Stromal transforming growth factor-beta 1 is crucial for reinforcing the invasive potential of low invasive cancer

Objective

Tumour cells alter the characteristics of the adjacent stroma to create a supportive microenvironment during cancer progression. In vitro and in vivo experiments were carried out to verify the role of stromal TGF-β1 in reinforcing of the invasive potential in low invasive cancer.

Materials and methods

Isolated NF or CAF was co-cultured with low invasive HSC-2 cells to evaluate whether stromal TGF-β1 induced PDPN expression by Transwell invasion and influenced tumour growth in orthotopic xenografts.

Results

Stimulation by TGF-β1 promoted PDPN expression and Transwell invasion through SMAD signalling as well as activation of Src, P38 mitogen activated protein kinase and extracellular regulated kinase 1/2. PDPN induction was TβRII-dependent. Tumour growth of HSC-2 OSCC in a mouse xenograft was intensified in the tumour CAF microenvironment.

Conclusions

Stromal TGF-β1 signalling promoted PDPN expression in cancer cells, thereby enhancing tumour growth and leading to a more invasive phenotype.     

The role of GPC5 in lung metastasis of salivary adenoid cystic carcinoma

Objective

Proteoglycans play a crucial role in salivary adenoid cystic carcinoma (SACC) tumorigenesis, neurotropic growth and lung metastasis. In this study, we investigated the role of GPC5 in the lung metastasis of SACC.

Design

The expression of heparan sulfate proteoglycans (HSPGs) was detected in SACC-M (high lung-metastatic cell line), SACC-2 (low lung-metastatic cell line) and SACC-83 (low lung-metastatic cell line) cells by relative quantitative Real-Time PCR. The expression of GPC5 was detected by immunofluorescence and Western blot analysis in SACC-M, SACC-2 and SACC-83 cells, and by immunohistochemical analysis in primary tumours from 16 cases of SACC patients with or without lung metastasis. GPC5 expression was silenced in SACC-M cells, cell proliferation and tumour growth was evaluated by MTT assay and nude mice model, respectively.

Results

Expression of most HSPGs was decreased in SACC-M cells, but GPC5 expression increased 3.24-fold and 815.69-fold (more than 3-fold) in SACC-M compared with SACC-2 and SACC-83 cells, respectively. Immunohistochemical analysis showed higher expression of GPC5 in SACC with lung metastasis compared to SACC without lung metastasis. The lung metastasis of SACC-M cells in nude mice was obviously decreased after GPC5 silencing (P < 0.05), although GPC5 silencing had no significant effect on SACC-M cell proliferation.

Conclusion

GPC5 may contribute to lung metastasis of SACC.     

Overexpression of S100A4 as a biomarker of metastasis and recurrence in oral squamous cell carcinoma

S100A4, a biomarker of epithelial mesenchymal transition (EMT), plays an important role in invasion and metastasis by promoting cancer cell motility. In oral squamous cell carcinoma (OSCC), metastasis results in 90% of cancer associated mortality.

Objective

To investigate the role of S100A4 expression as an important component of the epithelial mesenchymal transition (EMT) program in oral squamous cell carcinoma (OSCC).

Material and Methods

S100A4 protein expression was assessed semi-quantitatively by immunohistochemistry in 47 histologically confirmed cases of oral squamous cell carcinoma (OSCC) and 10 normal oral mucosal biopsies. The association between the S100A4 overexpression and the aggressive features of OSCC were analyzed by X2 test.

Results

Moderate to strong cytoplasmic expression of S100A4 was observed in 30 out of 47 specimens of OSCC (64%). Overexpression of S100A4 was significantly associated with the clinical stage, lymph node involvement, metastases, pattern of invasion and recurrence (p<0.05).

Conclusion

S100A4 expression represents an important biomarker of prognostic significance that may be used to identify a subset of patients at high risk of invasion and metast     

Adhesive performance of a multi-mode adhesive system: 1-Year in vitro study

Objectives

The aim of this study was to investigate the adhesive stability over time of a multi-mode one-step adhesive applied using different bonding techniques on human coronal dentine. The hypotheses tested were that microtensile bond strength (μTBS), interfacial nanoleakage expression and matrix metalloproteinases (MMPs) activation are not affected by the adhesive application mode (following the use of self-etch technique or with the etch-and-rinse technique on dry or wet dentine) or by ageing for 24 h, 6 months and 1 year in artificial saliva.

Methods

Human molars were cut to expose middle/deep dentine and assigned to one of the following bonding systems (N = 15): (1) Scotchbond Universal (3M ESPE) self-etch mode, (2) Scotchbond Universal etch-and-rinse technique on wet dentine, (3) Scotchbond Universal etch-and-rinse technique on dry dentine, and (4) Prime&Bond NT (Dentsply De Trey) etch-and-rinse technique on wet dentine (control). Specimens were processed for μTBS test in accordance with the non-trimming technique and stressed to failure after 24 h, 6 months or 1 year. Additional specimens were processed and examined to assay interfacial nanoleakage and MMP expression.

Results

At baseline, no differences between groups were found. After 1 year of storage, Scotchbond Universal applied in the self-etch mode and Prime&Bond NT showed higher μTBS compared to the other groups. The lowest nanoleakage expression was found for Scotchbond Universal applied in the self-etch mode, both at baseline and after storage. MMPs activation was found after application of each tested adhesive.

Conclusions

The results of this study support the use of the self-etch approach for bonding the tested multi-mode adhesive system to dentine due to improved stability over time.

Clinical significance

Improved bonding effectiveness of the tested universal adhesive system on dentine may be obtained if the adhesive is applied with the self-etch approach.     

MMP-7 and TIMP-1, New Targets in Predicting Poor Wound Healing in Apical Periodontitis

Introduction

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions.

Methods

Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy.

Results

Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus.

Conclusions

MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development.     

Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development

Objective

To evaluate the expression of matrix metalloproteinase inducer (EMMPRIN) and its correlation with the expression of matrix metalloproteinases (MMPs)-1, -2 and -9 during the development of periapical lesion in mice.

Methods

Periapical lesions were induced in the lower first molars of mice and after 7, 14, 21 and 42 days the mandibles were removed. The periapical lesions were measured by micro-computed tomography. The expression of EMMPRIN, MMPs-1, -2, and -9 genes were determined by real-time RT-PCR. The location and expression of EMMPRIN and MMPs were evaluated by immunohistochemistry.

Results

At 14 days, the periapical lesion area was higher than at 7 days. At 21 and 42 days no statistically significant bone loss was observed in comparison to 14 days. The control group showed discrete and occasional EMMPRIM, MMP-1, -2 and -9 immunostaining in the periodontal ligament fibroblasts. At 7, 14, 21 and 42 days intense immunoexpression was observed for EMMPRIN, MMPs-1, -2 and -9 in the region adjacent to the apical foramen. The EMMPRIN immunoexpression was higher at 7, 14, 21 and 42 days compared with the control. There was a positive correlation between gene expression of EMMPRIN and MMPs in the active phase of periapical lesion development.

Conclusion

There is a high expression of EMMPRIM mainly by the inflammatory infiltrate in the region adjacent to the apical foramen during periapical lesion development. Furthermore, the positive correlation with MMP-1, -2, and -9 during the first days after periapical lesion induction indicates that EMMPRIM may be involved in the active phase of periapical lesions development.     

Human gingival fibroblasts function is stimulated on machined hydrided titanium zirconium dental implants

Objectives

The aim of this study was to evaluate the influence of different titanium zirconium (TiZr) alloy surfaces on primary human gingival fibroblasts (HGF) for improved soft tissue integration of dental implants.

Methods

TiZr polished, machined and machined + HCl/H₂SO₄ acid-etched surfaces were modified by cathodic polarization and/or HNO₃/HF acid etching. Contact angle of surfaces was measured. The influence of modified TiZr surfaces on HGF was evaluated through the analysis of cell number, morphology, recovery after a wound (wound healing assay) and the expression of several genes, including matrix metalloproteinase-1 (MMP1) and metallopeptidase inhibitor-1 (TIMP1).

Results

Modification of TiZr surfaces decreased its hydrophilicity. Hydride implementation on TiZr surfaces via cathodic polarization increased TIMP1 expression and decreased MMP1/TIMP1 mRNA ratio. Cathodic polarization of machined surfaces promoted cell attachment. Cells on machined and machined + cathodic polarization surfaces grew aligned to the microgrooves whereas on all polished surfaces they grew randomly. Acid etching of polished and machined surfaces did not improve HGF function.

Conclusions

Hydride implementation on TiZr machined surfaces may be used as new dental implant material for improved soft tissue integration.

Clinical significance

Enhancing dental implant surfaces’ bioactivity by hydride implementation may promote soft tissue attachment and sealing around the implant and reduce peri-implantitis related to ECM-destruction compared with conventional machined surfaces.     

Gingival fibroblasts from periodontitis patients exhibit inflammatory characteristics in vitro

Objective

Gingival fibroblasts (GFs) are an important regulatory cell type in the progression of periodontitis. This study aimed to compare the expression levels of genes associated with inflammation, extracellular matrix degradation and bone destruction in GFs isolated from healthy and periodontitis subjects in the absence and presence of Porphyromonas gingivalis.

Designs

Primary GFs from healthy (n = 10) and periodontitis subjects (n = 10) were stimulated in vitro with viable P. gingivalis ATCC 49417 and 3 clinical isolates of P. gingivalis with type II fimbriae from one healthy subject (KUMC-H1) and two periodontitis patients (KUMC-P1, -P2). The mRNA expression of proinflammatory cytokines (interleukin (IL)-6, IL-8, IL-1B), anti-inflammatory cytokines (IL-4, IL-10), matrix metalloproteinase (MMP)-1 and 2, tissue inhibitor matrix metalloproteinase (TIMP)-3 and osteoprotegerin (OPG) were assessed using real-time PCR. The levels of IL-6, IL-1β and TIMP-3 protein were measured by an enzyme-linked immunosorbent assay.

Results

The mRNA expression of IL-6, IL-1B and TIMP-3 was higher in the periodontitis group compared with the healthy group, whereas IL-4 expression was higher in the healthy group both in the absence and presence of the P. gingivalis strains. The expression levels of IL-6, IL-1β and TIMP-3 protein were also higher in the periodontitis group in the absence and/or presence of the P. gingivalis strains. There was inter-strain variability among P. gingivalis strains in the ability to induce expression of the proinflammatory cytokines, MMPs and OPG and in the ability to degrade IL-6 protein.

Conclusion

High expression of proinflammatory cytokines and TIMP-3 and low expression of IL-4 can be a signature of GFs associated with periodontitis.     

MMPs activity and bond strength in deciduous dentine–resin bonded interfaces

Objectives

To determine effect of ageing on deciduous dentine–resin interfaces bond strength and the metalloproteinases (MMPs) activity at the hybrid layer compared to permanent dentine.

Methods

Microtensile bond strength (MTBS) tests were performed in human deciduous and permanent dentine after 24 h, 3 and 6 months using an etch and rinse adhesive. C-terminal telopeptide concentrations (ICTP) were calculated, in order to determine MMPs mediated collagen degradation at the hybrid layer.

Results

The highest MMPs-mediated collagen degradation values occurred in phosphoric acid demineralized dentine, ICTP values were similar for deciduous and permanent dentine after 1 week. Resin infiltration decreased collagen degradation in both dentins and ICTP values were similar to those attained by for untreated dentine. In resin infiltrated and untreated dentine specimens collagen degradation was always higher for deciduous dentine. At 24 h, MTBS was higher in permanent dentine. After ageing MTBS decreased and performed similarly in both dentins.

Conclusions

Higher collagenollytic activity is found in deciduous than in permanent dentine. At 24 h, collagen cleavage by MMPs at the hybrid layer is higher in deciduous dentine leading to a lower MTBS.

Clinical significance

The presence of resin monomers reduced collagen degradation when applied on demineralized dentine, but exerted protection was lower in deciduous dentine.     

Stabilization of dentin matrix after cross-linking treatments,in vitro

Objectives

To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen.

Methods

Dentin beams were obtained from human molars and completely demineralized in 10 wt% H₃PO₄ for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n = 10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance.

Results

EDC was able to significantly increase collagen stiffness in 60 s. 10% GA groups obtained the highest E values after both 30 and 60 s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1 M or 2 M EDC showed the lowest MMP activity.

Significance

Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.     

The inhibition effect of non-protein thiols on dentinal matrix metalloproteinase activity and HEMA cytotoxicity

Objectives

Phosphoric acid (PA) etching used in etch-and-rinse adhesives is known to activate host-derived dentinal matrix-metalloproteinases (MMPs) and increase dentinal permeability. These two phenomena will result, respectively; in degradation of dentine-adhesive bond and leaching of some monomers especially 2-hydroxyethyl methacrylate (HEMA) into the pulp that would negatively affect the viability of pulpal cells. This study is the first to investigate the inhibitory effect of non-protein thiols (NPSH); namely reduced glutathione (GSH) and N-acetylcysteine (NAC) on dentinal MMPs and compare their effects on HEMA cytotoxicity.

Methods

Dentine powder was prepared from human teeth, demineralized with 1% PA and then treated with 2% GSH, 2% NAC or 2% chlorhexidine (CHX). Zymographic analysis of extracted proteins was performed. To evaluate the effect of GSH, NAC and CHX on HEMA cytotoxicity, solutions of these compounds were prepared with or without HEMA and rat pulpal cells were treated with the tested solutions for (6 and 24 h). Cells viability was measured by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity data were analysed by one-way ANOVA and Tukey post hoc tests (p < 0.05).

Results

The inhibitory effect of GSH and NAC on dentinal MMPs was confirmed. GSH showed similar effectiveness to NAC regarding HEMA cytotoxicity inhibition.

Conclusion

NPSH were effective to inhibit dentinal MMPs and HEMA cytotoxicity.

Clinical significance

The tested properties of NPSH provide promising clinical use of these agents which would enhance dentine-bond durability and decrease post-operative sensitivity.     

Increased expression of Toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma

Objective

Toll-like receptor-9 (TLR-9), a new member of the interleukin-1 receptor superfamily, was recently found to have a high level of expression in many carcinoma specimens. The objective of this study was to examine the TLR-9 expression and its role in tumour cell proliferation in oral squamous cell carcinoma.

Materials and methods

Western blot and immunohistochemistry were used to detect TLR-9 protein in oral squamous cell carcinoma (OSCC) Tca-8113 cell lines and clinical specimens (n = 60). The relationship between TLR-9 expression and clinicopathologic features was analysed. Cell proliferation and inflammatory chemokines secretion were tested by MTT and ELISA methods respectively.

Results

Results showed that TLR-9 expression level was higher in OSCC tissues than in paired adjacent normal tissues (P < 0.01), and the expression level of TLR-9 was significantly associated with tumour size (P = 0.001), tumour clinical stage (P = 0.003) and Ki-67 expression (P < 0.01). In vitro results also suggested that stimulation of Tca-8113 cells with TLR-9 agonist CpG-ODN could significantly increase tumour cell proliferation as well as subsequent IL-1α and IL-6 secretions (P < 0.01), which could be partially inhibited by usage of anti-TLR-9 protein.

Conclusions

It was therefore hypothesized that increased expression of TLR-9 may be of great value in assessing the development of OSCC, and could be used as a new target for OSCC prevention and therapy in future.     

Effect of carbodiimide (EDC) on the bond stability of etch-and-rinse adhesive systems

Objective

Recent studies supported the use of protein cross-linking agents during bonding procedures to inactivate endogenous dentin proteases, preventing dentin collagen degradation thus improving bond durability. The aim of this study was to evaluate the effect of a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-containing conditioner on the stability of the adhesive interface created by two etch-and-rinse adhesives.

Methods

Human dentin was etched with 35% phosphoric acid, treated with 0.3 M EDC-containing conditioner followed by a three-step or a two-step etch-and-rinse adhesive. Adhesives were applied to control specimens without EDC pre-treatment. Specimens were subjected to microtensile bond strength test and pulled to failure after 24 h or 1 year of storage and interfacial nanoleakage expression was evaluated and quantified by light microscopy. Additionally, to investigate endogenous dentin matrix metalloproteinase activity a zymographic assay was performed on protein extracts obtained from phosphoric-acid-etched dentin powder with or without EDC treatment.

Results

The use of the EDC-containing conditioner did not affect immediate bond strength to dentin but contributed to preserve the bond strength after 1 year (p < 0.05) for both tested adhesives. No difference was found in the interfacial nanoleakage expression that increased after aging irrespective from the treatment. EDC pre-treatment inhibited dentin endogenous MMPs as assayed with the zymography.

Significance

In conclusion, the results of the study provide proof that EDC can produce long-term inactivation of MMPs in acid-etched dentin matrices contributing to bond strength preservation over time. Future studies are needed to support the use of EDC in vivo.     

Investigation of cancer-associated fibroblasts and p62 expression in oral cancer before and after chemotherapy

Purpose

The aim of this study is to investigate the expression of the autophagy protein p62 in oral squamous cell carcinoma (OSCC) cells before and after chemotherapy. We also detected cancer-associated fibroblasts (CAFs) in these OSCC samples to explore the roles of p62 and CAFs in chemotherapy.

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells

Objectives

This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism.

Design

Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA).

Results

While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found.

Conclusion

The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.     

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression

Objectives

Death domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.

Materials and methods

RT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.

Results

Daxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.

Conclusion

Our data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.

Clinical relevance

Daxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.

     

Evaluation of pretreatment serum interleukin-6 and tumour necrosis factor alpha as a potential biomarker for recurrence in patients with oral squamous cell carcinoma

Background

Oral squamous cell carcinoma (OSCC) constitutes 3 percent of all cancers with predominant occurrence in middle aged and elderly males. Tumour recurrence worsens disease prognosis and decreases quality of life in patients with OSCC. Proinflammatory cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α) have been suggested to play a certain role in variety of tumours. The aim of this study was to investigate the relationship of pretreatment serum IL-6 and TNF-α levels on tumour recurrence in patients with OSCC in order to identify potential biomarkers for the early detection of disease recurrence.

Material and Methods

The patients with newly diagnosed OSCC were treated and followed from the first visit from November 2006 until January 2008. Serum IL-6 and TNF-α concentrations were measured. The records of the patients were re-examined in July 2012 and data were recorded about cancer characteristics and tumour recurrence. Disease free survival was analyzed by Kaplan-Meier survival curves, log rank test and Cox proportional hazards regression.

Results

Serum IL-6 was shown as an independent risk factor for tumour recurrence.

Conclusions

Pretreatment serum IL-6 concentration may be a useful biomarker for identification of OSCC patients with increased risk of the disease recurrence. Key words: Serum IL-6, serum TNF-α, oral cancer, recurrence.     

Down-regulation of Inflammatory Mediator Synthesis and Infiltration of Inflammatory Cells by MMP-3 in Experimentally Induced Rat Pulpitis

Introduction

Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo.

Methods

Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3.

Results

NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo.

Conclusions

These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.     

In Vitro Regulation of CCL3 and CXCL12 by Bacterial By-products Is Dependent on Site of Origin of Human Oral Fibroblasts

Introduction

Production of chemokines by tissue resident cells is one of the main mechanisms involved in the inflammatory infiltrate formation during inflammation. The specific ability of fibroblasts from different oral tissues such as gingiva, periodontal ligament, and dental pulp from permanent and deciduous teeth in producing the chemokines CCL3 and CXCL12 under stimulation by bacterial products commonly found in endodontic infections was investigated.

Methods

Cultures of fibroblasts from gingiva and periodontal ligament as well as from dental pulp from permanent and deciduous teeth were established by using an explant technique and stimulated with increasing concentrations of Escherichia coli lipopolysaccharide (EcLPS) and Enterococcus faecalis lipoteichoic acid (EfLTA) for 1, 6, and 24 hours. Supernatants were tested for CCL3 and CXCL12 by enzyme-linked immunosorbent assay.

Results

In general, CCL3 production was induced by EcLPS in the 4 fibroblast subtypes and by EfLTA in fibroblasts from gingiva and periodontal ligament. Constitutive CXCL12 synthesis decreased in all fibroblast subtypes especially under stimulation with EcLPS. Fibroblast from permanent deciduous teeth was the cell type presenting the most expressive reduction in CXCL12 release by both stimuli. On the basis of computational matching of CXCL12 mRNA with the microRNAs miR-141 and miR-200a, their expression was also investigated. Although detected in the fibroblasts, these molecules remained unaltered by bacterial by-product stimulation.

Conclusions

EcLPS and EfLTA induced the production of CCL3 and unbalanced the synthesis of CXCL12 in a manner dependent on the specific tissue origin of fibroblasts.