Light-emitting diode effects on combined decellularization of tracheae. A novel approach to obtain biological scaffolds

PURPOSE: To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal. METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase). RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present. CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues. .

Efeito da criopreservação e/ou da descelularização na matriz extracelular de condutos valvados porcinos / Effects of cryopreservation and/or decellularization on extracellular matrix of porcine valves

O objetivo desse estudo foi avaliar a morfologia de valvas pulmonares porcinas criopreservadas e/ou descelularizadas para determinar uma solução que remova as células, sem promover danos à matriz extracelular. Valvas pulmonares porcinas foram incubadas por 24h em soluções de deoxicolato de sódio 1% e de dodecil sulfato de sódio 0,1% e 0,3%, com ou sem criopreservação adicional. A avaliação foi feita com microscopia óptica (hematoxilina eosina, orceína acética ou Gomori) e por morfometria. A efetividade das soluções foi variável, mas os melhores resultados foram obtidos com enxertos frescos descelularizados com dodecil sulfato de sódio 0,1%.

The objective of this study was to evaluate the morphology of decelluarized and/or cryopreserved porcine pulmonary valves, to determine a solution capable of completely remove the cells without damaging the extracellular matrix. Porcine pulmonary valves were incubated for 24 hs in sodium deoxicholate 1% or sodium dodecyl sulfate 0.1 and 0.3%, with or without associated cryopreservation. Evaluation was done with optical microscopy (Hematoxilin-Eosin, Acetic Orcein and Gomori) and with morphometric analysis. The effectiviness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.

Morphological and ultrastructural characteristics of extracellular matrix changes in oral squamous cell carcinoma.

Background: The biology of oral squamous cell carcinoma (OSCC), including its progression from dysplasia to carcinoma, "field effects", genetic changes in tumor associated mucosa (TAM) and effect of matrix metalloproteinases in breaking down of matrix proteins to facilitate invasion, has been well documented. However, what remains to be done is to extrapolate this knowledge to improve patient care. Aim: The aim of this study was to observe the extracellular matrix (ECM) changes with the routine histochemical stains available to most histopathologists. Materials and Methods: The study includes 72 cases of OSCC in which the tumor and adjacent normal appearing areas were sampled to study the ECM changes with hematoxylin and eosin (H and E) and Verhoeff's-Van Gieson elastic stain (VVG). Results: Basophilic fragmentation of collagen (H and E) and clumped short elastic fibers (VVG) were seen in 12 (16.7%) cases. Of the remaining cases, 18 (25%) had a dense lymphocytic infiltrate and had no demonstrable elastic fibers. Those cases with H and E changes were further studied and compared with normal mucosa for ultrastructural changes. The ultrastructural study demonstrated an increase in oxytalan, elaunin and elastic fibers and decrease in collagen fibers with some transformation changes associated with OSCCs and lymph node metastasis. Conclusion: Changes in transformation of collagen to elastic fibers and also the loss of both the fibers in areas of lymphocytic infiltration possibly indicate degradation of ECM fibers by factors released from the lymphocytes or tumor cells and the limiting effect on the tumor by ECM remodeling.

Interlinkages in the tunica media of goat aorta / Interrelaciones en la túnica media de la aorta de cabra

