Morphological and molecular analysis of the collagen fibers in inflammatory process

Collagen makes up one third of the total protein in humans, being formed by the connection of three polypeptide chains arranged in a triple helix. This protein has fundamental importance in the formation of extracellular matrix of connective tissue. This study aimed to analyze the structural changes of collagen, which are resulting from inflammatory processes in oral mucosa, and to make the comparative analysis between the histopathology and the Raman spectra. The samples of tissues with inflammatory fibrous hyperplasia (IFH) and normal mucosa (NM) were evaluated by Raman Spectroscopy, hematoxylin-eosin and Massons trichrome stain. The histological analysis in both stains showed differences in collagen fibers, which was presented as thin fibers and arranged in parallel direction in NM and as collagen fibers are thick, mature and not organized, showing that these types of stain show morphological changes of collagen in IFH. The Raman Spectroscopy discriminate the groups of NM and IFH based on vibrational modes of proline, hydroxiproline and CH3, CH2. The histological stains only shows information from morphological data, and can be complemented by Raman spectra. This technique could demonstrate that inflammatory process caused some changes in collagen structure which is related to aminoacids such as proline and hidroxyproline. © 2011 SPIE-OSA.

Histochemical and ultrastructural analysis of hepatic glycogen and collagen fibers in alloxan-induced diabetic rats submitted to long-term physical training

Alterations in liver functions are common among diabetic patients, and many symptoms in the liver have been reported, including changes in glycogen stores and in the amount of collagen fibers. The practice of physical training and its morphological effects in this organ, however, are scarcely studied. In order to observe the morphological effects of alloxan-induced diabetes and the alterations arising from the practice of long-term chronic physical training in the liver, samples were collected and processed, and then analyzed by means of the histochemical techniques Periodic Acid-Schiff and Picrosirius-Hematoxylin, and ultrastructural cytochemical test of Afzelius. Through evaluation of the tissue, it was observed a drastic reduction in hepatic glycogen stores of sedentary diabetics, recovered in trained diabetic rats. Furthermore, it was detected a decrease in the content of perisinusoidal collagen fibers in the diabetic liver, also recovered due to the development of a training protocol. On ultrastructural level, cytochemical analysis confirmed the loss of glycogen and the recovery obtained by training. In conclusion, the practice of a long-term chronic physical training protocol may be considered an important assistant in the treatment of diabetes, mitigating the occurrence of possible damages to liver tissue. © 2011 Elsevier Ltd.

Evaluation of tissue reaction, cell viability and cytokine production induced by Sealapex Plus

The aim of this study was to investigate the effects of mineral trioxide aggregate (MTA), Sealapex, and a combination of Sealapex and MTA (Sealapex Plus) on the reaction of subcutaneous connective tissue of rats, and on cell viability and cytokine production in mouse fibroblasts. The tissue reaction was carried out with dentin tubes containing the materials implanted in the dorsal connective tissue of rats. The histological analysis was performed after 7 and 30 days. Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts to evaluate the cell viability by MTT assay. ELISA assays were also performed after 24 h of exposure of the mouse fibroblasts to set material disks. Histopathologic examination showed Von Kossa-positive granules that were birefringent to polarized light for all the studied materials at the tube openings. No material inhibited the cell viability in the in vitro test. It was detected IL-6 production in all root-end filling materials. MTA and Sealapex Plus induced a slight raise of mean levels of IL-1β. The results suggest that Sealapex Plus is biocompatible and stimulates the mineralization of the tissue.

Pulp tissue dissolution when the use of sodium hypochlorite and EDTA alone or associated

