Failure of decellularized porcine small intestinal submucosa as a heart valved conduit.

OBJECTIVE: Decellularized extracellular matrix made from porcine small intestinal submucosa, commercially available as CorMatrix (CorMatrix Cardiovascular, Inc, Roswell, Ga) is used off-label to reconstruct heart valves. Recently, surgeons experienced failures and words of caution were raised. The aim of this study was to evaluate decellularized porcine small intestinal submucosa as right-sided heart valved conduit in a xenogeneic animal model. METHODS: A pulmonary valve replacement was performed with custom-made valved conduits in 10 lambs and 10 sheep (1 month [3 lambs and 3 sheep], 3 months [3 lambs and 3 sheep], 6 months [4 lambs and 4 sheep]). Valve function was assessed after implantation and before the animal was put to death. Explanted conduits were inspected macroscopically and analyzed using immunohistochemistry and scanning electron microscopy. They also underwent mechanical testing and testing for biochemical composition. RESULTS: All valved conduits were successfully implanted. Five sheep and 2 lambs died due to congestive heart failure within 2 months after surgery. In the animals that died, the valve leaflets were thickened with signs of inflammation (endocarditis in 4). Five sheep and 8 lambs (1 month: 6 out of 6 animals, 3 months: 4 out of 6 animals, 6 months: 3 out of 8 animals) survived planned follow-up. At the time they were put to death, 5 lambs had significant pulmonary stenosis and 1 sheep showed severe regurgitation. A well-functioning valve was seen in 4 sheep and 3 lambs for up to 3 months. These leaflets showed limited signs of remodeling. CONCLUSIONS: Fifty percent of sheep and 20% of lambs died due to valve failure before the planned follow-up period was complete. A well-functioning valve was seen in 35% of animals, albeit with limited signs of tissue remodeling at ≤3 months after implantation. Further analysis is needed to understand the disturbing dichotomous outcome before clinical application can be advised.

In vivo functional assessment of a novel degradable metal and elastomeric scaffold-based tissue engineered heart valve.

OBJECTIVE: Ideal heart valve solutions aim to provide thrombosis-free durability. A scaffold-based polycarbonate urethane urea tissue-engineered heart valve designed to mimic native valve microstructure and function was used. This study examined the acute in vivo function of a stented tissue-engineered heart valve in a porcine model. METHODS: Trileaflet valves were fabricated by electrospinning polycarbonate urethane urea using double component fiber deposition. The tissue-engineered heart valve was mounted on an AZ31 magnesium alloy biodegradable stent frame. Five 80-kg Yorkshire pigs underwent open tissue-engineered heart valve implantation on cardiopulmonary bypass in the pulmonary position. Tissue-engineered heart valve function was echocardiographically evaluated immediately postimplant and at planned study end points at 1, 4, 8, and 12 hours. Explanted valves underwent biaxial mechanical testing and scanning electron microscopy for ultrastructural analysis and thrombosis detection. RESULTS: All 5 animals underwent successful valve implantation. All were weaned from cardiopulmonary bypass, closed, and recovered until harvest study end point except 1 animal that was found to have congenital tricuspid valve dysplasia and that was euthanized postimplant. All 5 cases revealed postcardiopulmonary bypass normal leaflet function, no regurgitation, and an average peak velocity of 2 m/s, unchanged at end point. All tissue-engineered heart valve leaflets retained microstructural architecture with no platelet activation or thrombosis by scanning electron microscopy. There was microscopic evidence of fibrin deposition on 2 of 5 stent frames, not on the tissue-engineered heart valve. Biaxial stress examination revealed retained postimplant mechanics of tissue-engineered heart valve fibers without functional or ultrastructural degradation. CONCLUSIONS: A biodegradable elastomeric heart valve scaffold for in situ tissue-engineered leaflet replacement is acutely functional and devoid of leaflet microthrombosis.

On the bending properties of porcine mitral, tricuspid, aortic, and pulmonary valve leaflets.

