Immunocytochemical detection of dentine matrix protein 1 in experimentally induced reactionary and reparative dentine in rat incisors

Objective: Although the general mechanisms of dentinogenesis are understood, several aspects regarding tertiary dentine formation still deserve investigation, especially regarding the presence and distribution of some noncollagenous matrix proteins. As dentine matrix protein 1 (DMP 1) is present in primary dentine, it is possible that this protein may also be present in the dentine matrix secreted after injury, but there are no immunocytochemical studies attempting its detection in tertiary dentine. The aim of this study was to examine the ultrastructural immunolocalization of DMP 1 in the tertiary dentine after extrusion of the rat incisor. Study design: Upper incisors were extruded 3 mm and then repositioned into their sockets. After several periods, the incisors were fixed and processed for transmission electron microscopy and for immunocytochemistry for DMP 1. Results: Extrusion yielded both types of tertiary dentine, which varied in aspect and related cells. DMP 1 was found in the mineralized matrix of all types of dentine, presenting high affinity for collagen, but rare colloidal gold particles over predentine. DMP 1 was evident in the supranuclear region and inside the nucleus of some odontoblast-like cells. Conclusion: The observed association between DMP 1 and collagen seem to be essential for reactionary and reparative dentine formation. (C) 2010 Elsevier Ltd. All rights reserved.

Early alveolar bone regeneration in rats after topical administration of simvastatin

Objectives. The aim of this study was to ultrastructurally examine the influence of simvastatin on bone healing in surgically created defects in rat mandibles. Study design. Bone defects 0.8 mm in diameter were created in the buccal aspect of first mandibular molar roots and filled with 2.5% simvastatin gel, while the controls were allowed to heal spontaneously. The rats were humanely killed 7, 9, 11, or 14 days postoperatively, and the specimens were processed for scanning and transmission electron microscopy, as well as for colloidal gold immunolabeling of osteopontin. Results. The regenerated alveolar bone in the simvastatin-treated defects presented smaller marrow spaces, and the collagen fibrils were regularly packed exhibiting a lamellar bone aspect. Osteopontin was present through the bone matrix during the wound healing and alveolar bone regeneration. Conclusion. The present study provides evidence that a single topical application of 2.5% simvastatin gel improves the quality of the new bone and decreases bone resorption. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112: 170-179)

The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices

The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium. Objective. This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days. Methods. Dentin beams (1 mm x 2 mm x 6 mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus (E) (3-point bending, 15% strain) the beams were divided into 5 groups (n = 11/group) and incubated at 37 degrees C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA. Results. Both the storage medium and the storage time showed significant effects on E, mass loss and HOP release (p < 0.05). The incubation in CM resulted in relatively rapid and significant (p < 0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower (p < 0.05) than in the control CM medium, the recommended storage medium. Conclusions. The storage solutions used to age resin-dentin bonds should be buffered solutions that contain both calcium and zinc. The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs. (c) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Long-term effects of developmental exposure to di-n-butyl-phthalate (DBP) on rat prostate: Proliferative and inflammatory disorders and a possible role of androgens

In the present study we evaluated the toxic effects on the male adult rat prostate of DBP exposure during fetal and lactational periods, because although many studies have addressed the influence of phthalates on the male reproductive system, only a few have discussed their possible effects on prostate development. Pregnant females were distributed into two experimental groups: Control (C) and Treated (T). The females of the T group received DBP (100 mg/kg, by gavage) from gestation day 12 to postnatal day 21, while C rats received the vehicle (corn oil). In adulthood (90 days old), the animals were euthanized. The serum and testicular testosterone levels were measured. Ventral prostate was removed and weighed. Distal segment fragments of the ventral prostate were fixed and processed for histochemistry and immunohistochemistry to detect androgen receptor (AR) and Ki67 antigens. Protein extraction from ventral prostate fragments was performed for AR immunoblotting and Gelatin zymography for MMP-2 and MMP-9 (MMP, metalloproteinase). Stereological and histopathological analyses were also performed. Serum and testicular testosterone levels and prostate weight were comparable between groups. In the T group the relative proportions (%) of epithelial (C=32.86; T=42.04*) and stromal (C=21.61; T=27.88*) compartments were increased, while the luminal compartment was decreased (C=45.54; T=30.08*), *p < 0.05. In T, disseminated inflammatory infiltrate in the stroma, associated or not with epithelial dysplasia and PIN (Prostatic Intraepithelial Neoplasia), was observed. Increases in AR expression, proliferation index and metalloproteinase 9 (MMP-9) activity were noted in T animals. In some T animals, collagen fibrils accumulated adjacent to the epithelium. As far as we are aware, this is the first report in the literature showing that phthalates could play a role in proliferative and inflammatory disorders of the rat prostate. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Three-dimensional orthotropic viscoelastic finite element model of a human ligament

