Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells.

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality.

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.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Engineering patient-specific valves using stem cells generated from skin biopsy specimens.

BACKGROUND: Pediatric patients requiring valve replacement will likely require reoperations due to a progressive deterioration of valve durability and limited repair and growth potential. To address these concerns, we sought to generate a biologically active pulmonary valve using patient-specific valvular cells and decellularized human pulmonary valves. METHODS: We generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblast cells. We then differentiated iPSCs to mesenchymal stem cells (iPCSs-MSCs) using culture conditions that favored an epithelial-to-mesenchymal transition. Next, decellularized human pulmonary heart valves were seeded with iPCS-MSCs using a combination of static and dynamic culture conditions and cultured up to 30 days. RESULTS: The iPSCs-MSCs displayed cluster of differentiation CD105 and CD90 expression exceeding 90% after four passages and could differentiate into osteocytes, chondrocytes, and adipocytes (n = 4). Consistent with an MSC phenotype, iPSCs-MSCs lacked expression of CD45 and CD34. Compared with bone marrow MSCs, iPSCs-MSC proliferated more readily by twofold but maintained a gene expression profile exceeding 80% identical to bone marrow MSCs. In repopulated pulmonary valves compared with decellularized pulmonary valves, immunohistochemistry demonstrated increased cellularity, α-smooth muscle actin expression, and increased presence of extracellular matrix components, such as proteoglycans and glycosaminoglycans, suggesting sustained cell function and maturation. CONCLUSIONS: Our results demonstrate the feasibility of constructing a biologically active human pulmonary valve using a sustainable and proliferative cell source. The bioactive pulmonary valve is expected to have advantages over existing valvular replacements, which will require further validation.

Eighteen-month outcome of pulmonary valve stent implantation by direct right ventricle puncture: an animal study.

OBJECTIVE: The purpose of this study was to investigate the feasibility and safety of pulmonary valve implantation via direct right ventricle puncture. METHODS: A standard thoracotomy and direct right ventricle puncture were performed in 8 healthy sheep to implant the pulmonary valve stents. Animals were followed up for 18 months. RESULTS: Three sheep died within the first 4 months after stent placement. The remaining 5 animals survived. After 18 months, examinations by color echocardiography, 64-slice computed tomography scan, and cardiac catheter showed an ideal position of each stent. The function of the pulmonary valves and hearts was not different compared with the preoperative conditions of the sheep. Anatomic examination revealed that the stent was covered by a layer of endothelial tissue with no stent fracture or valvular calcification. The histologic evaluation of the stent and surrounding tissue showed that the surface of the stent was smooth and covered by a complete layer of endothelial cells without obvious infiltration of inflammatory cells. The vascular wall was integrative without tear phenomenon in each layer of tissue. CONCLUSIONS: These results show that pulmonary valve stents can be implanted via direct right ventricle puncture. Further studies evaluating xenograft valve material and the effect of implantation in vivo are needed.

Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells.

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.

Operação de Ross com homoenxertos valvares decelularizados: resultados de médio prazo / Ross Operation with decelularized pulmonary allografts: medium-term results

OBJETIVO: Avaliar os resultados de médio prazo do uso de homoenxertos decelularizados na Operação de Ross. MÉTODOS: Entre janeiro de 2003 e fevereiro de 2007, 68 pacientes foram submetidos à Operação de Ross com homoenxertos decelularizados. Quarenta e oito pacientes eram do sexo masculino, com idade media de 30,3±11,2 anos. A decelularização foi feita com Ácido Deoxicólico (DOA), em 35 casos, e com Dodecilsulfato de Sódio (SDS), em 33. Para a comparação dos gradientes, foram selecionados 68 pacientes pareados pela idade, e que usaram homoenxertos criopreservados. Todos os pacientes realizaram ecocardiograma antes da alta e estão sendo avaliados anualmente. Oito pacientes tiveram controle por ressonância nuclear. Em dois pacientes reoperados, foi possível fazer análise histológica de um segmento do conduto pulmonar. RESULTADOS: Houve um (1,4 por cento) óbito imediato. Na evolução tardia, houve duas reoperações e um óbito. Os gradientes imediatos variaram de 4 a 29mmHg (m=10,3±5,5), e apresentaram elevação para 16,5± 12,2mmHg (min= 4, max = 45) aos 24 meses. Quando comparados com o grupo criopreservado, não houve diferenças significativas. Entretanto, houve tendência a melhores resultados em homoenxertos decelularizados com SDS após 12 meses de evolução. A análise histológica revelou reendotelização e repovoamento parcial da camada média com células autógenas. Não houve insuficiência pulmonar progressiva. Os dados de ressonância magnética demonstraram menor tendência de retração dos condutos decelularizados. CONCLUSÕES: O uso de homoenxertos decelularizados foi seguro, com bons resultados até quatro anos de evolução. Houve tendência a menores gradientes tardios nos homoenxertos decelularizados com SDS após 12 meses.

OBJECTIVE: To evaluate the medium-term results (4 years) of decelularized allografts during Ross Operation. METHODS: From January 2003 to February 2007, 68 patients underwent Ross Operation with decelularized allografts. Forty eight were male and the mean age was 30.3±11.2 years. Decelularization was done with deoxicolic acid (DOA) in 35 cases and with sodium dodecylsulfate (SDS) in 33. For comparison of the gradients, 68 patients with cryopreserved allografts and matched for age were selected. All patients had a control echo before hospital discharge and annually thereafter. In addition, eight patients had MRI studies. In two patients, samples of the conduit wall were analyzed by histological analysis. RESULTS: There was one (1.4 percent) early death. In the late follow-up, there were two reoperations for endocarditis and one late death. The early gradients varied between 4 29 mmHg (m= 10.3± 5.5mmHg) and exhibited an increase to 16.5±12.2 mmHg (min=4, max=45) at 24 months postoperatively. There were no significant differences when compared to the cryopreserved group. There was, however, a tendency towards lesser gradients in the SDS decelularized group after 12 months. Histological analysis revealed partial reendothelization and progressive repopulation of the tunica media with autogenous cells. There was no progressive pulmonary insufficiency. The MRI results showed a lesser tendency to shrinkage in the decelularized conduits. CONCLUSIONS: The use of decelularized allografts was safe and with good medium-term results up to 4 years. There was a tendency to lower late gradients in the SDS decelularized allografts after 12 months.

Ross Operation with decellularized pulmonary allografts: medium-term results.

OBJECTIVE: To evaluate the medium-term results (4 years) of decellularized allografts during Ross Operation. METHODS: From January 2003 to February 2007, 68 patients underwent Ross Operation with decellularized allografts. Forty eight were male and the mean age was 30.3+/-11.2 years. Decellularization was done with deoxicolic acid (DOA) in 35 cases and with sodium dodecylsulfate (SDS) in 33. For comparison of the gradients, 68 patients with cryopreserved allografts and matched for age were selected. All patients had a control echo before hospital discharge and annually thereafter. In addition, eight patients had MRI studies. In two patients, samples of the conduit wall were analyzed by histological analysis. RESULTS: There was one (1.4%) early death. In the late follow-up, there were two reoperations for endocarditis and one late death. The early gradients varied between 4 29 mmHg (m= 10.3+/- 5.5 mmHg) and exhibited an increase to 16.5+/-12.2 mmHg (min=4, max=45) at 24 months postoperatively. There were no significant differences when compared to the cryopreserved group. There was, however, a tendency towards lesser gradients in the SDS decellularized group after 12 months. Histological analysis revealed partial reendothelization and progressive repopulation of the tunica media with autogenous cells. There was no progressive pulmonary insufficiency. The MRI results showed a lesser tendency to shrinkage in the decellularized conduits. CONCLUSIONS: The use of decellularized allografts was safe and with good medium-term results up to 4 years. There was a tendency to lower late gradients in the SDS decellularized allografts after 12 months.

Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2.

In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-beta2, individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF- and TGFbeta2-induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGFbeta2, a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGFbeta2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGFbeta2 but were resistant to endothelial-inducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves.

Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells.

OBJECTIVE: We compared 3 different decellularization protocols in porcine heart valves for efficiency of complete cell removal and potential for recellularization. METHODS: Porcine aortic and pulmonary roots were treated with trypsin, sodium-dodecyl-sulphate, or a new method using 0.25% tert-octylphenyl-polyoxyethylen in combination with sodium-deoxycholate. After a subsequent ribonuclease digestion, specimens were seeded with in vitro expanded human saphenous vein endothelial cells and myofibroblasts. RESULTS: After treatment with trypsin and subsequent ribonuclease digestion, endothelial attachment took place; however, xenogenic cells were still visible within the matrix. Unexpectedly, when human cells were seeded onto specimens that had been decellularized with sodium-dodecyl-sulphate, the matrices were surrounded by nonviable endothelial cell fragments, indicating a toxic influence of the ionic detergent; 0.25% tert-octylphenyl-polyoxyethylen together with sodium-deoxycholate completely removed porcine cells and enabled host recellularization. CONCLUSION: Compared with trypsin and sodium-dodecyl-sulphate involving decellularization procedures, reported to be effective in cell removal and susceptible to recellularization with human cells, only the porcine matrix treated with a new detergent-based decellularization method using 0.25% tert-octylphenyl-polyoxyethylen/sodium-deoxycholate followed by nuclease digestion presented an excellent scaffold for recellularization with human cells.

