Comparative experimental study between L-Hydro treated pulmonary homograft and fresh pulmonary homograft.

OBJECTIVE: In an effort to make available homografts preserved in a simpler and less costly way, we evaluated the polyethyleneglycol, L-Hydro (LH) method, that consists in the controlled extraction of antigenic substances and the incorporation of anti-inflammatory and anti-thrombotic agent. METHODS: We substituted the pulmonary trunk in ten ovines, seven received LH treated pulmonary homografts and three, fresh pulmonary homografts, orthotopically implanted and followed-up for 320 days. Ovines where evaluated by means of laboratory tests, echocardiographic exams. At the 320 days, were euthanized, hemodynamic, radiology, macroscopic, optic/electronic microscopic, scanning/transmission evaluations were performed. Results were analyzed by Student t test of independent samples for continuous data, by variance analysis of repeated measures, and by Fisher exact test for categorical data. RESULTS: We couldn't establish relevant differences in clinical evolution and laboratory tests between groups. Echocardiogram revealed difference in pulmonary medium gradient, which was significant 10 months follow-up, higher in the control group. Radiologic and macroscopic evaluations didn't established differences. In the optic/electronic microscopic evaluation, liner and interstitial cells were equally found in both groups. The cell liner percent calculated in both groups was similar. Cellularity nodules were observed only infresh homograft group. CONCLUSIONS: These data indicate that both groups presented similar clinical/hemodynamic performances. The LH group's echocardiogram presented a better performance. It also presented histological evidences of interstitial and endothelial cell repopulation. In the macro/optic and electronic microscopic analysis, group L-H presented macroscopy/histological structure and ultra-structural similar to the fresh group, with the exception of nodules with higher interstitial cellularity, present only in the fresh homograft group.

Estudo experimental comparativo do enxerto homólogo pulmonar tratado pelo processo L-Hydro com o homoenxerto pulmonar a fresco / Comparative experimental study between L-Hydro treated pulmonary homograft and fresh pulmonary homograft

OBJETIVO: Buscando novas formas de preservação de tecidos utilizamos o polietileno-glicol, método L-Hydro (LH), que consiste na extração controlada de substâncias antigênicas e incorporação de agente antinflamatório e antitrombótico. MÉTODOS: Em dez carneiros jovens, substituímos o tronco pulmonar, em sete, por homoenxertos pulmonares (HP) tratados pelo processo L-H e, em três, por HP a fresco, implantados ortotopicamente, seguidos por 320 dias. Os carneiros foram avaliados por exames laboratoriais e ecocardiográficos. Ao cabo dos 320 dias foram sacrificados, procedendo-se à avaliação hemodinâmica, radiológica, macro/microscópica, óptica e eletrônica, varredura e transmissão. Resultados foram analisados pelo teste t de Student de amostras independentes para dados contínuos, análise de variância para medidas repetidas, pelo teste exato de Fisher para dados categóricos. RESULTADOS: Evolução clínica e exames laboratoriais não conseguiram estabelecer diferenças significativas entre os grupos. Ecocardiograma revelou diferença quanto ao gradiente médio pulmonar, significativa aos 10 meses, maior no grupo controle. Avaliação radiológica e macroscópica não estabeleceu diferenças. Na avaliação microscópica, óptica/eletrônica, células de revestimento e intersticiais foram encontradas nos dois grupos igualmente. O porcentual de revestimento celular calculado nos dois grupos foi semelhante. Nódulos de celularidade foram observados somente no grupo de homoenxertos a fresco. CONCLUSÕES: Estes dados indicam que os dois grupos apresentaram desempenho clínico e hemodinâmico semelhante. Ao ecocardiograma o grupo LH apresentou melhor desempenho, e evidências histológicas de repopulação celular intersticial e endotelial. Na análise macro/microscópica, óptica/eletrônica, o grupo L-Hydro apresentou macroscopia, estrutura histológica e ultraestrutural semelhante ao homoenxerto fresco, à exceção de nódulos de maior celularidade intersticial, presentes apenas no homoenxerto a fresco.

OBJECTIVE: In an effort to make available homografts preserved in a simpler and less costly way, we evaluated the polyethyleneglycol, L-Hydro (LH) method, that consists in the controlled extraction of antigenic substances and the incorporation of anti-inflammatory and anti-thrombotic agent. METHODS: We substituted the pulmonary trunk in ten ovines, seven received LH treated pulmonary homografts and three, fresh pulmonary homografts, orthotopically implanted and followed-up for 320 days. Ovines where evaluated by means of laboratory tests, echocardiographic exams. At the 320 days, were euthanized, hemodynamic, radiology, macroscopic, optic/electronic microscopic, scanning/transmission evaluations were performed. Results were analyzed by Student t test of independent samples for continuous data, by variance analysis of repeated measures, and by Fisher exact test for categorical data. RESULTS: We couldn't establish relevant differences in clinical evolution and laboratory tests between groups. Echocardiogram revealed difference in pulmonary medium gradient, which was significant 10 months follow-up, higher in the control group. Radiologic and macroscopic evaluations didn't established differences. In the optic/electronic microscopic evaluation, liner and interstitial cells were equally found in both groups. The cell liner percent calculated in both groups was similar. Cellularity nodules were observed only infresh homograft group. CONCLUSIONS: These data indicate that both groups presented similar clinical/hemodynamic performances. The LH group's echocardiogram presented a better performance. It also presented histological evidences of interstitial and endothelial cell repopulation. In the macro/optic and electronic microscopic analysis, group L-H presented macroscopy/histological structure and ultra-structural similar to the fresh group, with the exception of nodules with higher interstitial cellularity, present only in the fresh homograft group.

Stentless valves treated by the L-hydro process in the aortic position in sheep.

Calcification of glutaraldehyde-treated bioprosthetic heart valves is a major cause of long-term failure. We studied porcine aortic valves treated by the L-Hydro process and implanted into 14 juvenile sheep (group 1). Another 10 sheep were implanted with glutaraldehyde-treated porcine bioprostheses (group 2). The animals were sacrificed after 150 days and the explanted valves were analyzed for calcification. Hemodynamic measurements by echocardiography and angiography were carried out prior to sacrifice. Macroscopic analysis showed calcification and loss of mobility of the leaflets in all group 2 implants and in one group 1 implant. Light microscopy showed foci of calcification in all group 2 implants and in 3 valves from group 1. A significant reduction in the level of calcification was found in porcine bioprostheses treated by the L-Hydro process and implanted into the juvenile sheep model.

Comparative study of the L-hydro process and glutaraldehyde preservation.

Commercial bioprosthetic heart valves are commonly preserved in glutaraldehyde and are cytotoxic to host cells, preventing spontaneous endothelialization. The aim of this study was to demonstrate the potential for in vivo endothelialization of bioprostheses treated by the L-Hydro process which consists of mild extraction of antigenic substances and incorporation of antiinflammatory and antithrombotic agents. Seven stented porcine heart valves treated by the L-Hydro process and 3 glutaraldehyde-fixed porcine heart valves were implanted in the mitral position in juvenile sheep. The valves were evaluated by echocardiography, angiography, histology, and histochemistry. No hemodynamic differences were observed, but scanning and transmission electron microscopy showed nearly complete coverage by endothelial cells of all leaflets in the L-Hydro-treated valves after 5 months of implantation. The endothelial cells were in direct contact with the underlying collagen and expressed von Willebrand-related antigens. The surfaces of the glutaraldehyde-treated valves were covered by fibrin, macrophages, calcium, and thrombotic material; only sparse endothelial cells were observed and contact with the underlying tissue was incomplete. These data indicate that L-Hydro-treated porcine valves are capable of inducing spontaneous endothelialization.

Halofuginone inhibits serum-stimulated pericardial tissue retraction in vitro.

We developed an in vitro model of tissue contraction in which living pericardium, in response to serum, contracted and the cells in situ expressed proliferating cell nuclear antigen (PCNA) and synthesized collagen. Here we evaluated the effects of halofuginone on these serum-stimulated pericardial tissue responses. Parietal pericardium was incubated with media containing increasing doses of halofuginone and evaluated for tissue contraction, evident by tissue curling. Proliferation was measured by MTS metabolism and PCNA expression. Furthermore, collagen synthesis was compared between samples incubated with halofuginone, cytochalasin B, cytochalasin D, aphidicolin, or cytosine arabinoside (AraC), using Masson's trichrome and the monoclonal antibody to sheep type I procollagen, SP1. D8. Halofuginone inhibited tissue contraction, cellular proliferation, and collagen synthesis in a dose-dependent manner. In contrast, cytochalasin B, cytochalasin D, aphidicolin, and AraC, shown previously to prevent cellular proliferation, did not prevent type I collagen synthesis. Halofuginone has been implicated as an agent in the prevention of wound-healing fibrosis. This study suggests that halofuginone may have an added benefit in the inhibition of pericardial tissue contraction, which appeared to be related to the synthesis of type I procollagen.

Improving the results of small-bore vascular graft testing in rabbits: a technical primer.

Appropriate models to evaluate the in vivo behavior of small-diameter grafts are varied. To evaluate the behavior of small-diameter, bovine-derived grafts in the arterial circulation, we chose the rabbit abdominal aorta model. In the development of our procedure, we evaluated several models published in the literature, with unsatisfactory results. The high incidence of postoperative mortality and morbidity led us to modify published methods to incorporate cautious surgical technique and mild systemic hypothermia with cross-clamp times shorter than 30 min, as well as perioperative administration of agents with metabolic, rheologic, and neuroprotective properties. These modifications enabled us to achieve 100% operative survival with a very low incidence of postoperative paralysis. The presented model will be used for further evaluation of small-diameter grafts in our laboratory.

Cellular expression of PCNA and procollagen in vital and ethanol-treated autologous pericardial implants in sheep.

BACKGROUND AND AIMS OF THE STUDY: Cardiovascular surgeries involving repair or reconstruction of heart valve leaflets with vital autologous pericardium have shown detrimental healing outcomes, mainly fibrosis with retraction. It is proposed that cells intrinsic to the pericardial implants may contribute to this fibrosis by becoming activated to proliferate and synthesize type I collagen. METHODS: Vital and ethanol-treated autologous pericardium were implanted as rectangular flaps bisecting the lumen in the descending aorta of sheep to simulate a heart valve leaflet. Implants recovered at 5, 10, 15, and 30 days were evaluated immunohistologically for expression of PCNA and procollagen. RESULTS: In ethanol-treated pericardium, concentrations of activated cells shifted from the fibrin layers on the periphery of implants at days 5 and 10 to cells internal to the implant at days 15 and 30. In contrast, concentrations of activated cells in vital pericardium shifted from cells within the implants at days 5 and 10 to the fibrin deposits overlaying the implants at days 15 and 30. CONCLUSION: Different distributional patterns of activated cells were observed between vital and ethanol-treated pericardial flap implants. These different patterns may be important in understanding the cause of the detrimental healing outcome observed with vital autologous pericardial flap implants.

An in vitro model of pericardial tissue healing.

INTRODUCTION: A previous study in our laboratory showed that a flap of fresh autologous pericardium bisecting the aorta of sheep retracted and became fibrotic. Histologic analyses suggested that activated cells within the pericardium contributed to the retraction of the implant. Here we report the development of an in vitro model to investigate the effects of serum on cellular proliferation and cell-mediated tissue contraction. METHODS: Sections of living and ethanol-treated sheep pericardium were incubated with 0.5%, 5%, 10%, 20%, and 50% serum in medium for up to 8 days and evaluated for cellular proliferation and tissue contraction. These serum-stimulated events were further evaluated in the presence of Mitomycin C, Cytochalasin B and D, Aphidicolin, AraC, and Cycloheximide. RESULTS: Cellular proliferation and cell-mediated tissue contraction were induced by serum in a dose-dependent manner. Expression of PCNA was suppressed in the presence of Cytochalasin B, Cytochalasin D, Aphidicolin, and AraC. Tissue contraction was prevented by Cycloheximide. Mitomycin C inhibited both proliferation and tissue contraction. Ethanol-treated tissue, which was absent of living cells, did not respond to stimulation with serum. CONCLUSIONS: An in vitro model was developed to study the responses of cells within pericardial tissues to stimulation by serum. In this model, serum induced cellular proliferation and tissue contraction. Different chemical inhibitors independently modulated these serum-stimulated events. Pre-existing cells within pericardial tissues might respond to stimulus through differential pathways. This model may help to develop methods to make autologous pericardium a clinically useful biomaterial.

Serum components stimulate pericardial tissue contraction.

BACKGROUND AND AIMS OF THE STUDY: Previous experiments have demonstrated the retraction and fibrosis of vital autologous pericardial flap implants in the descending aorta of sheep. An in-vitro model of pericardial tissue contraction was developed that showed healing reactions similar to those observed in the fresh in-vivo flap. Here, the component(s) of serum that stimulate tissue contraction were partially characterized. The molecular weight range and stability (heat and protease resistance) of the serum component(s) are described. Tissue contraction also requires de-novo protein synthesis. METHODS: Sections (1 cm2) of sheep pericardium were incubated with fractionated, heat-treated, or protease-treated fetal bovine serum for 14 days. In addition, SDS-PAGE protein profiles were generated using tissues incubated with and without cycloheximide for up to 12 days. RESULTS: Tissue contraction was observed in molecular weight serum fractions > or =5 kDa, as well as in samples incubated with heat and protease-treated serum. SDS-PAGE showed the appearance of a protein band after day 4 during the process of tissue contraction that was absent in samples incubated with cycloheximide. CONCLUSION: Serum fractions > or =5 kDa stimulated protein synthesis and pericardial tissue contraction. The active component(s) was shown to be heat stable, but partially sensitive to protease. The addition of cycloheximide to the culture medium, shown previously to prevent pericardial tissue contraction, inhibited de-novo synthesis of the protein that appeared during the process of tissue contraction.