Extracellular matrix of porcine fibrous pericardium: morphological and biochemical study

Orientador: Benedicto Campos VidalTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Tecido pericardial tem sido utilizado na confecção de biopróteses empregadas na reparação de diferentes lesões. Entretanto, calcificações e falência mecânica têm sido as principais causas de durabilidade limitada de biopróteses cardíacas fabricadas com pericárdio bovino. Nesse trabalho foi realizado um estudo do pericárdio fibroso porcino em sua estrutura microscópica e sua natureza bioquímica. A morfologia geral e a arquitetura das fibras colágenas e elásticas foram estudadas em tecido seccionado e também em montagens totais, corados por diferentes métodos histoquímicos e analisados em microscopia de luz convencional, de polarização, de fluorescência e confocal. O estudo bioquímico da matriz pericardial foram realizados de acordo com procedimentos bioquímicos pertinentes. O pericárdio mostrou-se um tecido altamente celularizado com características de tecido jovem. Os feixes colagênicos apresentaram-se arranjados em camadas multidirecionalmente orientadas, formando uma rede de trama fechada, com um número maior de fibras orientando-se obliquamente, partindo da região central inferior em direção súpero-lateral esquerda em relação ao coração. Uma discreta predominância de fibras colágenas no sentido base-ápice foi verificada na região anterior direita do pericárdio. A matriz extracelular apresentou-se pouco metacromática. Observaram-se finas fibrilas elásticas intimamente associadas às fibras colágenas, entrelaçadas em direções diversas ou paralelas entre si. Extração comparativa de componentes da matriz não revelou diferenças entre proteínas extraídas das regiões direita e esquerda e também entre material fresco e congelado. A análise em SDS-PAGE revelou dezesseis proteínas com valores de massa molecular aparente entre 11 e 109 kDa. Análises realizadas sugerem que o GAG encontrado possivelmente é o dermatam sulfato. Os dados obtidos podem subsidiar novos procedimentos direcionados à obtenção de biomembranas melhor preparadas e funcionalmente mais adequadas à construção de biopróteses.Abstract: Pericardial tissue has been used to construct bioprostheses employed in the repair different kinds of injuries. However, calcification and mechanical failure have been the main causes of limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work a study has been conducted on the porcine fibrous pericardium and its microscopic structure and its biochemical nature. The general morphology and the architecture of the collagen and elastic fibers have been studied in sectioned tissue and also total assemblies, stained by diverse histochemical methods and analyzed by conventional light, polarizing light, fluorescence and confocal microscopy. Biochemical study of the pericardial matrix was conducted according to pertinent procedures. The pericardium was shown as a highly cellularized tissue with a vascular tree very ramified and collagen fibers arranged in multidirectionally oriented layers. A predominant direction was verified, with a larger number of fibers obliquely oriented, starting at the lower central region towards high-lateral-left relatively to the heart. A discreet predominance of collagen fibers is the base-apex direction was verified in the right-front region of the pericardium. Extracellular matrix with little metachromasy was observed, indicating small quantities of acid glycosaminoglycans. Fine elastic fibrils were observed, intimately associated to collagen fibers, interlaced in various directions or parallel to themselves. No differences were found between proteins extracted from the right or left regions and between fresh or frozen material. The quantities of extracted GAGs were too small to be detected by the method used. The SDS-PAGE showed proteins with values of apparent molecular mass between 11 and 109 kDa included two polydisperse bands around 71 and 85kDa. The ectrophoretic analysis showed that the found GAG is possibly dermatan sulfate.DoutoradoHistologiaDoutor em Biologia Celular e Estrutura

Identification Of Elastic Fibers And Lamellae In Porcine Pericardium And Aorta By Confocal, Fluorescence And Polarized Light Microscopy.

Pericardial connective tissue has been used to construct bioprostheses to repair various types of injuries, including aortic wall repairs. The arrangement and the distribution of elastic and collagen staple fibers are related directly to the biomechanical properties of the tissue and thus determine the choice tissue for the construction of bioprostheses. Although elastic fibers can be visualized using several histochemical methods, the specificity and mechanism of binding involved remain to be clarified. In this work, we compared the elastic net of the porcine pericardial matrix with that of the aortic wall using 1-anilino-8-naphthalene sulfonate (ANS) and dansyl chloride (DCl) as fluorescent probes and the permanganate-bisulfite-toluidine (PBT) method. Polarized light and fluorescence microscopy were simultaneously used to analyze the tissues. Some samples also were examined using confocal microscopy. Aorta and pericardium treated with ANS and DCl showed elastic fibers and lamellae with an intense blue fluorescence. When stained with the PBT method, the aortic elastic lamellae were clearly metachromatic and, under polarized light, they showed a greenish birefringence. DCl provided clearer fluorescent labelling of elastic fibers when examined using confocal microscopy.108125-3

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin