In situ zymography and immunolabeling in fixed and decalcified craniofacial tissues.

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.

Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress.

Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker of activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug.

SEM Study of apical morphological alterations in primary teeth with vital and necrotic pulps.

This study evaluated, by SEM, the morphology of human primary teeth roots. Twenty-four teeth were divided into 3 groups: pulp vitality (group I) and pulp necrosis without (group II) and with apical periodontitis (group III). Roots were analyzed by the presence of periodontal ligament (PDL) fibers and resorption areas. In groups I and II, presence of PDL fibers and absence of resorption were observed in all cases (100%), while all specimens (100%) of group III showed no PDL fibers and resorption areas. In conclusion, there are morphological differences in the apical region of primary teeth with different pulpal and periapical pathologies.

Interferon-gamma, interleukin-10, Intercellular adhesion molecule-1, and chemokine receptor 5, but not interleukin-4, attenuate the development of periapical lesions.

This study examines the role of Th1 (interferon-gamma [IFN-gamma]) and Th2 (interleukin-4 [IL-4] and IL-10) cytokines, an intercellular adhesion molecule (ICAM-1), and a chemokine receptor (CCR5) in the pathogenesis of periapical lesions at different stages of development in knockout mice. For lesion induction, the first molar was opened and inoculated with 4 bacterial strains and left open to the oral environment. After 21 and 42 days, the IFN-gamma, IL-10, ICAM-1, and CCR5 knockout animals presented periapical lesions larger than those of wild-type animals. There was no statistically significant difference between periapical lesions induced in IL-4 knockout and wild-type animals during the periods evaluated. Our findings suggest an important role for IFN-gamma, IL-10, ICAM-1, and CCR5 in the pathogenesis of experimentally induced pulp infection and bone destruction as endogenous suppressor of periapical lesion development, whereas IL-4 appears to present a nonsignificant effect on periapical lesion modulation.

Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured l929 fibroblasts.

Because chlorhexidine (CHX) has been recommended as either an endodontic irrigant or root canal dressing, this study aimed to characterize, in vivo, the lesion induced by injections of CHX in the paw of mice at selected time intervals (24 and 48 hours and 7 and 14 days) and, in vitro, the mode of cell death, necrosis and/or apoptosis, and the cellular stress caused by exposition of cultured L929 fibroblasts to ascending concentrations of CHX for 24 hours. CHX injected in the subplantar space of the hind paw of mice induced severe toxic effects, as evidenced by necrotic changes in the epidermis, dermis, and subcutaneous tissue in association with reactive inflammatory response, particularly at higher concentrations. In addition, in cultured fibroblasts, CHX induced apoptosis at lower concentrations and necrosis at higher concentrations and increased expression of heat-shock protein 70, an indicator of cellular stress. Taken together, these findings suggest that CHX may have an unfavorable effect on the resolution of apical periodontitis.

Biomineralization of polyanionic collagen-elastin matrices during cavarial bone repair.

The polyanionic collagen-elastin matrices (PCEMs) are osteoconductive scaffolds that present high biocompatibility and efficacy in the regeneration of bone defects. In this study, the objective was to determine if these matrices are directly mineralized during the osteogenesis process and their influence in the organization of the new bone extracellular matrix. Samples of three PCEMs, differing in their charge density, were implanted into critical-sized calvarial bone defects created in rats and evaluated from 3 days up to 1 year after implantation. The implanted PCEMs were directly biomineralized by osteoblasts as shown by ultrastructural, histoenzymologic, and morphologic analysis. The removal of the implants occurred during the bone remodeling process. The organization of the new bone matrix was evaluated by image texture analysis determining the Shannon's entropy and the fractal dimension of digital images. The bone matrix complexity decreased as the osteogenesis progressed approaching the values obtained for the original bone structure. These results show that the PCEMs allow faster formation of new bone by direct biomineralization of its structure and skipping the biomaterial resorption phase.

Biocompatibility of anionic collagen matrix as scaffold for bone healing.

The basic approach to the treatment of bone defects involves the use of scaffolds to favor tissue growth. Although several bioscaffolds have been proposed for this purpose, the search for new and enhanced materials continues in an attempt to address the drawbacks of the present ones. Modifying current materials can be a fast and cheap way to develop new ones. Among them, type I collagen allows its structure to be modified using relatively simple techniques. By means of an alkaline treatment, anionic collagen with enhanced piezoelectric properties can be obtained through hydrolysis of carboxyamides groups of asparagine and glutamine residues from collagen in carboxylic. The process applied to a raw source of collagen, bovine pericardium, provided a sponge-like structure, with heterogeneous pore size, and, moreover, the complete removal of interstitial cells. For the evaluation of the biocompatibility of such matrices, they were implanted in surgically created bone defects in rat tibias. Empty defects served as controls. This experimental model allowed a preliminary evaluation of the osteoconductiveness of the matrices. The histological results presented a low inflammatory response and bone formation within a short period of time, similar to that of controls. The low cost of production associated to the biocompatibility and osteoconductivity performance make the anionic collagen matrices promising alternatives for bone defects treatment.

