CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Early systemic cellular immune response in children and young adults receiving decellularized fresh allografts for pulmonary valve replacement.

OBJECTIVES: The longevity of homografts is determined by the activation of the recipients' immune system resulting from allogenic antigen exposition. Fresh decellularized pulmonary homografts (DPH) have shown promising early results in pulmonary valve replacement in children and young adults and could potentially avoid significant activation of the immune system, as more than 99% of the donor DNA is removed during the decellularization process. While the humoral immune response to decellularized allografts has been studied, detailed information on the more significant cellular immune response is currently lacking. METHODS AND RESULTS: Peripheral blood samples were obtained from patients undergoing pulmonary valve replacement with DPH before, after, and for approximately 3 years after implantation. Absolute counts and percentages of mature T- (CD3(+)), B- (CD19(+)), and natural killer- (CD16(+)/CD56(+)) cells, as well as T helper- (CD4(+)) and cytotoxic T-cell- (CD8(+)) subsets, were determined by fluorescence-activated cell sorting (FACS). Between May 2009 and September 2013, 199 blood samples taken from 47 patients with a mean age at DPH implantation of 16.6±10.8 years were analyzed. The hemodynamic performance of DPH was excellent in all but one patient, and no valve-related deaths or conduit explantations were observed. The short-term follow up revealed a significant postoperative decrease in cell counts of most subtypes with reconstitution after 3 months. Continued assessment did not show any significant deviations in cell counts from their baseline values. CONCLUSION: The absence of cellular immune response in patients receiving DPH supports the concept that decellularization can provide a basis for autologous regeneration.

Bioengineered human and allogeneic pulmonary valve conduits chronically implanted orthotopically in baboons: hemodynamic performance and immunologic consequences.

OBJECTIVE: This study assesses in a baboon model the hemodynamics and human leukocyte antigen immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds. METHODS: Fourteen baboons underwent pulmonary valve replacement, 8 with decellularized and conditioned (bioengineered) pulmonary valves derived from allogeneic (N = 3) or xenogeneic (human) (N = 5) hearts; for comparison, 6 baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel-reactive serum antibodies (human leukocyte antigen class I and II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon rank-sum tests. RESULTS: All animals survived and thrived, exhibiting excellent immediate implanted valve function by transesophageal echocardiograms. Over time, reference valves developed a smaller effective orifice area index (median, 0.84 cm(2)/m(2); range, 1.22 cm(2)/m(2)), whereas all bioengineered valves remained normal (effective orifice area index median, 2.45 cm(2)/m(2); range, 1.35 cm(2)/m(2); P = .005). None of the bioengineered valves developed elevated peak transvalvular gradients: 5.5 (6.0) mm Hg versus 12.5 (23.0) mm Hg (P = .003). Cryopreserved valves provoked the most intense antibody responses. Two of 5 human bioengineered and 2 of 3 baboon bioengineered valves did not provoke any class I antibodies. Bioengineered human (but not baboon) scaffolds provoked class II antibodies. C1q(+) antibodies developed in 4 recipients. CONCLUSIONS: Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.

Elimination of alpha-gal xenoreactive epitope: alpha-galactosidase treatment of porcine heart valves.

BACKGROUND AND AIM OF THE STUDY: Porcine heart valves are among the most widely used tissue valves in clinical heart valve implantation. However, immunologic responses have been implicated as potential causes of the limited durability of xenograft heart valves. The study aim was to determine the effectiveness of alpha-galactosidase treatment used to degrade the major xenoreactive antigens found in xenograft heart valves. METHODS: Fresh porcine heart valves and pericardium treated with alpha-galactosidase were studied to evaluate the xenoreactive galactose (alpha1,3) galactose (alpha-gal) antigen. Removal of the alpha-gal epitope from the porcine heart valve was monitored via 3,3'-diaminobenzidine staining intensity, while the removal of alpha-gal from N-glycans on porcine heart valves treated with recombinant alpha-galactosidase was determined either qualitatively or quantitatively by mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The porcine pericardium was used for monitoring the change in mechanical properties after alpha-galactosidase treatment. In addition, the biomechanical modification property of collagen fiber rearrangement on tissue was assessed using transmission electron microscopy (TEM). RESULTS: Following a 24-h incubation at pH 7.2, 4 degrees C, employing 0.1 U/ml of Bacteroides thetaiotaomicron-derived recombinant alpha-galactosidase, the enzyme effectively removed the alpha-gal epitopes expressed on porcine heart valves. The identification type of alpha-gal N-glycan on fresh aortic valve, aortic wall, pulmonary valve, and pulmonary wall was 7.1%, 10.3%, 6% and 8%, respectively. In the presence of alpha-galactosidase treatment, alpha-gal-containing N-glycans were converted into alpha-gal-negative N-glycans. Likewise, alpha-gal-containing N-glycans were not detected when MALDI-TOF MS quantitative analysis was used. Furthermore, no significant difference was observed in the mechanical properties and findings from TEM in alpha-galactosidase-treated porcine pericardial tissue when compared to fresh porcine pericardium. CONCLUSION: Alpha-galactosidase can effectively remove the alpha-gal epitope from porcine heart valves and pericardium. This may possibly alleviate harmful xenoreactive immunologic responses by alpha-gal, without adversely affecting the biomechanical properties of the alpha-galactosidase-processed tissue.

Evaluation of humoral immune response to donor HLA after implantation of cellularized versus decellularized human heart valve allografts.

We have evaluated the development of antibodies in response to donor allograft valve implant in patients who received cellularized and decellularized allografts and determined possible immunogenic epitopes considered responsible for antibodies reactivity. Serum samples from all recipients who received cellularized allografts or decellularized allografts were collected before valve replacement and at 5, 10, 30 and 90 days post-operatively and frozen until required. Tests were performed using the Luminex-based single human leukocyte antigen (HLA)-A, -B, -C and HLA-DR, -DQ antigen microsphere assay. To determine possible immunogenic epitopes, we used the HLAMatchmaker (HLAMM) software if applicable. Decellularized grafts elicited lower levels of anti-HLA class I and II antibody formation after implantation than cellularized allografts. All patients from cellularized group presented donor-specific antibodies class I and II within 3 months of observation period. In HLAMM analysis, the cellularized group had significantly higher numbers of immunogenic epitopes than decellularized group for both class I and II (p: 0.002 - cl I / p: 0.009 - cl II / p: 0.004 - cl I and II). Our findings demonstrate that the anti-HLA antibodies detected in the cellularized group were against donor HLA possible immunogenic epitopes and that in the decellularized group the anti-HLA antibodies were not against donor HLA possible immunogenic epitopes. These findings lead us to suggest that choosing sodium dodecyl sulfate decellularization process is the best alternative to decrease the immunogenicity of allograft valve transplant.

Structural integrity of collagen and elastin in SynerGraft® decellularized-cryopreserved human heart valves.

SynerGraft® (SG) decellularized-cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.

Expression of A and B blood group antigens on cryopreserved homografts.

BACKGROUND: The contribution of ABO blood group mismatching on the rate of cardiac homograft failure is uncertain. It has been shown that there is lack of expression of A and B blood group antigens on fresh and cryopreserved homograft valves. However, expression of these antigens on the homograft vessel conduit has not been reported. METHODS: Unused portions of cryopreserved pulmonary artery homografts (n = 16) were immunohistochemically stained and examined for expression of endothelial cell marker CD31 and A and B blood group antigens. The staining pattern for each antigen was described. Comparison of homograft donor blood group (as determined by immunostaining) to the blood group reported by the homograft supplier was made. RESULTS: Staining of CD31 and A and B blood group antigens was poor on the luminal surface (tunica intima) of the homograft conduit but strong in the vaso vasorum of the tunica media and adventitia. Homograft donor blood group was consistent with the reported donor blood group in 15 of 16 specimens (4 group A, 3 group B, 8 group O). In one specimen (reported as group O), we detected strong expression of CD31 and A antigen on the endothelium of the vaso vasorum. CONCLUSIONS: Cryopreserved homografts strongly express A and B blood group antigens on well-preserved endothelial surfaces of the medial and adventitial vaso vasorum. The significance of this finding with regard to the immunologic response to mismatched ABO blood group antigens and homograft longevity is uncertain.