The arrangement and interconnections between various components of the aortic wall influence its physicomechanical properties and functional alterations that occur in disease and ageing. The goat is a suitable model for studying cardiovascular disease, but details of the intrinsic organization of its aorta are unknown. This study therefore investigated the histomorphology of aortic tunica media in the goat by transmission electron microscopy. Sixteen healthy juvenile and adult domestic male goats (capra hircus) purchased from livestock farms in the outskirts of Nairobi were used in the study. The animals were euthanized with overdose of sodium pentabarbitone 20mg/kg, and fixed with 3 percent phosphate buffered glutaraldehyde solution by gravimetric perfusion. Specimens obtained from the thoracic aorta (T9) were post fixed in osmium tetroxide, and prepared for durcupan embedding. Ultrathin sections stained with uranyl acetate/lead citrate were examined by EM 201 Phillips © electron microscope. Elastic and collagen fibres were structurally interconnected. Elastic lamellae, collagen and elastic fibres were linked to smooth muscle cells through areas of high electron density while smooth muscle cells were interconnected various inter cellular connections. The physical interlinkages between the components of the tunica media confer plasticity, adaptability and flexibility to the aortic wall enabling it to function as a mechanically homogenous structure. Disruptions of this structure in atherosclerosis and aging may disturb the vascular integrity and predispose to aneurysm formation.

Las relaciones e interconexiones entre los distintos componentes de la pared aórtica influyen en sus propiedades fisicomecánicas y en las alteraciones funcionales que se producen en la enfermedad y el envejecimiento. La cabra es un modelo adecuado para el estudio de las enfermedades cardiovasculares, pero los detalles de la organización propia de la aorta son desconocidos. Por tanto, se investigó la histomorfología de la túnica media aórtica en la cabra mediante microscopía electrónica de transmisión. Fueron utilizadas 16 cabras (Capra hircus) domésticas macho, jóvenes y adultas sanas, adquiridas en las explotaciones ganaderas en las afueras de Nairobi fueron utilizadas. Los animales fueron sacrificados con una sobredosis de 20 mg/kg de pentobarbital sódico, y se fijaron con una solución de fosfato de glutaraldehído al 3 por ciento por perfusión gravimétrica. Las muestras obtenidas de la aorta torácica (T9) fueron puestas en tetróxido de osmio, y se prepararon para inclusión en durcupan. Secciones ultrafinas teñidas con acetato de uranilo y citrato de plomo fueron examinados por microscopio electrónico EM 201 Phillips©. Fibras elásticas y colágenas estaban interconectadas estructuralmente. Láminas elásticas, de colágeno y fibras elásticas estaban conectadas a células de músculo liso a través de áreas de alta densidad de electrones, mientras que, las células musculares lisas estaban interconectados entre diferentes conexiones celulares. Las interconexiones físicas entre los componentes de la túnica media confieren plasticidad, adaptabilidad y flexibilidad a la pared aórtica, lo que le permite funcionar como una estructura mecánica homogénea. Las interrupciones de estas estructuras en la aterosclerosis y el envejecimiento pueden alterar la integridad vascular y predisponer a la formación de aneurismas.

Collagen analysis in human tooth germ papillae

The extracellular matrix (ECM) performs a very important role in growth regulation and tissue differentiation and organization. In view of this, the purpose of this study was to analyze the collagen, the major organic component of dental pulp ECM, in papillae of human tooth germs in different developmental phases. The maxillas and mandibles of 9 human fetuses ranging from 10 to 22 weeks of intrauterine life were removed and 16 tooth germs (1 in the cap stage, 8 in the early bell stage and 7 in the late bell stage) were obtained. The pieces were processed for histological analysis and stained with hematoxylin-eosin, Masson's Trichrome and picrosirius staining technique. Both types of collagen in the dental papilla were only detected by the picrosirius staining technique under polarized light microscopy. Type III collagen was detected in all specimens. Type I collagen was present in focal areas of the dental papilla only in some specimens. In conclusion, the findings of this study showed that type III collagen is a regular component of the papillae of human tooth germs whereas type I collagen is present in a significantly lesser amount.