Purpose: The aim of the present study was to evaluate the tissue dissolving capacity of various concentrations of sodium hypochlorite either alone or in combination with 17% EDTA. Methods: Eighty bovine pulp fragments were prepared, and their weight was determined using a precision balance. Each pulp fragment was immersed for 2 hours in a solution/mixture that was based on the following groups: G1-saline solution; G2-0.5% NaOCl; G3-1.0% NaOCl; G4-2.5% NaOCl; G5-17% EDTA; G6-0.5% NaOCl+17% EDTA; G7-1.0% NaOCl+ 17% EDTA; and G8-2.5% NaOCl+17% EDTA. The final weight was measured, and the weight loss was calculated. A statistical analysis was performed using either the Student's t-test for paired samples or an ANOVA and Tukey tests (P<0.05 was considered to be significant). Results: We measured a significant difference between the sample weight before and after treatment for each of the tested groups (P<0.05). The 2.5% sodium hypochlorite solution (G4) completely dissolved the pulp tissue within the test period. NaOCl+EDTA was less effective than sodium hypochlorite alone at dissolving the pulp tissue (P<0.05), and EDTA alone (G5) did not markedly dissolve the pulp tissue. Conclusion: Using EDTA together with NaOCl reduced the tissue dissolving properties compared with NaOCl alone, regardless of the concentration of NaOCl that was used. © 2011 Só et al.; licensee EDIPUCRS.

Biocompatibility analysis of Bioglass® 45S5 and Biosilicate® implants in the rabbit eviscerated socket

Purpose: Bioactive glass and bioactive glass-ceramic cone implants were placed in the rabbit eviscerated socket to assess their biocompatibility. Methods: Fifty-one Norfolk albino rabbits underwent evisceration of the right eye followed by implantation of cones made from Bioglass® 45S5 (control group) and two types of bioactive glass-ceramic (Biosilicate®), a single- and a two-phase bioactive glass-ceramic implants into the scleral cavity. Postoperative reactions, animal behavior and socket conditions were monitored daily. Clinical exam, biochemical evaluations, and orbit computed tomographic scan were done at 7, 90, and 180 days post-procedure. After that, the animals were euthanized, and the orbital content was removed and prepared to light microscopy with morphometric evaluation and scanning electron microscopy examination. Statistical analysis was done by parametric and non-parametric analysis of variance, complemented by Dunn's and Tukey's tests (p<0.05). Results: All animals did not develop systemic toxicity throughout the experimental period and also did not have orbit infection, implant migration or extrusion. Morphological analysis demonstrated pseudocapsule around all implants. Bioglass® and single-phase Biosilicate® implants induced less inflammation and pseudocapsule formation than two-phase Biosilicate® cones. Seven days post-procedure, the inflammatory reaction was intense and gradually decreased throughout the experiment. Tissue reaction was least intense in animals receiving Bioglass® implants. Conclusions: We observe discrete differences among the studied materials, with best responses obtained with use of Bioglass® 45S5 and single-phase Biosilicate®. The authors agree these implants might be useful in the management of the anophthalmic socket. © 2012 Informa Healthcare USA, Inc.

Comparative study of the healing process when using Vicryl®, Vicryl Rapid®, Vicryl Plus®, and Monocryl® sutures in the rat dermal tissue

Introduction: Various types of sutures are available in the market with different constitutions. However, there is a lack of research to assess and quantify the behavior of these materials. Resources and materials: This study comes benchmark wires polyglactin 910 (Vicryl ®), irradiated polyglactin 910 (Vicryl Rapid ®), polyglactin 910 treated with triclosan (Vicryl Plus ®), and poliglecaprone 25 (Monocryl ®). For this, we used 40 rats that were divided into two groups, underwent two skin incisions longitudinal 2-cm long. In Group A, simple interrupted sutures using irradiated polyglactin 910 on the right and left side of polyglactin 910, and in group B, polyglactin 910 with triclosan on the right and the left poliglecaprone 25 were made. At 2, 7, 14, and 28 days after surgery, the ten animals were killed per period, and the samples were processed for histomorphologic and histometric analysis. Conclusions: The results demonstrated that the wire poliglecaprone 25 showed better biological response, with less inflammatory infiltrates and rapid organization of connective tissue. © 2012 Springer-Verlag Berlin Heidelberg.

Evaluation epidermal p53 immunostaining by digital image analysis

Digital techniques have been developed and validated to assess semiquantitatively immunohistochemical nuclear staining. Currently visual classification is the standard for qualitative nuclear evaluation. Analysis of pixels that represents the immunohistochemical labeling can be more sensitive, reproducible and objective than visual grading. This study compared two semiquantitative techniques of digital image analysis with three techniques of visual analysis imaging to estimate the p53 nuclear immunostaining. Methods: Sixty-three sun-exposed forearm-skin biopsies were photographed and submitted to three visual analyses of images: the qualitative visual evaluation method (0 to 4 +), the percentage of labeled nuclei and HSCORE. Digital image analysis was performed using ImageJ 1.45p; the density of nuclei was scored per ephitelial area (DensNU) and the pixel density was established in marked suprabasal epithelium (DensPSB). Results: Statistical significance was found in: the agreement and correlation among the visual estimates of evaluators, correlation among the median visual score of the evaluators, the HSCORE and the percentage of marked nuclei with the DensNU and DensPSB estimates. DensNU was strongly correlated to the percentage of p53-marked nuclei in the epidermis, and DensPSB with the HSCORE. Conclusion: The parameters presented herein can be applied in routine analysis of immunohistochemical nuclear staining of epidermis. © 2012 John Wiley & Sons A/S.

Histological analysis of the osseointegration of Ti-30Ta dental implants after surface treatment

Metallic biomaterials are used to reinforce or to restore the form and function of hard tissues. Implants and prosthesis are used to replace shoulders, knees, hips and teeth. When these materials are inserted in bone several biological reactions happen. This process can be associated to surface properties (topography, roughness and surface energy). In this work, the influence of biomimetic surface treatment in the osseointegration of Ti-30Ta dental implants was evaluated. Ingots were obtained from titanium and tantalum by using an arc-melting furnace. They were submitted to heat treatment at 1,100°C for 1 h, cooled in water and cold worked by swaging. Then, screw-shaped implants (2.0 mm diameter by 2.5 mm length) were manufactured and they were implanted in a rat's femur. Animals were divided into two groups: untreated (control group) and treated (biomimetic surface treatment). They were sacrificed 30 days after implantation. For histological analysis, implants with surrounding tissue were removed and immersed in formaldehyde. Samples were embedded in polymethyl methacrylate and after polymerization, cut with a saw, polished and mounted on glass slides. The results obtained suggest that biomimetic surface treatment was able to promote an increase osseointegration on the surface of dental implants. © Springer-Verlag Berlin Heidelberg 2013.

Evaluation of effect of cyclosporine A on the bone tissue with induced periodontal disease to ligature in rats

The administration of cyclosporine A (CsA) has been associated with significant bone loss and increased bone remodeling. The present investigation was designed to evaluate the effects of CsA on alveolar bone of rats subjected to experimental periodontitis, using histomorphometric and histological analysis. Twenty-four rats were divided into groups with 6 animals each: 1, control; 2, rats with ligature around the lower first molars; 3, rats with ligature around the lower first molars and that were treated with 10 mg CsA/kg of body weight/d; and 4, rats treated with 10 mg CsA/kg of body weight/d. At the end of 30 days, rats were humanely killed and subjected to a histological processing, with analysis of the distance cemento-enamel junction and alveolar bone crest, bone area, eroded bone area, and cemento surface. All of them were assessed at the mesial region of the alveolar bone. The CsA therapy combined with ligature placement decreased bone area and increased the eroded bone area around the tooth surface. The results at the histological analysis showed the same combination and changes. Therefore, in spite of the lack of a direct effect on the alveolar bone height, the CsA therapy intensified the imbalance of the alveolar bone homeostasia in a rat model of experimental periodontitis. © 2013 Elsevier Inc.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

The Vampirome: Transcriptome and proteome analysis of the principal and accessory submaxillary glands of the vampire bat Desmodus rotundus, a vector of human rabies

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~. 200. million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology. Biological significance: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology. © 2013.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Is characterizing the digital forensic facial reconstruction with hair necessary? A familiar assessors' analysis

