Francisco Diniz Affonso da Costa

Decellularized heterografts versus cryopreserved homografts: experimental study in sheep model

OBJECTIVES The aim of this study is to assess the biological behaviour of porcine decellularized heterografts (Desc group) compared with cryopreserved homografts (Crio group) implanted in juvenile sheep. METHODS Decellularized porcine pulmonary heterografts were implanted in five animals and cryopreserved pulmonary homografts in another five. The animals were followed-up for a mean of 280 +/- 14 days. The valve diameter was measured by echocardiography, which was performed at the 30th postoperative day, and before the explantation. The valves were also assessed macroscopically. Histological evaluation was performed using H.E., Gomori and Weigert staining. Immunohistochemistry specified different cell types (Factor VIII, CD3, Vimentin and CD68). Calcium quantity was analyzed using atomic absortion spectometry. RESULTS There was one death in the Desc group due to endocarditis. The valves of Crio group showed decrease in the cellularity whereas the valves of Desc group showed matrix repopulation with endothelial and interstitial cells. Loss of collagen density and disarrangement of the normal fiber architecture was observed in Crio group. Calcium content demonstrated higher levels on the cusps and conduits in Crio group comparatively with Desc group. (P=0.016). The mean valvular diameter at the explantation was significantly increased (P=0.025) in the Desc group. CONCLUSIONS Decellularized heterografts had a different biological behaviour when compared to cryopreserved homografts and become repopulated by cells with fibroblasts and endothelial cells characteristics. The matrix was preserved and some regenerative potential was present.

Controle microbiológico em valvas cardíacas humanas

OBJECTIVE To evaluate, from microbiological point of view, the valves processed by Human Heart Valve Bank of Santa Casa de Misericórdia of Curitiba for use in cardiovascular surgeries. METHODS The processing of 1,671 valves, accomplished within the period of time between July 1999 and June 2004, was evaluated. Out of the valves and the solutions involved in the process, samples were collected and spread in culture mediums, such as fluid thioglycollate medium, tryptic soy broth and Sabouraud broth, for incubation during 14 days, using a modified methodology based on the Farmacopéia Brasileira 1988 (Brazilian Pharmacopeia) and USP 1990 (United States Pharmacopeia). The samples in which growing was observed were submitted to microbian identification. RESULTS In a set of 1,671 samples, 92% were considered proper for use under microbiological point of view, since they did not display microbian contamination. The remaining 8% were rejected for clinical use because of contamination in some stage of the valve processing. CONCLUSION From the Analysis of the results, it was observed the importance of microbiological control in human grafts, in order to avoid using microbiologically contaminated valves in patients submitted to cardiovascular surgery.

Experimental study with deccelularized porcine heterografts: the prosthesis of the future

OBJETIVO: Avaliar, comparativamente, o comportamento de homoenxertos valvares criopreservados e de heteroenxertos descelularizados implantados na via de saida do ventriculo direito de carneiros jovens, assim como relatar a experiencia clinica inicial com homoenxertos descelularizados em pacientes submetidos a operacao de Ross. METODO: No grupo A, foram implantados quatro homoenxertos pulmonares criopreservados e no Grupo B, quatro heteroenxertos porcinos valvares descelularizados com acido deoxicolico. Em cada grupo, dois animais foram sacrificados no 3o mes e dois no 5o mes de pos-operatorio. As pecas foram avaliadas macroscopicamente e por radiografias, alem de exame microscopico com coloracoes HE, Tricromico de Mallory e Sirius Red. A experiencia clinica com quatro pacientes submetidos a operacao de Ross com emprego de homoenxertos valvares descelularizados e relatada. RESULTADOS: Todos os animais sobreviveram. Os ecocardiogramas demonstraram boa funcao valvar nos dois grupos. Embora as cuspides valvares em ambos os grupos tivessem aspecto macroscopico normal, a mensuracao de calcio e o estudo radiologico demonstraram inicio de calcificacao focal nos homoenxertos criopreservados, mas nao nos heteroenxertos descelularizados. Esses achados foram confirmados por exames microscopicos. Os homoenxertos criopreservados demonstraram perda de sua celularidade, enquanto que os heteroenxertos descelularizados demonstraram repopulacao progressiva da matriz colagena com fibroblastos, assim como reendotelizacao. Os quatro pacientes operados tiveram evolucao pos-operatoria imediata satisfatoria, com funcao normal dos homoenxertos. CONCLUSOES: Heteroenxertos valvares descelularizados foram progressivamente repopulados por celulas autogenas e exibiram minima tendencia a calcificacao no modelo estudado. Esses resultados sugerem que homoenxertos descelularizados possam ter alguma capacidade regenerativa e com isso ter durabilidade superior aos homoenxertos criopreservados convencionais.

