Uso de la matriz extracelular como material para ingeniería de tejidos

Introducción: La evaluación de injertos vasculares de submucosa de intestino delgado para la regeneración de vasos sanguíneos ha producido una permeabilidad variable (0-100%) que ha sido concurrente con la variabilidad en las técnicas de fabricación. Metodología: Investigamos los efectos de fabricación en permeabilidad y regeneración en un diseño experimental de 22factorial que combino: 1) preservación (P) o remoción (R) de la capa estratum compactum del intestino, y 2) deshidratada (D) o hidratada (H), dentro de cuatro grupos de estudio (PD, RD, PH, RH). Los injertos fueron implantados en las Arterias Carótidas de porcinos (ID 4.5mm, N=4, 7d). Permeabilidad, trombogenicidad, reacción inflamatoria, vascularización, infiltración de fibroblastos, perfil de polarización de macrófagos y fuerza tensil biaxial fueron evaluadas. Resultados: Todos los injertos PD permanecieron permeables (4/4), pero tuvieron escasa vascularización e infiltración de fibroblastos. El grupo RD permaneció permeable (4/4), presentó una extensa vascularización e infiltración de fibroblastos, y el mayor número del fenotipo de macrófagos (M2) asociado a regeneración. El grupo RH presentó menor permeabilidad (3/4), una extensa vascularización e infiltración de fibroblastos, y un perfil dominante de M2. El grupo PH presentó el menor grado de permeabilidad, y a pesar de mayor infiltración celular que PD, exhibió un fenotipo de macrófagos dominante adverso. La elasticidad de los injertos R evolucionó de una manera similar a las Carótidas nativas (particularmente RD, mientras que los injertos P mantuvieron su rigidez inicial. Discusión: Concluimos que los parámetros de fabricación afectan drásticamente los resultados, siendo los injertos RD los que arrojaron mejores resultados.

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.

Keratinocyte growth factor: a new mesothelial targeted therapy to reduce postoperative pericardial adhesions

Background: Several methods have been utilized to prevent pericardial and retrosternal adhesions, but none of them evaluated the mesothelial regenerative hypothesis. There are evidences that the mesothelial trauma reduces pericardial fibrinolytic capability and induces an adhesion process. Keratinocyte growth factor (KGF) has proven to improve mesothelial cells proliferation. This study investigated the influence of keratinocyte growth factor in reducing post-surgical adhesions. Methods: Twelve pigs were operated and an adhesion protocol was employed. Following a stratified randomization, the animals received a topical application of KGF or saline. At 8 weeks, intrapericardial adhesions were evaluated and a severity score was established. The time spent to dissect the adhesions and the amount of sharp dissection used, were recorded. Histological sections were stained with sirius red and morphometric analyses were assessed with a computer-assisted image analysis system. Results: The severity score was lower in the KGF group than in the control group (11.5 vs 17, p = 0.005). The dissection time was lower in the KGF group (9.2 +/- 1.4 min vs 33.9 +/- 9.2 min, p = 0.004) and presented a significant correlation with the severity score (r = 0.83, p = 0.001). A significantly less sharp dissection was also required in the KGF group. Also, adhesion area and adhesion collagen were significantly tower in the KGF group than in the control group. Conclusion: The simulation of pericardial cells with KGF reduced the intensity of postoperative adhesions and facilitated the re-operation. This study suggests that the mesothelial regeneration is the new horizon in anti-adhesion therapies. (C) 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 C after the exposure to triethylamine vapors.

THERMAL BEHAVIOR OF IN VITRO MINERALIZED ANIONIC COLLAGEN MATRICES

In the present study porcine skin and bovine pericardium were used as a source of type I collagen. Both were submitted to an alkaline treatment and mineralized by the alternate soaking method. Thermal stability and extent of mineralization have been investigated using DSC and TG. After alkaline hydrolysis there is a decrease in thermal stability but mineralization stabilizes collagen structure. Thermogravimetric data have shown that the amount of hydroxyapatite present in bovine pericardium matrix (45%) was greater than on porcine skin matrix (20%). Presence of hydroxyapatite was confirmed by EDX.