The atrioventricular valve leaflets (mitral and tricuspid) are different from the semilunar valve leaflets (aortic and pulmonary) in layered structure, ultrastructural constitution and organization, and leaflet thickness. These differences warrant a comparative look at the bending properties of the four types of leaflets. We found that the moment-curvature relationships in atrioventricular valves were stiffer than in semilunar valves, and the moment-curvature relationships of the left-side valve leaflets were stiffer than their morphological analog of the right side. These trends were supported by the moment-curvature curves and the flexural rigidity analysis (EI value decreased from mitral, tricuspid, aortic, to pulmonary leaflets). However, after taking away the geometric effect (moment of inertia I), the instantaneous effective bending modulus E showed a reversed trend. The overall trend of flexural rigidity (EI: mitral > tricuspid > aortic > pulmonary) might be correlated with the thickness variations among the four types of leaflets (thickness: mitral > tricuspid > aortic > pulmonary). The overall trend of the instantaneous effective bending modulus (E: mitral < tricuspid < aortic < pulmonary) might be correlated to the layered fibrous ultrastructures of the four types of leaflets, of which the fibers in mitral and tricuspid leaflets were less aligned, and the fibers in aortic and pulmonary leaflets were highly aligned. We also found that, for all types of leaflets, moment-curvature relationships are stiffer in against-curvature (AC) bending than in with-curvature bending (WC), which implies that leaflets tend to flex toward their natural curvature and comply with blood flow. Lastly, we observed that the leaflets were stiffer in circumferential bending compared with radial bending, likely reflecting the physiological motion of the leaflets, i.e., more bending moment and movement were experienced in radial direction than circumferential direction.

Successful matrix guided tissue regeneration of decellularized pulmonary heart valve allografts in elderly sheep.

In vivo repopulation of decellularized allografts with recipient cells leads to a positive remodeling of the graft matrix in juvenile sheep. In light of the increasing number of heart valve replacements among older patients (>65 years), this study focused on the potential for matrix-guided tissue regeneration in elderly sheep. Pulmonary valve replacement was performed in seven-year old sheep using decellularized (DV), decellularized and CCN1-coated (RV), or decellularized and in vitro reendothelialized pulmonary allografts (REV) (n=6, each group). CCN1 coating was applied to support re-endothelialization. In vitro re-endothelialization was conducted with endothelial-like cells derived from peripheral blood. Echocardiograms of all grafts showed adequate graft function after implantation and at explantation 3 or 6 months later. All explants were macroscopically free of thrombi at explantation, and revealed repopulation of the allografts on the adventitial side of valvular walls and proximal in the cusps. Engrafted cells expressed vimentin, sm α-actin, and myosin heavy chain 2, while luminal cell lining was positive for vWF and eNOS. Cellular repopulation of valvular matrix demonstrates the capacity for matrix-guided regeneration even in elderly sheep but is not improved by in vitro endothelialization, confirming the suitability of decellularized matrix for heart valve replacement in older individuals.

Impact of γ-irradiation on extracellular matrix of porcine pulmonary valves.

BACKGROUND: The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components. METHODS: Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry. RESULTS: Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry. CONCLUSION: γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.

Effects of cryopreservation and/or decellularization on extracellular matrix of porcine valves.

The objective of this study was to evaluate the morphology of decellularized and/or cryopreserved porcine pulmonary valves, to determine a solution capable of completely remove the cells without damaging the extracellular matrix. Porcine pulmonary valves were incubated for 24 hs in sodium deoxicholate 1% or sodium dodecyl sulfate 0.1 and 0.3%, with or without associated cryopreservation. Evaluation was done with optical microscopy (Hematoxilin-Eosin, Acetic Orcein and Gomori) and with morphometric analysis. The effectiveness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.

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.

Efeito da criopreservação e/ou da descelularização na matriz extracelular de condutos valvados porcinos / Effects of cryopreservation and/or decellularization on extracellular matrix of porcine valves

O objetivo desse estudo foi avaliar a morfologia de valvas pulmonares porcinas criopreservadas e/ou descelularizadas para determinar uma solução que remova as células, sem promover danos à matriz extracelular. Valvas pulmonares porcinas foram incubadas por 24h em soluções de deoxicolato de sódio 1% e de dodecil sulfato de sódio 0,1% e 0,3%, com ou sem criopreservação adicional. A avaliação foi feita com microscopia óptica (hematoxilina eosina, orceína acética ou Gomori) e por morfometria. A efetividade das soluções foi variável, mas os melhores resultados foram obtidos com enxertos frescos descelularizados com dodecil sulfato de sódio 0,1%.

The objective of this study was to evaluate the morphology of decelluarized and/or cryopreserved porcine pulmonary valves, to determine a solution capable of completely remove the cells without damaging the extracellular matrix. Porcine pulmonary valves were incubated for 24 hs in sodium deoxicholate 1% or sodium dodecyl sulfate 0.1 and 0.3%, with or without associated cryopreservation. Evaluation was done with optical microscopy (Hematoxilin-Eosin, Acetic Orcein and Gomori) and with morphometric analysis. The effectiviness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.

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.

Measurements of the diameters of the great arteries and semi-lunar valves of chick and mouse embryos.

The great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 mum x 2 mum x 2 mum and 3 mum x 3 mum x 3 mum) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.