Immunologically untreated fresh xenograft implantation in a pig-to-goat model.

Immunologically untreated frozen-stored xenografts show controlled rejection and repair due to reduced cellularity in our previous study. To clarify the issue, we compared results obtained using fresh and frozen-stored xenografts. Porcine pulmonary valved conduits were prepared without immunologic treatment and implanted in the right ventricular outflow tract of goats under cardiopulmonary bypass immediately after harvest without frozen storage (the fresh group) or after frozen storage (-70 degrees C for 3 - 7 days) (the frozen group). Four goats were assigned to be raised for 3 (N = 1) or 6 (N = 3) months postoperatively in each group. One goat in the frozen group assigned for the 6-month observation expired at 2 months postoperatively because of thrombotic occlusion of the pulmonary valve. The other goats survived until the scheduled sacrifice. According to echocardiographic findings, one animal sacrificed at 3 months in the frozen group showed more than a moderate degree of pulmonary regurgitation, but all animals in the fresh group showed less than a mild degree. On gross examination, leaflets were slightly better preserved in the fresh group and aneurysmal dilatation of the pulmonary artery was observed at 3 months only in the frozen group. Microscopically, pulmonary arteries showed less severe degenerative changes in the fresh group. Moreover, in the fresh group, viable donor valve cells were still present until 3 months postoperatively (though it disappeared at 6 months postoperatively) and a linear endothelial lining of viable host cells was prominent on all leaflets, whereas this was not the case in the frozen group. In conclusion, fresh xenografts preserved cellular viability and showed fewer degenerative changes than frozen-stored xenografts. Thus, immunologically untreated fresh xenografts could provide a potential valve substitute with distinctive advantages in terms of self-healing potential as compared with frozen-stored xenografts.

Genipin blues: an alternative non-toxic crosslinker for heart valves?

BACKGROUND AND AIM OF THE STUDY: One approach in tissue-engineering involves the implantation of decellularized, xenogenic scaffolds, with the expectation of repopulation in vivo. However, a major limitation of this method is the propensity to induce a strong immune host response. The study aim was to mitigate this immunogenicity by employing a crosslinking treatment with genipin. METHODS: Porcine matrices were prepared using a detergent-enzymatic treatment and fixed in 0.01% or 0.001% aqueous genipin. The mechanical properties of the matrices were monitored by tensile strength testing. The survival of chicken fibroblasts was used to determine cell-friendliness of the matrices. Non-fixed, decellularized biological scaffolds (n = 3) were implanted in a sheep model and compared to an equal number of genipin-fixed scaffolds (n = 6). Matrices implanted in the pulmonary position were explanted after six weeks and examined using light and transmission electron microscopy. The antibody reaction against porcine tissue in sheep serum was also determined. RESULTS: Statistically significant differences were found between non-fixed leaflets, 0.001% genipin-and 0.6% glutaraldehyde (GA)-fixed leaflets for work to maximum load (non-fixed 0.00646 J; genipin-fixed 0.00509 J; GA-fixed 0.00543 J) and stiffness (non-fixed 9281 N/m; genipin-fixed 16214 N/m; GA-fixed 14401 N/m). Genipin-treated matrices were not cytotoxic. For all concentrations of genipin a high proportion of viable cells was present (79-100%). Low-dose GA (10 microg/ml) showed a distinct cytotoxicity (24.8% viability). At explant, an intense chronic inflammatory response was observed in non-fixed matrices, in contrast to genipin-fixed scaffolds. The sheep serum showed a marked decrease in IgG response in both 0.001% and 0.01% genipin-fixed matrices (IgG 30 and 20, respectively) when compared to non-fixed matrices (IgG 40). CONCLUSION: Genipin crosslinking of the matrices attenuated, but did not eliminate, the inflammatory host reaction. Whether genipin treatment might extend the durability of xenogenic scaffolds remains to be investigated.

