Analysis of oral mucosa erosive-ulcerative lesions by reflectance confocal microscopy.

In vivo Reflectance Confocal Microscopy (RCM) allows to optically biopsy vital tissues, non-invasively and in real time. It results in horizontal virtual slices at a microscopic resolution and correlating with conventional histopathology. The aim of the present work is to describe RCM cellular and architectural findings in oral mucosae affected by erosive-ulcerative diseases, thus highlighting in vivo the wellknown histological peculiarities. A series of conventionally diagnosed Recurrent Aphthous stomatitis (RAS) and Pemphigus Vulgaris (PV) erosive and/or ulcerative oral lesions underwent RCM imaging to establish the application of RCM imaging to this kind of inflammatory non-tumoral lesions. A total of 12 RAS-related lesions and 8 PV-related lesions were considered. RCM imaging was capable to visualize their microscopic peculiarities, mainly inflammatory infiltrate, vessel dilation (RAS) and acantholytic cells, intraepithelial clefts and inflammatory cell carpets (PV). Despite RCM may result unnecessary to diagnose oral lesions referred to RAS and PV, its capability to highlight their main microscopic features could be advantageously used to monitor the healing or worsening of the clinical situation as well as the responsiveness/refractoriness to therapy.

Association between denture stomatitis, candida species and diabetic status.

Candida species are commensal yeasts of the oral cavity, which, under predisposing systemic and/or local circumstances, are responsible for a wide variety of clinical manifestations, globally known as oral candidiasis. Candida-associated denture stomatitis is an oral candidiasis particularly affecting the oral mucosa covered by a dental prosthesis, with several degree of severity. Diabetics suffer oral candidiasis more frequently than healthy individuals do and if they are denture wearers, the risk increases. Since various controversies still remain regarding the interrelationship among diabetes, oral Candida spp. strains involved in denture stomatitis and the presence of dentures, the present review aims to investigate the differences in Candida species frequencies and degree of denture stomatitis severity existing among diabetic and non- diabetic individuals, with and without dentures.

Noma: a reappraisal in Western countries - are HIV-negative immunocompetent adult patients safe?

Noma is an ancient Greek term referring to a gangrenous disease that destroys soft and hard tissues of oral and perioral structures. This disease has been known since ancient times and persisted in Europe until World War 2, while in the present day it is almost exclusively found in sub-Saharan Africa.

Spider Zygoma: a new implant rehabilitation technique for atrophic maxilla.

OAtrophic maxilla is a challenge in dental implant surgery, and new strategies are needed. We present a new minimally-invasive approach, called "Spider Zygoma", consisting of implant-supported prosthesis with the addition of customized maxillofacial plates and screws on surface of zygomatic bone. A 3D-model of the edentulous upper jaw was used as preoperative model. Two customized bone plates were created and used as guide for placement of implants and zygomatic screws. Although this is only a pilot study, this new surgical technique seems to be safe and accurate, confirmed by the maintenance of good aesthetic and functional results after 5-year follow-up.

Use of supercharged cover screw as static magnetic field generator for bone healing, 1st part: in vitro enhancement of osteoblast-like cell differentiation.

Since 1979, Pulsed electromagnetic fields (PEMFs) have been approved by the Food and Drug Administration as an effective method in the treatment of non-unions. As well as PEMFs, also static magnetic fields (SMFs) have been widely investigated in orthopaedic studies. Even if the exact mechanism of action is not well understood, a large number of studies showed specific effects both at cellular and tissue levels. As bone fracture healing and osseointegration share the same biological events, the application of magnetic field stimulation in order to facilitate the osseointegration process has been suggested. In this study we investigated the proliferation rate and gene expression profile of MG63 osteoblastic-like cells after a 24, 48 and 72-hour SMF stimulation, generated by a small, customized cover screw-shaped neodymium-iron-bore magnet placed in the inner cavity of a dental implant. As a result, we found that the application of a SMF to osteoblastic-like cells does slightly decrease cell proliferation rate while enhancing the expression of those genes correlated to differentiation and mineralization. Our findings represent, to our knowledge, the first clinical ready technique for dental implants showing the ability of SMF to promote the osteogenesis process in vitro.

Use of supercharged cover screw as static magnetic field generator for bone healing, 2nd part: in vivo enhancement of bone regeneration in rabbits.

In 1979, Pulsed electromagnetic fields (PEMFs) were approved by the Food and Drug Administration as an effective method in the treatment of non-unions. As well as PEMFs, also static magnetic fields (SMFs) have been widely investigated in orthopaedic studies. Even if the exact mechanism of action is not well understood, a large number of studies showed specific effects both at cellular and tissue levels. As bone fracture healing and osseointegration share the same biological events, the application of magnetic field stimulation in order to facilitate the osseointegration process has been suggested. In this study we investigated BIC and newly formed bone volume around dental implants inserted in the tibia of New Zealand rabbits after SMF stimulation, generated by a small-customized cover-screw-shaped neodymium-iron-bore magnet placed in the inner cavity of dental implants. As a result, we found that the SMF field generated around dental implants enhanced bone healing in the animal model. Our findings represent, to our knowledge, the first ready clinical technique for dental implants showing the ability of SMF to promote the osteogenesis process in vivo.

Pokemon proto-oncogene in oral cancer: potential role in the early phase of tumorigenesis.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) represents about 90% of all oral neoplasms with a poor clinical prognosis. To improve survival of OSCC patients, it is fundamental to understand the basic molecular mechanisms characterizing oral carcinogenesis. Dysregulation of oncogenes and tumor suppressor genes seems to play a central role in tumorigenesis, including malignant transformation of the oral cavity. MATERIALS AND METHODS: We analyzed the expression levels of the pro-oncogenic transcription factor Pokemon through real-time PCR, Western blot and immunohistochemistry in tumor, and normal oral tissue samples obtained from 22 patients with OSCC. The relationship between tumor characteristics and the level of Pokemon intratumor expression was also analyzed. RESULTS: Pokemon was significantly downregulated in OSCC. In particular, both mRNA and protein levels (tumor vs normal tissue) inversely correlated with histological grading, suggesting its potential role as a prognostic factor for OSCC. Moreover, a significant inverse correlation was found between Pokemon protein expression levels (OSCC vs normal oral mucosa) and tumor size, supporting the hypothesis that Pokemon could play an important role in the early phase of tumor expansion. CONCLUSION: This work shows that reduced expression of Pokemon is a peculiar feature of OSCC. Additional studies may establish the effective role of Pokemon in oral tumorigenesis.

Pre-clinical evaluation of a new coral-based bone scaffold.

Coral is used worldwide for bone reconstruction. The favorable characteristics that make this material desirable for implantation are (i) osteoinduction, (ii) and osteoconduction. These proprieties have been demonstrated by in vivo studies with animal models and clinical trials over a twenty-year period. Also poly(2-hydroxyethylmethacrylate) [poly(HEMA)] is a widely used biomaterial. By using coral and poly(HEMA), a scaffold for bone reconstruction application has been recently synthesized. Cytological, histological and genetic analyses were performed to characterize this new alloplastic material. Four samples were analyzed: (a) white coral (WC), (b) red coral (RC), (c) WC plus polymer (WCP) and (d) RC plus polymer (RCP). Quantification of mitochondrial dehydrogenase activity by MTT assay was performed as indirect detector of cytotoxicity. In vivo effects were revealed by implanting corals and coral-based polymers in rabbit tibia. Samples were collected after 4 weeks and subjected to histological analysis. To evaluate the genetic response of cells to corals and coral-derived polymers an osteoblastlike cell line (i.e. MG63) was cultured in wells containing (a) medium, (b) medium plus corals and (c) medium plus two types of scaffolds (RCP or WCP). RNAs extracted from cells were retro-transcribed and hybridized on DNA 19.2K microarrays. No cytotoxicity was detected in corals and coral-based biopolymers. No inflammation or adverse effect was revealed by histological examination. By microarray analysis 154 clones were differentially expressed between RC and WC (81 up and 73 down regulated) whereas only 15 clones were repressed by the polymer. Histological evaluation not only confirmed that coral is a biocompatible material, but also that the polymer has no adverse effect. Microarray results were in agreement with cytological and histological analyses and provided further data regarding the genetic effects of RC, WC and the new polymer.

Novel hydroxyapatite biomaterial covalently linked to raloxifene.