Flow-dependent re-endothelialization of tissue-engineered heart valves.

BACKGROUND AND AIM OF THE STUDY: The generation of a functional, non-immunogenic, non-thrombogenic construct based on autologous cells seeded onto an acellular extracellular matrix is the major goal in heart valve tissue engineering. The study aim was to identify culturing conditions required to achieve a stable endothelial cell (EC) layer under physiological flow conditions, a prerequisite for the requested characteristics. METHODS: Eleven detergent-decellularized ovine pulmonary valves (PVs) were statically reseeded in special bioreactors with ovine venous ECs (1.2x10(7) cells per valve). The dynamic culture was started with 0.1 l/min in eight bioreactors. In four bioreactors the initial flow rate was slow, and increased by 0.1 l/min twice each day until maximal flow was 0.5 l/min and pulsation rate (PR) was 20 beats/min; in four other bioreactors the flow was increased by 0.7 l/min/day and reached 2.0 l/min with a PR of 50 beats/min. The mean system pressure was maintained at 25 +/- 5 mmHg during the whole dynamic cultivation in both groups. Three statically reseeded valves served as baseline. After achieving maximal appointed flow, the valves were investigated morphologically (hematoxylin and eosin staining, electron microscopy, von Willebrand factor, endothelial nitric oxide synthase immunostaining) and for metabolic activity (MTS assay). RESULTS: After reseeding, the endothelium appeared on the luminal surface of the PV as a non-confluent monolayer. Moderate pulsatile circulation induced complete confluence of EC monolayers on both cusp sides and the pulmonary wall. A high flow rate led to a partial loss of cells on the wall surface with large defects, and to complete cell wash-off from cusps. Cusp and wall metabolic activity was significantly higher after culture under moderate flow (p < 0.001) than in other groups, and was absent from cusps in high-flow bioreactors. CONCLUSION: Moderate pulsatile flow with small increments stimulates EC proliferation on the ovine decellularized valve scaffold. A rapid increase in bioreactor flow to physiological levels leads to significant damage of the reseeded endothelium and complete loss of cusp cellularity. This effect may be responsible for the in-vivo failure of static reseeded tissue-engineered valves exposed to physiological hemodynamic forces.

Decellularization does not eliminate thrombogenicity and inflammatory stimulation in tissue-engineered porcine heart valves.

BACKGROUND AND AIM OF THE STUDY: In tissue engineering of heart valves using decellularized xenogenic valves, it has been suggested that cell elimination would result in a biologically inert matrix. The aim of this in-vitro investigation was to evaluate different decellularization methods in regard to the completeness of cell removal, inflammatory response, and thrombocyte activation. METHODS: Decellularized porcine Synergraft valves were compared with porcine pulmonary conduits decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630 and ribonuclease. Completeness of decellularization was evaluated with staining for nuclei and alpha-Gal epitope. Decellularized heart valves with and without seeding with endothelial cells (ECs) were incubated with human platelet-rich plasma and stained for CD41 and PAC-1 to evaluate thrombocyte activation. Samples were processed for laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Migration of human monocytic cells towards extracted valve proteins was tested. RESULTS: In contrast to the Synergraft, complete cell removal and elimination of the alpha-gal epitope was achieved with the new decellularization method. Numerous adherent and activated platelets were found on the decellularized matrix. This was inhibited by seeding with ECs. Even in completely cell-free valve tissue extracellular matrix proteins attracted human monocytic cells as in early inflammation, depending on whether porcine or human tissue was used. CONCLUSION: Important differences were found in the decellularization efficacy of treatment methods. However, even complete elimination of cells and their remnants did not result in a biologically inert matrix. The decellularized porcine heart valve matrix has the potential to attract inflammatory cells and to induce platelet activation. These findings suggest that it will be important to control the different inflammation-stimulating factors if porcine tissues are to be used successfully in tissue engineering.

Impact of cryopreservation on extracellular matrix structures of heart valve leaflets.