Time-related histopathologic analyses of immunologically untreated porcine valved conduits implanted in a porcine-to-goat model.

This study was performed to evaluate the clinical feasibility of use of immunologically nontreated xenogenic valves, using a porcine-to-goat pulmonary valved conduit implantation model. Porcine pulmonary valve conduits were prepared with no specific immunological treatment and implanted in the right ventricular outflow tract of goats under cardiopulmonary bypass. The goats were assigned at predetermined intervals (1 day, 1 week, and 3, 6, and 12 months) as two animals for each interval. Echocardiographic examinations of the valves were performed before sacrifice. Upon retrieving the xenograft specimens, they were inspected visually and microscopically. Ten of the 12 animals survived the predetermined observation periods. Variable degrees of pulmonary regurgitation were the main findings on echocardiographic evaluations. On gross examination of the explanted specimens, all leaflets, except in one animal that prematurely died, were fairly well preserved. They were slightly shortened but free of thrombosis or vegetation. Aneurysmal dilatations of the anterior wall of the implanted pulmonary artery were observed in one of 12-month-survival animals and in another one of 3-month-survival animals. Microscopically, the three components of implanted xenografts (the pulmonary artery, valve, and infundibulum) were shown to be gradually replaced with host cells in time, while maintaining structural integrity. The nuclei of the donor tissue disappeared through pyknosis and karyolysis. In conclusion, immnunologically untreated xenogenic pulmonary valved conduits can be an alternative potential as valve substitutes with distinctive advantages of providing self-healing potential, despite a few problems observed in the current study such as occurrences of pulmonary regurgitation and sporadic cases of aortic aneurysm.

Presence and elimination of the xenoantigen gal (alpha1, 3) gal in tissue-engineered heart valves.

Tissue engineering of heart valves promises to create functional autologous tissue with the potential for regeneration and growth without the limitations of current heart valve prostheses. The appropriate valve matrix is essential. Porcine heart valves are attractive because of their anatomical similarity. Decellularization is used for antigen reduction. The efficacy of published protocols varies, however. The absence of a specific immunological or unspecific inflammatory reaction is mandatory. The porcine cell-specific alpha-Gal epitope is known to be responsible for hyperacute rejection in xenotransplantation. In tissue engineering residual alpha-Gal epitope may induce severe inflammation in humans and may lead to graft failure. In this study porcine pulmonary conduits were decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630, and ribonuclease treatment and were compared with specimens of the commercially available porcine decellularized SynerGraft regarding cell removal and elimination of the alpha-Gal epitope. In addition, samples of a porcine bioprosthesis were examined for the presence of the alpha-Gal epitope. In conclusion, we describe for the first time the presence of the alpha-Gal epitope in clinically used porcine bioprostheses and the first generation of a commercial tissue-engineered heart valve. In contrast, complete cell and alpha-Gal removal was achieved by a decellularization procedure developed by our group.

Factors influencing the survival of cryopreserved homografts. The second homograft performs as well as the first.

