O paradoxo do cálcio foi pela primeira vez citado em 1966 por Zimmerman et al. A partir daí, ganhou grande interesse por parte da comunidade científica internacional devido ao fato da ausência do íon cálcio produzir na célula muscular cardíaca dano semelhante à lesão de isquemia-reperfusão. Apesar de não serem conhecidos todos os mecanismos envolvidos no processo da lesão celular no paradoxo do cálcio, a conexão intercelular mantida somente pelo nexus parece ter papel chave na fragmentação celular. A adição de pequenas concentrações de cálcio, bloqueadores de canal de cálcio, hiponatremia ou hipotermia são importantes para evitar que haja lesão celular no momento da reperfusão com soluções com concentração fisiológica de cálcio.

The calcium paradox was first mentioned in 1966 by Zimmerman et al. Thereafter gained great interest from the scientific community due to the fact of the absence of calcium ions in heart muscle cells produce damage similar to ischemia-reperfusion. Although not all known mechanisms involved in cellular injury in the calcium paradox intercellular connection maintained only by nexus seems to have a key role in cellular fragmentation. The addition of small concentrations of calcium, calcium channel blockers, and hyponatraemia hypothermia are important to prevent any cellular damage during reperfusion solutions with physiological concentration of calcium.

The aim of this study was to evaluate the effect of scaffolds native or polyanionic collagen matrix (submitted to alkaline treatment for 48 or 96 hours, PCM48 or PCM96, respectively) on the repair of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats undergoing hormone replacement therapy. The physical and mechanical characteristics of bone were analyzed. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas. The percent mineral matter and bone mineral density of the femurs were lower in ovariectomized rats. The mechanical strength of newly formed bone was greater in the area receiving the PCM96 scaffolds compared to the area implanted with the native scaffolds. The PCM96 scaffold is the best choice for bone repair in animals with hormone deficiency since it promotes faster bone growth and good mechanical strength.

This work reports the covalent immobilization of tetracycline and rolitetracycline over anionic collagen membranes and the drug release studies as an effort to develop a two stage drug release based on diffusion (fast release) and on the rate of membrane biodegradation (slow release). Independent from casting conditions antibiotics incorporated by dispersion were released in the range from 80 to 100% within 7 hours in concentrations significantly higher than those described for the prevention of bacterial growth. Antibiotic release within this period was predominantly diffusion controlled. Covalent immobilization by a modified azide procedure occurred with preservation of collagen structure independently from pH of casting and reaction conditions. Its expected that anionic collagen membranes with dispersed and covalently bound rolitetracycline or tetracycline, in association with conventional therapy, may significantly reduce membrane induced infections observed post-implantation, one of the major problem associated with periodontal ligaments reconstruction by the Guided Tissue Regeneration procedure.

Major problems with valve bioprostheses are associated with progressive structural deterioration and calcification, directly associated with the use of glutaraldehyde (GA). This work describes the effects of GA processing and borate/glutamic acid buffer treatment on the mechanical, thermal and morphological properties of 0.5% GA crosslinked bovine pericardium (BP). The results showed that while the treatment of 0.5% GA crosslinked BP with borate/glutamic acid significantly improves the mechanical properties, it had no visible effect on surface morphology. Better surface preservation was only achieved for BP pre-treated with a lower GA concentration followed by the conventional treatment (0.5% GA). Improvements in mechanical properties probably arises from structural changes probably involving the depolymerization of polymeric GA crosslinks and an increase electrostatic interaction due to covalent binding of glutamic acid to free carbonyl groups (Schiff base).The results indicate that the treatment GA crosslinked BP with borate/glutamic acid buffer may be an attractive procedure for the manufacture of heart valve bioprostheses.

