A mechanism for localized lignin deposition in the endodermis.

The precise localization of extracellular matrix and cell wall components is of critical importance for multicellular organisms. Lignin is a major cell wall modification that often forms intricate subcellular patterns that are central to cellular function. Yet the mechanisms of lignin polymerization and the subcellular precision of its formation remain enigmatic. Here, we show that the Casparian strip, a lignin-based, paracellular diffusion barrier in plants, forms as a precise, median ring by the concerted action of a specific, localized NADPH oxidase, brought into proximity of localized peroxidases through the action of Casparian strip domain proteins (CASPs). Our findings in Arabidopsis provide a simple mechanistic model of how plant cells regulate lignin formation with subcellular precision. We speculate that scaffolding of NADPH oxidases to the downstream targets of the reactive oxygen species (ROS) that they produce might be a widespread mechanism to ensure specificity and subcellular precision of ROS action within the extracellular matrix.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-β1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.

Role of MT1-MMP in cancer dissemination : insights from the real time imaging of tumors

Abstract : Matrix metalloproteinases (MMPs) are thought to play a major role in the tumor dissemination process as they degrade all components of the extracellular matrix. However, failure of clinical trials testing broad MMP inhibitors in cancer led to the consensus that a better understanding of the MMP biology was required. Using intravital multiphoton laser scanning microscopy, we developed an in vivo model to observe tumor dissemination and extracellular matrix remodeling in real time. We show that the matrix-modifying hormone relaxin increases tumor associated fibroblast interaction with collagen fibers by inducing integrin beta-1 expression. This causes changes in the collagen network that are mediated by MMP-8 and MT1-MMP. Also, we show that MMP-mediated collagen remodeling in vivo requires a direct contact between stationary tumor associated fibroblasts (TAFs) and collagen fibers. As MMPs are expressed in the tumor and stromal compartment of breast cancers we determined the importance of Membrane-type 1 MMP (MT1-MMP) from each compartment for cancer progression. We find that tumor-MT1-MMP promotes the invasion of the blood vasculature and blood-borne metastasis in vivo by enhancing tumor cell migration and endothelial basement membrane degradation. Interestingly, stromal-MT1-MMP cannot compensate for the lack of tumor-MT1-MMP but promotes peritumor collagen I remodeling. Thus, the function of MT1-MMP is context dependent and we identify the different but complementary roles of tumor and stromal MT1-MMP for tumor dissemination. Finally, we translate our preclinical findings in to human breast cancer samples. We show that tumor-MT1-MMP expression correlates with tumor invasion of the blood vasculature in ER-PR-HER2- breast cancers and that MT1-MMP expression increases with cancer progression. MT1-MMP could thus represent an interesting therapeutic target for the prevention of blood vasculature invasion in these tumors. Resumé : Les matrix metalloproteinases (MMPs) semblent jouer un rôle majeur pour la dissémination tumorale en raison de leur capacité à dégrader l'ensemble des composants de la matrice extracellulaire (MEC). Néanmoins, les résultats décevants des études cliniques testant les inhibiteurs des MMP ont conduit à la notion qu'une compréhension plus précise de la biologie des MMP était requise. Dans ce travail de thèse, nous avons développé un modèle murin qui permet d'observer simultanément la dissémination tumorale ainsi que les modifications de la MEC en temps réel. Nous démontrons que le traitement de tumeurs par l'hormone relaxin augmente l'interaction des fibroblastes tumoraux avec les fibres de collagène via l'intégrine beta-1. Nous montrons que cette interaction favorise et est nécessaire à la dégradation des fibres de collagène par MMP-8 et MT1-MMP. Ensuite, étant donné que les MMPs sont exprimées dans les cellules tumorales et stromales des cancers du sein, nous nous sommes intéressés au rôle de la MMP membranaire type 1 (MT1-MMP) exprimée dans chacun de ces compartiments. Nous démontrons que MT1-MMP dérivant des cellules tumorales favorise leur invasion dans les vaisseaux sanguins par la dégradation de la membrane basale vasculaire. De manière inattendue, nous montrons que l'expression de MT1-MMP par le compartiment stromal ne peut compenser le manque de MT1-MMP dans le compartiment tumoral. Néanmoins, nos résultats prouvent que MT1-MMP dérivant du compartiment stromal est impliqué dans la dégradation de collagène peritumorale. La fonction de la protéine MT1-MMP varie donc selon le compartiment tumoral d'origine. Finalement, nous avons testé nos résultats pré cliniques chez l'humain. Dans des biopsies de cancer du sein nous montrons une corrélation entre l'expression de MT1-MMP dans les cellules tumorales et l'invasion de vaisseaux sanguins par des tumeurs ER-PR-HER2-. MT1-MMP pourrait donc être une cible intéressante pour la prévention de dissémination vasculaire de ces tumeurs

Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.

