Decellularized tissue-engineered heart valves (DTEHVs) showed remarkable results in translational... more Decellularized tissue-engineered heart valves (DTEHVs) showed remarkable results in translational animal models, leading to recellularization within hours after implantation. This is crucial to enable tissue remodeling. To investigate if the presence of scaffold remnants before implantation is responsible for the fast recellularization of DTEHVs, an in vitro mesofluidic system was used. Human granulocyte and agranulocyte fractions were isolated, stained, brought back in suspension, and implemented in the system. Three different types of biomaterials were exposed to the circulating blood cells, consisting of decellularized tissue-engineered constructs (DTECs) with or without scaffold remnants or only bare scaffold. After 5 h of testing, the granulocyte fraction depleted faster from the circulation than the agranulocyte fraction. However, only granulocytes infiltrated into the DTEC with scaffold, migrating toward the scaffold remnants. The agranulocyte population, on the other hand, w...
The creation of a living heart valve is a much-wanted alternative for current valve prostheses th... more The creation of a living heart valve is a much-wanted alternative for current valve prostheses that suffer from limited durability and thromboembolic complications. Current strategies to create such valves, however, require the use of cells for in vitro culture, or decellularized human- or animal-derived donor tissue for in situ engineering. Here, we propose and demonstrate proof-of-concept of in situ heart valve tissue engineering using a synthetic approach, in which a cell-free, slow degrading elastomeric valvular implant is populated by endogenous cells to form new valvular tissue inside the heart. We designed a fibrous valvular scaffold, fabricated from a novel supramolecular elastomer, that enables endogenous cells to enter and produce matrix. Orthotopic implantations as pulmonary valve in sheep demonstrated sustained functionality up to 12 months, while the implant was gradually replaced by a layered collagen and elastic matrix in pace with cell-driven polymer resorption. Our ...
There is limited information about age-specific structural and functional properties of human hea... more There is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation-but more pronounced in aortic valves-the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.
Recent studies on decellularized tissue engineered heart valves (DTEHVs) showed rapid host cell r... more Recent studies on decellularized tissue engineered heart valves (DTEHVs) showed rapid host cell repopulation and increased valvular insufficiency developing over time, associated with leaflet shortening. A possible explanation for this result was found using computational simulations, which revealed radial leaflet compression in the original valvular geometry when subjected to physiological pressure conditions. Therefore, an improved geometry was suggested to enable radial leaflet extension to counteract for host cell mediated retraction. In this study, we propose a solution to impose this new geometry by using a constraining bioreactor insert during culture. Human cell based DTEHVs (n = 5) were produced as such, resulting in an enlarged coaptation area and profound belly curvature. Extracellular matrix was homogeneously distributed, with circumferential collagen alignment in the coaptation region and global tissue anisotropy. Based on in vitro functionality experiments, these DTEHVs showed competent hydrodynamic functionality under physiological pulmonary conditions and were fatigue resistant, with stable functionality up to 16 weeks in vivo simulation. Based on implemented mechanical data, our computational models revealed a considerable decrease in radial tissue compression with the obtained geometrical adjustments. Therefore, these improved DTEHV are expected to be less prone to host cell mediated leaflet retraction and will remain competent after implantation.
The collagen architecture is the major determinant of the function and mechanical behavior of car... more The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure that mimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/ width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.
Aims: Tissue engineering is an innovative method to restore cardiovascular tissue function by imp... more Aims: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs) are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT). We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor b1 (TGFb1). Methods and Results: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFb1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFb1 before exposing them to strain led to more homogenous matrix production. Conclusions: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.
Despite recent progress, mechanical behavior of tissue-engineered heart valves still needs improv... more Despite recent progress, mechanical behavior of tissue-engineered heart valves still needs improvement when native aortic valves are considered as a benchmark. Although it is known that cyclic straining enhances tissue formation, optimal loading protocols have not been defined yet. To obtain a better understanding of the effects of mechanical conditioning on tissue development, mechanical behavior of tissue constructs should be monitored and controlled during culture. However, currently used methods for mechanical characterization (e.g., tensile and indentation tests) are destructive and are only performed at the end-stage of tissue culture. In this study, an inverse experimental-numerical approach was developed that enables a noninvasive and nondestructive assessment of mechanical properties of engineered heart valves. The applied pressure and volumetric deformation of an engineered heart valve were measured during culture, and served as input for the estimation of mechanical properties using a computational model. To validate the method, six heart valves were cultured, and the mechanical properties obtained from the inverse experimental-numerical approach were in good agreement with uniaxial tensile test data. The method provides a real-time, noninvasive and nondestructive functionality and quality check of tissue-engineered heart valves and can be used to monitor and control the evolution of mechanical properties during tissue culture.
Journal of the American College of Cardiology, 2010
The aim of this study was to demonstrate the feasibility of combining the novel heart valve repla... more The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials. Background Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential. Methods Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed. Results The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source. Conclusions This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices.
Journal of the American College of Cardiology, 2014
Objectives: This study sought to evaluate long-term in vivo functionality, host cell repopulation... more Objectives: This study sought to evaluate long-term in vivo functionality, host cell repopulation, and remodeling of "off-the-shelf" tissue engineered transcatheter homologous heart valves. Background: Transcatheter valve implantation has emerged as a valid alternative to conventional surgery, in particular for elderly high-risk patients. However, currently used bioprosthetic transcatheter valves are prone to progressive dysfunctional degeneration, limiting their use in younger patients. To overcome these limitations, the concept of tissue engineered heart valves with self-repair capacity has been introduced as next-generation technology. Methods: In vivo functionality, host cell repopulation, and matrix remodeling of homologous transcatheter tissue-engineered heart valves (TEHVs) was evaluated up to 24 weeks as pulmonary valve replacements (transapical access) in sheep (n = 12). As a control, tissue composition and structure were analyzed in identical not implanted TEHVs (n = 5). Results: Transcatheter implantation was successful in all animals. Valve functionality was excellent displaying sufficient leaflet motion and coaptation with only minor paravalvular leakage in some animals. Mild central regurgitation was detected after 8 weeks, increasing to moderate after 24 weeks, correlating to a compromised leaflet coaptation. Mean and peak transvalvular pressure gradients were 4.4 ± 1.6 mm Hg and 9.7 ± 3.0 mm Hg, respectively. Significant matrix remodeling was observed in the entire valve and corresponded with the rate of host cell repopulation. Conclusions: For the first time, the feasibility and long-term functionality of transcatheter-based homologous off-the-shelf tissue engineered heart valves are demonstrated in a relevant pre-clinical model. Such engineered heart valves may represent an interesting alternative to current prostheses because of their rapid cellular repopulation, tissue remodeling, and therewith selfrepair capacity. The concept of homologous off-the-shelf tissue engineered heart valves may therefore substantially simplify previous tissue engineering concepts toward clinical translation.
Preclinical studies of tissue-engineered heart valves (TEHVs) showed retraction of the heart valv... more Preclinical studies of tissue-engineered heart valves (TEHVs) showed retraction of the heart valve leaflets as major failure of function mechanism. This retraction is caused by both passive and active cell stress and passive matrix stress. Cell-mediated retraction induces leaflet shortening that may be counteracted by the hemodynamic loading of the leaflets during diastole. To get insight into this stress balance, the amount and duration of stress generation in engineered heart valve tissue and the stress imposed by physiological hemodynamic loading are quantified via an experimental and a computational approach, respectively. Stress generation by cells was measured using an earlier described in vitro model system, mimicking the culture process of TEHVs. The stress imposed by the blood pressure during diastole on a valve leaflet was determined using finite element modeling. Results show that for both pulmonary and systemic pressure, the stress imposed on the TEHV leaflets is comparable to the stress generated in the leaflets. As the stresses are of similar magnitude, it is likely that the imposed stress cannot counteract the generated stress, in particular when taking into account that hemodynamic loading is only imposed during diastole. This study provides a rational explanation for the retraction found in preclinical studies of TEHVs and represents an important step towards understanding the retraction process seen in TEHVs by a combined experimental and computational approach.
DOI to the publisher's website. • The final author version and the galley proof are versions of t... more DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:
To optimize the mechanical properties and integrity of tissue-engineered aortic heart valves, it ... more To optimize the mechanical properties and integrity of tissue-engineered aortic heart valves, it is necessary to gain insight into the effects of mechanical stimuli on the mechanical behavior of the tissue using mathematical models. In this study, a finite-element (FE) model is presented to relate changes in collagen fiber content and orientation to the mechanical loading condition within the engineered construct. We hypothesized that collagen fibers aligned with principal strain directions and that collagen content increased with the fiber stretch. The results indicate that the computed preferred fiber directions run from commissure to commissure and show a strong resemblance to experimental data from native aortic heart valves.