Descrição anátomo-topográfica do coração da paca (Agouti paca)

Background: The domestic animals heart is a conical hollow viscera, surrounded by pericardium, laterally compressed, accompanying the thorax shape. Atriums constituted the heart basis and their auricles partially bound the initial portion of the aorta and pulmonary trunk. In mammals, heart is kept suspended in the thoracic cavity and the pericardic sac is fixed dorsally by great veins and arteries roots, and ventrally fixed to the sternum, although its fixation to the diaphragm varies among species. This paper aimed to describe morphological aspects of the heart of the paca, the second biggest Brazilian rodent.Materials, Methods & Results: There were used 12 hearts of adult pacas for this study, obtained from the UNESP, Jaboticabal, SP, which died due fights or anesthesia during bandages or radiograph exams. The thoracic aorta was filled with colored latex and the animal was set in a 10% formaldehyde solution for at least 72 hours. The thoracic cavity was dissected and hearts individualized and measured with a paquimeter, lateromedially, craniocaudally and dorsoventrally. The paca heart is placed between the first and fifth intercostal space (ICS), in a craniocaudal oblique position; its basis is craniodorsally positioned, on the middle third between the first and second ICS and its apex is located near the sternodiaphragmatic joint, on the fifth ICS, tilted to the left antimere. The heart is surrounded by pericardium, which from ventrocaudally is originated the sternopericardic ligament, that continues as phrenopericardic ligament. At the heart basis, the rising of the pulmonary trunk was observed and the conus arteriosus formed a typical projection. The aorta also rised from the heart basis and its arch, which was caudally curved, crossed dorsally the pulmonary trunk; the right cranial and caudal cava veins drained to the right atrium. There is a left cranial cava vein, which surrounded the left atrium and joined the right caudal cava vein on the right atrium. The azygos vein joins the right cranial cava vein and four pulmonary veins drained to the left atrium. At palpation, a hard structure on the rising of the aorta was observed, similarly to a cartilaginous tissue, which would be part of the cardiac skeleton. The left and right coronary arteries were observed in all hearts.Discussion: The paca heart is anatomica and topographically similar to those of domestic mammals, differing from them for being placed one intercostal space more cranial and due to the presence of two cranial cava veins, the left and the right ones, besides the presence of the caudal cava vein. This vascular description is similar to that of small rodents, as rats and mice. In paca heart, the sinus venous, the terminal crest, the oval fossa, the atrioventricular valvae, the papillary muscles and tendinous cords, besides smooth atriums and auricles covered by pectinate muscles, were observed. The sternopericardic ligament, which is dorsally elongated as phrenopericardic ligament, is similar to the one present in humans, pigs, castors, and different from the one observed in carnivorous, that presents the phrenopericardic ligament and from the one of horses and ruminants, which present the sternopericardic ligament.

Comparação entre pericárdio bovino preservado em glicerina e malha de poliéster no reparo de falhas da parede abdominal em ratos

OBJETIVO: Comparar uma malha comercial de poliéster com o pericárdio bovino preservado em glicerina na reconstituição de defeitos da parede abdominal. MÉTODOS: Foram utilizados 30 ratos, divididos em dois grupos eqüitativos. Efetuou-se uma excisão retangular de 2,5 x 2 cm, incluindo toda a musculatura abdominal e peritônio. No grupo I a parede abdominal foi reparada com malha de poliéster e no grupo II com pericárdio bovino conservado em glicerina. Os animais foram sacrificados aos 15, 60 e 90 dias de pós-operatório, sendo o local cirúrgico avaliado macroscópica e histologicamente. RESULTADOS: Os animais do grupo I apresentaram aderências mais severas e em maior número quando comparados aos do grupo II; porém, sem comprometimento funcional. A análise histológica revelou incorporação dos tecidos aos implantes, com maior resposta fibroblástica nos animais do grupo I. CONCLUSÃO: A malha de poliéster oferece maior resistência estrutural e resposta fibroblástica mais intensa; contudo, promove grande quantidade de aderências às vísceras abdominais, quando comparada ao pericárdio bovino.

