Decellularized Heart Valves Research Articles

After decellularization of porcine heart valves: no non‐Gal antigeneic epitopes detectable by non conditioned human sera

BackgroundPatients that undergo heart valve replacement surgery with glutaraldehyde fixated porcine grafts do not face acute rejection as recipients of native porcine organs and tissues do, however, grafts calcify over time and fail after 10–20 years in adults and 3–4 years in children. Responsible for hyperacute and acute rejection of unfixated tissue are preformed antibodies that recognize xenoantigens. Major targets are carbohydrates like the αGal epitope or Neu5Gc linked to proteins as well as to lipids on cells and extracellular matrix. To overcome the limited availability of decellularized allogeneic heart valve grafts, which show the ability to remodel and to grow by ingrowth of autologous cells, a characteristics lacking in glutaraldehyde fixated heart valve grafts, we want to develop a porcine derived decellularized matrix that is well tolerated in humans.MethodsThe generation of such optimal porcine heart valves matrices is dependent on a successful elimination of bound xenoantigens. To quantify the amount of xenoantigens present on decellularized matrix, we established an inhibition ELISA by exposing human IgG to crushed matrix and measuring unbound IgG in an ELISA set up. High levels of measured IgG means low levels of xenoantigens present on the decelllularized matrix. To enhance the specificity of IgG tested in the ELISA, human sera were perfused through porcine kidneys, followed by intensive washing and elution of bound xenoantibodies. Total amount of IgG and αGal specific antibodies was monitored to control the purification steps. Inhibition ELISA was performed with matrices derived from Landrace pigs, GalT‐KO pigs and humans. Binding of total IgG as well as αGal specific antibodies was assayed.ResultsThe amount of total IgG binding correlates with the amount of αGal specific antibodies binding to decellularized porcine matrix indicating the presence of remaining αGal epitopes on the porcine matrix after decellularization. However, decellularized GalT‐KO pig and decellularized human matrix showed no binding of αGal specific antibodies and comparable amounts of total IgG binding.ConclusionOur results indicate that only αGal epitopes present on decellularized porcine heart valve matrixes are recognized by preformed xenoantibodies while no other xenoantigens are detected. Therefore, further evaluation focusing on induction of immunogeneic reactions has to be conducted in an in vivo model like the humanized mouse utilizing decellularized heart valve material derived from GalT‐KO pigs.

Reduction of xeno‐antigens in porcine pulmonary heart valves by decellularization and glycolytic enzymatic treatment

BackgroundHeart valve replacement therapy with allogenic decellularized heart valve matrices allows an in vivo autologisation by repopulation of the foreign matrix with autologous cells. This method, which is superior based on experimental evidence to traditional heart valve replacements either of mechanical or glutaraldehyde fixated heart valve grafts, is current object of a multicentric international clinical trial (ESPOIR) granted by the EU. To circumvent the limited availability of allogenic heart valve matrices, we aim to the reduction of immunogenic epitops common to xenogeneic materials by high efficient decellularization paralleled by enzymatic treatment of the glycocalyx.MethodsPorcine pulmonary heart valves of landrace pigs harvested at the local slaughterhouse were subjected to detergent and proteinase based decellularization protocols widely described in the literature. In a second step decellularized matrices were exposed to α1‐3 Galactosidase or PNGase F digestion. Alterations in αGal epitope levels and glycocalyx structures were investigated by inhibitory ELISAs with anti‐αGal antibodies (M86) and IgGs of unconditioned human sera on crushed matrix, and histochemical stains on cryosections exploiting lectins as isolectin B4 (staining αGal), wheat germ agglutinin (staining GalNAc), and Datura stramonium lectin (staining GlcNAc). Equally treated GalT‐KO pig and human pulmonary artery tissues served as controls.ResultsInhibitory ELISA results show that all decellularization protocols reduced αGal epitope levels significantly, some down to <30% of native porcine tissue. Further reduction down to control levels was achieved by α1‐3 Galactosidase digestion. The absence of preformed non‐ αGal xenoantibodies in human IgG of unconditioned donors is evident by equal binding of human IgG to decellularized tissues of GalT‐KO and human origin.Histochemistry reveals that decellularization results in reduced Isolectin B4 (αGal) and WGA (GalNAc) staining, but not of DSL (GlcNAc) staining. Additional enzymatic treatment of decellularized matrix with PNGase F reduces binding of all lectins, whereas digestion with α1‐3 Galactosidase only affects Isolectin B4 staining (αGal).ConclusionThe reduction of αGal epitope levels is strongly dependent on the decellularization method. A further decrease can be achieved by enzymatic digestion of carbohydrates present on the decellularized matrix. Sugar structures of the glycocalyx are believed to be strongly immunogenic, therefore, their general removal by PNGase F digestion may produce non‐immunogenic heart valve matrices. However, the lack of preformed human non‐ αGal xenoantibodies in non conditioned sera requires the testing for immunogenicity in an in vivo system. Thus, in future decellularized and enzymatically treated GalT‐KO matrices will be tested in a humanized mouse model.

Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules

Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.

085 Freeze-dried cells and tissues

Cryopreservation and freeze drying are widely accepted as the preferred techniques for long-term storage of biological materials in medical applications. Whereas cryopreserved samples are typically stored in liquid nitrogen, dried samples can be stored at room temperature, which has clear advantages for banking and transport. Freeze-drying is widely used to preserve biomolecules and macromolecular assemblies, and has been implicated as a method to preserve cells and tissues in the dry state. Freeze-drying involves a freezing and a drying step both imposing stress on biomaterials. Freezing may cause irreversible structural changes in biomolecules due to ice crystal formation, osmotic dehydration, and mechanical forces. Drying causes removal of bound water surrounding biomolecules. Non-reducing disaccharides have been shown to stabilize biomolecules during drying by forming direct hydrogen bonding interactions and by forming a glassy state. Attempts to stabilize cells in the dry state have focused on disaccharides as main stabilizing compound. The major challenge in using disaccharides for drying of cells is to overcome the impermeability of cell membranes to disaccharides, to provide intracellular protection. Freeze-drying of tissues has not been widely explored. Decellularized tissues, however, can be freeze-dried and used as matrix for tissue regeneration. In this presentation, feasibility of freeze-drying for stabilization of cells and decellularized tissues in the dry state will be discussed. Various methods to incorporate disaccharides into mammalian cells will be highlighted including a novel method making use of freezing-induced membrane phase transitions. This is illustrated for platelets, red blood cells, and sperm. Furthermore, it is shown how disaccharides can be used for freeze-drying of decellularized heart valve scaffolds. Diffusion of protective agents in the scaffolds has been studied by Fourier transform infrared spectroscopy, and the effects of freeze-drying on biomechanical and ultrastructural properties are shown. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. wolkers@imp.uni-hannover.de

The Effect of Heparin-VEGF Multilayer on the Biocompatibility of Decellularized Aortic Valve with Platelet and Endothelial Progenitor Cells

The application of polyelectrolyte multilayer films is a new, versatile approach to surface modification of decellularized tissue, which has the potential to greatly enhance the functionality of engineered tissue constructs derived from decellularized organs. In the present study, we test the hypothesis that Heparin- vascular endothelial growth factor (VEGF) multilayer film can not only act as an antithrombotic coating reagent, but also induce proliferation of endothelial progenitor cells (EPCs) on the decellularized aortic heart valve. SEM demonstrated the adhesion and geometric deformation of platelets. The quantitative assay of platelet activation was determined by measuring the production of soluble P-selectin. Binding and subsequent release of heparin and VEGF from valve leaflets were assessed qualitatively by laser confocal scanning microscopy and quantitatively by ELISA methods. Human blood derived EPCs were cultured and the adhesion and growth of EPCs on the surface modified valvular scaffolds were assessed. The results showed that Heparin-VEGF multilayer film improved decellularized valve haemocompatibility with respect to a substantial reduction of platelet adhesion. Release of VEGF from the decellularized heart valve leaflets at physiological conditions was sustained over 5 days. In vitro biological tests demonstrated that EPCs achieved better adhesion, proliferation and migration on the coatings with Heparin-VEGF multilayer film. Combined, these results indicate that Heparin-VEGF multilayer film could be used to cover the decellularized porcine aortic valve to decrease platelet adhesion while exhibiting excellent EPCs biocompatibility.

Xenogeneic decellularized heart valve matrices in the sheep model: A histo-morphologic analysis of a nine-month follow up study

Objectives: Allogenic decellularized heart valves demonstrate superior durability compared to commercial available bioprostetic grafts. In comparison, xenogenic decellularized grafts, showed severe degeneration in vivo. The aim of our study was the generation of alternative xenogenic decellularized grafts to overcome the limited availability of allogeneic grafts by an improved decellularization protocol.