Human pulmonary valve endothelial cells express functional adhesion molecules for leukocytes.

BACKGROUND AND AIM OF THE STUDY: Histopathological studies of rejected orthotopic heart transplants suggest that cardiac valve endothelium is spared the inflammatory cell infiltration and tissue damage that occurs in the myocardium. To test whether this apparent protection from leukocyte invasion might be an inherent feature of the valve endothelium, leukocyte adhesion molecule expression and function were analyzed in human pulmonary valve endothelial cells (HPVEC). Use of cultured HPVEC allowed delineation of the potential contribution of functional adhesion molecules from the contribution of hemodynamic forces exerted on the leaflet surface in vivo METHODS AND RESULTS: HPVEC express E-selectin, ICAM-1, and VCAM-1 in response to the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) similarly to other types of cultured human endothelial cells. In a static cell adhesion assay, E-selectin-mediated adhesion of HL-60 cells, a human promyelocytic leukemia cell line, and U937 cells, a human monocytic cell line, was determined in cells treated with TNF-alpha for 5 h. After 24 h of TNF-alpha, adhesion of U937 cells to HPVEC was mediated primarily by VCAM-1, consistent with the high expression of VCAM-1 and diminished expression of E-selectin at 24 h. CONCLUSION: These results demonstrate that HPVEC express functional leukocyte adhesion molecules in vitro and suggest that cardiac valve endothelium is competent to initiate leukocyte adhesion. Thus, other factors, such as the hemodynamic forces exerted on the valve, may contribute to the apparent protection from inflammatory cell infiltration in vivo.

NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells.

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.

Recellularization of heart valve grafts by a process of adaptive remodeling.

The objective of this study was to investigate if function and durability of connective tissue grafts stems from in vivo revascularization and recellularization. Viability is important for durable valve performance, demonstrated by pulmonary autografts. A pattern of in vivo recellularization occurs in xenogeneic or allogeneic heart valves decellularized prior to implantation, dictated by the tissue matrix and functional biomechanics. Porcine or sheep heart valves were decellularized with the SynerGraft antigen reduction process (a common treatment process to remove all histologically demonstrable leaflet cells), and implanted as pulmonary (n = 11) or aortic valve (n = 9) replacements in sheep. Sheep allograft pulmonary valves (n = 4) were implanted as pulmonary valve replacements. Recellularization was evaluated histologically after 3, 4, 5, 6, and 11 months, with cell phenotypes identified using specific antibodies. SynerGraft heart valves were progressively recellularized beginning with an initial cellular infiltrate, and subsequent repopulation with mature interstitial cells. This process occurs in the conduit and then in the leaflet, and is associated with revascularization of the graft. Functional, fully developed fibrocytes, actively synthesizing type I procollagen (antibody probe) were present within 3 months. As the process matured cell density and distribution became similar to native valve leaflets with localization of smooth muscle actin positive cells at the ventricularis/spongiosa interface. After 11 months, leaflet explants had no detectable inflammatory cells, were as much as 80% repopulated, and had a distribution of smooth muscle actin positive cells similar to that of the natural leaflet. SynerGraft- treated heart valve implants are repopulated by a process typical of adaptive remodeling following implantation. This antigen reduction treatment is the first successful tissue engineering effort obtaining an implant with mature recipient cells capable of matrix protein synthesis. Normal early valve function and durability is maintained.

Cell composition of the human pulmonary valve: a comparative study with the aortic valve--the VESALIO Project. Vitalitate Exornatum Succedaneum Aorticum labore Ingegnoso Obtinebitur.

BACKGROUND: Cell populations present in human semilunar valves have not been investigated thoroughly. The aim of this study was to characterize the cell phenotypes in pulmonary valve leaflets (PVL) in comparison with aortic (AVL) valve leaflets. METHODS: AVL and PVL were dissected from hearts (n = 4) harvested from transplanted patients. Leaflets were processed for immunocytochemistry analysis and Western blotting procedures using a panel of monoclonal antibodies specific for cytoskeletal/contractile antigens. RESULTS: The fibrosa and the ventricularis layers of AVL had a higher cellularity than PVL. In PVL and AVL most cells were reactive for vimentin and nonmuscle (NM) myosin, though vimentin-positive cells were more abundant in AVL than in PVL. Sparse cells positive to anti-smooth muscle (SM) alpha-actin, calponin, and anti-SM myosin antibodies were found only at the outer edge of fibrosa. In Western blotting, AVL and PVL extracts were shown to be equally reactive for vimentin, SM alpha-actin, and NM myosin, whereas both valves were negative for SM myosin and SM22. CONCLUSIONS: Three distinct cell phenotypes have been identified in both valves: fibroblasts, myofibroblasts, and fetal-type SM cells whose distribution is specifically related to the valve layers. Although PVL and AVL cell populations differ quantitatively, some minor qualitative differences exist for vimentin and NM myosin distribution. These data are essential for studies aimed at repopulating valve scaffolds by using tissue engineering technology.