OBJETIVO: Neste estudo, tivemos como objetivo comparar a resistência mecânica do pericárdio decelularizado com o pericárdio convencional, assim como avaliar sua capacidade de induzir resposta inflamatória em modelo experimental com ratos. MÉTODO: Dividimos os pericárdios em: Grupo I - pericárdio submetido a tratamento convencional com glutaraldeído e Grupo II - pericárdio submetido a tratamento de decelularização, previamente ao tratamento convencional. Após o processamento químico, as amostras do Grupo II foram histologicamente avaliadas para confirmar a eficácia da decelularização. A seguir, apenas para análise da resistência mecânica por testes de tração e de desnaturação térmica, os pericárdios foram divididos em: grupo 1 (pericárdio convencional com critérios de aprovação), grupo 2 (pericárdio convencional com critérios de reprovação) e grupo 3 (pericárdio decelularizado). A capacidade de induzir resposta inflamatória foi avaliada em estudo experimental em 50 ratos Wistar, os quais foram submetidos a implante subcutâneo de fragmentos dos pericárdios. Nossa terceira etapa de avaliação consistiu em confeccionar três biopróteses com o pericárdio decelularizado e que foram submetidas à avaliação hidrodinâmica, juntamente com uma bioprótese convencional de teste. RESULTADOS: A análise histológica inicial demonstrou decelularização completa. A resistência mecânica mostrou diferença significativa com relação às variáveis "tensão de ruptura" e "índice de tenacidade". Não encontramos diferença quanto à atividade inflamatória em modelo experimental com ratos. O desempenho hidrodinâmico foi semelhante e todas biopróteses atingiram a marca de 150 milhões de ciclos. A avaliação histológica ao fim da ciclagem mostrou padrão microscópico habitual, não havendo ruptura ou fragmentação anormal induzida por estresse mecânico. CONCLUSÃO: A decelularização mantém a resistência física do pericárdio, além de não induzir resposta inflamatória diferente daquela habitualmente encontrada no pericárdio convencional.

INTRODUCTION: In this paper, our objective was to compare the decellularized and conventional pericardium mechanical resistance and also its capability of inducing inflammatory response in an animal experimental model. METHOD: In order to study these properties, we divided the pericardia into two groups: Group I - pericardium conventionally treated with GTA - and Group II - pericardium previously decellularized and then treated with GTA in the conventional way. After the chemical treatment, Group II samples were histologically evaluated to confirm the efficacy of the decellularization process. Then, only for the analysis of mechanical resistance, pericardia were divided in: Groups 1 (conventional pericardia with criteria of approval), 2 (conventional pericardial with criteria of disapproval) and 3 (decellularized pericardia). The capacity of inducing inflammatory response was tested in a rat experimental model with 50 Wistar rats, in which rats of each group received patches of the pericardia in the abdomen. Our third step of analysis was manufacturing three decellularized pericardium bioprosthesis which were submitted to hydrodynamic evaluation together with a conventional bioprosthesis test. RESULTS: The histological analysis showed complete decellularization. Mechanical resistance gave statistical differences in the "tension of rupture" and "tenacity index" tests. We found no difference in the inflammatory activity in the animal model. Hydrodynamic performance was similar and all prostheses reached 150 million cycles. The final histological analysis assessed the standard microscopic pattern, and no rupture or abnormal fragmentation caused by mechanical stress. CONCLUSION: The decellularization technique maintains the physical resistance of the pericardium when compared with the conventionally prepared pericardium. And also, there was no difference in both groups regarding to inflammatory response studied in the animal model.

Este trabalho descreve a determinação de parâmetros para o desenvolvimento de um processo contínuo de tratamento do pericárdio bovino com glutaraldeído, para a produção de válvulas cardíacas. Os resultados mostraram que o tempo de reação do glutaraldeído com o pericárdio bovino é dependente da concentração e para concentrações de 0,001 à 0,05%, no intervalo de 1 à 2 horas, compatível com o desenvolvimento de um processamento industrial de reticulação contínua. Sob condições estequiométricas a impermeabilização e a heterogeneidade da reação do glutaraldeído com o pericárdio bovino foi verificada ser concentração dependente, com heterogeneidade parcial ou totalmente removida, pelo tratamento com tampão glicina/borato.

This work describes the determination of parameters for the development of a continuous process for the glutaraldehyde processing of bovine pericardium intended for cardiac valve manufacture. The results showed that glutaraldehyde reaction time with bovine pericardium is concentration dependent and for concentrations from 0.001 to 0.05%(w/w), in the range from 1 to 2 hours, compatible with the development of a continuous industrial crosslinking processing. Under stoichiometric conditions impermeabilization and the heterogeneity of glutaraldehyde reaction with PB was found to be concentration dependent, with heterogeneity partially or totally removed by the treatment with glycin/borate buffer.

This work describes the selective hydrolysis of carboxyamide groups of asparagine and glutamine of collagen matrices for the preparation of negatively charged collagen biomaterials. The reaction was performed in the presence of chloride and sulfate salts of alkaline and alkaline earth metals in aqueous dimethylsulfoxide solution and, selectively hydrolysis of carboxyamide groups of collagen matrices was promoted without cleavage of the peptide bond. The result is a new collagen material with controlled increase in negative charge content. Although triple helix secondary structure of tropocollagen was preserved, significative changes in thermal stabilities were observed in association with a new pattern of tropocollagen macromolecular association, particularly in respect microfibril assembly, thus providing at physiological pH a new type of collagen structure for biomaterial preparation, characterized by different charge and structural contents .