Simvastatin treatment does not reduce the in vivo degeneration of decellularized heart valve prostheses

Objectives: All present biological heart valve prostheses are prone to progressive in vivo degeneration, which can be partially impaired by decellularization. According to in vitro reports, the administration of statins may provide an additional beneficial effect, whereas clinical trials have failed to prove a positive in vivo result.

Heart valve transplantation: the urgent need for non‐immunogenic porcine heart valve matrices

Heart valve replacement is a life‐saving procedure conducted worldwide approximately 300 000 times per year. Used mechanical devices and bioprosthetic valves, however, exhibit severe drawbacks. They either request for lifelong anticoagulation therapy, or display an early failure due to calcification and stenosis. Furthermore, their use in pediatric surgery requests reoperations since these prosthesis lack the ability to grow. An optimal heart valve substitute that exhibits unlimited durability, perfect hemodynamics (no need for anticoagulation) and the capacity to grow has been identified by implantations of decellularized allogenic heart valve matrices. Such grafts are revitalized in vivo by cells of the recipient, which form a functional endothelium and a living interstitium. However, the remaining obstacle is the limited availability of human donor heart valves. Until now, xenogenic substitutes (porcine origin) failed due to acute or chronic rejection when implanted into humans as well as in the sheep model. Thus, we aim to generate a porcine derived decellularized matrix, free of immunogenic epitopes, for endogenous tissue regeneration and tissue engineering.In a first step, we developed analytical tests to quantify residual α(1,3)Gal epitopes, known to be responsible for hyper‐acute rejection reactions, and other xenoantigens detected by natural human anti‐pig antibodies, which are present on matrices treated by our standard decellularization protocol. Since extracellular matrix is insoluble with employed the inhibitory ELISA technique using the commercial available anti‐αGal antibody M86 and natural anti‐pig antibodies isolated from the serum of healthy volunteers. In brief, porcine kidneys were perfused with human plasma. Unbound antibodies were washed away and bound xenoantibodies were eluted using 3.5 M NaSCN. After dialysis against PBS antibodies were used to perform the inhibitory ELISA as follows. Native as well as decellularized heart valve tissue was crushed in liquid nitrogen and incubated with a defined amount of antibodies. Unbound antibodies were separated from matrix bound antibodies by centrifugation and quantified. M86 ELISA was performed using αGal‐BSA (Dextra) as solid antigen and HRP conjugated goat anti‐mouse IgM antibody as detection antibody. In the case of natural human anti‐pig antibodies, the total amount of human IgG was quantified using a human IgG ELISA (Mabtech).In our experiments comparing native and decellularized porcine heart valves, we found that standard decellularization removes already 70% of the αGal epitopes. In heart valves from α1,3‐galactosyltransferase KO (GalT‐KO) pigs the binding of M86 antibody is comparable to the extent as binding of M86 is found in human pulmonary artery tissue. In respect to general xenoantigens reduced binding of xenoantibodies after decellularization was detected. However, human as well as GalT‐KO pig derived tissue bind less xenoantibodies as tissue derived from normal pigs. Comparing only GalT‐KO and human tissue, the human tissue binds less xenoantibodies indicating the existence of non‐Gal antigens on GalT‐KO tissue.In conclusion, we found strong evidence that xenoantigens including αGal and non‐Gal xenoantigens are not only present on cells and cell debris but also on decellularized heart valve matrixes that are in fact insoluble extra cellular matrix proteins. Thus, decellularization causes a reduction of the antigenicity, however, the degree of this removal might be dependent on the decellularization protocol. Modification of the current decellularization protocol as well as enzymatic treatment against glycocalyx structures, which are believed to be hard candidates as xenoantigens may result in an additional reduction.

Platelet gel supernatant as a potential tool to repopulate acellular heart valves

The aim of this study was to repopulate decellularized heart valve matrices with ovine mesenchymal stem cells (oMSCs) by the use of platelet gel (PG) supernatant, a storage vehicle for growth factors. oMSCs were exposed to different concentrations of PG-released supernatant and cell proliferation was evaluated using the MTS assay. oMSC motility and invasiveness were assayed using a Boyden chamber. A quantitative sandwich enzyme immunoassay was used to examine amounts of bFGF and TGF-β1 in the PG supernatant. Repopulation of acellular heart valve matrices was stimulated by seeding matrices with oMSCs supplemented with the PG supernatant. The most significant increase in proliferation induced by PG supernatant appeared at 1 × 10(5) plts/ml concentration. Higher concentrations evoked reduction of the stimulatory process. oMSC motility was most significantly stimulated at 1 × 10(6) plts/ml. Stimulating invasiveness of oMSCs needed the much higher concentration of 2 × 10(6) plts/ml. Immunoassays revealed that sheep PG supernatant contains 184.8 pg/ml bFGF and 60.5 ng/ml TGF-β1. Moreover, repopulation of acellular heart valve matrices was significantly enhanced by PG supernatant addition and resulted in upregulation of the myofibroblast marker alpha-smooth muscle actin. Growth factors released from platelets had the potential to induce cell repopulation in a heart valve tissue engineering procedure, through stimulation of mesenchymal stem-cell migration and invasion.

Physiological Performance of a Detergent Decellularized Heart Valve Implanted for 15 Months in Vietnamese Pigs: Surgical Procedure, Follow‐up, and Explant Inspection

This study features the longest experimental follow-up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X-100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals (n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL-treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham-operated group (n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL-treated valves during follow-up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue-engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self-supporting substitute.

The adhering junctions of valvular interstitial cells: molecular composition in fetal and adult hearts and the comings and goings of plakophilin-2 in situ, in cell culture and upon re-association with scaffolds

The interstitial cells of cardiac valves represent one of the most frequent cell types in the mammalian heart. In order to provide a cell and molecular biological basis for the growth of isolated valvular interstitial cells (VICs) in cell culture and for the use in re-implantation surgery we have examined VICs in situ and in culture, in fetal, postnatal and adult hearts, in re-associations with scaffolds of extracellular matrix (ECM) material and decellularized heart valves. In all four mammalian species examined (human, bovine, porcine and ovine), the typical mesenchymal-type cell-cell adherens junctions (AJs) connecting VICs appear as normal N-cadherin based puncta adhaerentia. Their molecular ensemble, however, changes under various growth conditions insofar as plakophilin-2 (Pkp2), known as a major cytoplasmic plaque component of epithelial desmosomes, is recruited to and integrated in the plaques of VIC-AJs as a major component under growth conditions characterized by enhanced proliferation, i.e., in fetal heart valves and in cell cultures. Upon re-seeding onto decellularized heart valves or in stages of growth in association with artificial scaffolds, Pkp2 is - for the most part - lost from the AJs. As Pkp2 has recently also been detected in AJs of cardiac myxomata and diverse other mesenchymal tumors, the demonstrated return to the normal Pkp2-negative state upon re-association with ECM scaffolds and decellularized heart valves may now provide a safe basis for the use of cultured VICs in valve replacement surgery. Even more surprising, this type of transient acquisition of Pkp2 has also been observed in distinct groups of endothelial cells of the endocardium, where it seems to correspond to the cell type ready for endothelial-mesenchymal transition (EMT).

Protein secondary structure and solvent accessibility of proteins in decellularized heart valve scaffolds

Decellularized heart valve scaffolds can be used for pulmonary heart valve replacements in heart surgery. In order to assess quality parameters of heart valves prior to implantation into patients various techniques can be applied, including ultrastructural evaluation and biomechanical testing. Fourier transform infrared spectroscopy (FTIR) was used here to study protein secondary structure and solvent accessibility in decellularized heart valve matrices. FTIR was used to study proteins in the three main types of structures in decellularized heart valves: leaflet, pulmonary artery wall and heart muscle. The amide-I band region was used to reveal differences in the overall protein secondary structure amongst tissues. Leaflet material contains a relatively high contribution of α-helical structures, whereas artery matrix contains a relatively high content of triple-helix and β-sheet structures. Solvent accessibility of tissue proteins was studied by exposing material to D2O. The exchange of hydrogen of the NH amide-II bond for deuterium originating from D2O was visible as an increase in the area of the amide-II band (1500– 1400 cm−1). The amide-I band also depicted changes in shape and position during incubation in D2O: a low frequency band at around 1625 cm−1 increased as a function of time. Remarkable differences in hydrogen-to-deuterium exchange kinetics and protein solvent accessibility were observed among the three types of heart valve matrices: proteins in artery matrix are highly accessible to solvent, whereas proteins in leaflet and heart muscle structures are relatively inaccessible to solvent and show slow hydrogen/deuterium exchange. The differences in protein solvent accessibility in the different types of matrices likely reflect differences in scaffold porosity.

Layer-by-Layer Incorporation of Growth Factors in Decellularized Aortic Heart Valve Leaflets

Aortic heart valve disease is a growing health problem and a tissue-engineered aortic heart valve could be a promising therapy. In this paper, decellularized porcine aortic heart valve leaflets are used as scaffolds and loaded with growth factor and heparin via layer-by-layer electrostatic deposition (LbL technique) with the final purpose to stimulate and control cellular processes. Binding and subsequent release of heparin and basic fibroblast growth factor (bFGF) from aortic valve leaflets were assessed qualitatively by immunohistochemistry and quantitatively by radioactive labeling methods. It was observed that the amount of heparin and bFGF bound to aortic heart valve leaflets was directly proportional to the concentration of heparin and bFGF in the incubation medium. Release of heparin and bFGF from the decellularized heart valve leaflets at physiological conditions was sustained over 4 days while preserving the biological activity of the released growth factor.

Crosslinking effect of Nordihydroguaiaretic acid (NDGA) on decellularized heart valve scaffold for tissue engineering

Decellularized heart valve scaffolds possess many desirable properties in valvular tissue engineering. However, their current applications were limited by short durability, easily structural dysfunction and immunological competence. Although crosslinking with chemical reagents, such as glutaraldehyde (GA), will enhance the mechanical properties, the low long-term stability and cytotoxicity of the scaffolds remains potential problem. Nordihydroguaiaretic acid (NDGA) is a bioactive natural product which is able to crosslink collagen and was proven to be effective in preparation of scaffold for tendon tissue engineering. In this paper, NDGA crosslinked decellularized heart valve scaffolds demonstrated higher tensile strength, enzymatic hydrolysis resistance and store stability than the non-crosslinked ones. Its mechanical properties and cytocompability were superior to that of GA-crosslinked heart valve matrix. Below the concentration of 10 microg/ml, NDGA has no visible cytotoxic effect on both endothelial cells (EC) and valvular interstitial cells (VIC) and its cytotoxicity is much less than that of GA. The LC50 (50% lethal concentration) of NDGA on ECs and VICs are 32.6 microg/ml and 47.5 microg/ml, respectively, while those of GA are almost 30 times higher than NDGA (P < 0.05). ECs can attach to and maintain normal morphology on the surface of NDGA-crosslinked valvular scaffolds but not GA-crosslinked ones. This study demonstrated that NDGA-crosslinking of decellularized valvular matrix is a promising approach for preparation of heart valve tissue engineering scaffolds.

Endothelialization of Heart Valve Matrix Using a Computer-Assisted Pulsatile Bioreactor

Although calcification remains as the main clinical concern associated with bioprosthetic heart valve replacement surgery, there is evidence that tissue deterioration leads to thromboembolism. In such instances, measures that prevent thrombosis may be beneficial. To minimize thrombosis, endothelialization of the valve surface before implantation has been proposed to facilitate coverage. In this study we aimed to define the optimal flow parameters for the endothelialization of decellularized heart valves using endothelial progenitor cell (EPC)-derived endothelial cells (ECs). We assessed the thrombogenic characteristics of the endothelialized heart valve surface using a bioreactor. EPC-derived ECs were seeded on decellularized porcine valve scaffolds. A computer-controlled bioreactor system was used to determine the optimal flow rates. Successful endothelialization was achieved by preconditioning the cell-seeded valves with stepwise increases in volume flow rate up to 2 L/min for 7 days. We show that decellularized valve scaffolds seeded with EPC-derived ECs improved the anti-thrombotic properties of the valve, whereas the scaffolds without ECs escalated the coagulation process. This study demonstrates that preconditioning of ECs seeded on valve matrices using a bioreactor system is necessary for achieving uniform endothelialization of valve scaffolds, which may reduce thrombotic activity after implantation in vivo.

