based approach. Depth of invasion (DOI) and resection margins (RM) were measured, and each case was graded according to the malignancy grading system as described by Anneroth et al. and Bryne et al. on two different sites of the tumor (surface and invasion front; TS and IF). RESULTS: Parameters that could be identified as significant predictors of OS and RFS were UICC cancer stage (p = 0.009 and p = 0.012); pT-stage as defined in the 7th edition (p = 0.029 and 0.015) and, after restaging using DOI, 8th edition (p = 0.023 and p = 0.005) of the TNM classification of malignant tumors; the presence of lymphonodular metastases (LM) (p = 0.004 and p = 0.011); degree of keratinization (p = 0.029 and p = 0.042); and pattern of growth (p = 0.029 and p = 0.024) at the TS after applying a binary scale for both parameters. Also, when directly comparing the most extreme subgroups (scores 1 and 4) of lymphoplasmacytic infiltration at the IF, there was a significant difference in OS (p = 0.046) and RFS (p = 0.005). Invasion of blood vessels (p = 0.013) and perineural invasion (p = 0.023) were significantly associated with a lower OS. Age lower than 60 years (univariate p = 0.029, multivariate p = 0.031), infiltration of lymphatic vessels (p = 0.003), infiltration of nerves (p = 0.010), pT-stage (8th edition) (p = 0.014), degree of keratinization at the IF (p = 0.033), and nuclear polymorphism at the IF (p = 0.043) after conversion to a binary scale were found to be significant prognostic parameters regarding the presence of LM. DOI evolved as a significant predictor for OS (p = 0.006), RFS (p = 0.003), and LM (p = 0.032) in metric and grouped analysis. CONCLUSIONS: The current evaluation revealed depth of invasion as strongest histologic predictor of metastatic tumor growth, overall survival, and relapse-free survival in OSCC, confirming the current adaption of the T-classification. Other distinct histologic grading parameters investigated during this study can give valuable indications of a tumor's potential aggressiveness, but the exact site, mode, and procedure need further exploration. CLINICAL RELEVANCE: Integrating measurement of DOI also into the pretherapeutic staging process could aid in treatment planning.

Lymph node ratio as a predictor for outcome in oral squamous cell carcinoma: a multicenter population-based cohort study.

OBJECTIVES: Recently, multiple studies addressed the importance of lymph node ratio (LNR) in specifying patients' risk of disease recurrence in various malignancies. The present study examines the prognostic significance of LNR in predicting outcome of oral squamous cell carcinoma (OSCC) patients after surgical treatment with curative intent. METHODS: Here, we describe a retrospective population-based cohort with 717 patients previously diagnosed with OSCC. Histopathologically verified lymph node metastasis was diagnosed in 290 patients. Among these patients, we evaluated the impact of LNR on overall survival (OAS) and recurrence-free survival (RFS) in uni- as well as multivariate analysis. RESULTS: A median cutoff (0.055) in LNR was found to significantly predict outcome in OSCC patients. Five-year OAS was 54.1% in patients with a low LNR, whereas a high LNR was associated with a 5-year OAS of 33.3% (p < 0.001). Similar results were detected for RFS with a 5-year survival rate of 49.8% (LNR low) and 30.3% (LNR high) (p = 0.002). Results were confirmed in multivariate Cox regression which substantiated the importance of LNR in predicting survival in OSCC patients. CONCLUSIONS: LNR was shown to be an independent prognostic factor for outcome of OSCC in a population-based cohort in uni- as well as multivariate analysis. Hereby, a LNR ≥ 0.055 predicted a shorter OAS and RFS in our cohort. CLINICAL RELEVANCE: Besides established histopathological factors, LNR can be used as a reliable predictor of outcome in OSCC and might therefore be further applied in evaluating adjuvant treatment after resection in curative intention.

Impact of Leptin on Periodontal Ligament Fibroblasts during Mechanical Strain.

Orthodontic treatment to correct dental malocclusions leads to the formation of pressure zones in the periodontal ligament resulting in a sterile inflammatory reaction, which is mediated by periodontal ligament fibroblasts (PDLF). Leptin levels are elevated in obesity and chronic inflammatory responses. In view of the increasing number of orthodontic patients with these conditions, insights into effects on orthodontic treatment are of distinct clinical relevance. A possible influence of leptin on the expression profile of PDLF during simulated orthodontic mechanical strain, however, has not yet been investigated. In this study, PDLF were exposed to mechanical strain with or without different leptin concentrations. The gene and protein expression of proinflammatory and bone-remodelling factors were analysed with RT-qPCR, Western-blot and ELISA. The functional analysis of PDLF-induced osteoclastogenesis was analysed by TRAP (tartrate-resistant acid phosphatase) staining in coculture with human macrophages. Pressure-induced increase of proinflammatory factors was additionally elevated with leptin treatment. PDLF significantly increased RANKL (receptor activator of NF-kB ligand) expression after compression, while osteoprotegerin was downregulated. An additional leptin effect was demonstrated for RANKL as well as for subsequent osteoclastogenesis in coculture after TRAP staining. Our results suggest that increased leptin concentrations, as present in obese patients, may influence orthodontic tooth movement. In particular, the increased expression of proinflammatory factors and RANKL as well as increased osteoclastogenesis can be assumed to accelerate bone resorption and thus the velocity of orthodontic tooth movement in the orthodontic treatment of obese patients.