Decellularization as an anticalcification method in stentless bovine pericardium valve prosthesis: a study in sheep

OBJECTIVE The objective was to analyze the decellularization process with SDS in glutaraldehyde-preserved bovine pericardium as an anticalcification method in a circulatory sheep model. METHODS The valved tubs were implanted in pulmonary artery position in sheep by 180 days. The animals were divided in two groups of 8 animals: control group--glutaraldehyde-preserved bovine pericardium and the study group--decellularized bovine pericardium with 0,1% SDS and glutaraldehyde-preserved. After explantation the tubs were analized by x-ray macroscopy, hematoxilin-eosin, alizarin-red and Russel-Movatz pentacromic histology. The calcium content was measured by flame atomic absorption spectrometry. RESULTS There was no early mortality, but two animals in each group died during the study. All cusps in the control group were severely calcified and in some points in the conduits, while the decellularized group did not show macroscopic calcification. Data were proved by x-ray and histologycal exams. The matrix was preserved in histologycal analysis in decellularized group, without gross calcification. The wall conduits calcium content was 35,25 ± 42,13 µg/mg in the control group versus 15,75 ± 10,44 µg/mg in the decellularized one: in the cusps was 264,4 ± 126,16 µg/mg in control group versus 94,29 ± 27,05 µg/mg in decellularized group (P = 0,009). CONCLUSION The decellularization with 0.1% SDS was effective as an anticalcification method in bovine pericardial grafts implanted in a sheep circulatory model for 180 days.

Valvas cardíacas obtidas por engenharia de tecidos: a mais nova geração de próteses biológicas

Hospital Charite, Universidade Humboldt, Berlim, Alemanha e Santa Casa deMisericordia, Pontificia Universidade Catolica do Parana, CuritibaCorrespondencia: Francisco Diniz Affonso da Costa – Rua Henrique Coelho Neto, 5582200-120 – Curitiba, PR – E-mail: fcosta@mps.com.brRecebido para publicacao em 12/11/01Aceito em 18/2/02

Cinco anos de experiência com a operação de Ross: o que aprendemos?

OBJETIVO: Analisar a evolucao clinica e funcional de 96 pacientes submetidos a substituicao da valva aortica por auto-enxerto pulmonar. CASUISTICA E METODOS: De maio/95 a marco/2000, 96 pacientes com media de idades de 25,4±11,4 anos foram consecutivamente submetidos a substituicao da valva aortica por auto-enxerto pulmonar. O diagnostico pre-operatorio mais comum foi de insuficiencia aortica de origem reumatica e 89% encontravam-se em classe funcional II ou III. Todos realizaram ecocardiograma bidimensional com Doppler e 42 tambem foram submetidos a cateterismo cardiaco antes da operacao. O auto-enxerto foi implantado pela tecnica de substituicao total da raiz aortica em 85 casos, como cilindro intraluminal em seis e em posicao subcoronariana nos demais. A reconstrucao da via de saida do ventriculo direito foi feita com homoenxertos pulmonares (84) ou aorticos (12), conservados em solucao nutriente com antibioticos (34) ou criopreservados (62). Antes da alta hospitalar, todos repetiram o exame ecocardiografico e 30 foram submetidos a cateterismo cardiaco. Atraves deles, foi feita avaliacao do desempenho hemodinâmico dos auto e homoenxertos, assim como da funcao e massa ventricular esquerda. No periodo de acompanhamento, os pacientes foram examinados e repetiram o ecocardiograma a cada seis meses. Vinte pacientes, com tempo de evolucao superior a seis meses, submeteram-se a ecocardiografia de stress com dobutamina, para estudar o desempenho hemodinâmico dos auto e homoenxertos em condicao de exercicio. RESULTADOS: A mortalidade hospitalar foi de 6,2%. Apos um tempo medio de 32,1 meses (1 - 58), 98,9% dos pacientes estao vivos. A incidencia de pacientes livres de tromboembolismo, endocardite, disfuncao do auto-enxerto, disfuncao do homoenxerto, reoperacoes mitrais e de qualquer tipo de complicacao apos 41 meses e de 100%, 100%, 97,1%, 96,5%, 93,9% e 87,5%, respectivamente. O auto-enxerto pulmonar teve desempenho hemodinâmico fisiologico, com media de gradiente medio de 3,8±3,3 mmHg. O grau de insuficiencia valvar foi negligenciavel. Mesmo em condicao de exercicio, os gradientes nao se elevaram de forma significativa, com media de gradiente medio de 6,8±3,8 mmHg. A funcao hemodinâmica dos homoenxertos foi excelente na fase imediata, com gradiente medio de 4,1±4,6 mmHg. Houve, entretanto, discreto aumento desses gradientes na fase tardia, com media de 13,9±10 mmHg. Durante o exercicio, houve aumento significativo destes gradientes, que foram para 33,6±18,9 mmHg. Diâmetro do homoenxerto e idade do receptor tiveram correlacao inversa com o gradiente pulmonar tardio. O indice de massa ventricular diminui de 180±68 g/m2 no pre-operatorio para 117±32 g/m2 na fase tardia. A funcao ventricular no pos-operatorio tardio foi normal tanto em repouso como em exercicio na maioria dos pacientes. CONCLUSOES: O implante do auto-enxerto pulmonar em posicao aortica pode ser feito com baixa mortalidade imediata. A sobrevida tardia foi excelente, com baixa incidencia de complicacoes. O desempenho hemodinâmico dos auto-enxertos pulmonares foi proximo ao fisiologico tanto em repouso como em exercicio. Houve significativa regressao da massa ventricular esquerda, e, os indices de funcao ventricular tambem foram normais na fase tardia de pos-operatorio. Apesar dos homoenxertos da via de saida do ventriculo direito apresentarem desempenho hemodinâmico adequado na fase imediata, o aparecimento de discretos gradientes na fase tardia foi frequente.