Tissue response to polyanionic collagen: elastin matrices implanted in rat calvaria

The tissue response to polyanionic collagen matrices, prepared from bovine pericardium and implanted subperiosteally in rat calvaria, was studied. The materials were implanted in 72 male rats (Rattus norvegicus, albinus, Holtzman), randomly divided into four groups: GI-MBP hydrolyzed for 24 h; GII-MBP hydrolyzed for 36 h; GIII-MBP hydrolyzed for 48 h; GIV-native M BP. The materials were explanted after 15, 30 and 60 days and analyzed by routine histological procedures. Except for group IV (native bovine pericardium), polyanionic collagen from groups GI, GII and GIII showed low inflammatory reaction associated with bone formation, partially or completely integrated to the cranial bone; group GIV was characterized by an intense inflammatory reaction with occasional dystrophic mineralization and with occasional bone formation at 60 days when there was a decrease in the inflammatory reaction. Thus, the MBP from groups I, II and III were biologically compatible, enhancing bone formation with a slight delay at 60 days in GII. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Detecção de anticorpo anticoração em camundongos Balb/c imunizados com Streptococcus mutans

Anticorpos para antígenos cardíacos foram analisados por ELISA em 14 soros de camundongos Balb/c hiperimunizados com Streptococcus mutans, inativado pelo formaldeído. Os níveis de anticorpos da classe IgG anticoração e antimiosina elevaram-se significativamente nos animais imunizados quando comparados com os controles, especialmente no grupo A, imunizado e reestimulado com antígenos solúveis de S. mutans. Neste grupo, os resultados do Western Blot mostraram reatividade com miosina cardíaca e uma banda de 35 kDa. A análise histológica dos corações dos animais do grupo B, imunizado e reestimulado com antígenos de superfície do microrganismo, demonstrou a presença de degeneração celular, tipo hidrópica e hialina e focos inflamatórios constituídos de linfócitos e macrófagos no miocárdio e pericárdio. Os resultados deste trabalho reforçam a hipótese da existência de mimetismo antigênico entre tecido cardíaco e S. mutans e chamam a atenção para o risco de desenvolvimento de anticorpos reativos com antígenos próprios induzidos por vacina anticárie com componentes estreptocócicos.

Comparação entre a lidocaína e a acupuntura no tratamento da taquicardia ventricular induzida com dopamina em equinos anestesiados com halotano

Os efeitos da lidocaina e da acupuntura nos pontos bilaterais associados ao pericárdio 6 (Pc6-Neiguan) e ao coração 7 (C7-Shenmen), no tratamento da taquicardia ventricular (TV) induzida por dopamina em equinos anestesiados com halotano, foram avaliados e comparados. Seis equinos, distribuídos em três grupos: grupo-controle (GC), grupo tratado com acupuntura (GA) e grupo tratado com lidocaína (GL), foram anestesiados três vezes cada, com intervalo de uma semana entre cada anestesia. Avaliaram-se os parâmetros cardiovasculares (frequência cardíaca, pressão arterial e eletrocardiografia), os respiratórios (frequência respiratória, capnografía, saturação de hemoglobina e hemogasometria) e o escore de recuperação. A dose arritmogênica da dopamina (DAD) foi determinada a partir da infusão de 70µg/kg/min IV durante 10 minutos, sem interrupção, preenchendo o critério arritmogênico: quatro ou mais complexos ventriculares prematuros seguidos, com duração de pelo menos 15 segundos ou TV sustentada. O tempo médio de aparecimento da DAD ou da TV foi de 6,05±0,45 minutos nos animais não tratados, e a TV se reverteu espontaneamente aos 2,7±0,2 minutos. O grupo tratado com acupuntura reverteu a TV no tempo médio de 1,8±0,2 (P<0,05) em relação ao grupo tratado com lidocaina 1,3±0,2 (P<0,01). Os tratamentos mostraram-se eficientes na reversão TV.

Implantação intra-orbital, após a enucleação transpalpebral, de resina acrílica ou pericárdio em coelhos

Dezoito coelhos, Norfolk, fêmeas, com 45 dias de idade foram divididos em três grupos de seis animais e submetidos a enucleação transpalpebral. Os animais do grupo I receberam na cavidade orbitaria acrílico auto-polimerizável, os do grupo II pericárdio eqüino conservado em glicerina e os do grupo III foram mantidos como controle. Para avaliação macroscópica e histopatológica das cavidades orbitarias, três animais de cada grupo foram sacrificados com 30 e 60 dias após a implantação. Apesar da resina ter sido aplicada na fase pastosa, na qual a alta temperatura que ocorre durante a polimerizaçâo pode ser lesiva aos tecidos, foi o produto que apresentou os melhores resultados.