Ligaments undergo finite strain displaying hyperelastic behaviour as the initially tangled fibrils present straighten out, combined with viscoelastic behaviour (strain rate sensitivity). In the present study the anterior cruciate ligament of the human knee joint is modelled in three dimensions to gain an understanding of the stress distribution over the ligament due to motion imposed on the ends, determined from experimental studies. A three dimensional, finite strain material model of ligaments has recently been proposed by Pioletti in Ref. [2]. It is attractive as it separates out elastic stress from that due to the present strain rate and that due to the past history of deformation. However, it treats the ligament as isotropic and incompressible. While the second assumption is reasonable, the first is clearly untrue. In the present study an alternative model of the elastic behaviour due to Bonet and Burton (Ref. [4]) is generalized. Bonet and Burton consider finite strain with constant modulii for the fibres and for the matrix of a transversely isotropic composite. In the present work, the fibre modulus is first made to increase exponentially from zero with an invariant that provides a measure of the stretch in the fibre direction. At 12% strain in the fibre direction, a new reference state is then adopted, after which the material modulus is made constant, as in Bonet and Burton's model. The strain rate dependence can be added, either using Pioletti's isotropic approximation, or by making the effect depend on the strain rate in the fibre direction only. A solid model of a ligament is constructed, based on experimentally measured sections, and the deformation predicted using explicit integration in time. This approach simplifies the coding of the material model, but has a limitation due to the detrimental effect on stability of integration of the substantial damping implied by the nonlinear dependence of stress on strain rate. At present, an artificially high density is being used to provide stability, while the dynamics are being removed from the solution using artificial viscosity. The result is a quasi-static solution incorporating the effect of strain rate. Alternate approaches to material modelling and integration are discussed, that may result in a better model.

Differential expression and distribution of syndecan-1 and-2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell-cell and cell-matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell-cell and cell-matrix interactions, while syndecan-2 showed a predilection to associate with cell-matrix interactions during hard tissue formation.

Comparisons between bone and cementum compositions and the possible basis for their layered appearances

In humans, age estimation from the adult skeleton represents an attempt to determine chronological age based on growth and maturational events. In teeth, such events can be characterized by appositional growth layers in midroot cementum. The purpose of this study was to determine the underlying cause of the layered microstructure of human midroot cementum. Whether cementum growth layers are caused by changes in relative mineralization, collagen packing and/or orientation, or by variations in organic matrix apposition was investigated by subjecting midroot sections of human canine teeth to analysis using polarized light and scanning electron microscopy (SEM). Polarized light was used to examine transverse midroot sections in both mineralized and demineralized states. Mineralized sections were also reexamined following subsequent decollagenization. Polarized light was additionally used in the examination of mineralized sections taken transversely, longitudinally, and obliquely from the same tooth root. From the birefringence patterns it was concluded that collagen orientation does not change with varying section plane. Instead, the mineral phase was most responsible for the birefringence of the cementum. SEM studies suggested that neither collagen packing nor collagen orientation change across the width of the cementum, confirming and validating the results of the polarized light examination. Also, SEM analysis using electron backscatter and the electron probe suggested no changes in the mean atomic number density, calcium, phosphate, and sulfur levels across the width of the cementum. Therefore, we conclude that crystalline orientation and/or size is responsible for the layered appearance of cementum. (Bone 30:386-392; 2002) (C) 2002 by Elsevier Science Inc. All rights reserved.

