Repopulation of decellularised porcine pulmonary valves in the right ventricular outflow tract of sheep: Role of macrophages.

The primary objective was to evaluate performance of low concentration SDS decellularised porcine pulmonary roots in the right ventricular outflow tract of juvenile sheep. Secondary objectives were to explore the cellular population of the roots over time. Animals were monitored by echocardiography and roots explanted at 1, 3, 6 (n = 4) and 12 months (n = 8) for gross analysis. Explanted roots were subject to histological, immunohistochemical and quantitative calcium analysis (n = 4 at 1, 3 and 12 months) and determination of material properties (n = 4; 12 months). Cryopreserved ovine pulmonary root allografts (n = 4) implanted for 12 months, and non-implanted cellular ovine roots were analysed for comparative purposes. Decellularised porcine pulmonary roots functioned well and were in very good condition with soft, thin and pliable leaflets. Morphometric analysis showed cellular population by 1 month. However, by 12 months the total number of cells was less than 50% of the total cells in non-implanted native ovine roots. Repopulation of the decellularised porcine tissues with stromal (α-SMA+; vimentin+) and progenitor cells (CD34+; CD271+) appeared to be orchestrated by macrophages (MAC 387+/ CD163low and CD163+/MAC 387-). The calcium content of the decellularised porcine pulmonary root tissues increased over the 12-month period but remained low (except suture points) at 401 ppm (wet weight) or below. The material properties of the decellularised porcine pulmonary root wall were unchanged compared to pre-implantation. There were some changes in the leaflets but importantly, the porcine tissues did not become stiffer. The decellularised porcine pulmonary roots showed good functional performance in vivo and were repopulated with ovine cells of the appropriate phenotype in a process orchestrated by M2 macrophages, highlighting the importance of these cells in the constructive tissue remodelling of cardiac root tissues.

Decellularization as a method to reduce calcification in bovine pericardium bioprosthetic valves.

OBJECTIVES: Decellularization is an alternative method for processing biological tissues with decreased antigenicity and resistance to calcification. The aim of this study was to characterize the properties of decellularized (dCell) bovine pericardium fixed with 0.1% glutaraldehyde (GA) and to evaluate outcomes of bioprosthetic valves constructed with this tissue when implanted in the mitral position of juvenile sheep. METHODS: Bioprosthetic mitral valves were constructed with fresh bovine pericardium fixed in 0.5% GA (control group) or dCell bovine pericardium fixed in 0.1% GA (study group). Before implantation, samples were submitted to histological (haematoxylin-eosin, Movat and 4',6-diamidino-2-phenylindole), biochemical (residual deoxyribonucleic acid and α-gal epitopes) and biomechanical characterization. Valves were implanted (n = 8 in each group) as a mitral valve replacement for 180 days in sheep and explants were re-evaluated histologically and for calcification with radiological studies and calcium content determination. RESULTS: Unimplanted dCell pericardia exhibited a well-preserved extracellular matrix with absence of cells, a 77% reduction in deoxyribonucleic acid levels and with no detectable α-gal epitopes. When compared to controls, they had lower ultimate tensile strength (7.3 ± 5.4 vs 10.2 ± 3.0 mPa, P = 0.04) and greater percentage elongation in the longitudinal direction (29 ± 6.5% vs 23.8 ± 5.1%, P = 0.02). After 180 days in mitral position, dCell valves showed pliable leaflets without macroscopic signs of calcification. Histologically, dCell leaflets had intact collagen fibres, better tissue remodelling and a significant 89% reduction in calcium content. CONCLUSIONS: This study demonstrates that bioprosthetic valves constructed with dCell bovine pericardium fixed in low GA concentration were resistant to calcification and may thereby improve long-term durability of the tissue.

Human Heart Explant-Derived Extracellular Vesicles: Characterization and Effects on the In Vitro Recellularization of Decellularized Heart Valves.

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.

Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches.

Tissue-engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long-term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)-based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose-derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM-cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.

In vitro evaluation of bovine pericardium after a soft decellularization approach for use in tissue engineering.

Pericardial membrane derived from bovine heart tissues is a promising source of material for use in tissue-engineering applications. However, tissue processing is required for its use in humans due to the presence of animal antigens. Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the bovine pericardium (BP) after a soft decellularization process with a 0.1% sodium dodecyl sulfate (SDS) solution, with the aim to remove xenoantigens and preserve extracellular matrix (ECM) bioactivity. The decellularization process promoted a mean reduction of 77% of the amount of DNA in the samples in which cell nuclei staining was undetectable. The ECM content was maintained as mostly preserved after decellularization as well as its biomechanical properties. In addition, the decellularization protocol has proven to be efficient in removing the xenoantigen alpha-gal, which is responsible for immune rejection. The decellularized BP was noncytotoxic in vitro and allowed human adipose-derived stem cell (hASC) adhesion. Finally, after 7 days in culture, the tissue scaffold became repopulated by hASCs, and after 30 days, the ECM protein pro-collagen I was seen in the scaffold. Together, these characteristics indicated that soft BP decellularization with 0.1% SDS solution allows the acquirement of a bioactive scaffold suitable for cell repopulation and potentially useful for regenerative medicine.

Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination.

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

20 years experience with the Ross operation in middle-aged patients: the autologous principle is still alive.

Objectives: Review our long-term results with the Ross operation in middle-aged patients. Methods: Between 1995 and 2016, 129 consecutive patients (106 males); mean age (47.2 ± 5.2 years) underwent a Ross operation. Right ventricular outflow tract (RVOT) reconstruction was performed with cryopreserved (n = 45) or decellularized allografts (n = 84). Mean follow-up was 8.4 ± 5.3 years (0.1 20.5 years). We analyzed early and late mortality, as well as valve related events and the need for reoperations. Results: Early mortality was 1.6% and late survival was 87.6% at 16 years. There were 4 reoperations on the pulmonary autograft (96% freedom at 16 years) and 2 on the pulmonary allografts (99% freedom at 16 years). The 16-year freedom from more than mild aortic insufficiency (AI) and a late root diameter >45 mm was 64% and 71%, respectively. Patients with the preoperative diagnosis of AI are at greater risk for these complications. Among the allografts, decellularized allografts showed superior freedom from structural valve dysfunction. Conclusions: The Ross operation in this cohort was associated with long-term survival similar to the general population and low incidence of reoperations. Patients with the preoperative diagnosis of AI are at increased risk for late autograft insufficiency and root dilatation. Decellularized allografts presented the best results for reconstruction of the RVOT. These results support the conclusion that the Ross operation has an important role in the treatment of middle-aged patients with aortic valve disease, especially those with pure aortic stenosis.

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.

Efeito da descelularização com SDS na prevenção da calcificação em pericárdio bovino fixado em glutaraldeído: estudo em ratos / Effect of SDS-based decelullarization in the prevention of calcification in glutaraldehyde-preserved bovine pericardium: study in rats

OBJETIVO: Avaliar a descelularização com SDS como tratamento anticalcificante em pericárdio bovino fixado em glutaraldeído. MÉTODOS: Peças de 0,5 cm² foram implantadas em modelo subcutâneo de 18 ratos por até 90 dias. Foram formados quatro grupos: grupo GDA: pericárdio fixado em glutaraldeído 0,5% (GDA), grupo GDA-GL: pericárdio fixado em GDA + ácido glutâmico (GL) 0,2%, grupo D-GDA: pericárdio descelularizado (D) com SDS 0,1% e fixado em GDA e grupo D-GDA-GL: pericárdio descelularizado + GDA + ácido glutâmico 0,2%. Cada animal recebeu enxertos dos quatro grupos. Os explantes foram realizados com 45 e 90 dias. As avaliações foram: análise histológica com as colorações hematoxilina-eosina e alizarina-red, análise morfométrica e quantificação de cálcio por espectrometria de absorção atômica. RESULTADOS: O padrão de infiltrado inflamatório foi o mesmo nos quatro grupos, sendo mais intenso nos grupos GDA e GDA-GL aos 45 dias, ficando mais evidente aos 90 dias. O conteúdo de cálcio aos 45 dias foi de 32,52 ± 3,19 µg/ mg no grupo GDA; 22,12 ± 3,87 µg/mg no grupo GDA-GL; 1,06 ± 0,38 µg/mg no grupo D-GDA e 3,99 ± 5,78 µg/mg no grupo D-GDA-GL (P< 0,001). Aos 90 dias, foi de 65,91 ± 24,67 µg/mg no grupo GDA; 38,37 ± 13,79 µg/mg no grupo GDA-GL; 1,24 ± 0,99 µg/mg no grupo D-GDA e 30,54 ± 8,21 µg/mg no grupo D-GDA-GL (P< 0,001). O grupo D-GDA foi o único que não apresentou progressão da calcificação de 45 para 90 dias (P=0,314). CONCLUSÃO: A descelularização com SDS reduziu o processo inflamatório e inibiu a calcificação em pericárdio bovino implantado em modelo subcutâneo de ratos até 90 dias.