Este trabalho descreve um novo processo para a reticulação de matrizes de colágeno com glutaraldeído para a preparação de materiais para a confecção de válvulas cardíacas biológicas, e consiste no tratamento do pericárdio bovino com concentrações progressivamente crescentes com este reagente no intervalo de concentração entre 0,005 e 0,5% em tampão fosfato, pH 7,4. O perfil da reação com glutaraldeído, quando comparado com o procedimento convencional, foi mais homogêneo e os materiais obtidos apresentaram propriedades térmicas e mecânicas similares. Entretanto, mostraram-se significativamente mais estáveis à degradação enzimática, principalmente em relação à tripsina (17 x), que é devido provavelmente à formação de ligações de reticulação mais homogêneas e/ou em maior densidade. Os resultados mostram que metodologia seqüencial estudada, associada ao tratamento convencional, pode constituir-se em uma técnica de pré-tratamento promissora para a preparação de materiais mais eficientes para confecção de biopróteses.

This work describes a new process for crosslinking of collagen matrices with glutaraldehyde for the preparation of improved material for biological heart valve manufacture which consists in the treatment of bovine pericardium with low and progressive increasing concentrations of glutaraldehyde from 0.005 to 0.5% in phosphate buffer, pH 7.4. The crosslinking reaction profile was more homogeneous compared to the conventional procedure and the material was characterized by a higher stability towards enzyme degradation, particularly in respect to trypsin hydrolysis by a factor of 17 times, with the required thermal and elastic properties. This increased enzyme stability is probably due to a more homogeneous crosslink distribution and/or increased density within the collagen matrix. The results showed that the sequential procedure may be a potential and efficient pre-treatment technique for the preparation of bovine pericardium derived materials for the manufacture of bioprosthesis.

Anionic collagen: calcium phosphate composite was obtained by controlled mixing of collagen and calcium phosphate until the consistence of a past. Material was characterized by a Ca/P ratio of 1.55, with a X-ray diffraction pattern similar to that for hydroxyapatite. Differential Scanning Calorimetry showed that the protein is not denatured under the processing conditions. Scanning Electronic Microscopy showed that the mineral phase are regularly covered with collagen fibers, indicating that anionic collagen is efficient in the preparation of stable form of calcium phosphate ceramic paste.

Este trabalho descreve o efeito da ransana, um polissacarídeo bacteriano, sobre géis de colágeno aniônico. A interação colágeno:ransana ocorreu independentemente do pH, mesmo com baixas concentrações de ransana, e os materiais obtidos no estado sólido foram caracterizados por serem mais estáveis térmicamente à medida em que se aumenta a concentração do polissacarídeo. Nenhuma alteração na estrutura secundária em tripla hélice do tropocolágeno foi observada. O efeito mais significativo da ransana sobre os géis aniônicos de colágeno foi um aumento significativo da viscosidade, e as variações observadas em função de pH e temperatura sugerem que nesta interação, não estão envolvidas forças de natureza eletrostática ou hidrofóbica. Micrografias de colágeno aniônico e colágeno aniônico:ransana mostraram a presença de estruturas vesiculares, diferente do padrão fibrilar característico de colágeno nativo. Um modelo de interação baseado na ação da ransana sobre a água estruturada, associada à organização macromolecular do colágeno em solução é proposto. Mais importante, géis de colágeno aniônico:ransana mostraram uma estabilidade térmica compatível com aquelas desejáveis para um biomaterial injetável de colágeno, evitando o uso do glutaraldeído como agente de estabilização.

This work reports the effects of rhamsan, a bacterial polysaccharide, over anionic collagen gels. Collagen: rhamsan interaction occurred independently from pH even at extremely low collagen:rhamsan ratios and materials were characterized in the solid state by an increase in thermal stability with an increase in polysaccharide concentration. No disruption of the triple helix secondary structure of tropocollagen was observed. The most striking effect of rhamsan addition over anionic collagen gels was the high increase in viscosity. A dependence study on pH and temperature suggests that electrostatic or hydrophobic forces are probably not involved in the process. Anionic collagen and anionic collagen: rhamsan gel micrographs at neutral pH showed the presence of spherical vesicles, different from the expected fibrillar pattern of native collagen. A model based on the action of rhamsan over structured water associated to collagen assembly in solution is proposed. Most important, anionic collagen:rhamsan gels showed a thermal stability compatible for its use as an injectable collagen biomaterial, thus avoiding the use o glutaraldehyde as an stabilizing agent.

Este trabalho descreve um processo para hidrólise seletiva de grupos carboxamidas de resíduos de asparagina e glutamina presentes em matrizes colagênicas, para a obtenção de colágeno aniônico para produção de novos biomateriais. Os resultados mostraram que em meio alcalino e na presença de metais alcalinos e alcalinos terrosos, os grupos carboxamidas da proteína são seletivamente hidrolizados para dar origem a pH neutro, a materiais de colágeno com um aumento de 106 de carga negativas, em relação à matrizes não modificadas. Embora não tenham sido observadas alterações na estrutura secundária da proteína ou na organização microfibrilar da matriz, modificações significativas foram observadas no padrão da sub-periodicidade do período D da estrutura microfibrilar, que provavelmente são devidas a um aumento da intensidade de interações eletrostáticas.

This work describes a process for the selective hydrolysis of carboxamide groups of asparagine and glutamine found in collagen polymeric matrices for the preparation of new charged collagen biomaterials. The results showed that under alkaline conditions and in the presence of alkaline and alkaline earth metals, protein carboxamide side chain groups were selectively hydrolyzed to produce collagen matrices with net increase of 106 negative charges at neutral pH, in comparison to native matrices. Although no changes were observed at the secondary protein structure or at the microfibrillar level of collagen matrix organization, significative changes were detected in the pattern of the sub-periodicity of the D-period, probably due to an increase in electrostatic interaction.

A maioria dos implantes valvulares cardíacos realizados no Brasil é representada pelas válvulas de pericárdio bovino, seguidas por próteses porcinas. Na avaliação de válvulas biológicas, deve-se considerar: desempenho hidrodinámico, resistência à fadiga e processo de calcificação. No presente estudo, foi avaliado o desempenho hidrodinámico de biopróteses de pericárdio bovino (Biopro-PB-Braile Biomédica) comparativamente às válvulas porcinas (Biopro-PP-Braile Biomédica) através do gradiente médio transvalvular. Os testes hidrodinámicos foram realizados em próteses de diâmetros variando de 19 a 35 mm, submetidas ao Sistema Duplicador de Pulsos Shelhigh (Shelhigh Inc.). O volume de ejeção foi mantido constante em 90 ml, com freqüência de pulso de 60, 70,80, 90 e 100 ciclos por minuto, possibilitando fluxos entre 5 e 9 litros por minuto, equivalentes a fluxos contínuos aproximados de 8 a 18 litros por minuto. Houve tendência à diminuição dos gradientes pressóricos à medida em que aumenta o diâmetro externo das próteses. O gradiente pressórico médio encontrado em próteses de pericárdio bovino foi significativamente menor que o de próteses porcinas (p<0,01), para todos os diâmetros estudados. Em vista da grande variabilidade dos protocolos de testes encontrados na literatura, reforça-se a necessidade de testes padronizados para avaliação hidrodinâmica de próteses valvulares, visando comparação adequada entre os diferentes modelos existentes.

Most of the cardiac valve implantations in Brazil are represented by bovine pericardial valves, followed by the porcine prostheses. In the evaluation of biological valves, the following should be taken into consideration: hydrodynamic performance resistance to fatigue and calcification process. In this study, the hydrodynamic performance of bovine pericardial bioprostheses (Biopro-BP-Braile Biomedica) was evaluated comparatively to porcine valves (Biopro-PP-Braile Biomedica), through the transvalvular medium gradient. The hydrodynamic tests were made on prostheses varying from 19 to 35 mm in diameter, which underwent the pulse duplicator system Shelhigh (Shelligh Inc.). The ejection volume was constantly kept at 90 ml. The pulse frequencies varied between 60 and 100 cycles per minute, allowing flows between 5 and 9 liters per minute, equivalent to approximate continuos flows of 8 to 18 liters per minute. There was tendency to decrease the gradients, in proportion to increase of the bioprostheses external diameters. The medium pressure gradient found in the bovine pericardial prostheses was significantly smaller than that in the porcine prostheses (p<0.01), for all diameters studied. Respecting the large variability of protocols of tests in the literature for hydrodynamic evaluation of valve prostheses, the necessity of standard tests becomes stronger, seeking adequate comparison among the several valves.