Anchor or Accelerate – A Study on Cancer Cell Adhesion and Motility

Cell migration and adhesion to the extracellular matrix (ECM) are crucial in many biological and pathological processes such as morphogenesis, tissue repair, inflammatory responses, survival, and cancer. Cell-matrix adhesion is mediated by the integrin family of transmembrane receptors, which not only anchor cells to their surroundings, but also transmit bidirectional signalling at the cell surface and couple the ECM to the cytoskeleton. Another group of adhesion receptors are the syndecan proteoglycans, which engage the ECM and possess signalling activity in response to a variety of ligands. Cell migration is a complex process that requires spatial and temporal coordination of adhesion, cell contractility, intracellular traffic of integrins, and matrix turnover by matrix metalloproteinases (MMPs). Thus, integrins and syndecans, as well as MMPs, play essential roles in cancer cell migration and invasion. The understanding of the cooperation of syndecans and integrins was broadened in this thesis study. The results reveal that syndecan-1 functions in concert with 21 integrin in cell adhesion to collagen, whereas syndecan-4 is essential in 21 integrin-mediated matrix contraction. Finally, oncogenic K-Ras was shown to regulate 21 integrin, membrane-type 1 MMP, and syndecan-1 and -4 expression and their cooperation in cell invasion. Epithelial-mesenchymal transition (EMT) is fundamental during embryogenesis and organ development. Activation of EMT processes, including the upregulation of mesenchymal intermediate filament protein vimentin, has also been implicated in the acquisition of a malignant phenotype by epithelial cancer cells. Members of the protein kinase C (PKC) superfamily are involved in cell migration and various integrindependent cellular functions. One aim of this work was to shed light on the role of vimentin in the regulation of integrin traffic and cell motility. In addition, the mechanism by which vimentin participates in EMT was investigated. The results show that integrin recycling and motility are dependent on the PKC–mediated phosphorylation of vimentin. In addition, vimentin was found to be a positive regulator of EMT and regulate the expression of several migratory genes. Specifically, vimentin governs the expression of receptor tyrosine kinase Axl, which is implicated in tumour growth and metastasis. Taken together, the findings described in this thesis reveal novel aspects of the complex interplay between distinct cellular components: integrins, syndecans, and the vimentin cytoskeleton, which all contribute to the regulation of human cancer cell adhesion, migration, and invasion.

Cellular and matrix interactions during the development of T lymphocytes

The thymus contains an extensive extracellular matrix. Although thymocytes express integrins capable of binding to matrix molecules, the functional significance of the matrix for T cell development is uncertain. We have shown that the matrix is associated with thymic fibroblasts which are required for the CD44+ CD25+ stage of double negative (CD4-8-) thymocyte development. The survival of cells at this stage is dependent on IL-7 and we propose that the role of fibroblasts is to present, via the matrix, IL-7 to developing T cells.

Role of gelatinases MMP-2 and MMP-9 in tissue remodeling following acute lung injury

Acute lung injury is characterized by a severe disruption of alveolo-capillary structures and includes a variety of changes in lung cell populations. Evidence suggests the occurrence of rupture of the basement membranes and interstitial matrix remodeling during acute lung injury. The dynamic equilibrium of the extracellular matrix (ECM) under physiological conditions is a consequence of the balance between the regulation of synthesis and degradation of ECM components. Matrix metalloproteinases (MMPs) represent a group of enzymes involved in the degradation of most of the components of the ECM and therefore participate in tissue remodeling associated with pathological situations such as acute lung injury. MMP activity is regulated by proteolytic activation of the latent secreted proenzyme and by interaction with specific tissue inhibitors of metalloproteinases. This review details our knowledge of the involvement of MMPs, namely MMP-2 and MMP-9, in acute lung injury and acute respiratory distress syndrome.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

Use of the spray chilling method to deliver hydrophobic components: physical characterization of microparticles

Food industry has been developing products to meet the demands of increasing number of consumers who are concerned with their health and who seek food products that satisfy their needs. Therefore, the development of processed foods that contain functional components has become important for this industry. Microencapsulation can be used to reduce the effects of processing on functional components and preserve their bioactivity. The present study investigated the production of lipid microparticles containing phytosterols by spray chilling. The matrices comprised mixtures of stearic acid and hydrogenated vegetable fat, and the ratio of the matrix components to phytosterols was defined by an experimental design using the mean diameters of the microparticles as the response variable. The melting point of the matrices ranged from 44.5 and 53.4 ºC. The process yield was melting point dependent; the particles that exhibited lower melting point had greater losses than those with higher melting point. The microparticles' mean diameters ranged from 13.8 and 32.2 µm and were influenced by the amount of phytosterols and stearic acid. The microparticles exhibited spherical shape and typical polydispersity of atomized products. From a technological and practical (handling, yield, and agglomeration) points of view, lipid microparticles with higher melting point proved promising as phytosterol carriers.

L’étude de l’interaction entre les chondrocytes et le collagène modifié par le 4-Hydroxynonénal : implication dans le développement de l’arthrose

OBJECTIF: Récemment, nous avons démontré que la modification du collagène type II (Col II) par le 4-hydroxynonénal (HNE), un produit de la peroxydation lipidique, est augmentée dans le cartilage arthrosique sans qu’on sache la signification de cette augmentation dans la pathogenèse de l’arthrose. L’objectif de cette étude vise à démontrer que cette modification affecte l’interaction chondrocytes/matrice extracellulaire (MEC) et en conséquence induit des changements phénotypiques et fonctionnels de ces cellules. METHODES: Des plaques de culture ont été préalablement cotées avec du Col II puis traitées avec du HNE (0.1-2 mM) excepté le puits contrôle. Les chondrocytes ont été ensuite ensemencés puis incubés pendant 48 heures. La viabilité des cellules est évaluée par le test MTT. Le Western blot est utilisé pour mesurer l’expression des molécules d’adhésion (l’ICAM-1 et l’intégrine α1β1), de la cyclooxygenase-2 (COX-2), du Col II ainsi que la phosphorylation de la p38 MAPK, ERK1/2 et NF-κB-p65. La RT-PCR en temps réel est utilisée pour mesurer l’expression de l’ARNm de l’ICAM-1, des intégrines α1β1, de la COX-2 et de la métalloprotéinases-13 (MMP-13). La détermination de l’expression de l’ICAM-1 à la surface des cellules est réalisée par cytométrie de flux. Des kits commerciaux ont servi pour mesurer le niveau de la MMP-13, de la prostaglandine E2 (PGE2), de l’activité de la caspase-8 et de la phosphorylation de la p38 MAPK, ERK1/2 et NF-κB-p65. RESULTATS: La modification du Col II par 0.2 mM HNE induit significativement l’expression des molécules d’adhésion telles que l’ICAM-1 et l’intégrine α1β1, de la MMP-13 sans avoir un effet sur la morphologie, la survie et le phénotype cellulaires. Nos résultats montrent aussi une forte augmentation de la phosphorylation de la p38 MAPK, d’ERK1/2 et de NF-κB-p65. Cependant, la modification du Col II par 2 mM HNE affecte la morphologie et la viabilité cellulaires et induit l’activité de la caspase-8. Elle inhibe fortement l’expression des integrines α1β1 et du Col II ainsi que la phosphorylation de l’ERK1/2 et de NF-κB-p65, mais par contre, induit significativement la production de la COX-2 et son produit la PGE2 ainsi que la phosphorylation de la p38 MAPK. Fait intéressant, le prétraitement des complexes HNE/Col II par 0.1 mM de carnosine empêche les changements phénotypiques et fonctionnels des chondrocytes. CONCLUSION : Ces nouveaux résultats suggèrent le rôle important de la modification du Col II par le HNE dans l’arthrose, en affectant le phénotype et le fonctionnement cellulaires des chondrocytes. La carnosine, par sa capacité de neutraliser le HNE, a révélé d’être un agent promoteur dans le traitement de l’arthrose.

The inflammatory role of angiogenic growth factors : a neutrophil perspective

De par sa présence dans tous les vaisseaux sanguins, l'endothélium joue un rôle clef dans le processus d’hémostase, tant par sa libération de facteurs anticoagulants que par ses changements protéiques qui permettent à l’organisme de déclencher la réparation tissulaire. La fonction anticoagulante de l’endothélium peut être mise en défaut en cas d’atteinte de son intégrité, entrainant la formation de thrombus, le rejet précoce de greffes ou encore l’induction de l’athérosclérose. L’intégrité de l’endothélium est donc capitale pour la prévention de nombreuses maladies cardiovasculaires. Chez l’adulte, les cellules endothéliales (CE), normalement quiescentes, sont rapidement activées en cas d’hypoxie ou d’inflammation, leur permettant ainsi d’amorcer le processus angiogénique comme suit: Tout d’abord, l’induction de l’hyperperméabilité vasculaire permet l’extravasation des protéines plasmatiques. Ensuite, la dégradation de la lame basale par des métalloprotéases permet aux CE de se détacher, de proliférer, de migrer et de s’organiser pour former l’ébauche du futur vaisseau. La dernière étape consiste en la maturation du vaisseau, c’est-à-dire son recouvrement par des cellules murales, telles que les cellules musculaires lisses et les péricytes. Ces processus sont régulés par de nombreux facteurs angiogéniques tels que les membres de la famille Notch, du vascular endothelial growth factor (VEGF), du fibroblast growth factor (FGF), des angiopoïétines, et des matrix metalloproteases (MMP). L’angiogenèse pathologique, soit une insuffisance ou un excès de vascularisation, est impliquée dans les blessures chroniques, les accidents cardiovasculaires, les pathologies coronariennes artérielles, les pathologies tumorales, l’arthrite rhumatoïde, la rétinopathie diabétique, l’athérosclérose, le psoriasis et l’asthme. Ces pathologies sont souvent issues d’une dérégulation de l’activité endothéliale, fréquemment observée conjointement à l’expression continue de molécules d’adhésion leucocytaires, à l’augmentation de la perméabilité vasculaire, et aux anomalies de la vasoréactivité. L’activation non-contrôlée de l’endothélium entraîne ainsi une inflammation chronique et la formation de structures vasculaires anarchiques. Les premiers leucocytes à répondre à l’appel inflammatoire sont les neutrophiles. Equippées d’une panoplie de produits antibactériens puissants mais aussi nocifs pour les tissus qui les entourent, ces cellules polylobées participent à chaque étape du processus inflammatoire, depuis l’induction de l’hyperperméabilité vasculaire jusqu’à la résolution. En effet, grâce à leurs récepteurs, les neutrophiles détectent et interprètent les signaux biochimiques présents dans la circulation et à la surface de l’endothélium, et libèrent aussi leurs propres médiateurs tels le VEGF, les MMP, et l’interleukine-8 (IL-8), dont les effets sont à la fois paracrines et autocrines. Existent-ils d’autres modulateurs typiques de la fonction endothéliale capables d’influencer le comportement des neutrophiles? En effet, notre laboratoire a démontré que chez l’humain, une stimulation directe aux angiopoïétines incitait les neutrophiles à adhérer aux CE, à migrer, à synthétiser et à relâcher l’IL-8, voire même à vivre plus longtemps. La présence du récepteur des angiopoïétines, Tie2, à la surface des neutrophiles laisse présager que la famille possèderait d’autres fonctions leucocytaires encore non-identifiées. Par ailleurs, dans un modèle classique de l’angiogenèse in vivo (matrigel), nous avons observé que sous l’effet du FGF1 et 2, les ébauches des nouveaux vaisseaux étaient parfois accompagnées d’une infiltration de cellules granulocytaires. Ainsi, en partant de ces observations, l’objectif de nos études (présentées ci-après) était d’approfondir nos connaissances sur la relation entre neutrophiles et facteurs angiogéniques, notamment les FGF et les angiopoïétines. Par tests in vitro, nous avons confirmé que les neutrophiles humains exprimaient plusieurs récepteurs du FGF (FGFR1-4) d’une façon hétérogène, et qu’ils migraient vers un gradient des ligands FGF1 et 2. Par ailleurs, nous nous sommes intéressés aux voies de signalisation inflammatoires activées par les ligands FGF1, FGF2, Ang1 et Ang2. Grâce à une stratégie génique ciblant 84 gènes inflammatoires, nous avons identifié plusieurs cibles d’intérêt touchées par Ang1, dont certains membres de la famille de l’IL-1, alors qu’aucun des gènes testés n’avait changé de façon significative sous l’effet des FGF ou d’Ang2. Suite à des cinétiques approfondies, nous avons démontré qu’Ang1 stimulait la transcription de l’ARN messager de l’IL-1β, et augmentait simultanément la quantité de protéine immature (pro-IL-1β; inactive) et clivée (IL-1β « mature »; active). En parallèle, Ang1 augmentait la sécrétion de l’antagoniste naturel de l’IL-1β, l’IL-1RA, sans pour autant stimuler la relâche de l’IL-1β. A l’instar des endotoxines bactériennes dont les effets liés à l’IL-1 dépendaient de la kinase p38, ceux d’Ang1 découlaient presque entièrement des voies de signalisation du p42/44.

Isolation and characterization of glycosaminoglycans and a study of its bioactive potential in two commercially important species of cephalopods, Loligo duvauceli and Sepia pharaonis

The present study is aimed at the isolation and characterization of glycosaminoglycans from selected tissues of two commercially important species of cephalopods;squid,Loligo duvauceli and cuttlefish,Sepia pharaonis,keeping in view of the aforementioned benefits on the utilization of waste generated during processing.The cephalopod GAGs may also be expected to have an effect on various physiological functions based on the results obtained from GAGs from other sources.In addition,knowledge of the chemical structure of macromolecules that constitute major components of extracellular matrix(ECM) will be helpful in understanding their interactions with other matrix components.

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)