Impact of Spatially Heterogeneous Trop-2 Expression on Prognosis in Oral Squamous Cell Carcinoma.

Oral cancer often presents with aggressive behavior and a high risk of recurrence and metastasis. For oral squamous cell carcinoma (OSCC), which is the most frequent histological subtype, therapy strategies include surgery, radiation therapy, chemotherapy, immune checkpoint inhibitors, and EGFR inhibitors. Recently, a Trop-2 antibody-drug conjugate (ADC) has been approved in the United States of America for the treatment of advanced triple-negative breast cancer. However, this ADC has also been tested in other solid tumors including head & neck squamous cell carcinoma. The prognostic impact of Trop-2 has already been reported for several cancers. We studied the prognostic influence of Trop-2 protein expression on OSCC patients' survival. The cohort comprised n = 229 OSCC patients with available archived tumor tissue and corresponding non-neoplastic oral mucosa tissue. Using immunohistochemistry, we investigated Trop-2 expression in both the central and peripheral regions of each tumor and in corresponding non-neoplastic oral mucosa. In patients suffering from OSCC with combined high central and low peripheral Trop-2 expression, five-year overall survival (OS) was 41.2%, whereas 55.6% of OSCC patients who presented lower central and/or higher peripheral tumoral Trop-2 expression were alive after five years (p = 0.075). In multivariate Cox regression, the expression pattern of high central tumoral and lower peripheral Trop-2 expression was significantly correlated with impaired OS (HR = 1.802, 95%-CI: 1.134-2.864; p = 0.013) and recurrence-free survival (RFS) (HR = 1.633, 95%-CI: 1.042-2.560; p = 0.033), respectively, when adjusting for co-variables. Hence, Trop-2 may serve as an independent prognostic biomarker in OSCC. In subsequent studies, the pathophysiological meaning of downregulated Trop-2 expression in the OSCC periphery has to be analyzed.

The role of HIF-1α in nicotine-induced root and bone resorption during orthodontic tooth movement.

BACKGROUND: In orthodontic tooth movement (OTM), pseudo-inflammatory processes occur that are similar to those of nicotine-induced periodontitis. Previous studies have shown that nicotine accelerates OTM, but induces periodontal bone loss and dental root resorption via synergistically increased osteoclastogenesis. This study aimed to investigate the role of hypoxia-inducible factor 1 alpha (HIF-1α) in nicotine-induced osteoclastogenesis during OTM. MATERIALS/METHODS: Male Fischer-344 rats were treated with l-Nicotine (1.89 mg/kg/day s.c., N = 10) or NaCl solution (N = 10). After a week of premedication, a NiTi spring was inserted to mesialize the first upper left molar. The extent of dental root resorption, osteoclastogenesis, and HIF-1α protein expression was determined by (immuno)histology, as well as bone volume (BV/TV) and trabecular thickness (TbTh) using µCT. Receptor activator of nuclear factor of activated B-cells ligand (RANK-L), osteoprotegerin (OPG), and HIF-1α expression were examined at the protein level in periodontal ligament fibroblasts (PDLF) exposed to pressure, nicotine and/or hypoxia, as well as PDLF-induced osteoclastogenesis in co-culture experiments with osteoclast progenitor cells. RESULTS: Nicotine favoured dental root resorptions and osteoclastogenesis during OTM, while BV/TV and TbTh were only influenced by force. This nicotine-induced increase does not appear to be mediated by HIF-1α, since HIF-1α was stabilized by force application and hypoxia, but not by nicotine. The in vitro data showed that the hypoxia-induced increase in RANK-L/OPG expression ratio and PDLF-mediated osteoclastogenesis was less pronounced than the nicotine-induced increase. CONCLUSIONS: Study results indicate that the nicotine-induced increase in osteoclastogenesis and periodontal bone resorption during OTM may not be mediated by hypoxic effects or HIF-1α stabilization in the context of nicotine-induced vasoconstriction, but rather by an alternative mechanism.