Since raloxifene, a drug used in osteoporosis therapy, inhibits osteoclast, but not osteoblast functions, it has been suggested to improve recovery during implant surgery. The present paper describes an effective method to link raloxifene, through a covalent bond, to a nano-Hydroxyapatite-based biomaterial by interfacing with (3-aminopropyl)-Triethoxysilane as assessed by Infra Red-Fourier Transformed (IR-FT) spectroscopy and Scanning Electron Microscope (SEM). To evaluate the safety of this modified new material, the vitality of osteoblast-like cells cultured with the new biomaterial was then investigated. Raloxifene-conjugated HAbiomaterial has been shown to be a safe material easy to obtain which could be an interesting starting point for the use of a new functional biomaterial suitable in bone regeneration procedures.

Osteopontin, osteocalcin and OB-cadherin expression in Synthetic nanohydroxyapatite vs bovine hydroxyapatite cultured Osteoblastic-like cells.

Calcium phosphate ceramics have been applied in bone replacement for several decades due to their excellent biocompatibility, bioactivity, osteo-conductivity and mechanical strength. Several studies have demonstrated that porous hydroxyapatite (HA) is an excellent scaffold for osteogenic proliferation and differentiation of the osteoprogenitor cells. However, different methods of synthesis and production of HA ceramic-based materials may have considerable effect on the physical and biological properties. In the present work, two hydroxyapatite-based materials, a natural hydroxyapatite ceramic of bovine origin and a synthetic nano-cristalline hydroxyapatite were tested in vitro with MG63 cell line. The results displayed that both the materials demonstrated a good biocompatibility. The immunocytochemical stain revealed a different positivity of the osteogenic markers between the cultures with the biomaterials, and the control culture. Western blot data confirmed the immunocytochemical stain. Both the materials tested in the present study demonstrated a good biocompatibility with the osteoblastic cells allowing, at the same time, the osteogenic differentiation, and they may be useful in clinical use.

Expression of poly(ADP-ribose) polymerase in bone regeneration.

Poly(ADP-ribose) polymerase (PARP) is a 116kDa enzyme catalysing the synthesis of ADP-ribose polymers from NAD+. PARP is activated in response to DNA strand breaks and plays a critical role in the maintenance of genomic integrity. However, considering its role also in transcription, proliferation as well as apoptosis in biological process, in the present study the role of PARP in bone regeneration was evaluated, in particular in bone cell proliferation and differentiation processes. Thus, formalin fixed paraffin embedded specimens of 10 human bone samples after sinus lift were collected and investigated by immunohistochemistry using a mouse monoclonal anti-human PARP antibody. PARP was expressed in cells with morphological features of osteoblasts in the areas of new bone formation at the junction between mineralized and unmineralized tissue, between osteoid tissue and bone. Few osteoclasts were observed and showed only focal nuclear expression of PARP, while osteocytes showed no positivity for PARP. Our data showed an overall involvement of PARP enzyme in human bone tissues, in particular during bone regeneration process.

Multiple 3D inversion recovery imaging for volume T1 mapping of the heart.

In this article, a three-dimensional inversion recovery sequence was optimized with the aim of generating in vivo volume T(1) maps of the heart using a 1.5-T MR system. Acquisitions were performed before and after gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA) administration in one patient with hypertrophic cardiomyopathy and in two healthy volunteers. Data were acquired with a multishot fast field echo readout using both ECG and respiratory triggers. A dedicated phantom, composed of four solutions with different T(1) values, was positioned on the subjects' thoracic region to perform patient-specific calibration. Pixel based T(1) maps were calculated with a custom Matlab(®) code. Phantom measurements showed a good accuracy of the technique and in vivo T(1) estimation of liver, skeletal muscle, myocardium, and blood resulted in good agreement with values reported in the literature. Multiple three-dimensional inversion recovery technique is a feasible and accurate method to perform T(1) volume mapping.

p63 expression in laryngeal squamous cell carcinoma is related to tumor extension, histologic grade, lymph node involvement and clinical stage.

To analyse the relationship of the immunohistochemical p63 expression with tumoral extent, histologic grade, lymph node involvement and clinical stage in laryngeal squamous cell carcinoma (LSCC), a series of 81 patients with primary LSCC treated by primary surgery was retrospectively evaluated. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissue blocks from surgical samples. Clinicopathologic data were correlated with the p63 staining results. Differences in p63 immunoreactivity between the different groups were compared using both parametric analysis of variance (ANOVA) and non-parametric Kruskal-Wallis test. Statistical significance was set at p less than 0.05. All statistical analyses were performed using the R statistical package. We found a statistically significant association between p63 protein expression and increase of tumor extension (T1 vs T3), of histological grading, of level of lymph node involvement (N0 vs N1 and N2), and clinical stage (I vs IV). Our findings suggest that abnormal expression of p63 may be involved in the early phases of laryngeal tumorigenesis and this oncoprotein might become a useful predictor of clinical outcome.

Adhesion and growth of osteoblast-like cells on laser-engineered porous titanium surface: expression and localization of N-cadherin and beta-catenin.

Response of different types of cells on biomaterials is crucial for the applications of tissue engineering and regenerative medicine. It is recognized that cell behaviour depends largely on material surface characteristics. The purpose of this study was to define the biologic response of MG63 cells to the innovative patented surface SYNTHEGRA. MG63 morphology and distribution on the three different titanium disk surfaces (sandblasted, smooth, and laser-treated) were evaluated by microscopy analysis after staining with hematoxylin and eosin. Cell adhesion was determined by crystal violet assay at 48 h while proliferation and cytotoxicity were performed by MTT assay at 24, 48, 72 and 240 h. The expression and localization of N-cadherin and beta-catenin were studied by immunofluorescence and confocal microscopy. At 48 h the adhesion was similar in all titanium surfaces, no difference in cell viability were observed in all titanium disks when compared with controls, while the cell growth on laser-treated disks was significantly higher at 240 h than at 24 and 72 h. Morphological analysis show that cells are aligned along the grooves and inside the cavities. beta-catenin signal appeared more diffuse and localized underneath the cell membrane, while N-cadherin signal was fainter in cells grown on SYNTHEGRA surface. This work put into evidence the performance of newly designed laser-micromachined surface for adhesion, growth and distribution of human osteoblast-like cells. SYNTHEGRA surface inducing modification of N-cadherin and beta-catenin expression and localization, are suggestive of cells undergoing differentiation towards osteocytes and could be particularly suited for immediate load implant procedures.

Oral Syphilis: a retrospective analysis of 12 cases and a review of the literature.

OBJECTIVE: To present a retrospective analysis of multicentre case series of oral syphilis and a review of relevant literature. SUBJECTS AND METHODS: A PUBMED search was carried out from 1950 to 2011. Clinical records of patients with exclusive/prevalent oral manifestations of syphilis were collected and examined in three independent hospitals. RESULTS: Of 23 reports describing 34 patients were detected through the review (35% primary, 56% secondary, and 9% tertiary disease), describing unspecific ulcers (59%), mucosal patches (23%), keratosis (6%), pseudomembranes (3%), and gumma (9%). Multicentre case series revealed 12 patients with oral syphilis, of which 17%, 58%, and 25% with, respectively, primary, secondary, and tertiary lesions. Clinically, patients showed white patches (17%), blistering mucositis (8%), chronic unspecific ulcers with/without skin lesions (50%), gumma (17%), and necrosis of the dorsum of the tongue (8%). Oral bullae and tongue necrosis are never described in the current review. CONCLUSIONS: Diagnosis of syphilis remains a challenge because of the multiform and polymorphous clinical pattern at onset and its ability to imitate different diseases. It is mandatory to include syphilis in the differential diagnosis of unusual oral lesions. Diagnosis of oral lesions of syphilis is often difficult, and biopsy is required in controversial cases.

Aspirin challenge/desensitisation before coronary stenting in subjects with history of hypersensitivity. A pragmatic approach.

BACKGROUND: Aspirin hypersensitivity may represent a major problem in patients with ischemic coronary disease who need a stenting procedure. In those patients, clinically unsettled reasonably quick desensitisation procedures are needed. In our study we attempted to select the most suitable procedure on the basis of characteristics and severity of ASA hypersensitivity. METHODS: Thirty patients with a history of mild reactions to anti-inflammatory doses of aspirin (> 325 mg) were considered at low risk and underwent a tolerance test in 5 steps. Thirty-one patients, with a history of severe reactions to anti-platelet doses of aspirin 0 mg) underwent a slow desensitisation in 12 steps, reaching a cumulative dose of 150 mg ASA in 220 minutes. RESULTS: In the first group, 29 patients tolerated the challenge. One developed urticaria, thus underwent challenge/desensitisation and achieved tolerance. In the second group, 3 patients did not tolerate the procedure and had to discontinue. CONCLUSION. Our approach to aspirin hypersensitivity in patients needing coronary stenting, based on a severity stratification, allowed to achieve an effective tolerance to aspirin in the majority of subject in a reasonable short time.

Lymphovascular invasion as a prognostic tool for oral squamous cell carcinoma: a comprehensive review.

Oral cancer is the most common malignancy of the head and neck region, characterized by a poor prognosis. Novel prognostic markers are needed to better stratify these patients. Lymphovascular invasion (LVI) has been included in the eighth edition of the AJCC Cancer Staging Manual as an additional prognostic factor, but its influence on the recurrence risk and lymph node metastasis is relatively understudied. This is a comprehensive review of the literature on the clinical and prognostic role of LVI in oral cancer. A relevant search of the PubMed, Scopus, and Web of Science databases yielded 29 articles that satisfied the inclusion criteria. Findings indicated that LVI is an independent negative prognostic factor in oral cancer patients and appears to be associated with cervical lymph node metastasis and loco-regional recurrence. Notably, in oral tongue cancer, survival outcomes progressively worsen when LVI is associated with other adverse pathological features, especially in the early stages. Therefore, these patients could benefit from elective neck dissection and/or adjuvant therapy. The high variability of LVI prevalence hinders the comparison of literature results. Several methodological limitations were found to be present in the collected articles, including the lack of a rigorous definition for LVI, the difficult detection in routine histological section, the presence of potential confounders, the retrospective nature, and an inadequate sample size in most studies. Therefore, it is necessary to conduct prognostic studies using standardized methods to define and quantify LVI.

p53 and BCL-2 over-expression inversely correlates with histological differentiation in occupational ethmoidal intestinal-type sinonasal adenocarcinoma.

Despite their histological resemblance to colorectal adenocarcinoma, there is little information on the molecular events involved in the pathogenesis of intestinal-type sinonasal adenocarcinoma (ITACs). The aim of this paper is to evaluate the possible role of TP53 and Bcl-2 gene defects in ITAC by investigating the immunohistochemical expression of TP53 and Bcl-2 gene products in a group of ethmoidal ITACs associated with occupational exposure. A retrospective study on 15 patients with pathological diagnosis of primary ethmoidal ITAC was conducted. Representative formalin-fixed, paraffin wax-embedded block from each case was selected for immunohistochemical studies using the antibodies against p53 and Bcl-2. Clinical-pathological data were also correlated with the staining results. The results of immunohistochemical examination demonstrated that poorly differentiated cases showed a higher percentage of p53 and Bcl-2 expressing cells in comparison to well-differentiated cases. No correlation was found with other clinico-pathological parameters, including T, stage and relapses. The relationship between up-regulation of p53 and Bcl-2 and poorly differentiated ethmoidal adenocarcinoma suggests a role of these genes, in combination with additional genetic events, in the pathogenesis of ITAC.

Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression.

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.

Reciprocal altered expression of E-cadherin and P-cadherin in mucous membrane pemphigoid.

E- and P- cadherins are involved in the selective adhesion of epidermal cells. To gain insight into the role of cadherins on the acantholysis of keratinocytes and further investigate the pathogenesis of Mucous Membrane Pemphigoid, we examined the expression of P-cadherin and E-cadherin, in normal human oral mucosa, lesional and peri-lesional mucosa in MMP. Twenty-nine samples from paraffin-embedded specimens of MMP were used for the study. Five specimens of healthy oral mucosa were evaluated as control group. To evaluate the E- and P-Cadherin expression, a mean percentage of positive cells was determined from the percentage of positive cells derived from the analysis of 100 cells in ten random areas at x400 magnification. It was observed that E-cadherin was weakly and discontinuously expressed on the epithelial layers of pemphigoid mucosa, while it was intensively expressed on all keratinocytes in normal human skin. In contrast, P-cadherin was strongly expressed throughout the entire epidermal layer in MMP samples, although its expression is restricted to the basal cell layer in normal human skin. Statistical analyses showed that the percentage of E-cadherin positive cells in the epithelium of pemphigoid cases was significantly decreased compared with that in normal human mucosa. There was a significant increase in the percentage of P-cadherin positive cells in the epithelial layers of MMP compared with normal human mucosa. The present study showed that there is downregulation of E-cadherin expression and upregulation of P-cadherin expression in MMP mucosa, which may be involved in the pathogenesis of MMP.