Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

This study showed that bone marrow mononuclear cell pre-seeding had detrimental effects on functionality and in situ remodeling of bioresorbable bisurea-modified polycarbonate (PC-BU)-based tissue-engineered heart valves (TEHVs) used as transcatheter pulmonary valve replacement in sheep. We also showed heterogeneous valve and leaflet remodeling, which affects PC-BU TEHV safety, challenging their potential for clinical translation. We suggest that bone marrow mononuclear cell pre-seeding should not be used in combination with PC-BU TEHVs. A better understanding of cell-scaffold interaction and in situ remodeling processes is needed to improve transcatheter valve design and polymer absorption rates for a safe and clinically relevant translation of this approach.

Transcatheter valve replacement indication is currently being extended to younger and lower-risk patients. However, transcatheter prostheses are still based on glutaraldehyde-fixed xenogeneic materials. Hence, they are prone to calcification and long-term structural degeneration, which are particularly accelerated in younger patients. Tissue-engineered heart valves based on decellularized in vitro grown tissue-engineered matrices (TEM) have been suggested as a valid alternative to currently used bioprostheses, showing good performance and remodeling capacity as transcatheter pulmonary valve replacement (TPVR) in sheep. Here, we first describe the in vitro development of human cell-derived TEM (hTEM) and their application as tissueengineered sinus valves (hTESVs), endowed with Valsalva sinuses for TPVR. The hTEM and hTESVs were systematically characterized in vitro by histology, immunofluorescence, and biochemical analyses, before they were evaluated in a pulse duplicator system under physiological pulmonary pressure conditions. Thereafter, transapical delivery of hTESVs was tested for feasibility and safety in a translational sheep model, achieving good valve performance and early cellular infiltration. This study demonstrates the principal feasibility of clinically relevant hTEM to manufacture hTESVs for TPVR.

Objective: Initially developed for inoperable cases, transcatheter valve replacement (TVR) is being extended to lower-risk and younger patients. Tissue engineering aims at solving the degenerative complications of current bioprostheses compatible with TVR by providing a replacement that transforms into a living valve upon implantation. Methods: We used cell-free bioresorbable elastomeric tissue engineered heart valves (TEHVs) for transcatheter pulmonary valve replacement in a preclinical sheep model. To favour in-vivo remodelling via paracrine signalling, we developed a one-step procedure prior to implantation to test the role of autologous bone marrow mononuclear cells (BMMNCs) pre-seeding on the TEHVs. For each group (cell-free, pre-seeded), 4 (n=2) and 24 (n=3) weeks follow-up in sheep were performed (total n=10). Results: Cell-free TEHVs showed stable functionality and good remodelling potential for up to 24 weeks, demonstrating integration into the native tissues, cellular infiltration, collagen and elastin deposition, and initial scaffold degradation. Despite its feasibility, the BMMNCs pre-seeding had detrimental effects on valve functionality and tissue remodelling. Upon implantation and up to 4 weeks, the BMMNC-seeded valves showed sufficient functionality, but fusion of contiguous leaflets was already evident in the 4 weeks explants (n=2). Starting at 12 weeks, moderate regurgitation was observed, and worsened over time leading to pre-term sacrifice at 18 weeks (n=2). Histological evaluation revealed abundant presence of αSMA-positive cells, calcified nodules, and signs of leaflet thickening and retraction. Independently on the pre-seeding, leaflet-specific remodelling, in terms of thickening, cellular infiltration, and elastin deposition, was evident for all the TEHVs. Conclusions: Taken together, these results suggest that, albeit feasible, BMMNC-seeding is associated with maladaptive valve remodelling. Further studies should investigate the causes for the observed leaflet-dependent remodelling mechanisms to increase the safety profile of the valve, before achieving a competitive and clinically relevant valve replacement solution.

Valvular heart disease is a major cause of morbidity and mortality worldwide. Current heart valve prostheses have considerable clinical limitations due to their artificial, nonliving nature without regenerative capacity. To overcome these limitations, heart valve tissue engineering (TE) aiming to develop living, native-like heart valves with self-repair, remodeling, and regeneration capacity has been suggested as next-generation technology. A major roadblock to clinically relevant, safe, and robust TE solutions has been the high complexity and variability inherent to bioengineering approaches that rely on cell-driven tissue remodeling. For heart valve TE, this has limited long-term performance in vivo because of uncontrolled tissue remodeling phenomena, such as valve leaflet shortening, which often translates into valve failure regardless of the bioengineering methodology used to develop the implant. We tested the hypothesis that integration of a computationally inspired heart valve design into our TE methodologies could guide tissue remodeling toward long-term functionality in tissue-engineered heart valves (TEHVs). In a clinically and regulatory relevant sheep model, TEHVs implanted as pulmonary valve replacements using minimally invasive techniques were monitored for 1 year via multimodal in vivo imaging and comprehensive tissue remodeling assessments. TEHVs exhibited good preserved long-term in vivo performance and remodeling comparable to native heart valves, as predicted by and consistent with computational modeling. TEHV failure could be predicted for nonphysiological pressure loading. Beyond previous studies, this work suggests the relevance of an integrated in silico, in vitro, and in vivo bioengineering approach as a basis for the safe and efficient clinical translation of TEHVs.

Heart valve replacement is often the only solution for patients suffering from valvular heart disease. However, currently available valve replacements require either life‐long anticoagulation or are associated with valve degeneration and calcification. Moreover, they are suboptimal for young patients, because they do not adapt to the somatic growth. Tissue‐engineering has been proposed as a promising approach to fulfil the urgent need for heart valve replacements with regenerative and growth capacity. This review will start with an overview on the currently available valve substitutes and the techniques for heart valve replacement. The main focus will be on the evolution of and different approaches for heart valve tissue engineering, namely the in vitro, in vivo and in situ approaches. More specifically, several heart valve tissue‐engineering studies will be discussed with regard to their shortcomings or successes and their possible suitability for novel minimally invasive implantation techniques. As in situ heart valve tissue engineering based on cell‐free functionalized starter materials is considered to be a promising approach for clinical translation, this review will also analyse the techniques used to tune the inflammatory response and cell recruitment upon implantation in order to stir a favourable outcome: controlling the blood–material interface, regulating the cytokine release, and influencing cell adhesion and differentiation. In the last section, the authors provide their opinion about the future developments and the challenges towards clinical translation and adaptation of heart valve tissue engineering for valve replacement. Copyright © 2016 John Wiley & Sons, Ltd.

Heart valve replacement is often the only solution for patients suffering from valvular heart disease. However, currently available valve replacements require either life-long anticoagulation or are associated with valve degeneration and calcification. Moreover, they are suboptimal for young patients, because they do not adapt to the somatic growth. Tissue-engineering has been proposed as a promising approach to fulfil the urgent need for heart valve replacements with regenerative and growth capacity. This review will start with an overview on the currently available valve substitutes and the techniques for heart valve replacement. The main focus will be on the evolution of and different approaches for heart valve tissue engineering, namely the in vitro, in vivo and in situ approaches. More specifically, several heart valve tissue-engineering studies will be discussed with regard to their shortcomings or successes and their possible suitability for novel minimally invasive implantation techniques. As in situ heart valve tissue engineering based on cell-free functionalized starter materials is considered to be a promising approach for clinical translation, this review will also analyse the techniques used to tune the inflammatory response and cell recruitment upon implantation in order to stir a favourable outcome: controlling the blood-material interface, regulating the cytokine release, and influencing cell adhesion and differentiation. In the last section, the authors provide their opinion about the future developments and the challenges towards clinical translation and adaptation of heart valve tissue engineering for valve replacement. Copyright (c) 2016 John Wiley & Sons, Ltd.

Data on in vitro engineered "off the shelf" matrices support the concept of endogenous cellular repopulation driving the graft's remodeling via immune-mediated response. This seems important to further accelerate the cell reconstitution and may play a crucial role when mononuclear cells are used. Nevertheless, studies on decellularized xenogeneic grafts showed only limited host cell repopulation post-implantation. This study aims at a systematic comparison of reseeding methods (dripping, injection, bathing in a cell suspension and combined puncturing-dripping method) to define the most efficient technique enhancing recellularization of tissue engineered vascular matrices (patches, vessels, small diameter and standard size valves) prior implantation. The constructs were analyzed histologically, biochemically and biomechanically. Various preconditioning treatments (wet, lyophilized and air-dried) combined with reseeding methods demonstrated the highest cell loading efficiency, despite applied crimping and flow stress, of lyophilization followed by puncturing-dripping technique. This novel seeding method allows for an efficient, time-saving graft reseeding that can be used within a one-step cardiovascular clinical intervention. (C) 2018 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Tissue-engineered heart valves with self-repair and regeneration properties may overcome the problem of long-term degeneration of currently used artificial prostheses. The aim of this study was the development and in vivo proof-of-concept of next-generation off-the-shelf tissue-engineered sinus valve (TESV) for transcatheter pulmonary valve replacement (TPVR). Transcatheter implantation of off-the-shelf TESVs was performed in a translational sheep model for up to 16 weeks. Transapical delivery of TESVs was successful and showed good acute and short-term performance (up to 8 weeks), which then worsened over time most likely due to a non-optimized in vitro valve design. Post-mortem analyses confirmed the remodelling potential of the TESVs, with host cell infiltration, polymer degradation, and collagen and elastin deposition. TESVs proved to be suitable as TPVR in a preclinical model, with encouraging short-term performance and remodelling potential. Future studies will enhance the clinical translation of such approach by improving the valve design to ensure long-term functionality.

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 results offer new perspectives for endogenous heart valve replacement starting from a readily-available synthetic graft that is compatible with surgical and transcatheter implantation procedures.

Aims: The objective was to implant a stented decellularised tissue-engineered heart valve (sdTEHV) percutaneously in an animal model, to assess its in vivo functionality and to examine the repopulation and remodelling of the valvular matrix by the recipient's autologous cells.Methods and results: Prototypes of sdTEHV were cultured in vitro, decellularised and percutaneously implanted into the pulmonary position in 15 sheep. Functionality was assessed monthly by intracardiac echocardiography (ICE). Valves were explanted after eight, 16 or 24 weeks and analysed macroscopically, histologically and by electron microscopy. Implantation was successful in all animals. Valves showed normal pressure gradients throughout the study. Due to a suboptimal design with small coaptation area, stent ovality led to immediate regurgitation which continuously increased during follow-up. Analyses revealed complete endothelialisation and rapid cellular repopulation and remodelling of the entire matrix. Valves were free from endocarditis, calcification and graft rejection.Conclusions: sdTEHV can be safely implanted percutaneously. The fast autologous recellularisation and the extensive matrix remodelling demonstrate the valve's potential as a next-generation percutaneous prosthesis with the capacity for tissue self-maintenance and longevity. Regurgitation may be prevented by valve design optimisation.

In regenerative medicine, adult stem cells are the most promising cell types for cell-based therapies. As a new source for multipotent stem cells, human adipose tissue has been introduced. These so called adipose tissue-derived stem cells (ADSCs) are considered to be ideal for application in regenerative therapies. Their main advantage over mesenchymal stem cells derived from other sources, e.g. from bone marrow, is that they can be easily and repeatable harvested using minimally invasive techniques with low morbidity. ADSCs are multipotent and can differentiate into various cell types of the tri-germ lineages, including e.g. osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Interestingly, ADSCs are characterized by immunosuppressive properties and low immunogenicity. Their secretion of trophic factors enforces the therapeutic and regenerative outcome in a wide range of applications. Taken together, these particular attributes of ADSCs make them highly relevant for clinical applications. Consequently, the therapeutic potential of ADSCs is enormous. Therefore, this review will provide a brief overview of the possible therapeutic applications of ADSCs with regard to their differentiation potential into the tri-germ lineages. Moreover, the relevant advancements made in the field, regulatory aspects as well as other challenges and obstacles will be highlighted.

The incidence of severe valvular dysfunctions (e.g., stenosis and insufficiency) is increasing, leading to over 300,000 valves implanted worldwide yearly. Clinically used heart valve replacements lack the capacity to grow, inherently requiring repetitive and high-risk surgical interventions during childhood. The aim of this review is to present how different tissue engineering strategies can overcome these limitations, providing innovative valve replacements that proved to be able to integrate and remodel in pre-clinical experiments and to have promising results in clinical studies. Upon description of the different types of heart valve tissue engineering (e.g., in vitro, in situ, in vivo, and the pre-seeding approach) we focus on the clinical translation of this technology. In particular, we will deepen the many technical, clinical, and regulatory aspects that need to be solved to endure the clinical adaptation and the commercialization of these promising regenerative valves.

BACKGROUNDDecellularized tissue-engineered heart valves (TEHVs) are under investigation as alternative for current heart valve prostheses with the potential to rapidly repopulate with cells within the body. Ideally, these valves are stented for transapical or minimally invasive delivery. It is unclear if and how the matrix of these valves remodels under in vivo hemodynamic loading conditions and in the presence of a stent. Here, we study the evolution of collagen orientation and tissue maturation in the wall of stented decellularized TEHVs with time after implantation.METHODS AND RESULTSIn a previous study, stented TEHVs based on rapidly degrading scaffolds were cultured in bioreactors, decellularized, and transapically implanted as pulmonary valve replacement in sheep. In the present study, collagen (re)orientation in the initially isotropic valvular wall was assessed using a fluorescent collagen probe combined with confocal imaging and image analysis of explanted tissue at 8, 16, and 24 weeks following implantation. Collagen tortuosity or waviness in the explants, as a measure of matrix maturity, was quantified using a Gabor wavelet method and compared with tortuosity in native sheep vascular wall tissue. Results indicate that on the luminal side of the valvular wall, fibers became aligned in circumferential direction, while tortuosity increased with implantation time, showing striking similarities with the native collagen structure after 24 weeks. On the outside of the wall, where the engineered tissue touches the stent, collagen fibers in the vicinity of the struts aligned along the struts, whereas collagen fibers in between struts were randomly oriented. Immunohistochemistry was performed to evaluate the presence of elastin and collagen type I and III. After 8 weeks, collagen types I and III were mostly present at the luminal side of the wall, whereas at 16 and 24 weeks, a homogenous distribution of collagen I and III was observed throughout the wall. Elastin was mostly expressed at the luminal side after 24 weeks. Biochemical assays showed that the amount of DNA (as a measure of cell number) increased significantly after 8 and 24 weeks, glycosaminoglycans increased significantly after 8, 16, and 24 weeks, and hydroxyproline, as a measure of collagen amount, increased significantly after 24 weeks compared to the controls.CONCLUSIONSThe collagen matrix in the wall of decellularized TEHVs shows clear structural remodeling and maturation with time. While collagen orientation rapidly remodels toward a native anisotropic architecture on the luminal side of the engineered valvular wall, it is dominated and guided by stent geometry on the outer side of the wall. Collagen tortuosity was increased with implantation time and was accompanied by an increase in elastin, especially on the luminal side of the vessel.