Calcification Resistance of Procyanidin-Treated Decellularized Porcine Aortic Valves In Vivo

Conventional glutaraldehyde fixation is conducive to calcification of bioprosthetic tissues. The aim of this study was to test calcification resistance of procyanidin-treated decellularized porcine aortic valve in a rat model. We performed cross-linking of the decellularized porcine aortic heart valves by procyanidins and observed morphologic performance and examined the tensile strength and cross-linking index. Then we implanted subcutaneous samples of procyanidin cross-linking decellularized valves, glutaraldehyde cross-linking decellularized valves, and decellularized valves in rats. The retrieved grafts were stained with hematoxylin-eosin and von Kossa and were analyzed with scanning electron microscopy and x-ray energy dispersive spectroscopy (EDS) after 21 and 63 days. After decellularized and cross-linking pretreatment, the procyanidin cross-linked leaflets were soft and stretchable. In addition, the cellular components of the porcine aortic heart valve leaflets were completely removed, and the extracelluar matrix was maintained completely. Examination of tensile strength revealed a significantly higher tissue resistance to tension in procyanidin cross-linked tissue than in other tissues, including the glutaraldehyde group (P< .05), even though the extents of cross-linking of each group were roughly the same at approximately 90%. Histopathologic examination showed that the procyanidin cross-linked valve matrix had no significant calcification, and there were no calcium peaks in the EDS profile of procyanidin cross-linked samples in the 21-day and 63-day rat studies. This study demonstrated that procyanidin cross-linked decellularized heart valves can resist calcification to some extent.

Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells

Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA). EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells. EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

Decellularized Xenogenic Heart Valves Reveal Remodeling and Growth Potentialin Vivo

We have developed an advanced tissue processing technique on porcine pulmonary heart valves for pulmonary valve replacement and its initial clinical application during the autograft operation according to Ross. The novel concept consists of a cell-free matrix achieved by deoxycholic acid treatment that is repopulated by host cells in vivo. Molecular biology, radioligand binding, and electron microscopy consistently showed that these valves are almost free of cellular components. Animal experiments and clinical investigations revealed excellent hemodynamic properties of the valves, no need for antithrombotic therapy, and repopulation by host cells without any signs of calcification. In juvenile sheep the internal diameter of the implanted valves significantly increased in growing animals by approximately 10 mm. The repopulation of the decellularized heart valves was found not only in sheep but also in humans, which indicates that the underlying mechanisms, presumably repair mechanisms, might be common in mammals. If these findings can be confirmed by others, they will lead to new concepts in the field of cardiovascular tissue engineering that will eliminate the need for in vitro construction of autologous heart valves.

Bioprosthetic Valves and Laudable Inflammation?

In this issue of Circulation , Dr Manji and colleagues1 from the University of Alberta (Canada) report a series of animal experiments designed to test the mythic role of gluteraldehyde in preventing recognition of bioprosthetic heart valve antigenicity with subsequent rejection and failure. Gluteraldehyde–cross-linked xenograft tissues (initially porcine) have been used in the manufacturing of stented (and now stentless) heart valves since 1970. Original theories for the clinical efficacy of gluteraldehyde were based on its ability to irreversibly cross-link collagen and thus to increase mechanical strength and durability over fresh untanned cardiovascular tissues. Porcine valve leaflets, bovine pericardium, equine pericardium, and bovine jugular vein, among other structures, have been treated this way for clinical applications. Since its introduction, however, limitations and unexpected consequences of gluteraldehyde and similar chemical treatments have been recognized clinically. As pointed out by the authors, durability is quite variable, tending to be better in older patients whereas younger patients suffer early deterioration, calcification, and fibrocalcific failure.2,3 The good news has been that the failures tend to be progressive rather than catastrophic, leading to semielective reoperations. Additionally, the xenograft bioprostheses have been extremely helpful in avoiding warfarin in the very young and the very old, reducing the risk of thromboembolism. The traditional explanation for the fibrocalcific degeneration of the nonvital gluteraldehyde-treated bioprosthetic heart valves has been a combination of physical and chemical effects leading to calcification and a fixation of the structural proteins that prevent protein recycling and renewal. Mechanical theories of fatigue-induced wear resulting in calcification have been proposed, especially along the flexion lines of the cusps. Others have proposed that the gluteraldehyde has serendipitously “masked” the antigenicity of xenograft proteins, retained cells, and cellular debris, thus prolonging the period to calcification. This well-designed series of studies buttresses other studies demonstrating that gluteraldehyde fixation …

Preclinical Testing of Tissue-Engineered Heart Valves Re-Endothelialized Under Simulated Physiological Conditions

The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-alpha-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-alpha-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.