Efeito da criopreservação e/ou da descelularização na matriz extracelular de condutos valvados porcinos

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%.The objective of this study was to evaluate the morphology of decellularized 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 effectiveness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.

Effect of SDS-based decelullarization in the prevention of calcification in glutaraldehyde-preserved bovine pericardium: study in rats

OBJECTIVE The aim of study was to investigate the SDS-based decellularization process as an anticalcification method in glutaraldehyde-preserved bovine pericardium in subcutaneous rat model. METHODS Pericardium samples with 0.5 cm² area were divide in four groups: group GDA: 0.5% glutaraldehyde-preserved pericardium (GDA); group GDA-GL: GDA + 0.2% glutamic acid (GL); group D-GDA: decellularized (D) pericardium with 0.1% SDS + GDA and group D-GDA-GL: decellularized pericardium + GDA + 0.2% glutamic acid. After this samples were implanted in 18 rats in subcutaneous position till 90 days. Each animal received samples of the four groups. The explants were performed at 45 and 90 days. The explants were subjected to histology in glass slides stained with hematoxilin-eosin and alizarin red, morphometry evaluation and the calcium content was measured by flame atomic absorption spectrometry. RESULTS The inflammatory infiltrate was the same in all groups, however more intense in GDA and GDA-GL groups in 45 days, increasing at 90 days. The calcium contents for 45 days were: 32.52 ± 3.19 µg/mg in GDA group; 22.12 ± 3.87 µg/ mg in GDA-GL group; 1.06 ± 0.38 µg/mg in D-GDA group and 3.99 ± 5.78 µg/mg in D-GDA-GL (P< 0.001). For 90 days were 65.91 ± 24.67 µg/mg in GDA group; 38.37 ± 13.79 µg/mg in GDA-GL group; 1.24 ± 0.99 µg/mg in D-GDA group and 30.54 ± 8.21 µg/mg in D-GDA-GL (P< 0.001). Only D-GDA did not show increase rates of calcium at 45 to 90 days (P=0.314). CONCLUSION SDS-based decellularization process reduced the inflammatory intensity and calcification in bovine pericardium in subcutaneous rat model for 90 days.Objective: The aim of this study was to investigate the SDS-based decellularization process as an anticalcification method in glutaraldehyde-preserved bovine pericardium in subcutaneous rat model. Methods: Pericardium samples with 0.5 cm 2 area and divided into four groups: GDA group: 0.5% glutaraldehydepreserved pericardium (GDA); GDA-GL group: GDA + 0.2% glutamic acid (GL); D-GDA group: decellularized (D) pericardium with 0.1% SDS + GDA, and D-GDA-GL group: decellularized pericardium + GDA + 0.2% glutamic acid. Afterwards these samples were implanted in 18 rats in subcutaneous position up to 90 days. Each animal received samples of the four groups. The explants were performed at 45 and 90 days. The explants were subjected to histology in glass slides stained with hematoxilin-eosin and alizarin red, morphometry evaluation and the calcium content was measured by flame atomic absorption spectrometry. Results: The standard of inflammatory infiltrate was the same in all groups, however more intense in GDA and GDAGL groups in 45 days, increasing at 90 days. The calcium contents for 45 days were: 32.52 ± 3.19 µg/mg in GDA group; 22.12 ± 3.87 µg/mg in GDA-GL group; 1.06 ± 0.38 µg/mg in D-GDA group and 3.99 ± 5.78 µg/mg in D-GDA-GL (P< 0.001). For 90 days were 65.91 ± 24.67 µg/mg in GDA group; 38.37 ± 13.79 µg/mg in GDA-GL group; 1.24 ± 0.99 µg/mg in D-GDA group and 30.54 ± 8.21 µg/mg in D-GDA-GL (P< 0.001). Only D-GDA did not show increase rates of calcium at 45 to 90 days (P=0.314). Conclusion: SDS-based decellularization process reduced the inflammatory intensity and calcification in bovine pericardium in subcutaneous rat model for 90 days.

Avaliação inicial de homoenxertos em posição pulmonar em crianças e adolescentes

Objective: Heart valve homografts - fresh or cryopreserved - are a good valvular substitute in infants and teenagers as they need no anticoagulation, have a higher resistance to infection and present a good hemodynamic performance. The objective of this report was to evaluate the initial postoperative results on pediatric subjects after homograft implant in pulmonary position. Methods: Fourteen homografts were implanted in pulmonary position from September, 1995 to December, 1997. Four of them were fresh (28.6%) and ten were cryopreserved (71.4%). Subjects ranged in age from 10 months to 17 years with 9 (64%) males and 5 (36%) females . The subjects were divided into two groups, as follows: A - aortic valve disease (Ross operation) and B - congenital heart disease. The diameter of the homografts ranged from 16 to 26 mm. The cardiopulmonary-bypass time ranged from 74 to 303 minutes and the aortic-occlusion time between 49 to 160 minutos. Results: A hospital death occurred. All the survivors were clinically, radiologically, electrocardiographycally and echocardiographycally monitored. There were 13 NYHA I patients and one NYHA II patients. The most common complications were low output syndrome, pericardial effusion and EKG abnormalities. All these ocurred in two patient but with a favorable outcome. Stenosis of the right ventricular outflow track was observed early on in two subjects with gradients of 44 and 23 mmHg. One of the pulmonary homografts was submitted to a successful balloon valvuloplasty due to a severe stenosis. The rheumatic patients are still receiving antibiotic therapy and without recurrence of the disease. Conclusion: Evaluation of the hemodynamics of the homografts, in pulmonary position, was satisfactory. One of the subjects has presented marked stenosis but, after a balloon valvuloplasty, his gradient decreased. Another patient has kept his preoperative NYHA classification. We believe was not correlated with the homograft (coagulation disorder). Its our belief that fresh and cryopreserved homografts are a satisfactory solution for the correction of congenital or acquired valve disease in infants and adolescents.

Operação de Ross: a intervenção ideal para pacientes aórticos jovens?

Background: Aortic valve prosthesis with adequate hemodynamic performance should allow more complete left ventricular mass regression and return left ventricular function back to normal. This possibly affects long term prognosis after aortic valve replacement. Objective: Assessment of hemodynamic performance of the pulmonary autograft in the aortic position and the regression of left ventricular mass after the Ross procedure. Material and Methods: Between May/95 and Mar/96, 45 patients with mean age of 27.1 years were submitted to a Ross procedure. Doppler echocardiography and cardiac catheterization were performed on all patients before hospital discharge to analyze the hemodynamic performance of the auto and homografts employed, as well as to evaluate left ventricular mass and function. Fourteen patients with follow-up longer than six months were sumitted to dobutamine stress echocardiography to study hemodynamic performance of the auto and homografts at during exercise. Results: Hospital mortality was 6%. After a mean follow-up of 12.8 months (1-23), there was one late sudden death. No valve related event was noted during this period. Immediate and late hemodynamic performance of the pulmonary autografts were normal with average mean gradients of 1.8 ± 0.6 mmHg and average maximum instantaneous gradients of 2.9 ± 0.9 mmHg. Valvular insufficiency was almost null. Even during exercise, gradients did not increase significantly with average mean gradients of 4.3 ± 2.5 mmHg and average maximum gradients of 10.4 ± 6.1 mmHg. Homografts used for right ventricular reconstruction exhibited excellent immediate hemodynamic performance. However, at late follow-up an increase in flow velocities was noted with an average of mean gradients of 10 ± 7.1 mmHg at rest and 26 ± 13.3 mmHg at exercise. Left ventricular mass index dropped from 168 ± 46 g/m2 preoperatively to 115 ± 32 g/m2 six months after the operation. Left ventricular function was normal at rest and during exercise in the marjority of patients. Conclusions: Given the normal hemodynamic performance of the autografts, the important decrease in left ventricular mass and the normal left ventricular function late postoperatively, the Ross procedure is considered the operation of choice for young patients who need aortic valve replacement.