Living tissues show an adaptive response to mechanical loading by changing their internal structu... more Living tissues show an adaptive response to mechanical loading by changing their internal structure and morphology. Understanding this response is essential for successful tissue engineering of load-bearing structures, such as the aortic valve. In this study, mechanically induced remodeling of the collagen architecture in the aortic valve was investigated. It was hypothesized that, in uniaxially loaded regions, the fibers aligned with the tensile principal stretch direction. For biaxial loading conditions, on the other hand, it was assumed that the collagen fibers aligned with directions situated between the principal stretch directions. This hypothesis has already been applied successfully to study collagen remodeling in arteries. The predicted fiber architecture represented a branching network and resembled the macroscopically visible collagen bundles in the native leaflet. In addition, the complex biaxial mechanical behavior of the native valve could be simulated qualitatively wi...
Accurate constitutive models are required to gain further insight into the mechanical behavior of... more Accurate constitutive models are required to gain further insight into the mechanical behavior of cardiovascular tissues. In this study, a structural constitutive framework for cardiovascular tissues is introduced that accounts for the angular distribution of collagen fibers. To demonstrate its capabilities, the model is applied to study the biaxial behavior of the arterial wall and the aortic valve. The pressure–radius relationships of the arterial wall accurately describe experimentally observed sigma-shaped curves. In addition, the nonlinear and anisotropic mechanical properties of the aortic valve can be analyzed with the proposed model. We expect that the current model offers strong possibilities to further investigate the complex mechanical behavior of cardiovascular tissues, including their response to mechanical stimuli.
Background— Previous attempts in heart valve tissue engineering (TE) failed to produce autologous... more Background— Previous attempts in heart valve tissue engineering (TE) failed to produce autologous valve replacements with native-like mechanical behavior to allow for systemic pressure applications. Because hypoxia and insulin are known to promote protein synthesis by adaptive cellular responses, a physiologically relevant oxygen tension and insulin supplements were applied to the growing heart valve tissues to enhance their mechanical properties. Methods and Results— Scaffolds of rapid-degrading polyglycolic acid meshes coated with poly-4-hydroxybutyrate were seeded with human saphenous vein myofibroblasts. The tissue-engineered constructs were cultured under normal oxygen tension (normoxia) or hypoxia (7% O 2 ) and incubated with or without insulin. Glycosaminoglycan production in the constructs approached that of native values under the influence of hypoxia and under the influence of insulin. Both insulin and hypoxia were associated with enhanced matrix production and improved me...
Background— Tissue engineering represents a promising approach for the development of living hear... more Background— Tissue engineering represents a promising approach for the development of living heart valve replacements. In vivo animal studies of tissue-engineered autologous heart valves have focused on pulmonary valve replacements, leaving the challenge to tissue engineer heart valves suitable for systemic application using human cells. Methods and Results— Tissue-engineered human heart valves were analyzed up to 4 weeks and conditioning using bioreactors was compared with static culturing. Tissue formation and mechanical properties increased with time and when using conditioning. Organization of the tissue, in terms of anisotropic properties, increased when conditioning was dynamic in nature. Exposure of the valves to physiological aortic valve flow demonstrated proper opening motion. Closure dynamics were suboptimal, most likely caused by the lower degree of anisotropy when compared with native aortic valve leaflets. Conclusions— This study presents autologous tissue-engineered h...
Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning proto... more Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning protocols in tissue-engineering of load-bearing cardiovascular structures. Mathematical models offer strong possibilities to gain insight into the mechanisms and mechanical stimuli involved in these remodelling processes. In this study, a framework is proposed to investigate remodelling of angular collagen fiber distribution in cardiovascular tissues. A structurally based model for collagenous cardiovascular tissues is extended with remodelling laws for the collagen architecture, and the model is subsequently applied to the arterial wall and aortic valve. For the arterial wall, the model predicts the presence of two helically arranged families of collagen fibers. A branching, diverging hammock-type fiber architecture is predicted for the aortic valve. It is expected that the proposed model may be of great potential for the design of improved tissue engineering protocols and may give further insight into the pathophysiology of cardiovascular diseases.
In tissue engineered heart valves, cell-mediated stress development during culture results in lea... more In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum
Decellularized tissue-engineered heart valves (DTEHVs) showed remarkable results in translational... more Decellularized tissue-engineered heart valves (DTEHVs) showed remarkable results in translational animal models, leading to recellularization within hours after implantation. This is crucial to enable tissue remodeling. To investigate if the presence of scaffold remnants before implantation is responsible for the fast recellularization of DTEHVs, an in vitro mesofluidic system was used. Human granulocyte and agranulocyte fractions were isolated, stained, brought back in suspension, and implemented in the system. Three different types of biomaterials were exposed to the circulating blood cells, consisting of decellularized tissue-engineered constructs (DTECs) with or without scaffold remnants or only bare scaffold. After 5 h of testing, the granulocyte fraction depleted faster from the circulation than the agranulocyte fraction. However, only granulocytes infiltrated into the DTEC with scaffold, migrating toward the scaffold remnants. The agranulocyte population, on the other hand, w...
The creation of a living heart valve is a much-wanted alternative for current valve prostheses th... more The creation of a living heart valve is a much-wanted alternative for current valve prostheses that suffer from limited durability and thromboembolic complications. Current strategies to create such valves, however, require the use of cells for in vitro culture, or decellularized human- or animal-derived donor tissue for in situ engineering. Here, we propose and demonstrate proof-of-concept of in situ heart valve tissue engineering using a synthetic approach, in which a cell-free, slow degrading elastomeric valvular implant is populated by endogenous cells to form new valvular tissue inside the heart. We designed a fibrous valvular scaffold, fabricated from a novel supramolecular elastomer, that enables endogenous cells to enter and produce matrix. Orthotopic implantations as pulmonary valve in sheep demonstrated sustained functionality up to 12 months, while the implant was gradually replaced by a layered collagen and elastic matrix in pace with cell-driven polymer resorption. Our ...
There is limited information about age-specific structural and functional properties of human hea... more There is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation-but more pronounced in aortic valves-the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.
Recent studies on decellularized tissue engineered heart valves (DTEHVs) showed rapid host cell r... more Recent studies on decellularized tissue engineered heart valves (DTEHVs) showed rapid host cell repopulation and increased valvular insufficiency developing over time, associated with leaflet shortening. A possible explanation for this result was found using computational simulations, which revealed radial leaflet compression in the original valvular geometry when subjected to physiological pressure conditions. Therefore, an improved geometry was suggested to enable radial leaflet extension to counteract for host cell mediated retraction. In this study, we propose a solution to impose this new geometry by using a constraining bioreactor insert during culture. Human cell based DTEHVs (n = 5) were produced as such, resulting in an enlarged coaptation area and profound belly curvature. Extracellular matrix was homogeneously distributed, with circumferential collagen alignment in the coaptation region and global tissue anisotropy. Based on in vitro functionality experiments, these DTEHVs showed competent hydrodynamic functionality under physiological pulmonary conditions and were fatigue resistant, with stable functionality up to 16 weeks in vivo simulation. Based on implemented mechanical data, our computational models revealed a considerable decrease in radial tissue compression with the obtained geometrical adjustments. Therefore, these improved DTEHV are expected to be less prone to host cell mediated leaflet retraction and will remain competent after implantation.
The collagen architecture is the major determinant of the function and mechanical behavior of car... more The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure that mimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/ width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.
Aims: Tissue engineering is an innovative method to restore cardiovascular tissue function by imp... more Aims: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs) are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT). We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor b1 (TGFb1). Methods and Results: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFb1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFb1 before exposing them to strain led to more homogenous matrix production. Conclusions: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.
Despite recent progress, mechanical behavior of tissue-engineered heart valves still needs improv... more Despite recent progress, mechanical behavior of tissue-engineered heart valves still needs improvement when native aortic valves are considered as a benchmark. Although it is known that cyclic straining enhances tissue formation, optimal loading protocols have not been defined yet. To obtain a better understanding of the effects of mechanical conditioning on tissue development, mechanical behavior of tissue constructs should be monitored and controlled during culture. However, currently used methods for mechanical characterization (e.g., tensile and indentation tests) are destructive and are only performed at the end-stage of tissue culture. In this study, an inverse experimental-numerical approach was developed that enables a noninvasive and nondestructive assessment of mechanical properties of engineered heart valves. The applied pressure and volumetric deformation of an engineered heart valve were measured during culture, and served as input for the estimation of mechanical properties using a computational model. To validate the method, six heart valves were cultured, and the mechanical properties obtained from the inverse experimental-numerical approach were in good agreement with uniaxial tensile test data. The method provides a real-time, noninvasive and nondestructive functionality and quality check of tissue-engineered heart valves and can be used to monitor and control the evolution of mechanical properties during tissue culture.
Journal of the American College of Cardiology, 2010
The aim of this study was to demonstrate the feasibility of combining the novel heart valve repla... more The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials. Background Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential. Methods Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed. Results The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source. Conclusions This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices.
Journal of the American College of Cardiology, 2014
Objectives: This study sought to evaluate long-term in vivo functionality, host cell repopulation... more Objectives: This study sought to evaluate long-term in vivo functionality, host cell repopulation, and remodeling of "off-the-shelf" tissue engineered transcatheter homologous heart valves. Background: Transcatheter valve implantation has emerged as a valid alternative to conventional surgery, in particular for elderly high-risk patients. However, currently used bioprosthetic transcatheter valves are prone to progressive dysfunctional degeneration, limiting their use in younger patients. To overcome these limitations, the concept of tissue engineered heart valves with self-repair capacity has been introduced as next-generation technology. Methods: In vivo functionality, host cell repopulation, and matrix remodeling of homologous transcatheter tissue-engineered heart valves (TEHVs) was evaluated up to 24 weeks as pulmonary valve replacements (transapical access) in sheep (n = 12). As a control, tissue composition and structure were analyzed in identical not implanted TEHVs (n = 5). Results: Transcatheter implantation was successful in all animals. Valve functionality was excellent displaying sufficient leaflet motion and coaptation with only minor paravalvular leakage in some animals. Mild central regurgitation was detected after 8 weeks, increasing to moderate after 24 weeks, correlating to a compromised leaflet coaptation. Mean and peak transvalvular pressure gradients were 4.4 ± 1.6 mm Hg and 9.7 ± 3.0 mm Hg, respectively. Significant matrix remodeling was observed in the entire valve and corresponded with the rate of host cell repopulation. Conclusions: For the first time, the feasibility and long-term functionality of transcatheter-based homologous off-the-shelf tissue engineered heart valves are demonstrated in a relevant pre-clinical model. Such engineered heart valves may represent an interesting alternative to current prostheses because of their rapid cellular repopulation, tissue remodeling, and therewith selfrepair capacity. The concept of homologous off-the-shelf tissue engineered heart valves may therefore substantially simplify previous tissue engineering concepts toward clinical translation.
Preclinical studies of tissue-engineered heart valves (TEHVs) showed retraction of the heart valv... more Preclinical studies of tissue-engineered heart valves (TEHVs) showed retraction of the heart valve leaflets as major failure of function mechanism. This retraction is caused by both passive and active cell stress and passive matrix stress. Cell-mediated retraction induces leaflet shortening that may be counteracted by the hemodynamic loading of the leaflets during diastole. To get insight into this stress balance, the amount and duration of stress generation in engineered heart valve tissue and the stress imposed by physiological hemodynamic loading are quantified via an experimental and a computational approach, respectively. Stress generation by cells was measured using an earlier described in vitro model system, mimicking the culture process of TEHVs. The stress imposed by the blood pressure during diastole on a valve leaflet was determined using finite element modeling. Results show that for both pulmonary and systemic pressure, the stress imposed on the TEHV leaflets is comparable to the stress generated in the leaflets. As the stresses are of similar magnitude, it is likely that the imposed stress cannot counteract the generated stress, in particular when taking into account that hemodynamic loading is only imposed during diastole. This study provides a rational explanation for the retraction found in preclinical studies of TEHVs and represents an important step towards understanding the retraction process seen in TEHVs by a combined experimental and computational approach.
DOI to the publisher's website. • The final author version and the galley proof are versions of t... more DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:
To optimize the mechanical properties and integrity of tissue-engineered aortic heart valves, it ... more To optimize the mechanical properties and integrity of tissue-engineered aortic heart valves, it is necessary to gain insight into the effects of mechanical stimuli on the mechanical behavior of the tissue using mathematical models. In this study, a finite-element (FE) model is presented to relate changes in collagen fiber content and orientation to the mechanical loading condition within the engineered construct. We hypothesized that collagen fibers aligned with principal strain directions and that collagen content increased with the fiber stretch. The results indicate that the computed preferred fiber directions run from commissure to commissure and show a strong resemblance to experimental data from native aortic heart valves.
Living tissues show an adaptive response to mechanical loading by changing their internal structu... more Living tissues show an adaptive response to mechanical loading by changing their internal structure and morphology. Understanding this response is essential for successful tissue engineering of load-bearing structures, such as the aortic valve. In this study, mechanically induced remodeling of the collagen architecture in the aortic valve was investigated. It was hypothesized that, in uniaxially loaded regions, the fibers aligned with the tensile principal stretch direction. For biaxial loading conditions, on the other hand, it was assumed that the collagen fibers aligned with directions situated between the principal stretch directions. This hypothesis has already been applied successfully to study collagen remodeling in arteries. The predicted fiber architecture represented a branching network and resembled the macroscopically visible collagen bundles in the native leaflet. In addition, the complex biaxial mechanical behavior of the native valve could be simulated qualitatively wi...
Accurate constitutive models are required to gain further insight into the mechanical behavior of... more Accurate constitutive models are required to gain further insight into the mechanical behavior of cardiovascular tissues. In this study, a structural constitutive framework for cardiovascular tissues is introduced that accounts for the angular distribution of collagen fibers. To demonstrate its capabilities, the model is applied to study the biaxial behavior of the arterial wall and the aortic valve. The pressure–radius relationships of the arterial wall accurately describe experimentally observed sigma-shaped curves. In addition, the nonlinear and anisotropic mechanical properties of the aortic valve can be analyzed with the proposed model. We expect that the current model offers strong possibilities to further investigate the complex mechanical behavior of cardiovascular tissues, including their response to mechanical stimuli.
Background— Previous attempts in heart valve tissue engineering (TE) failed to produce autologous... more Background— Previous attempts in heart valve tissue engineering (TE) failed to produce autologous valve replacements with native-like mechanical behavior to allow for systemic pressure applications. Because hypoxia and insulin are known to promote protein synthesis by adaptive cellular responses, a physiologically relevant oxygen tension and insulin supplements were applied to the growing heart valve tissues to enhance their mechanical properties. Methods and Results— Scaffolds of rapid-degrading polyglycolic acid meshes coated with poly-4-hydroxybutyrate were seeded with human saphenous vein myofibroblasts. The tissue-engineered constructs were cultured under normal oxygen tension (normoxia) or hypoxia (7% O 2 ) and incubated with or without insulin. Glycosaminoglycan production in the constructs approached that of native values under the influence of hypoxia and under the influence of insulin. Both insulin and hypoxia were associated with enhanced matrix production and improved me...
Background— Tissue engineering represents a promising approach for the development of living hear... more Background— Tissue engineering represents a promising approach for the development of living heart valve replacements. In vivo animal studies of tissue-engineered autologous heart valves have focused on pulmonary valve replacements, leaving the challenge to tissue engineer heart valves suitable for systemic application using human cells. Methods and Results— Tissue-engineered human heart valves were analyzed up to 4 weeks and conditioning using bioreactors was compared with static culturing. Tissue formation and mechanical properties increased with time and when using conditioning. Organization of the tissue, in terms of anisotropic properties, increased when conditioning was dynamic in nature. Exposure of the valves to physiological aortic valve flow demonstrated proper opening motion. Closure dynamics were suboptimal, most likely caused by the lower degree of anisotropy when compared with native aortic valve leaflets. Conclusions— This study presents autologous tissue-engineered h...
Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning proto... more Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning protocols in tissue-engineering of load-bearing cardiovascular structures. Mathematical models offer strong possibilities to gain insight into the mechanisms and mechanical stimuli involved in these remodelling processes. In this study, a framework is proposed to investigate remodelling of angular collagen fiber distribution in cardiovascular tissues. A structurally based model for collagenous cardiovascular tissues is extended with remodelling laws for the collagen architecture, and the model is subsequently applied to the arterial wall and aortic valve. For the arterial wall, the model predicts the presence of two helically arranged families of collagen fibers. A branching, diverging hammock-type fiber architecture is predicted for the aortic valve. It is expected that the proposed model may be of great potential for the design of improved tissue engineering protocols and may give further insight into the pathophysiology of cardiovascular diseases.
In tissue engineered heart valves, cell-mediated stress development during culture results in lea... more In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum
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Papers by frank baaijens