OBJECTIVE: To determine the life span of cryopreserved homografts implanted in the right ventricular outflow tract and the factors influencing it. METHODS: From 1989 through 2003, we reconstructed the pulmonary valve with 301 homografts in 272 patients (median age 13 years; range 4 days-69 years). Indications were tetralogy of Fallot (136), truncus (23), Rastelli repair (11), double outlet ventricle (13), endocarditis (5), and the Ross operation (84). Median follow-up was 5.7 years (range 0-14). We analyzed possible predictors of graft replacement by simple and multiple Cox regression. RESULTS: Actuarial survival was 96+/-1.2% at 1, 95+/-1.4% at 5, and 94+/-1.5% at 10 years follow-up. Three homografts were explanted because of endocarditis (excluded from the analysis). Freedom from explantation was 99.6+/-0.4% at 1, 94.5+/-1.7% at 5, and 81.8+/-4.1% at 10 years. Variables, significantly related to explantation in the univariate analysis, were younger age, small graft size, implantation in a non-anatomical position, the aortic donor homograft, a shorter aortic cross-clamp time and the implantation of a second homograft. In the multiple model, non-anatomical position (P=0.001), smaller graft size (P<0.0001) or younger age (on square root scale, P<0.0001) and clamp time (P=0.01) remain as independent risk factors. Immunological variables, like blood group incompatibility, implantation of a second homograft and short warm ischemic time were not significant. CONCLUSIONS: The life span of a cryopreserved homograft is determined by graft size (correlates with age) and the non-anatomic position (correlates with indication). In a specific patient, the second homograft performs as well as the first.

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon.

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

Mid-term findings on echocardiography and computed tomography after RVOT-reconstruction: comparison of decellularized (SynerGraft) and conventional allografts.

OBJECTIVE: The immune response against human-leucocyte-antigens on donor-cells may be an important factor contributing to the degeneration of allograft-valves. We have previously reported that the use of the decellularized allograft SynerGraft (CryoLife) reduces the immunologic response of the allograft-recipient. In this study we compare the echocardiographic and computed tomography angiographic (CTA) findings of SynerGrafts with conventional cryopreserved allografts. METHODS: 22 patients who received a pulmonary SynerGraft (SG-group) (21 during a Ross-procedure) underwent CTA and resting echocardiography (median: 10 months postoperatively). 47 randomly chosen patients who underwent a Ross-procedure served as controls (C-group) (median: 32 months postoperatively). RESULTS: Neither the pressure gradients (mean: SG=9+/-4 vs C=10+/-4mmHg; P=0.64) across the allograft, nor the effective orifice area (EOAI) (SG=0.93+/-0.80 vs C=0.93+/-0.42cm(2)/m(2); P=0.96) differed between the groups. The EOAI showed a significant correlation with the smallest allograft-conduit-area measured on CTA (r=0.81; P<0.001) which was most frequently (n=34) found in the proximal postvalvular tubular part of the conduit. Calcifications (n=11) or a fibroproliferative reaction (n=15) were rarely observed. Overall, there were no radiologic differences between the groups. On CTA, the smallest diameter of the allograft-conduits was significantly smaller than the diameter given on the cryopreservation protocol (SG=16+/-3 and C=17+/-3mm vs 25mm in both groups; P<0.001 each) whereas the diameter of the distal part of the allograft was not (SG=24+/-2, P=0.066, and C=25+/-3mm, P=0.82). CONCLUSIONS: Despite a significant shorter follow-up in the SynerGraft-group, no functional or radiologic differences were observed as compared to control-patients. The smallest diameter is located almost exclusively at the proximal level of allograft-conduits.

Immunological and echocardiographic evaluation of decellularized versus cryopreserved allografts during the Ross operation.

OBJECTIVE: Compare the immunological and echocardiographic data of decellularized versus cryopreserved allografts used for RVOT reconstruction during Ross operation. METHODS: From 16/01/03 thru 07/10/03, 20 Ross operations were performed using decellularized (n=11) or cryopreserved (n=9) allografts. Echocardiography was done at discharge, 1, 3, 6 and 12 months and annually thereafter. Samples for determination of antibodies against HLA class I and II were obtained preoperatively and at days 5, 10, 30, 90 and 180 postoperatively. These samples were tested by the ELISA method in LAT-M dishes (unspecific) for identification of circulating antibodies and the results expressed as mean sample values (Is=DO/cutoff). If positive, LAT-E (specific) was performed and PRA levels determined. RESULTS: There was no mortality. Cryopreserved allografts showed marked Is values elevations for class I and II antibodies which started at the first month and remained elevated up to 6 months. In contrast, of the patients receiving decellularized allografts, seven remained negative, two patients had only marginal elevation of class I antibodies and two patients showed abnormal elevations of PRA levels. This response happened earlier than in the cryopreserved group, starting on the 5th postoperative day and has returned to baseline levels in one case. Echocardiography showed mild, but significant, elevation of gradients in cryopreserved valves but none in the decellularized. CONCLUSIONS: Decellularized allografts had normal function up to 18 months and showed important reduction of the immunogenic response when compared to cryopreserved valves.