EM evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth.

The aim of this study was to evaluate the presence of bacterial biofilm on the external surface of the root apex in teeth with pulp necrosis, with and without radiographically visible periapical lesions, and in teeth with a vital pulp. Twenty-one teeth were extracted, eight with pulp necrosis and periapical lesions, eight with pulp necrosis without radiographically visible periapical lesions, and five with a vital pulp. The roots were sectioned, and the root apexes (+/- 3 mm) were processed for scanning electron microscope evaluation. The surface of the apical root was evaluated for the presence of microorganisms, root resorption, and biofilm. There were no microorganisms on the apical root surface of either teeth with pulp vitality or with pulp necrosis with no radiographically visible periapical lesions. Microorganisms were always present in teeth with pulp necrosis and radiographically visible periapical lesions. These included cocci, bacilli, and filaments and the presence of an apical biofilm. Apical biofilm is clinically important because microbial biofilms are inherently resistant to antimicrobial agents and cannot be removed by biomechanical preparation alone. This may cause failure of endodontic treatment as a consequence of persistent infection.

Effect of calcium hydroxide on bacterial endotoxin in vivo.

The aim of this study was the histopathological evaluation of apical and periapical tissues in dog teeth that were submitted to bacterial endotoxin, associated or not with calcium hydroxide. After removal of the pulp from 60 premolars, the teeth were divided into four groups and were filled with bacterial endotoxin (group 1), bacterial endotoxin plus calcium hydroxide (group 2), saline solution (group 3), or had induced periapical lesions with no treatment (group 4). After 30 days, animals were killed and the teeth processed histologically. The inflammatory infiltrate, the thickness of the periodontal ligament, and the presence of resorption areas were similar for groups 1 and 4. Groups 2 and 3 were similar to each other. It can be concluded that the bacterial endotoxin caused a periapical lesion and that calcium hydroxide detoxified the lipopolysaccharides in vivo.

Efficacy of polyanionic collagen matrices for bone defect healing.

Polyanionic collagen-elastin matrices (PCEMs) possess attractive properties, such as extra negative charges, piezoelectricity, and extra RGD sites, which could make them effective in the treatment of bone defects. Although they are biocompatible with the osteogenesis process, it is unknown if PCEMs could aid in the recovery of bone defects in challenging situations. To evaluate this hypothesis, three PCEMs, differing in their negative charge density, were implanted in rat calvarial defects. Specimens harvested at 3, 7, 15, 30, and 60 days after implantation were evaluated radiographically and histologically. Two matrices were able to sustain the osteogenesis process and quickly recover the lost bone structure. The third, and most electronegative, left matrix remnants amidst the areas of new bone. The control showed bone formation limited to the boundaries of the defect. These results suggest that some PCEMs might become a useful resource in the treatment of bone defects.

Cellular and molecular tissue response to triple antibiotic intracanal dressing.

INTRODUCTION: The aim of this study was to characterize the response of mouse subcutaneous tissue to triple antibiotic paste (TAP) using conventional light microscopy and real-time PCR (qRT-PCR). METHODS: Polyethylene tubes containing TAP or calcium hydroxide (CH) (ie, the control group) were implanted in mouse subcutaneous tissue. Animals that received empty tubes or no tubes were used as additional controls. After periods of 7, 21, and 63 days postimplantation, the specimens were removed and subjected to histologic processing. The number of inflammatory cells and vessels, vessel areas, vascular density, and relative percentage of collagen were evaluated. Gene expression of proinflammatory (interleukin-1 beta, tumor necrosis factor alpha, and interleukin 17) and anti-inflammatory (transforming growth factor beta) cytokines and angiogenic factors (vascular endothelial growth factor and hypoxia-inducible factor-1 alpha) was quantified by 7 and 21 days postimplantation. Results were analyzed using the Student t test, analysis of variance, and the Tukey test (α = 0.05). RESULTS: TAP induced an exuberant inflammatory and angiogenic response, with higher numbers of inflammatory cells, higher vascular area and density, and lower relative percentage of collagen compared with CH. In general, the expression of genes involved in inflammation and angiogenesis was higher in the TAP group compared with animals that received CH or empty tubes. CONCLUSIONS: The response of mouse subcutaneous tissue to TAP was characterized by exuberant and persistent inflammatory and angiogenic responses with no repair and high gene expression of biomarkers associated with inflammation and angiogenesis.