A matriz extracelular (MEC) tem um papel importante na regulação do crescimento e na diferenciação e organização dos tecidos. Com base nestes aspectos o objetivo do deste estudo foi analisar o colágeno, maior componente orgânico da MEC da polpa dentária, na papila de germes dentários humanos, em diferentes fases do desenvolvimento. Foram obtidos fragmentos de maxilas e mandíbulas de 9 fetos humanos com 10 a 22 semanas de vida intra-uterina, dos quais foram analisados 16 germes dentários (1 em estágio de capuz, 8 em estágio de campânula precoce e 7 em estágio de campânula tardia). Secções histológicas seriadas foram coradas com hematoxilina e eosina, tricrômico de Masson e técnica de coloração do picrosirius. Ambos os tipos de colágeno na papila dentária foram somente detectados pela técnica de coloração do picrosirius usando microscopia de luz polarizada. Colágeno tipo III foi detectado em todas as amostras. Colágeno tipo I estava presente em áreas focais da papila dental em algumas amostras. Concluiu-se que o colágeno tipo III mostrou-se um componente regular da papila de germes dentários humanos, enquanto o colágeno tipo I esteve presente em quantidade significativamente menor.

Cambios fenotípicos del epitelio mamario inducidos por su entorno celular / Phenotypical changes of mammary epithelium induce by its celullar surroundings

Se presenta una revisión bibliográfica relacionada con la funcionalidad de la matriz extracelular y sus distintos componentes en el sentido de gatillar y modular el proceso biológico de la diferenciación celular, con especial énfasis en los diferentes fenotipos adquiridos por las células pertenecientes al epitelio mamario de ratas mantenido en cultivo, como resultado de la inducción al proceso de diferenciación tanto en ausencia como en presencia de constituyentes de dicha matriz.

Collagen arrangement in hepatic granuloma in mice infected with Schistosoma mansoni: dependence on fiber radiation centers

The collagen structure of isolated and in situ liver granuloma from Swiss Webster mice infected with Schistosoma mansoni was sequentially and three-dimensionally analyzed during different times of infection (early acute, acute, transitional acute-chronic, and chronic phases) by laser scanning confocal microscopy and electron scanning variable vacuum microscopy. The initial granuloma structure is characterized by vascular collagen residues and by anchorage points (or fiber radiation centers), from where collagenous fibers are angularly shed and self-assembled. During the exudative-productive stage, the self-assembly of these fibers minimizes energy and mass through continuous tension and focal compression. The curvature or angles between collagen fibers probably depends on the fibroblastic or myofibroblastic organization of stress fibers. Gradually, the loose unstable lattice of the exudative-productive stage transforms into a highly packed and stable architecture as a result of progressive compactness. The three-dimensional architecture of granulomas provides increased tissue integrity, efficient distribution of soluble compounds and a haptotactic background to the cells

Influencia del 4-metil umbelliferil ß-D-xilosido sobre la matriz extracelular durante el desarrollo de la aorta en embriones de pollo / Influence of the 4-metyl umbelliferil ß-D-xilosido on the extracelullar during the develoment of the aortic in chick embryos

Se realizó un estudio ultraesructural e histoquímico del efecto que ejerce el 4-metil umbelliferil ß-D-xilósido (4MUX) sobre la pared de la aorta de embriones de pollo del estadío 36. Se inocularon huevos fértiles de gallina white legorn a los 2.5 días de desarrollo (estadío 17) con 0.1 ml de 4MUX. Sus efectos se observaron con técnicas de microscopía electrónica de barrido e histoquímicas. Se ha puesto de manifiesto que el 4MUX interfiere en el desarrollo normal de la aorta. Causando alteraciones en la distribución de elastina, colágeno y GAGs en los espacios intercelulares

Estudo histoquímico comparativo da matriz extracelular de tumores primários e metastáticos em ratos nude xenotransplantados com células KB / Histochemical comparative study of primary and metastatic tumors in nude rates xenotransplanted with KB cells

Estudou-se comparativamente, através de métodos histoquímicos, a expressäo dos componentes da matriz extracelular de tumores primitivos e metastáticos em ratos nude, xenotransplantados com células KB. Em ambas as neoplasias observou-se uma variabilidade tanto qualitativa como quantitativa dos componentes matriciais, coexistência de diferentes tipos de fibras, pouca representatividade de fibras elásticas de glicosaminoglicanas ácidas e sulfatadas e de polissacarídeos neutros, além da ausência de membrana basal