Immunological and histological evaluation of decellularized allograft in a pig model: comparison with cryopreserved allograft.

BACKGROUND AND AIM OF THE STUDY: The remodeling process of the decellularized allograft after implantation remains unclear. Herein, the hemodynamics, recellularization and immunological response of the decellularized allograft were evaluated at four weeks after implantation in a mini-pig model, and compared with a cryopreserved allograft. METHODS: Six porcine pulmonary allografts were harvested from mini-pigs, and cryopreserved for four weeks. In two pigs, the grafts were decellularized with Triton X solution, after which static reseeding of the valve surface was performed for 48 h with autologous endothelial cells harvested from a leg artery. Decellularization, but not reseeding, was carried out in two mini-pigs, and cryopreservation alone in two mini-pigs. Whilst under right heart bypass, the right ventricular outflow tract was replaced in six minipigs. The grafts were explanted after four weeks; analysis included direct pressure measurement, echocardiography, macroscopy, light microscopy with hematoxylin and eosin staining, and immunohistochemical studies to identify macrophages, T lymphocytes, and endothelial cells. RESULTS: Hemodynamically and macroscopically, there were no major differences between the three groups. In the cryopreservation-only group, immunohistochemistry showed an influx of macrophages, and T lymphocytes at the cusps. Endothelial cell coverage was found in the decellularized and decellularized + cell-seeded groups, but no macrophages and T lymphocytes were found at the cusps. CONCLUSION: Decellularization of the cryopreserved allograft may reduce the inflammatory response and improve its long-term durability.

Factors influencing late allograft valve failure.

Allograft valves are a valuable valve replacement substitute in the surgical management of heart valve disease. It remains the valve substitute of choice in the reconstruction of the right ventricular outflow tract in children with congenital heart disease and in the Ross procedure. However, its durability remains suboptimal, particularly in children. This article reviews the mechanisms and factors implicated in late allograft dysfunction, with a focus on the evidence for an immunological cause for allograft valve failure. Unravelling the mechanisms of allograft valve failure may allow modification of the allograft to improve its long-term durability.

Role of inflammation and ischemia after implantation of xenogeneic pulmonary valve conduits: histological evaluation after 6 to 12 months in sheep.

OBJECTIVE: Commercially available biological heart valve prostheses undergo degenerative changes, which finally lead to complete destruction. Here we evaluate the role of inflammation and ischemia after implantation of xenogeneic heart valve conduits (XPC) generated by novel concepts of tissue engineering. METHODS: Acellularized (a-)XPC and autologus re-seeded (s-)XPC were implanted into sheep. Samples were taken as follows: after acellularization (n = 2), after re-seeding (n = 2), 6 months (seeded/non-seeded: n = 3/5), 9 months (n = 2/5), and 12 months (n = 3/2) post implantation. Five native porcine conduits served as control. Using histological methods, samples were evaluated for pathological changes and existence/density of microvessels. RESULTS: Prior to implantation a-XPC were completely free of cells. Six months after implantation, leaflets and pulmonary arteries of s-XPC and a-XPC showed good endothelial surface coverage. Microvessel density within the myocardial cuffs and pulmonary vessel walls were comparable to control in all grafts. However, 6, 9 and 12 months after implantation pathological severe microvessel ingrowth, calcification and cellular infiltrations were observed on a-XPC and s-XPC valves, whereas myocardial cuffs and XPC-artery walls showed only mild degenerative alterations. CONCLUSIONS: Inflammatory reactions play a pivotal role in the degeneration of a-XPC and s-XPC. Thus, since ischemia seems to have little or no influence on this process, inflammation inductive factors should be the center of interest.