Response of mice connective tissue to intracanal dressings containing chlorhexidine.

Substances containing chlorhexidine (CHX) have been studied as intracanal medicaments. The aim of the present study was to characterize the response of mouse subcutaneous connective tissue to CHX-containing medications by conventional optical microscopy. The tissue response was evaluated by implanting polyethylene tubes containing one of the substances evaluated: Calen paste + 0.5% CHX, Calen + 2% CHX, 2% CHX gel, and Calen paste (control). After experimental periods of 7, 21, and 63 days, the implants (n = 10) were removed along with the subcutaneous connective tissue. Tissue samples were subjected to histological processing, and sections were stained with hematoxylin and eosin. Qualitative and quantitative analyses of the number of inflammatory cells, blood vessels, and vascularized areas were performed. Results were analyzed by ANOVA and Tukey tests with the significance level set at 5%. We concluded that Calen + 0.5% CHX led to reparative tissue response in contrast with Calen + 2% CHX and 2% CHX gel, which induced persistent inflammatory response, pointing to the aggressive nature of this mixture. When Calen + 2% CHX and 2% CHX gel were compared, the latter induced more intense inflammatory response.

Application of fluorescence microscopy on hematoxylin and eosin-stained sections of healthy and diseased teeth and supporting structures.

Destruction of dental tissue and supporting structures is usually microscopically assessed by routine hematoxylin and eosin (HE)-stained sections. This short communication is concerned with the potential role of fluorescence microscopy of HE-stained sections to study morphological aspects of intact and pathological teeth in dental research. This methodology improves the visualization of the anatomical structures of the intact teeth, especially anatomical features and periodontal ligament spatial distribution. This technique also improves the visualization of the root and bone resorption and the delineation of the periapical lesion extension. The fluorescence microscopy technique of HE-stained sections is an easy, reliable and inexpensive method that seems to be a useful tool for evaluating morphological aspects of intact and pathological teeth in dental research.

Effect of rotary or manual instrumentation, with or without a calcium hydroxide/1% chlorhexidine intracanal dressing, on the healing of experimentally induced chronic periapical lesions.

OBJECTIVE: To evaluate the healing of experimentally induced chronic periapical lesions in dogs at 30, 75, and 120 days after root canal instrumentation with rotary NiTi files or manual K-files, with or without a calcium hydroxide/1% chlorhexidine paste intracanal dressing. STUDY DESIGN: The second, third, and fourth mandibular premolars and the second and third maxillary premolars of 5 dogs (12 to 18 months of age, weighing 8 to 15 kg) were selected for treatment (a total of 82 root canals). After pulp removal, the root canals were left exposed to the oral cavity for 7 days to allow microbial contamination, after which the root canals were sealed with ZOE cement until periapical lesions were confirmed with radiography. Group I and II teeth were instrumented with manual K-files using the crown-down technique. In group III and IV teeth, NiTi rotary files were used. The apical delta was perforated by using #20 to #30 K-files at the length of the tooth, thus creating a standardized apical opening. The apical stop was enlarged to size 70, with 2.5% sodium hypochlorite irrigation at each file change. Teeth in groups II and IV were dressed with calcium hydroxide (Ca(OH) 2 )/1% chlorhexidine (CHX) paste for 15 days before root filling. Group I and III teeth did not receive an intracanal dressing. The access openings of the teeth were permanently restored with silver amalgam condensed on a glass ionomer cement base. Pairs of standardized periapical radiographs were taken at the beginning of the treatment (0 days) and at 30, 75, and 120 days after filling. RESULTS: There was no significant difference in the rate of radiographic healing of the periapical lesions between manual and rotary instrumentation. Radiographs taken at 120 days showed that the treatment with Ca(OH) 2 /1% CHX paste resulted in a significant reduction in mean size of the periapical lesions in comparison to single-session treatment. These findings were also true for histologic observations. CONCLUSION: The findings support the hypothesis that, regardless of the instrumentation technique (manual or rotary), the use of an intracanal dressing is important in the endodontic treatment of dog's teeth with experimentally induced chronic periapical lesions.

Estudo estereológico das alteraçöes determinadas pelo etanol nas glândulas salivares submandibular e parótida do rato / Stereological study of the alterations induced by ethanol on rat submandibular and parotid salivary glands

Foi realizado um estudo estereológico com o objetivo de determinar os efeitos do consumo prolongado de etanol na estrutura das glândulas salivares de ratos bem nutridos. Animais mantidos com uma dieta sólida semi-sintética enriquecida e álcool, correspondendo a 35 por cento de ingestäo calórica total, desenvolveram alteraçöes estruturais marcantes nas glândulas salivares. Foram observadas atrofia com fibrose intersticial e infiltraçäo gordurosa. Essas alteraçöes foram avaliadas estereologicamente