Processing and characterization of alpha-elastin electrospun membranes

Elastin isolated from fresh bovine ligaments was dissolved in a mixture of 1,1,1,3,3,3-Hexafluoro-2-propanol and water and electrospun into fiber membranes under different processing conditions. Fiber mats of randomly and aligned fibers were obtained with fixed and rotating ground collectors and fibrils were composed by thin ribbons whose width depends on electrospinning conditions; fibrils with 721 nm up to 2.12 m width were achieved. After cross-linking with glutaraldehyde, -elastin can uptake as much as 1700 % of PBS solution and a slight increase on fiber thickness was observed. The glass transition temperature of electrospun fiber mats was found to occur at ~ 80 ºC. Moreover, -Elastin showed to be a perfect elastomeric material, and no mechanical hysteresis was found in cycle mechanical measurements. The elastic modulus obtained for oriented and random fibers mats in a PBS solution was 330 ± 10 kPa and 732 ± 165 kPa, respectively. Finally, the electrospinning and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Cell culture results showed good cell adhesion and proliferation in the cross-linked elastin fiber mats.

Stability of the Transthyretin Molecule as a Key Factor in

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/ABeta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4- (3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Contribution to the histology of Biomphalaria glabrata

A combination of histological techniques applied to the study of Biomphalaria glabrata yielded some interesting new data about the histology of this snail, a major intermediate host of Schistosoma mansoni in Brazil. Three kinds of pigments were identified: a dark pigment which bleached following oxidation with potassium permanganate; a lipofuchsin-like, diastase-resistant PAS-positive pigment and an iron-containing pigment, probably related to hemosiderin. Calcium was detected in small deposits within the connective tissue and forming a dense core inside the chitinous radular teeth. The presence of fibrils, staining with sirius-red and birefringence under polarized light strongly suggest primitive collagen tissue. The radular apparatus appeared as a storing site for glycogen, while abundant Alcian-blue positive material (proteoglycans) was extremely concentrated in the radular sac.

Chondrogenic differentiation within magnetic-responsive multilayered liquified capsules containing collagen II/TFG-β3 coated microparticles

The use of stem cells is a promising therapeutic approach for the substantial challenge to regenerate cartilage. Considering the two prerequisites, namely the use of a 3D system to enable the chondrogenic differentiation and growth factors to avoid dedifferentiation, the diffusion efficiency of essential biomolecules is an intrinsic issue. We already proposed a liquified bioencapsulation system containing solid microparticles as cell adhesion sites1. Here, we intend to use the optimized system towards chondrogenic differentiation by encapsulating stem cells and collagenII-TGF-β3 PLLA microparticles. As a proof-of-concept, magnetite-nanoparticles were incorporated into the multilayered membrane. This can be a great advantage after implantation procedures to fixate the capsules in situ with the held of an external magnetic patch and for the follow-up through imaging. Results showed that the production of glycosaminoglycans and the expression of cartilage-relevant markers (collagen II, Sox9, aggrecan, and COMP) increased up to 28 days, while hypertrophic (collagen X) and fibrotic (collagen I) markers were downregulated. The presence of nanofibers in the newly deposited ECM was visualized by SEM, which resembles the collagen fibrils of native cartilage. The presence of the major constituent of cartilage, collagen II, was detected by immunocytochemistry and afranin-O and alcian blue stainings revealed a basophilic ECM deposition, which is characteristic of neocartilage. These findings suggest that the proposed system may provide a suitable environment for chondrogenic differentiation.

Notas sôbre a meiose de pachylis (Hemipt-Coreidae)

Spermatogonial chromosomes of Pachylis laticornis and Pachylis pharaonis begin anaphasic movement with both ends turned toward the same pole, maintaining this form util they reach the poles. This is a proof that they are provided with one kinetochore at each end. Additional proof for a longitudinal division of each longitudinal half of the anaphase chromosomes of the primary sper- matocytes is presented against the idea of a previous end-toend pairing at metaphase. The longitudinal split of the chromosomes of the secondary spermatocytes which used to be considered as tertiary split is therefore a true secondary split. The heterochromosome in both species passes undivided to one pole in the first division of the spermatocyte. In Pachylis laticornis it appears connected with the poles by means of two fibrils detached from each extremity, what may be considered as indicating a rather premature longitudinal spliting. The behavior of the heterochromosome of Pachylis pharaonis is highly interesting and affords one of the most beautiful evidences in favour of the dicentricity of the chromosomes. Really, in metaphase the heterochromosome appears at the equator of the cell with a more or less round shape. In the beginning of anaphase it becomes fusiform. As anaphase proceeds it distends itself between the autosomal plates forming a long fusiform bridge or sends toward the plates a thick chromosomal thread. The bulky part of the heterochromosome as it passes to one side it reincorporates the substance of the thread in this side. The thread in the other side, which becomes generally thiner, is left with its kinetochore in the cell at this side. The heterochromosome therefore becomes terminally monocentric in the first division of the spermatocyte. Some figures, however, suggest that the heterochromossome from time to time may pass with both kinetochores to one of the cells, as ordinarily happens in the case of Pachylis laticornis. Summing up, other things apart the behavior of the heterochromosome in both species studied here puts out of doubt the question of the existence of two terminally located kinetochores.

Estudis funcionals i de plegament dels dominis d'activació de les procarboxipeptidases

Els dominis d’activació (ADs) de les procarboxipeptidases de la subfamília A/B sempre han sorprès ja que representen una quarta part del proenzim. S’han realitzat alguns estudis per intentar descobrir-ne alguna possible funció alternativa, però no han estat fructífers. El descobriment de l’elevada velocitat de plegament del domini d’activació de la procarboxipeptidasa A2 humana, (ADA2h), emperò, va portar a proposar la possibilitat de que realitzessin una funció d’assistència al plegament del domini enzimàtic. Posteriorment, l’anàlisi del plegament d’ADA2h a pH baix va revelar la capacitat d’aquest domini per formar fibres amiloides, a més de demostrar que un increment de l’estabilitat proteica podia prevenir la formació d’aquests agregats. La profunda caracterització del plegament d’ADA2h va fer que aquesta proteïna fos un bon model amiloidogènic, de manera que es van proposar un seguit d’experiments que s’han desenvolupat en el present treball per tal de conèixer millor aquest procés. S’han dut a terme estudis cinètics d’agregació per tal de valorar la contribució dels diferents aminoàcids de la seqüència polipeptídica, utilitzant 29 variants puntuals d’ADA2h. Es va eliminar la contribució de l’estabilitat mitjançant la utilització d’urea, i per dicroïsme circular conjuntament amb un aparell de flux detingut, es van obtenir dues velocitats diferents, v1 i v2, que corresponen a la formació d’un intermediari i a la seva reorganització, respectivament. Experiments complementaris utilitzant espectroscòpia d’infraroig (IR) revelaren la reorganització de l’estat natiu (en aquest cas) per a donar la forma agregada. Les cinètiques d’IR van mostrar que ADA2h forma l’estructura _ típica de les fibres amiloides, previ desplegament les seves hèlixs-_. Finalment, s’han realitzat estudis de biocomputació per tal d’esbrinar possibles funcions alternatives dels ADs. Les superposicions estructurals semblen mostrar similaritat dels ADs amb dominis de reconeixement d’RNA (RRM). Aquesta hipòtesi s’ha comprovat experimentalment amb ADA4h, mostrant una dèbil, però existent, unió a RNA.

Morphological features of collagen degradation in advanced hepatic schistosomiasis of man

Optical and electron microscopical evidences of focal matrix degradation were frequently seen in liver sections taken from patients with periportal ("pipe-stem") fibrosis caused by schistosomiasis mansoni. Besides present of focal areas of rarefaction, fragmentation and dispersion of collagen fibers, the enlargend portal spaces also showed hyperplasia of elastic tisue and disarray of smooth muscle fibers following the destrution of portal vein branches. Ultrastructural cahnges represented by focal lytic and/or electron dense alterations of colagen fibrils were similar to those first seen in experimental material and designated as "chronic collagen degradation". Elastin and related microfibrils were also affected by focal condensation, fragmentation, distorsion and dissolution. Schistosome eggs were scanty in the tissue sections examined. Matrix degradation represented involuting changes related to the progressive diminution of parasite aggression, which occurs spontaneously with age or after cure by chemotherapy. Changes of focal matrix degradation now being described represent the basic morphological counterpart of periportal fibrosis involution documented clinically, especially by ultrasonography, in patients with hepatosplenic schistosomiasis submitted to curative chemotherapy.