The role of 25-hydroxyvitamin-D3 and vitamin D receptor gene in human periodontal ligament fibroblasts as response to orthodontic compressive strain: an in vitro study.

BACKGROUND: This study aimed to investigate, if different physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fibroblasts induced by simulated orthodontic compressive strain. METHODS: A pool of hPDL fibroblasts was treated in absence or presence of 25(OH)D3 in 3 different concentrations (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fibroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxylase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non-parametric tests were used with an alpha of 5%. RESULTS: RANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were differentially expressed during simulated orthodontic compressive strain (p < 0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean = 0.96 ± 0.68, mean = 1.61 ± 0.66 and mean = 1.86 ± 0.78, respectively) in comparison to the control (mean 2.58 ± 1.16) (p < 0.05). CYP2R1 gene expression was statistically modulated by the different 25(OH)D3 concentrations applied (p = 0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p < 0.05). CONCLUSIONS: Simulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regulate the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fibroblasts in the context of orthodontic compressive strain. Our study also suggests that single nucleotide polymorphisms in the VDR gene regulate VDR expression in periodontal ligament fibroblasts in the context of orthodontic compressive strain.

Genetic polymorphisms influence gene expression of human periodontal ligament fibroblasts in the early phases of orthodontic tooth movement.

Genetic polymorphisms could be involved in the individual rate of OTM (orthodontic tooth movement) corresponding to the clinical phenomenon of "slow movers" and "fast movers". This study evaluated, if genetic polymorphisms in RANK, RANKL, OPG, COX2 and IL6 are associated with the expression of RANKL, OPG, COX2 and IL6 by human periodontal ligament (hPDL) fibroblasts during OTM. Primary hPDL fibroblasts from periodontal connective tissue of teeth extracted from 57 human subjects for medical reasons were collected, isolated, cultivated and characterized. To simulate orthodontic forces in PDL pressure areas, a physiological compressive force of 2 g/cm2 was applied to the hPDL fibroblasts under cell culture conditions at 70% confluency for 48 h, using a glass disc. Thereafter we analysed relative expression of RANKL, OPG, COX2 and IL6 by RT-qPCR. We also performed genotyping analysis of seven genetic polymorphisms in RANK, RANKL, OPG, COX2 and IL6. Relative gene expression was compared among the genotypes. The genotype TT in polymorphism rs9594738 (RANKL) had a higher RANKL expression in the recessive model (p = 0.021; TT vs. CT + CC). For polymorphism rs9594738 (RANKL), in the recessive model, TT was associated with a higher RANKL/OPG expression ratio (p = 0.013; TT vs. CT + CC). In the dominant model, GG genotype in rs5275 (COX2) was associated with a lower gene expression of COX2 (p = 0.04; GG vs. AA + AG). Genetic polymorphisms in genes associated with OTM affect the relative force-induced upregulation of these genes in hPDL fibroblasts.

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

Simplified method for applying static isotropic tensile strain in cell culture experiments with identification of valid RT-qPCR reference genes for PDL fibroblasts.

BACKGROUND/OBJECTIVE: Periodontal ligament fibroblasts (PDLF) play an important mediating role in orthodontic tooth movement expressing various cytokines, when exposed to compressive or tensile strain. Here, we present a simplified and easy-to-handle, but reliable and valid method for simulating static isotropic tensile strain in vitro using spherical silicone cap stamps. Furthermore, we identify appropriate reference genes for data normalization in real-time quantitative polymerase chain reaction (RT-qPCR) experiments on PDLF subjected to tensile strain. MATERIALS AND METHODS: PDLF were cultivated on flexible bioflex membranes and exposed to static isotropic tensile strain of different magnitudes and timeframes. We determined cell number, cytotoxicity, and relative expression of proinflammatory genes cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). For normalization of RT-qPCR data, we tested the stability and validity of nine candidate reference genes with four mathematical algorithms (geNorm, NormFinder, comparative ΔCq, and BestKeeper) and ranked them based on their calculated expression stability. RESULTS: We observed no decrease in cell number or cytotoxic effect at any of the applied magnitudes and timeframes of tensile strain. At 16 per cent and 35 per cent tensile strain for 48 hours, we detected a significant increase in COX-2 and decrease in IL-6 gene expression. Highest stability was found for TBP (TATA-box-binding protein) and PPIB (peptidylprolyl isomerase A) in reference gene validation. According to the geNorm algorithm, both genes in conjunction are sufficient for normalization. In contrast to all other candidate genes tested, gene expression normalization of target gene COX-2 to reference genes EEF1A1, RPL22, and RNA18S5 indicated no significant upregulation of COX-2 expression. CONCLUSIONS: A strain magnitude of 16 per cent for 48 hours elicited the most distinct cellular response by PDLF subjected to static tensile isotropic strain by the presented method. TBP and PPIB in conjunction proved to be the most appropriate reference genes to normalize target gene expression in RT-qPCR studies on PDLF subjected to tensile strain.

Sodium-chloride-induced effects on the expression profile of human periodontal ligament fibroblasts with focus on simulated orthodontic tooth movement.

Increased salt (NaCl) consumption triggers chronic diseases such as hypertension or osteopenia. Its impact on orthodontic tooth movement and periodontitis, however, has not been investigated, although both processes are related to the immune system, with periodontal ligament fibroblasts (PDLFs) playing a key mediating role. Here, we investigated the impact of NaCl on the expression pattern of PDLFs in a model of simulated compressive orthodontic strain. Periodontal ligament fibroblasts were preincubated for 24 h with additional 0 or 40 mM NaCl and concurrently treated for another 48 h with or without compressive strain of 2 g cm-2 . We analyzed the expression of genes and proteins involved in orthodontic tooth movement by reverse transcription quantitative polymerase chain reaction (RT-qPCR), ELISA, and immunoblot. Co-culture experiments were performed to observe PDLF-mediated osteoclastogenesis. A higher (40 mM) concentration of NaCl in the culture medium resulted in increased secretion of prostaglandin, expression of alkaline phosphatase, and expression of genes involved in extracellular matrix remodeling, but decreased compression-induced expression of the interleukin-6 (IL6) gene. The 40 mM concentration of NaCl also enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) but reduced that of osteoprotegerin (OPG), resulting in upregulated PDLF-mediated osteoclastogenesis. A high NaCl concentration in the periodontal ligament, corresponding to a high-salt diet in vivo, may influence orthodontic tooth movement and periodontitis through increased secretion of prostaglandins by PDLFs and upregulated PDLF-mediated osteoclastogenesis, possibly accelerating orthodontic tooth movement and propagating periodontitis and periodontal bone loss.

Effects of the Highly COX-2-Selective Analgesic NSAID Etoricoxib on Human Periodontal Ligament Fibroblasts during Compressive Orthodontic Mechanical Strain.

Human periodontal ligament (hPDL) fibroblasts play a major role during periodontitis and orthodontic tooth movement, mediating periodontal inflammation, osteoclastogenesis, and collagen synthesis. The highly COX-2-selective NSAID etoricoxib has a favorable systemic side effect profile and high analgesic efficacy, particularly for orthodontic pain. In this in vitro study, we investigated possible side effects of two clinically relevant etoricoxib concentrations on the expression pattern of mechanically strained hPDL fibroblasts and associated osteoclastogenesis in a model of simulated orthodontic compressive strain occurring during orthodontic tooth movement. hPDL fibroblasts were incubated for 72 h under physiological conditions with etoricoxib at 0 µM, 3.29 µM, and 5.49 µM, corresponding to clinically normal and subtoxic dosages, with and without mechanical strain by compression (2 g/cm2) for the final 48 h, simulating conditions during orthodontic tooth movement in compressive areas of the periodontal ligament. We then determined gene and/or protein expression of COX-2, IL-6, PG-E2, RANK-L, OPG, ALPL, VEGF-A, P4HA1, COL1A2, and FN1 via RT-qPCR, ELISA, and Western blot analyses as well as apoptosis, necrosis, cell viability, and cytotoxicity via FACS, MTT, and LDH assays. In addition, hPDL fibroblast-mediated osteoclastogenesis was assessed by TRAP staining in coculture with RAW267.4 cells for another 72 h. Gene and protein expression of all evaluated factors was significantly induced by the mechanical compressive strain applied. Etoricoxib at 3.29 µM and 5.49 µM significantly inhibited PG-E2 synthesis, but not COX-2 and IL-6 gene expression nor RANK-L-/OPG-mediated osteoclastogenesis or angiogenesis (VEGF-A). Extracellular matrix remodeling (COL1A2, FN1) and bone anabolism (ALPL), by contrast, were significantly stimulated particularly at 5.49 µM. In general, no adverse etoricoxib effects on hPDL fibroblasts regarding apoptosis, necrosis, cell viability, or cytotoxicity were detected. Clinically dosed etoricoxib, that is, a highly selective COX-2 inhibition, did not have substantial effects on hPDL fibroblast-mediated periodontal inflammation, extracellular matrix remodeling, RANK-L/OPG expression, and osteoclastogenesis during simulated orthodontic compressive strain.

The NC11 domain of human collagen XVI induces vasculogenic mimicry in oral squamous cell carcinoma cells.

Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions.