ABSTRACT
Understanding cell geometric and mechanical properties is crucial to understanding how cells sense and respond to their local environment. Moreover, changes to cell mechanical properties under varied micro-environmental conditions can both influence and indicate fundamental changes to cell behavior. Atomic Force Microscopy (AFM) is a well established, powerful tool to capture geometric and mechanical properties of cells. We have previously demonstrated substantial functional and behavioral differences between aortic and pulmonary valve interstitial cells (VIC) using AFM and subsequent models of VIC mechanical response. In the present work, we extend these studies by demonstrating that to best interpret the spatially distributed AFM data, the use of spatial statistics is required. Spatial statistics includes formal techniques to analyze spatially distributed data, and has been used successfully in the analysis of geographic data. Thus, spatially mapped AFM studies of cell geometry and mechanics are analogous to more traditional forms of geospatial data. We are able to compare the spatial autocorrelation of stiffness in aortic and pulmonary valve interstitial cells, and more accurately capture cell geometry from height recordings. Specifically, we showed that pulmonary valve interstitial cells display higher levels of spatial autocorrelation of stiffness than aortic valve interstitial cells. This suggests that aortic VICs form different stress fiber structures than their pulmonary counterparts, in addition to being more highly expressed and stiffer on average. Thus, the addition of spatial statistics can contribute to our fundamental understanding of the differences between cell types. Moving forward, we anticipate that this work will be meaningful to enhance direct analysis of experimental data and for constructing high fidelity computational of VICs and other cell models.
Subject(s)
Aortic Valve/cytology , Cell Shape , Models, Statistical , Pulmonary Valve/cytology , Animals , Aortic Valve/physiology , Cells, Cultured , Microscopy, Atomic Force , Pulmonary Valve/physiology , Stress Fibers/ultrastructure , SwineABSTRACT
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.
Subject(s)
Aortic Valve/cytology , Multipotent Stem Cells/cytology , Pulmonary Valve/cytology , Tissue Scaffolds , Animals , Blood Cells/cytology , Cell Differentiation , Cells, Cultured , Endothelial Cells , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Gene Expression , Heart Valve Prosthesis Implantation , Mesenchymal Stem Cells/cytology , Multipotent Stem Cells/physiology , Nanostructures , Sus scrofa , Swine , Tissue Engineering/methodsABSTRACT
UNLABELLED: Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. STATEMENT OF SIGNIFICANCE: Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.
Subject(s)
Cryopreservation/methods , Pulmonary Valve/anatomy & histology , Pulmonary Valve/physiology , Animals , Biomechanical Phenomena , Cell Death , Materials Testing , Pulmonary Valve/cytology , Sus scrofa , Tensile StrengthABSTRACT
Interface biofunctionalization strategies try to enhance and control the interaction between implants and host organism. Decellularized extracellular matrix (dECM) is widely used as a platform for bioengineering of medical implants, having shown its suitability in a variety of preclinical as well as clinical models. In this study, specifically designed, custom-made synthetic peptides were used to functionalize dECM with different cell adhesive sequences (RGD, REDV, and YIGSR). Effects on in vitro endothelial cell adhesion and in vivo endothelialization were evaluated in standardized models using decellularized ovine pulmonary heart valve cusps (dPVCs) and decellularized aortic grafts (dAoGs), respectively. Contact angle measurements and fluorescent labeling of custom-made peptides showed successful functionalization of dPVCs and dAoGs. The functionalization of dPVCs with a combination of bioactive sequences significantly increased in vitro human umbilical vein endothelial cell adhesion compared to nonfunctionalized controls. In a functional rodent aortic transplantation model, fluorescent-labeled peptides on dAoGs were persistent up to 10 days in vivo under exposure to systemic circulation. Although there was a trend toward enhanced in vivo endothelialization of functionalized grafts compared to nonfunctionalized controls, there was no statistical significance and a large biological variability in both groups. Despite failing to show a clear biological effect in the used in vivo model system, our initial findings do suggest that endothelialization onto dECM may be modulated by customized interface biofunctionalization using the presented method. Since bioactive sequences within the dECM-synthetic peptide platform are easily interchangeable and combinable, further control of host cell proliferation, function, and differentiation seems to be feasible, possibly paving the way to a new generation of multifunctional dECM scaffolds for regenerative medicine.
Subject(s)
Cell Adhesion/physiology , Extracellular Matrix/chemistry , Heart Valves/cytology , Human Umbilical Vein Endothelial Cells/cytology , Pulmonary Valve/cytology , Tissue Engineering/methods , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , Male , Rats , Rats, Wistar , Sheep , Tissue Scaffolds/chemistryABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, CD/immunology , Bioprosthesis , Endothelial Progenitor Cells/immunology , Glycoproteins/immunology , Heart Valve Prosthesis , Peptides/immunology , Pulmonary Valve/cytology , AC133 Antigen , Cell Line , Cell Separation/methods , Cell-Free System/immunology , Cell-Free System/pathology , Humans , Pulmonary Valve/immunologyABSTRACT
Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.
Subject(s)
Graphite/chemistry , Platelet Activation/drug effects , Pulmonary Valve/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Cell Adhesion , Cell Cycle/drug effects , Graphite/pharmacology , Graphite/toxicity , Heart Valve Prosthesis , Humans , Materials Testing , Platelet Adhesiveness/drug effects , Tissue Engineering/instrumentationABSTRACT
Pediatric patients account for a small portion of the heart valve replacements performed, but a pediatric pulmonary valve replacement with growth potential remains an unmet clinical need. Herein we report the first tubular heart valve made from two decellularized, engineered tissue tubes attached with absorbable sutures, which can meet this need, in principle. Engineered tissue tubes were fabricated by allowing ovine dermal fibroblasts to replace a sacrificial fibrin gel with an aligned, cell-produced collagenous matrix, which was subsequently decellularized. Previously, these engineered tubes became extensively recellularized following implantation into the sheep femoral artery. Thus, a tubular valve made from these tubes may be amenable to recellularization and, ideally, somatic growth. The suture line pattern generated three equi-spaced leaflets in the inner tube, which collapsed inward when exposed to back pressure, per tubular valve design. Valve testing was performed in a pulse duplicator system equipped with a secondary flow loop to allow for root distention. All tissue-engineered valves exhibited full leaflet opening and closing, minimal regurgitation (<5%), and low systolic pressure gradients (<2.5 mmHg) under pulmonary conditions. Valve performance was maintained under various trans-root pressure gradients and no tissue damage was evident after 2 million cycles of fatigue testing.
Subject(s)
Bioprosthesis , Fibroblasts/transplantation , Heart Valve Prosthesis , Pulmonary Valve/cytology , Pulmonary Valve/growth & development , Tissue Engineering/methods , Animals , Cell-Free System , Cells, Cultured , Equipment Failure Analysis , Prosthesis Design , SheepABSTRACT
Decellularized allograft heart valves have been used as tissue-engineered heart valve (TEHV) scaffolds with promising results; however, little is known about the cellular mechanisms underlying TEHV neotissue formation. To better understand this phenomenon, we developed a murine model of decellularized pulmonary heart valve transplantation using a hemodynamically unloaded heart transplant model. Furthermore, because the hemodynamics of blood flow through a heart valve may influence morphology and subsequent function, we describe a modified loaded heterotopic heart transplant model that led to an increase in blood flow through the pulmonary valve. We report host cell infiltration and endothelialization of implanted decellularized pulmonary valves (dPV) and provide an experimental approach for the study of TEHVs using mouse models.
Subject(s)
Heart Valve Prosthesis , Heart Valves/physiology , Hemodynamics , Tissue Engineering/methods , Animals , Heart Transplantation , Heart Valves/diagnostic imaging , Heart Ventricles , Mice, Inbred C57BL , Models, Animal , Pressure , Pulmonary Valve/cytology , Pulmonary Valve/physiology , UltrasonographyABSTRACT
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.
Subject(s)
Bioprosthesis , Guided Tissue Regeneration , Heart Valve Prosthesis Implantation , Heart Valve Prosthesis , Pulmonary Valve/cytology , Pulmonary Valve/physiology , Allografts , Animals , Cells, Cultured , Endothelial Cells/cytology , Female , Guided Tissue Regeneration/methods , Heart Valve Prosthesis Implantation/methods , Pulmonary Valve/ultrastructure , Regeneration , Sheep , Tissue EngineeringABSTRACT
Decellularized heart valves can be used as starter matrix implants for heart valve replacement therapies in terms of guided tissue regeneration. Decellularized matrices ideally need to be long-term storable to assure off-the-shelf availability. Freeze-drying is an attractive preservation method, allowing storage at room temperature in a dried state. However, the two inherent processing steps, freezing and drying, can cause severe damage to extracellular matrix (ECM) proteins and the overall tissue histoarchitecture and thus impair biomechanical characteristics of resulting matrices. Freeze-drying therefore requires a lyoprotective agent that stabilizes endogenous structural proteins during both substeps and that forms a protective glassy state at room temperature. To estimate incubation times needed to infiltrate decellularized heart valves with the lyoprotectant sucrose, temperature-dependent diffusion studies were done using Fourier transform infrared spectroscopy. Glycerol, a cryoprotective agent, was studied for comparison. Diffusion of both protectants was found to exhibit Arrhenius behavior. The activation energies of sucrose and glycerol diffusion were found to be 15.9 and 37.7 kJ·mol(-1), respectively. It was estimated that 4 h of incubation at 37°C is sufficient to infiltrate heart valves with sucrose before freeze-drying. Application of a 5% sucrose solution was shown to stabilize acellular valve scaffolds during freeze-drying. Such freeze-dried tissues, however, displayed pores, which were attributed to ice crystal damage, whereas vacuum-dried scaffolds in comparison revealed no pores after drying and rehydration. Exposure to a hygroscopic sucrose solution (80%) before freeze-drying was shown to be an effective method to diminish pore formation in freeze-dried ECMs: matrix structures closely resembled those of control samples that were not freeze-dried. Heart valve matrices were shown to be in a glassy state after drying, suggesting that they can be stored at room temperature.
Subject(s)
Freeze Drying , Heart Valves/cytology , Sucrose/pharmacology , Animals , Calorimetry, Differential Scanning , Cryoprotective Agents/pharmacology , Diffusion , Glass , Heart Valves/drug effects , Pulmonary Valve/cytology , Pulmonary Valve/drug effects , Spectroscopy, Fourier Transform Infrared , Sus scrofa , Temperature , Tissue Scaffolds/chemistry , WaterABSTRACT
This study investigated cryopreserved pulmonary homograft (CPA) structural integrity after prolonged cold ischemic harvesting times in a juvenile sheep model. Three groups with different post-mortem cold ischemic harvesting times were studied, i.e. Group 1 (24 h, n = 10); group 2 (48 h, n = 10); group 3 (72 h, n = 10). In each group, 5 CPAs were studied in vitro after cryopreservation and thawing. The other 5 CPAs were implanted in juvenile sheep for a minimum of 180 days. Serology samples were obtained and echocardiography was performed before euthanasia. Hematoxylin and eosin (H&E), scanning electron microscopy (SEM), von Kossa, Picrosirius red, α-actin, immunohistochemistry [von Willebrand factor (vWF), CD4, CD31 and CD34] and calcium content analyses were performed on explanted CPAs. The in vitro and in vivo studies failed to demonstrate any change in tensile strength, Young's Modulus and thermal denaturation (Td) results between the groups. SEM demonstrated a reduction in endothelial cells (50 % at 24 h, 60.9 % at 48 h and 40.9 % at 72 h), but H&E could not demonstrate autolysis in any CPA in vitro. All cultures were negative. In the explanted groups, IgE, IgM and IgG results were inconclusive. Echocardiography demonstrated normal valve function in all groups. H&E and Picrosirius red staining confirmed tissue integrity. vWF, CD31 and CD34 staining confirmed a monolayer of endothelial cells in all explanted valves. Calcium content of explanted CPA leaflets was similar. This experimental study supports the concept of prolonging the cold ischemic harvesting time of cryopreserved homografts to reduce homograft shortage.
Subject(s)
Cold Ischemia/methods , Cryopreservation/methods , Graft Survival/physiology , Postmortem Changes , Pulmonary Valve/physiology , Pulmonary Valve/transplantation , Allografts , Animals , Elastic Modulus , Male , Pulmonary Valve/cytology , Sheep , Tensile StrengthABSTRACT
Non-linear and anisotropic heart valve leaflet tissue mechanics manifest principally from the stratification, orientation, and inhomogeneity of their collagenous microstructures. Disturbance of the native collagen fiber network has clear consequences for valve and leaflet tissue mechanics and presumably, by virtue of their intimate embedment, on the valvular interstitial cell stress-strain state and concomitant phenotype. In the current study, a set of virtual biaxial stretch experiments were conducted on porcine pulmonary valve leaflet tissue photomicrographs via an image-based finite element approach. Stress distribution evolution during diastolic valve closure was predicted at both the tissue and cellular levels. Orthotropic material properties consistent with distinct stages of diastolic loading were applied. Virtual experiments predicted tissue- and cellular-level stress fields, providing insight into how matrix-to-cell stress transfer may be influenced by the inhomogeneous collagen fiber architecture, tissue anisotropic material properties, and the cellular distribution within the leaflet tissue. To the best of the authors' knowledge, this is the first study reporting on the evolution of stress fields at both the tissue and cellular levels in valvular tissue and thus contributes toward refining our collective understanding of valvular tissue micromechanics while providing a computational tool enabling the further study of valvular cell-matrix interactions.
Subject(s)
Extracellular Matrix/metabolism , Finite Element Analysis , Pulmonary Valve/cytology , Stress, Mechanical , Animals , Anisotropy , Collagen/metabolism , Elasticity , Models, Cardiovascular , Pulmonary Valve/metabolism , SwineABSTRACT
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.
Subject(s)
Aortic Valve/chemistry , Calcium/analysis , Phosphorus/analysis , Pulmonary Valve/chemistry , Allografts , Animals , Aorta/chemistry , Aorta/cytology , Aortic Valve/cytology , Bioprosthesis , Calcinosis/prevention & control , Cryopreservation , DNA/isolation & purification , Heart Valve Prosthesis , Heterografts , Pulmonary Artery/chemistry , Pulmonary Artery/cytology , Pulmonary Valve/cytology , SheepABSTRACT
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.
Subject(s)
Pulmonary Valve/surgery , Skin/cytology , Stem Cell Transplantation , Tissue Engineering/methods , Biopsy , Cell Differentiation , Cells, Cultured , Humans , Pulmonary Valve/cytology , Skin/pathologyABSTRACT
Mathematical models can provide valuable information to assess and evaluate the mechanical behavior and remodeling of native tissue. A relevant example when studying collagen remodeling is the Ross procedure because it involves placing the pulmonary autograft in the more demanding aortic valve mechanical environment. The objective of this study was therefore to assess and evaluate the mechanical differences between the aortic valve and pulmonary valve and the remodeling that may occur in the pulmonary valve when placed in the aortic position. The results from biaxial tensile tests of pairs of human aortic and pulmonary valves were compared and used to determine the parameters of a structurally based constitutive model. Finite element analyzes were then performed to simulate the mechanical response of both valves to the aortic diastolic load. Additionally, remodeling laws were applied to assess the remodeling of the pulmonary valve leaflet to the new environment. The pulmonary valve showed to be more extensible and less anisotropic than the aortic valve. When exposed to aortic pressure, the pulmonary leaflet appeared to remodel by increasing its thickness and reorganizing its collagen fibers, rotating them toward the circumferential direction.
Subject(s)
Aorta/physiology , Mechanical Phenomena , Pulmonary Valve/physiology , Adolescent , Adult , Aorta/cytology , Aorta/metabolism , Biomechanical Phenomena , Child , Collagen/metabolism , Finite Element Analysis , Humans , Middle Aged , Pulmonary Valve/cytology , Pulmonary Valve/metabolismABSTRACT
Decellularized allografts offer potential as heart valve substitutes and scaffolds for cell seeding. The effects of decellularization on the quasi-static and time-dependent mechanical behavior of the pulmonary valve leaflet under biaxial loading conditions have not previously been reported in the literature. In the current study, the stress-strain, relaxation and creep behaviors of the ovine pulmonary valve leaflet were investigated under planar-biaxial loading conditions to determine the effects of decellularization and a novel post-decellularization extracellular matrix (ECM) conditioning process. As expected, decellularization resulted in increased stretch along the loading axes. A reduction in relaxation was observed following decellularization. This was accompanied by a reduction in glycosaminoglycan (GAG) content. Based on previous implant studies, these changes may be of little functional consequence in the short term; however, the long term effects of decreased relaxation and GAG content remain unknown. Some restoration of relaxation was observed following ECM conditioning, especially in the circumferential specimen direction, which may help mitigate any detrimental effects due to decellularization. Regardless of processing, creep under biaxial loading was negligible.
Subject(s)
Cryopreservation/methods , Extracellular Matrix/metabolism , Pulmonary Valve/cytology , Pulmonary Valve/physiology , Animals , Biomechanical Phenomena/physiology , Blood Vessel Prosthesis , Calorimetry, Differential Scanning , In Vitro Techniques , Materials Testing , Sheep , Time FactorsABSTRACT
In this study, structure and biomechanical properties of freeze-dried decellularized porcine pulmonary heart valves were investigated. Heart valves were dissected from porcine hearts. The tissues were decellularized and separated in three groups: (1) without lyoprotectant, (2) with 5% sucrose, and (3) with a mixture of 2.5% sucrose and 2.5% hydroxyl ethylene starch (HES), and then underwent freeze-drying. Freeze-drying in the absence of lyoprotectants caused an overall more disintegrated appearance of the histological architecture of the porcine valves, especially between the fibrosa and the ventricularis layers. Freeze-dried tissues with lyoprotectants have a looser network of collagen and elastic fibers with bigger pore sizes. Tissue freeze-dried in the absence of lyoprotecants had the largest pore sizes, whereas the tissue freeze-dried in the presence of protectants showed pores of intermediate sizes between the decellularized tissue and the unprotected freeze-dried samples. Tissue freeze-dried with sucrose alone displayed less porosity than tissue freeze-dried with the sucrose/HES mixture, whereas no significant differences in biomechanical properties were observed. Decellularization decreased the elastic modulus of artery tissue. The elastic modulus of freeze-dried tissue without protectants resembled that of decellularized tissue. The elastic modulus values of freeze-dried tissue stabilized by lyoprotectants were greater than those of decellularized tissue, but similar to those of native tissue.
Subject(s)
Freeze Drying/methods , Heart Valves/cytology , Heart Valves/physiology , Pulmonary Valve/cytology , Pulmonary Valve/physiology , Tissue Engineering/methods , Tissue Scaffolds , Animals , Biomechanical Phenomena , Elastic Modulus , Porosity , SwineABSTRACT
BACKGROUND: This study examines in vitro seeding of decellularized human pulmonary valves (hPVs) with human valve interstitial cells (hVICs) isolated from unrelated donor aortic valve leaflets. An assay was developed to assess seeding using precut uniform sized biopsies from whole hPVs for sequential evaluation of seeding efficiency, proliferation, and migration. MATERIALS AND METHODS: Scaffolds for seeding were created from decellularized hPVs using a reciprocating osmolality, double detergent, enzyme, multiple solvent protocol. hVICs seeded decellularized leaflet and sinus wall scaffolds were incubated in either static or cyclic pressure bioreactors. Low, medium, and high initial cell seeding "dosing" densities were assayed at subsequent three time points, using eight replicates each (n = 576 biopsies including manufactured scaffold controls). Metabolically viable seeded cells were quantified by MTT assay. Histology defined cell locations and morphology. RESULTS: After 24 h of static seeding with 2.5 × 10(5) cells (medium dose), 100 ± 13 cells/mm(2) (2.5%) attached to leaflets, compared with 193 ± 21 cells/mm(2) (8%) for sinuses. Subsequent 4 d in static culture yielded 894 ± 84 and 838 ± 50 cells/mm(2)versus pulsatile culture yielding 80 ± 12 and 79 ± 12 cells/mm(2) for leaflet and sinus, respectively. However, 76.0% ± 12.2% of cells in leaflets in the pulsatile bioreactor were subsurface as compared to 21.4% ± 3.9% in statically cultured leaflets (P < 0.001). CONCLUSION: Different seeding modes suggest a tradeoff between surface proliferation resulting in higher absolute cell numbers for static seeding versus fewer cells in a cyclic pressure bioreactor but with a greater percentage having migrated into the matrix. The medium seeding dose determined to be optimal is actually feasible for tissue engineering heart valves, and can be achieved by fairly traditional cell amplification methods.
Subject(s)
Pulmonary Valve/cytology , Tissue Engineering/methods , Cell Count , Cell Proliferation , HumansABSTRACT
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.
Subject(s)
Heart Valve Prosthesis Implantation/methods , Pulmonary Valve/surgery , Animals , Cardiac Catheterization , Cell Proliferation , Echocardiography, Doppler, Color , Endothelial Cells/pathology , Feasibility Studies , Female , Heart Valve Prosthesis , Heart Valve Prosthesis Implantation/instrumentation , Heart Ventricles , Male , Prosthesis Design , Pulmonary Valve/cytology , Pulmonary Valve/diagnostic imaging , Punctures , Sheep , Stents , Thoracotomy , Time Factors , Tomography, X-Ray ComputedABSTRACT
There is a significant gap in our knowledge of engineered heart valve tissue (EHVT) development regarding detailed three-dimensional (3D) tissue formation and remodeling from the point of in vitro culturing to full in vivo function. As a step toward understanding the complexities of EHVT formation and remodeling, a novel serial confocal microscopy technique was employed to obtain 3D microstructural information of pre-implant (PRI) and post-implant for 12 weeks (POI) EHVT fabricated from PGA:PLLA scaffolds and seeded with ovine bone-marrow-derived mesenchymal stem cells. Custom scaffold fiber tracking software was developed to quantify scaffold fiber architectural features such as length, tortuosity, and minimum scaffold fiber-fiber separation distance and scaffold fiber orientation was quantified utilizing a 3D fabric tensor. In addition, collagen and cellular density of ovine pulmonary valve leaflet tissue were also analyzed for baseline comparisons. Results indicated that in the unseeded state, scaffold fibers formed a continuous, oriented network. In the PRI state, the scaffold showed some fragmentation with a scaffold volume fraction of 7.79%. In the POI specimen, the scaffold became highly fragmented, forming a randomly distributed short fibrous network (volume fraction of 2.03%) within a contiguous, dense collagenous matrix. Both PGA and PLLA scaffold fibers were observed in the PRI and POI specimens. Collagen density remained similar in both PRI and POI specimens (74.2 and 71.5%, respectively), though the distributions in the transmural direction appeared slightly more homogenous in the POI specimen. Finally, to guide future 2D histological studies for large-scale studies (since acquisition of high-resolution volumetric data is not practical for all specimens), we investigated changes in relevant collagen and scaffold metrics (collagen density and scaffold fiber orientation) with varying section spacing. It was found that a sectioning spacing up to 25 µm (for scaffold morphology) and 50 µm (for collagen density) in both PRI and POI tissues did not result in loss of information fidelity, and that sectioning in the circumferential or radial direction provides the greatest preservation of information. This is the first known work to investigate EHVT microstructure over a large volume with high resolution and to investigate time evolving in vivo EHVT morphology. The important scaffold fiber structural changes observed provide morphological information crucial for guiding future structurally based constitutive modeling efforts focused on better understanding EHVT tissue formation and remodeling.