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% glutaraldehydepreserved 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.

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.

Descelularização como método anticalcificante em próteses valvares de pericárdio bovino sem suporte: estudo em ovinos / Decellularization as an anticalcification method in stentless bovine pericardium valve prosthesis: a study in sheep

OBJETIVO: Avaliar o processo de descelularização com dodecil sulfato de sódio (SDS) como método anticalcificante em próteses de pericárdio bovino fixadas em glutaraldeído, em modelo circulatório de ovinos. MÉTODOS: Tubos valvulados de pericárdio bovino foram implantados em posição pulmonar de ovinos por 180 dias. Os animais foram divididos em dois grupos com oito animais: grupo controle, com condutos de pericárdio fixado em glutaraldeído e grupo estudo, com pericárdio descelularizado com SDS 0,1% e posteriormente fixado em GDA. Os explantes foram submetidos à análise macroscópica, histológica com hematoxilina-eosina, alizarina-red e pentacrômico de Russel-Movatz, estudo radiológico e quantificação de cálcio com espectrometria de absorção atômica. RESULTADOS: Não houve mortalidade imediata, porém dois animais de cada grupo faleceram na evolução tardia. Os enxertos do grupo controle apresentavam intensa calcificação das cúspides e em algumas regiões dos condutos, enquanto que os enxertos descelularizados apresentavam-se preservados, sem calcificações macroscópicas evidentes. Esses resultados foram comprovados por análise histológica e radiográfica. Histologicamente, os enxertos descelularizados tiveram sua matriz melhor preservada e com diminuição acentuada da calcificação. O conteúdo de cálcio nos condutos foi de 35±42 µg/mg de tecido no grupo controle versus 15 ±10 µg/mg nos descelularizados. Nas cúspides valvares, esses valores foram de 264±126 µg/mg no grupo controle versus 94±27 µg/mg nos descelularizados (P=0,009). CONCLUSÃO: A descelularização com SDS 0,1% foi efetiva como método anticalcificante em condutos de pericárdio bovino implantados em modelo circulatório de ovinos por 180 dias.

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.

Evaluation of the biological behavior of decellularized pulmonary homografts: an experimental sheep model.

INTRODUCTION: The cryopreserved homograft is a good valve substitute due attributes like excellent hemodynamics, low incidence of thromboembolic events, infection resistance and good mid-term durability. However, progressive homograft degeneration and fibrocalcification may occur, particularly in the childhood and young adults. Their antigenicity triggers an immunological reaction that plays an important role in their degeneration and failure. The decellularization process was proposed to decrease this antigenicity. By the action of detergents and enzymes, this process removes all cellular components from the homograft matrix, diminishing immunogenicity and probably delaying its degeneration. OBJECTIVE: The objective of this experimental and descriptive study is to evaluate the biological and functional behavior of decellularized pulmonary homografts (Decell-H), treated by a sodium dodecil sulfate solution (0.1%), developed in our University (Pontifícia Universidade Católica do Paraná). For the characterization of Decell-H performance, parameters like recellularization, calcification, and echocardiographic data will be analyzed. METHODS: Eight juvenile sheep were submitted to the implantation of the Decell-H sutured into orthotopic position, through a left thoracotomy and with cardiopulmonary bypass support. They were followed-up clinically and by periodical echocardiograms until the explantation, which were performed in different time for every two sheep: seven, 30, 90 and 180 postoperative days. For histological analysis we used Hematoxilin-eosin, Movat and Alizarin-Red staining. RESULTS: The sheep reached their follow-up period in a good clinical state. There was no valve regurgitation or stenonis by the echocardiogram. The animals submitted to the explantation in 90 and 180 days had a significant somatic growth and these Decell-H(s) had a diameter increase, without central valve insufficiency. Histologically, all homografts preserved their extra-cellular matrix organization and were progressively recellularized, without calcification. CONCLUSION: In this experimental model, the Decell-H behaved as an excellent valve substitute.

Avaliação do comportamento biológico de homoenxertos valvares pulmonares descelularizados: estudo experimental em ovinos / Evaluation of the biological behavior of decellularized pulmonary homografts: an experimental sheep model

INTRODUÇÃO: Não havendo um substituto valvar ideal, os homoenxertos criopreservados são considerados uma boa opção, pelo excelente perfil hemodinâmico, baixa incidência de tromboembolismo, resistência a infecções e durabilidade a médio prazo. Porém, estão sujeitos à progressiva degeneração, especialmente em crianças e adultos jovens. Sua antigenicidade desencadeia uma resposta imunológica que contribui para sua degeneração, calcificação e falência. Para diminuir esta antigenicidade, desenvolveu-se o processo de descelularização. Pela ação de detergentes e enzimas, este processo remove os componentes celulares do homoenxerto, diminuindo sua imunogenicidade e, provavelmente, retardando sua degeneração. OBJETIVO: O objetivo deste estudo, experimental e descritivo, é analisar o comportamento histológico e funcional de homoenxertos pulmonares ovinos descelularizados (H-descel) por uma nova solução, composta principalmente de dodecil sulfato de sódio a 0,1 por cento e desenvolvida na PUCPR. Para caracterizar este comportamento, serão avaliados o repovoamento celular, a ocorrência de calcificação e a função valvar ao ecocardiograma. MÉTODOS: A amostra foi constituída de oito ovinos, submetidos ao implante de H-descel em posição ortotópica, através de uma toracotomia esquerda, com auxílio de circulação extracorpórea. Os animais foram acompanhados clinicamente e por ecocardiogramas periódicos até o explante, realizados em prazos predefinidos para cada dois animais: sete, 30, 90 e 180 dias. A análise histológica foi realizada por colorações Hematoxilina-eosina, Pentacrômio de Movat e Alizarina Red. RESULTADOS: Todos os animais sobreviveram ao procedimento e atingiram seus períodos de seguimento. Não houve insuficiência ou estenose destes enxertos ao ecocardiograma. Os animais submetidos aos explantes em 90 e 180 dias tiveram significativos ganhos ponderais e estes H-descel aumentaram de diâmetro, sem desenvolver insuficiência. À histologia, todos mantiveram a organização de sua matriz extracelular, foram progressivamente repovoados e não apresentaram calcificação. CONCLUSÃO: Neste modelo experimental, os H-descel mostraram-se excelentes substitutos valvares a médio prazo.

INTRODUCTION: The cryopreserved homograft is a good valve substitute due attributes like excellent hemodynamics, low incidence of thromboembolic events, infection resistance and good mid-term durability. However, progressive homograft degeneration and fibrocalcification may occur, particularly in the childhood and young adults. Their antigenicity triggers an immunological reaction that plays an important role in their degeneration and failure. The decellularization process was proposed to decrease this antigenicity. By the action of detergents and enzymes, this process removes all cellular components from the homograft matrix, diminishing immunogenicity and probably delaying its degeneration. OBJECTIVE: The objective of this experimental and descriptive study is to evaluate the biological and functional behavior of decellularized pulmonary homografts (Decell-H), treated by a sodium dodecil sulfate solution (0.1 percent), developed in our University (Pontifícia Universidade Católica do Paraná). For the characterization of Decell-H performance, parameters like recellularization, calcification, and echocardiographic data will be analyzed. METHODS: Eight juvenile sheep were submitted to the implantation of the Decell-H sutured into orthotopic position, through a left thoracotomy and with cardiopulmonary bypass support. They were followed-up clinically and by periodical echocardiograms until the explantation, which were performed in different time for every two sheep: seven, 30, 90 and 180 postoperative days. For histological analysis we used Hematoxilin-eosin, Movat and Alizarin-Red staining. RESULTS: The sheep reached their follow-up period in a good clinical state. There was no valve regurgitation or stenonis by the echocardiogram. The animals submitted to the explantation in 90 and 180 days had a significant somatic growth and these Decell-H(s) had a diameter increase, without central valve insufficiency. Histologically, all homografts preserved their extra-cellular matrix organization and were progressively recellularized, without calcification. CONCLUSION: In this experimental model, the Decell-H behaved as an excellent valve substitute.

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.

Análise do comportamento biológico de heteroenxertos descelularizados e homoenxertos criopreservados: estudo em ovinos / Decellularized heterografts versus cryopreserved homografts: experimental study in sheep model