Histologia da associação de membranas biológicas de origem bovina implantadas no subcutâneo de ratos

O trabalho teve por objetivo avaliar a capacidade de barreira da membrana de cortical óssea desmineralizada liofilizada bovina, testando sua permeabilidade e integridade, em associação com a membrana de pericárdio bovino liofilizado. em 15 ratos Wistar, machos, adultos, implantou-se no subcutâneo da região cérvico-torácica dorsal a combinação de duas membranas de cortical óssea desmineralizada tendo no interior a membrana de pericárdio bovino. Os animais (cinco ratos/grupo) foram submetidos à eutanásia aos 15, 30 e 60 dias de pós-operatório. A avaliação microscópica mostrou que, aos 15 dias de pós-cirúrgico, as membranas estavam íntegras, com exceção de pequenas áreas de reabsorção da membrana de cortical óssea junto aos locais referentes aos antigos canais nutritivos. Aos 30 dias, havia apenas restos da membrana de cortical óssea e, aos 60 dias, a sua total ausência. Baseados nos resultados aqui obtidos, é possível concluir que a membrana de cortical óssea bovina é rapidamente absorvida e não confere proteção à membrana de pericárdio bovino liofilizado.

Patologia do Coração na AIDS. Estudo de 73 Necropsias Consecutivas

Purpose - To study the incidence and the etiology of the cardiac lesions in AIDS patients. Methods - The autopsy protocols and the filled slides of the heart from 73 consecutive AIDS patients were reviewed. There were, at least, 2 slides of each heart stained by haematoxylin-eosin; when indicated, Ziehl-Nielsen, Gram and Gomori Grocott stains were used. Results - No cause of death was assigned to the heart. There was involvement of the heart in 66 (90%) cases. Marked atrophy of cardiac fibers with or without lipomatosis was observed in 38 patients. Interstitial infiltrates of myocardium were present in 38 necropsies and in 13 of these cases a probable pathogen was demonstrated: cryptococcus neoforms in three cases and mycobacteria tuberculosis, atypical mycobacteria, toxoplasma gondii, trypanosoma cruzi and cytomegalovirus in two cases each. Bacterial endocarditis was found in 4 autopsies and Kaposi sarcome in one. The pericardium was involved in 22 cases; in 12 there was only non specific mononuclear infiltration. Conclusion - Autopsy examination of the heart from AIDS patients revealed frequent pathologic involvement.

Structural characteristics of the superior venae cavae wall at the intrapericardium segment in adults and aging individuals

The authors admit that the conjunctive-muscular constitution of the walls of the superior venae cavae throughout the length of the intrapericardium segment and the debouchement in the right atrium, associated with the presence of a myocardium sheath can be made up of a functional device of sphincter form able to act on the control of the blood flow to the interior of the arterial chamber as well as stopping blood flow during the arterial systole. The fixation of the venous wall to the deep chamber of the serous pericardium, contributes to growth in resistance during the elevation of venous pressure, making possible the preservation of integrity of the superior venae cavae. The expansion of fasciculi of nodal fibers in the right wall of the superior venae cavae permits that the unlocked impulse in the S-A node also reaches the vascular wall contributing to the shortening and diminution in caliber of the vein.

Propriedades tensiométricas comparadas entre fragmentos do centro tendíneo do diafragma, pericárdio fibroso e peritônio parietal de bovinos não conservados e conservados em glicerina

The behaviour of the non conserved and 98% glycerin conserved specimens for periods of 30, 60 and 90 days of bovine diaphragma's tendinous center, fibrous pericardium and parietal peritoneum submitted to mechanical tests of traction, was observed in ten bovines between 30 months and 36 months of age, crossbreeds, males and females, collecting fragments of these aforesaid membranes in each animal. The diaphragma's tendinous center and parietal peritoneum did not suffered significant modification (p>0.05) in the values of tension when compared to the resistance tests of traction of non conserved and 98% glycerin conserved membranes. However, all the evaluated tissues showed significant increase (p£0.05) of the elongation values when conserved in 98% glycerin for until 90 days. It was also observed that fibrous pericardium is the one which supports greaters tensions. So, it to was concluded, that glycerin is efficient to the conservation of biological membranes besides modifying its mechanical properties.