Effect of storage temperature on cell viability in cryopreserved canine aortic, pulmonic, mitral, and tricuspid valve homografts.

We determined how long cryopreserved aortic, pulmonic, mitral, and tricuspid valve homografts could be stored in a deep freezer (-80 degrees C) without compromising fibroblast viability. Valves harvested from 20 anesthetized mongrel dogs were grouped into nonfrozen control, frozen and stored in liquid nitrogen (-196 degrees C), and frozen and stored in a deep freezer (-80 degrees C). Frozen groups were divided into subgroups and stored for 2, 4, 8, or 12 weeks. A leaflet of each valve was divided into three fragments, and fibroblast viability was analyzed by flow cytometry. Cell viability was defined as staining by fluorescent diacetate but not by propidium iodide. The viability of untreated control valves from all four sites was about 70%, decreasing to about 50% when treated with low doses of antibiotics. The viability of frozen valves stored in liquid nitrogen was about 45% without a significant difference among storage periods. The viability of valves frozen and stored in a deep freezer was significantly lower than for the liquid nitrogen group at 2 weeks for the mitral valve and at 4 weeks for other valves. These results suggest that homografts can be stored in a deep freezer for up to 2 weeks without deterioration.

Disproportionate enlargement of the pulmonary autograft in the aortic position in the growing pig.

PURPOSE: This study was aimed to demonstrate growth in the pulmonary autograft after transplantation to the aortic position. METHODS AND MATERIALS: In 20 piglets (weight 25.4 +/- 3.5 kg) (mean +/- standard deviation) a Ross operation was performed and in five piglets (weight 9.3 +/- 0.7 kg) (mean +/- standard deviation) the ascending aorta was replaced with a valveless pulmonary autograft. Animals were allowed to grow as much as possible. Postmortem explanted autografts were studied by direct measurements of the valve cusps in the Ross group and of the wall segments in the valveless autograft group. Measurements of the first group were compared with the values of a separate control group, and values of the second group were compared with values of samples taken at operation. RESULTS: In the Ross group, cuspal weight, height, and width increased significantly by comparison with body weight (p < or = 0.003). The rate of increase did not differ significantly from that of the control group with a native pulmonary valve. However, there was a rapid adaptation of the autograft valves resulting in a significantly higher mean cuspal weight, height, and width. In the valveless autograft group, wall circumference, thickness, and height increased significantly (p < or = 0.001). The circumference increased significantly more than that of the native pulmonary wall. Compared with the native aortic wall, the pulmonary autograft media showed retained pulmonary architecture on microscopic study. CONCLUSION: These data suggest that the dimensional increase of the pulmonary autograft in the aortic position in the growing pig is determined by growth and dilatation, that the valve mass increases more than that of the native pulmonary valve, and that the characteristic pulmonary microscopic architecture is retained.

Effect of cryopreservation on the presence of endothelial cells on human valve allografts.

Cryopreserved human allograft valves are useful in a variety of cardiac operations. The presence or absence of endothelial cells on allografts may be important in determining immunogenicity and ultimate graft longevity. The purpose of this study was to determine whether endothelial cells are present on cryopreserved human allografts. Portions of cryopreserved allografts (35 valve leaflets, 96 pieces of arterial wall) not used at operation were studied. For comparison, untreated tissues (44 valve leaflets, 46 pieces of arterial wall) were obtained from structurally normal hearts and lungs removed or inserted at the time of transplantation and from pathologic tissues obtained during operations for congenital heart defects. A monolayer of cells from the luminal surface of each specimen was harvested by means of a Hautchen preparation. The monolayer was stained with fluorescein isothiocyanate-labeled Ulex europaeus I, a lectin with strong affinity for human endothelium. Positive staining with fluorescein was considered to be evidence for the presence of human endothelium. Endothelial cells were observed on 21 of 131 (16%) cryopreserved allograft specimens and on 70 of 90 (78%) untreated tissues (p < 0.001). These results show that cryopreservation typically results in the loss of endothelium from aortic and pulmonary valve allografts. These findings may have important implications for the immunologic response of the host to allograft implantation.