OBJETIVO: Este estudo avalia o comportamento biológico dos heteroenxertos porcinos descelularizados (Grupo Desc) comparados com os homoenxertos criopreservados (Grupo Crio) implantados em carneiros jovens. MÉTODOS: Foram implantados em cinco animais heteroenxertos pulmonares porcinos descelularizados e em outros cinco, homoenxertos pulmonares criopreservados. Os animais apresentaram seguimento médio de 280 ± 14 dias. O diâmetro valvar foi medido por ecocardiografia, a qual foi realizada no 30º pós-operatório e antes do explante. As valvas foram também avaliadas macroscopicamente. A avaliação histológica foi realizada utilizando-se coloração de H.E., Gomori e Weigert e imunohistoquímica (Fator VIII, CD3, Vimentina e CD68). A quantificação de cálcio foi realizada utilizando-se espectrometria de absorção atômica. RESULTADOS: Houve um óbito no Grupo Desc por endocardite. As valvas do Grupo Crio apresentaram decréscimo na celularidade, enquanto que as valvas do Grupo Desc demonstraram repovoamento da matriz com células endoteliais e intersticiais. No grupo Crio, observou-se perda na densidade e desarranjo da arquitetura das fibras colágenas. A espectrometria de absorção atômica demonstrou maior calcificação no conduto e nas cúspides dos enxertos criopreservados quando comparados aos descelularizados (P=0,016). O diâmetro médio valvar no explante foi significantemente maior no Grupo Desc (P=0,025). CONCLUSÃO: Heteroenxertos descelularizados apresentam um comportamento biológico diferente quando comparados aos homoenxertos criopreservados, tornando-se repovoados por células com características de fibroblastos e células endoteliais. A matriz permaneceu bem preservada, o que possibilitou um processo de regeneração celular.

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

Eficácia do AlCl3 e etanol na prevenção da calcificação de fragmentos da parede aórtica porcina fixados em GDA / Efficacy of AlCl3 and ethanol in the prevention of calcification of fragments of porcine aortic wall fixed in GDA

OBJETIVO: Avaliar a eficácia do cloreto de alumínio, isoladamente ou em associação com o etanol, na prevenção da calcificação e da resposta inflamatória de fragmentos de parede aórtica porcina fixada em glutaraldeído (GDA), implantados no tecido subcutâneo de ratos jovens. MÉTODO: Utilizaram-se 15 ratos da linhagem Sprague-Dawley, em cujas telas subcutâneas foram implantados fragmentos de parede aórtica porcina, submetidos a três diferentes métodos de tratamento [grupos: I (GDA), II (GDA+alumínio), III (GDA+etanol+alumínio)]. Os explantes foram realizados com 15, 30 e 60 dias após as operações. Foram realizadas análises histológicas pelas colorações de hematoxilina & eosina (HE) e de alizarina, nos pHs de 4,2 e 7,0, e a dosagem de cálcio feita por espectroscopia de absorção atômica. RESULTADOS: Pelo HE, constatou-se que a matriz extracelular das paredes aórticas ficaram melhor preservadas nos explantes do grupo III. A intensidade da reação inflamatória intensa foi menor nesse grupo. Pela alizarina pH 4,2, o grupo II e III tiveram menores índices de calcificação comparado ao controle. Pela alizarina pH 7,0, o grupo III teve menor índice de calcificação comparado aos grupos I e II. Pela espectroscopia de absorção atômica, os níveis de cálcio foram semelhantes para os grupos II e III, mas significativamente menores do que os do grupo I. CONCLUSÃO: O tratamento com cloreto de alumínio diminuiu a calcificação dos fragmentos de parede aórtica porcina. O uso combinado do etanol com cloreto de alumínio foi ainda mais eficiente em inibir a calcificação, e também em diminuir a reação inflamatória.

OBJECTIVE: To evaluate the efficiency of aluminum chloride in isolation or associated with ethanol to prevent calcification and inflammatory reaction with fragments of porcine aortic wall fixed in glutaraldehyde (GDA) and subdermally implanted in young rats. METHOD: Fifteen Sprague-Dawley rats were studied. Three fragments of porcine aortic wall were implanted in the subdermal tissue. The fragments were previously subjected to three different methods of treatment: I (GDA), II (GDA + aluminum), III (GDA + ethanol + aluminum). Explantation was performed after fifteen, thirty and sixty days. Histological analysis was achieved using hematoxylin & eosin (HE) and alizarin-red at pHs of 4.2 and 7.0. Calcium content was determined by atomic absorbance spectroscopy. RESULTS: HE and alizarin red staining showed that the aortic wall extracellular matrix was best preserved in the fragments of Group III. The intensity of the inflammatory reaction was lower in this group. When stained with alizarin red at pH 4.2, Groups II and III had lower degrees of calcification compared with Group I. With alizarin red staining at pH 7.0, Group III demonstrated less calcification compared with Groups I and II. Atomic absorbance spectroscopy showed similar calcium levels for both Groups II and III, but significantly less than in Group I. CONCLUSION: Treatment with aluminum chloride inhibits calcification of fragments of aortic wall after implantation and reduces inflammatory reaction. The combined use of ethanol with aluminum chloride is more efficient to inhibit calcification and also to diminish inflammatory reaction.