Background: In the international scientific literature, there are few studies that emphasize the presence or absence of hair in forensic facial reconstructions. There are neither Brazilian studies concerning digital facial reconstructions without hair, nor research comparing recognition tests between digital facial reconstructions with hair and without hair. The miscegenation of Brazilian people is considerable. Brazilian people, and, in particular, Brazilian women, even if considered as Caucasoid, may present the hair in very different ways: curly, wavy or straight, blonde, red, brown or black, long or short, etc. For this reason, it is difficult to find a correct type of hair for facial reconstruction (unless, in real cases, some hair is recovered with the skeletal remains). Aims and methods: This study focuses on the performance of three different digital forensic facial reconstructions, without hair, of a Brazilian female subject (based on one international database and two Brazilian databases for soft facial-tissue thickness) and evaluates the digital forensic facial reconstructions comparing them to photographs of the target individual and nine other subjects, employing the recognition method. A total of 22 assessors participated in the recognition process; all of them were familiar with the 10 individuals who composed the face pool. Results and conclusions: The target subject was correctly recognized by 41% of the 22 examiners in the International Pattern, by 32% in the Brazilian Magnetic Resonance Pattern and by 32% in the Brazilian Fresh Cadavers Pattern. The facial reconstructions without hair were correctly recognized using the three databases of facial soft-tissue thickness. The observed results were higher than the results obtained using facial reconstructions with hair, from the same skull, which can indicate that it is better to not use hair, at least when there is no information concerning its characteristics. © 2013 Elsevier B.V. All rights reserved.

Comparison of different designs of implant-retained overdentures and fixed full-arch implant-supported prosthesis on stress distribution in edentulous mandible - A computed tomography-based three-dimensional finite element analysis

A finite element analysis was used to compare the effect of different designs of implant-retained overdentures and fixed full-arch implant-supported prosthesis on stress distribution in edentulous mandible. Four models of an human mandible were constructed. In the OR (O'ring) group, the mandible was restored with an overdenture retained by four unsplinted implants with O'ring attachment; in the BC (bar-clip) -C and BC groups, the mandibles were restored with overdentures retained by four splinted implants with bar-clip anchor associated or not with two distally placed cantilevers, respectively; in the FD (fixed denture) group, the mandible was restored with a fixed full-arch four-implant-supported prosthesis. Models were supported by the masticatory muscles and temporomandibular joints. A 100-N oblique load was applied on the left first molar. Von Mises (σvM), maximum (σmax) and minimum (σmin) principal stresses (in MPa) analyses were obtained. BC-C group exhibited the highest stress values (σvM=398.8, σmax=580.5 and σmin=-455.2) while FD group showed the lowest one (σvM=128.9, σmax=185.9 and σmin=-172.1). Within overdenture groups, the use of unsplinted implants reduced the stress level in the implant/prosthetic components (59.4% for σvM, 66.2% for σmax and 57.7% for σmin versus BC-C group) and supporting tissues (maximum stress reduction of 72% and 79.5% for σmax, and 15.7% and 85.7% for σmin on the cortical and trabecular bones, respectively). Cortical bone exhibited greater stress concentration than the trabecular bone for all groups. The use of fixed implant dentures and removable dentures retained by unsplinted implants to rehabilitate edentulous mandible reduced the stresses in the periimplant bone tissue, mucosa and implant/prosthetic components. © 2013 Elsevier Ltd.

Role of Ca, Fe, Cu and Zn in breast cancer: Study by X-ray fluorescence techniques and immunohistochemical analysis

In recent years, several studies have shown that concentrations of trace elements are altered in neoplastic breast tissues. However, the microenvironment and metabolic changes caused by tumors are complex and still not completely understood. Under this aspect, the combination of different techniques to investigate the role of trace elements in promoting and/or maintaining a tumor is interesting once the combination of information obtained by analytical techniques and immunohistochemical assays, associated with clinicopathological data, may allow a better metabolic understanding of trace elements in breast cancer. In this work, the role of the trace elements Ca, Fe, Cu and Zn in neoplastic breast tissues was investigated by X-ray fluorescence (XRF) techniques and immunohistochemical assays. We determined concentrations of Ca, Fe, Cu and Zn in normal and neoplastic breast tissues using energy dispersive XRF, and these values were used to set the positive or negative expression of elements in normal and neoplastic tissues. These expressions were correlated with the spatial distributions of trace elements (evaluated by micro-XRF) and with immunoexpression of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs and vascular endothelial growth factor. The results revealed that the expression of the trace elements Fe, Cu and vascular endothelial growth factor are related, indicating that higher levels of these elements can be associated with the angiogenic process in breast cancer. Also, associations between Ca, Zn and MMPs expression have been observed, possibly because of the fact that both metals are present in these proteins. © 2013 John Wiley & Sons, Ltd.