Estudo de células cultivadas do adenoma pleomórfico humano: análise do fenótipo celular frente à açäo de proteínas da matriz extra-celular, análise da distribuiçäo citoesqueletal do filamento intermediário vimentina / Study of cells cultivated from human pleomorphic adenoma: analysis of cellular phenotype under the action of proteins from the extracellular matrix, analysis of cytoskeletal distribution of vimentine intermediary filaments

Cultura primária de células derivadas do adenoma pleomórfico humano (AP2) foi estabelecida e utilizada em estudos de resposta à açäo de proteínas da matriz extra-celular (MEC). As células cultivadas foram caracterizadas como mio-epitelial símile por imunocitoquímica e microscopia eletrônica de transmissäo (MET). Células AP2 cresceram em contato com as seguintes proteínas da MEC: lamina, colágeno I, colágeno IV e membrana basal reconstituída (Matrigel). Laminina e colágenos tipos I e IV, quando aplicados individualmente, näo causaram efeito no fenótipo das células AP2. No entanto, células crescidas em Matrigel mostraram importantes alteraçöes fenotípicas, dependendo do modo de aplicaçäo do substrato. Células crescidas sobre finas camadas de Matrigel desenvolveram fenótipo estrelado, com prolongamentos delicados, longos e intercomunicantes, lembrando as células mio-epiteliais normais. Células crescidas dentro de massas de Matrigel formaram agrupamentos tri-dimensionais. Ao microscópio confocal e MET esses agrupamentos apresentaram dupla camada de células epitelióides delimitando espaços luminais. As células próximas aos lúmens eram cubóides, com vilosidades apicais e complexo juncional. Nosso trabalho forneceu uma evidência direta demonstrando que a formaçäo de estruturas luminais do adenoma pleomórfico somente ocorre quando suas células säo tri-dimensionalmente envoltas por membrana basal. Paralelamente a esse estudo, foi analisada a distribuiçäo do filamento intermediário vimentina no citoplasma de células AP2. Nessa célula, a vimentina distribui-se como filamentos pequenos, completamente segregados da rede principal. A maioria desses filamentos näo co-localiza com microtúbulos. Análise da relaçäo vimentina-microtúbulos nas células AP2 mostrou que essas estruturas somente interagem quando os filamentos de vimentina se estendem em direçäo à periferia da célula

Scanning electron microscopy of acinar cells of rat submandibular salivary glands

1. The submandibular salivary gland of rats was observed by high-resolution scanning electron microscopy employing the aldehyde-osmium-DMSO-osmium method. 2. The intracellular membranous components and sponge-like structures of basement membrane containing the fine collagen fibrils of acinar cells were clearly identified in three dimensional images. The granular endoplasmic reticulum and Golgi apparatus showed the luminal surface. The mitochondria were small, ranging in diameter from 0.3 to 0.5 µm, and revealed their cristae. The secretory granules ranged in diameter from 0.3 to 1.4 µm. ribosome granules were attached to the surfaces of cisterns, and measured 20 to 25 nm in diameter. 3. The contact areas between the acinar cells revealed numerous cytoplasmic protrusions. In the striated duct cells, the mitochondria were arranged vertically and surrounded by nasal infoldings of the plasma membranes. At high magnification, the mitochondrial cristae were visualized in their three-dimensional characteristics