BACKGROUND: Transplantation of cryopreserved allografts represents a well-established valve replacement option. Despite their clinical use for more than 40 years, the integrity of the extracellular matrix (ECM) of these valves after thawing has not been determined. The purpose of this study was to investigate and compare ECM structures of fresh and cryopreserved porcine heart valve leaflets with special emphasis on the condition of collagenous and elastic fibers. METHODS: Pulmonary valves were excised from unprocessed porcine hearts under sterile conditions. After treatment with antibiotics, the valves were incubated in a cryoprotective solution, cryopreserved stepwise, and stored at -196 degrees C for 1 week. Two groups of heart valves (fresh untreated and thawed cryopreserved [each, n = 8]) were analyzed using biochemical (collagen, elastin, desmosine), histologic (hematoxylin-eosin, Movat-pentachrome, resorcin-fuchsin), and immunohistochemical (antibodies against collagen I, III, IV, and elastin) methods. Near-infrared femtosecond multiphoton laser scanning microscopy and second harmonic generation were used for high-resolution three-dimensional imaging of ECM structures. RESULTS: Biochemical testing demonstrated similar amounts of collagen and desmosine, but a minor loss of elastin in the cryopreserved specimens. Conventional histology revealed almost comparable cell and ECM formations in fresh and cryopreserved valve leaflets. In contrast, laser-induced autofluorescence imaging showed substantial ultrastructural deterioration and disintegration of most collagenous structures. Second harmonic generation was not inducible. CONCLUSIONS: Conventional cryopreservation of heart valves is accompanied by serious alterations and destruction of leaflet ECM structures, specifically demonstrated by multiphoton imaging. Further in-depth studies to clarify the impact of alternative cryopreservation techniques proposed for clinical use, such as vitrification, are crucial.

Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy.

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.

Multiphoton autofluorescence imaging of intratissue elastic fibers.

Multiphoton induced blue/green autofluorescence by near infrared femtosecond laser pulses has been used to selectively image intratissue elastic fibers in native and tissue engineered (TE) viable heart valves without any invasive tissue removal, embedding, fixation, and staining. Elastic fibers could be clearly distinguished from collagenous structures which emit ultraviolet/violet radiation when excited with intense ultrashort pulses due to second harmonic generation. Deep-tissue three-dimensional imaging of elastic fibers with submicron spatial resolution was performed by optical sectioning of heart valves using a multiphoton laser scanning microscope in connection with a tunable 80 MHz femtosecond laser source. The technology was used to diagnose extracellular matrix structures and cell resettlement of TE heart valves prior implantation. This novel non-invasive method opens the general possibility of high-resolution in situ imaging of elastic fibers, collagen structures and intracellular organelles in living intact tissues without staining.

Impact of processing on surface structure of human cardiac valve allografts.

Methods of processing and cryopreservation are believed to be the most important factors of long term clinical performance of biological heart valve prostheses. That is why we decided to cooperate in evaluating the impact of current AHV (allograft heartvalve) bank protocol on valve tissue morphology. AHV harvested from "heart-beating" cadaveric donors, considered as a fresh tissue, were compared with valve samples from non-heart beating donors, samples stored in saline, samples treated with antibiotic solution, and finally with cryopreserved valves, stored in liquid nitrogen for months. All samples were dissected, dried with hexamethyldisilazane (HMDS) method, gold-coated, studied and photographed in scanning electron microscope Tesla BS 301. Different superficial patterns were found on ventricular and vascular surfaces of "fresh" semilunar valves. We were able to detect early changes of endothelium after harvesting, denudation of endothelial covering during preservation with and without freezing. Our alternative method of drying samples by HMDS method proved to be suitable for thin membranes of human semilunar valves. Scanning electron microscopy seems to be helpful for morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. We believe that further confrontation of morphological investigation with other methods helps us to develop more suitable protocol of handling AHV in heart valve banking.

Comparison of biomechanical and structural properties between human aortic and pulmonary valve.

OBJECTIVE: Pulmonary valve autografts have been reported as clinically effective for replacement of diseased aortic valve (Ross procedure). Published data about pulmonary valve mechanical and structural suitability as a long-term substitute for aortic valve are limited. The aim of this study was to compare aortic and pulmonary valve properties. METHODS: Experimental studies of biomechanical properties and structure of aortic and pulmonary valves were carried out on pathologically unchanged human heart valves, collected from 11 cadaveric hearts. Biomechanical properties of 84 specimens (all valve elements: cusps, fibrous ring, commissures, sinotubular junction, sinuses) were investigated using uniaxial tensile tests. Ultrastructure was studied using transmission and scanning electron microscopy. RESULTS: Ultimate stress in circumferential direction for pulmonary valve cusps is higher than for aortic valve (2.78+/-1.05 and 1.74+/-0.29 MPa, respectively). Ultimate stress in radial direction for pulmonary and aortic cusps is practically the same (0.29+/-0.06 and 0.32+/-0.04 MPa, respectively). In ultrastructural study, different layout and density in each construction element are determined. The aortic and pulmonary valves have common ultrastructural properties. CONCLUSIONS: Mechanical differences between aortic and pulmonary valve are minimal. Ultrastructural studies show that the aortic and pulmonary valves have similar structural elements and architecture. This investigation suggests that the pulmonary valve can be considered mechanically and structurally suitable for use as an aortic valve replacement.

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.