Use of lectins to evaluate the effects of GaAS softlaser on dog tendon

1. Lectins labeled with colloidal gold particles were used for the ultrastructural evaluation of the biological effects of GaAs softlaserirradiation on the healing of dog tendon wounds. 2. Six dogs were submitted to tenotomy and tenorrhaphy on the right and left hind legs. All animals received laser irradiation (4J/cm²) daily for ten days only on the left leg, and the right leg of the same animal was used as control. Biopsies were taken 11, 22 and 40 days after surgery. 3. Laser-irradiated and control tendon tissues were embedded in L.R. White resin and thin section were incubated in the presence of gold-labeled Arachis hypogaea (PNA), Canavalia ensiformes (Con A) and Triticum vulgare (WGA). 4. In general, the control and laser-irradiated tissues presented homogeneous and similar labelling of heterochromatin, rough endoplasmic reticulum and extracellular matrix. 5. We conclude that GaAs softlaser irradiation does not produce significant changes in the glycosylation of healing tendons

Ultra estrutura da matriz extracelular da polpa de dentes humanos hígidos / Ultrastructural study of the extracellular matrix of pulps of healthy human teeth

Considerando o papel fisiológico e biológico da matriz extracelular, propomo-nos a estudar a estrutura da rede de glicosaminoglicanas (GAGs) e proteoglicanas (PGs) no tecido conjuntivo de polpas de dentes molares humanos hígidos de pacientes ao redor de 20 anos. As polpas foram fixadas para microscopia eletrônica de transmissäo em soluçöes de glutaraldeído com vermelho de rutênio e com tetróxido de ósmio reduzido. A rede de GAGs e PGs foi constituída por grânulos de filamentos dispostos numa extensa malha uniforme, contínua e compacta, com nítido inter-relacionamento com a estrutura e posicionamento das fibrilas colágenas e com a superfície de células do tecido conjuntivo

Estudo morfológico e histoquímico em nível ultra-estrutural da corda tendínea de coraçöes de rato albino / Morphological and histochemical study in structural pattern of chordae tendinae of hearts from albino rat

Fragmentos de cordas tendíneas dos complexos valores mitral e tricúspide de ratos albinos foram observados ao microscópio eletrônico de transmissäo com o objetivo de estudar os componentes da matriz extracelulair. Foram utilizados dois tipos de fixador, um contendo ácido tânico para demonstrar a presença de fibras dos sistemas colágeno e elástico, e outro contendo vermelho de rutênio para a visualizaçäo de proteuglicans. Alguns fragmentos foram tratados previamente pela colagenase ou pela tripisina antes de serem fixados pelo glutaraldeído com vermelho de rutênio. Foi observado que as cordas tendíneas de rato säo revestidas por um endotélio contínuo que repousa sobre uma camada de conjuntivo contendo fibroblastos e esparsas fibras de colágeno, compondo a zona esponjosa. Tal camada circunda uma zona central (zaza fibrosa) constituída de grossos feixes de colágeno onde se encontram vários fibroblastos. Tanto na zona esponjosa como na fibrosa foram detectadas inúmeras fibras com padräo ultra-estrutural de fibras elaunínicas, os quais, juntamente com o colágeno, desempenham funçöes de resistência mecânica ao transmitirem as forças de traçäo exercidas pelo músculo papilar às bordas das válvulas. Adicionalmente, foram observadas as relaçöes morfológicas entre proteoglicans, fibrilas de colágeno e microfibrilas elaunínicas, tendo sido enfatizada a importância do equilíbrio de funçöes entre os diversos componentes da matriz extracelular concorrendo para o perfeito funcionamento deste complexo morfofuncional na fisiologia da corda tendínea.

Gelatinous transformation of bone marrow.

Gelatinous transformation of bone marrow is usually encountered in patients of anorexia nervosa. We report two cases of gelatinous transformation of the marrow, one without any detectable cause and the other associated with visceral leishmaniasis.

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-346) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2x 10***4 and 5x 10***4 blood forms respectively). The rate of morality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100mg/k.b.w/day, for 60 days, or with the MK-436(3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinovulation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of mitritial components and collagen fibers, macrophages and fibroblasts appeared at the ultrastructural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated mice, parasitologically cured, presented clear-cut regression of the inflamatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombia