Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices.

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve's functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1ß secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1ß secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

Macrophage-extracellular matrix interactions: Perspectives for tissue engineered heart valve remodeling.

In situ heart valve tissue engineering approaches have been proposed as promising strategies to overcome the limitations of current heart valve replacements. Tissue engineered heart valves (TEHVs) generated from in vitro grown tissue engineered matrices (TEMs) aim at mimicking the microenvironmental cues from the extracellular matrix (ECM) to favor integration and remodeling of the implant. A key role of the ECM is to provide mechanical support to and attract host cells into the construct. Additionally, each ECM component plays a critical role in regulating cell adhesion, growth, migration, and differentiation potential. Importantly, the immune response to the implanted TEHV is also modulated biophysically via macrophage-ECM protein interactions. Therefore, the aim of this review is to summarize what is currently known about the interactions and signaling networks occurring between ECM proteins and macrophages, and how these interactions may impact the long-term in situ remodeling outcomes of TEMs. First, we provide an overview of in situ tissue engineering approaches and their clinical relevance, followed by a discussion on the fundamentals of the remodeling cascades. We then focus on the role of circulation-derived and resident tissue macrophages, with particular emphasis on the ramifications that ECM proteins and peptides may have in regulating the host immune response. Finally, the relevance of these findings for heart valve tissue engineering applications is discussed.

Endothelial Progenitor Cell-Based in vitro Pre-Endothelialization of Human Cell-Derived Biomimetic Regenerative Matrices for Next-Generation Transcatheter Heart Valves Applications.

Hemocompatibility of cardiovascular implants represents a major clinical challenge and, to date, optimal antithrombotic properties are lacking. Next-generation tissue-engineered heart valves (TEHVs) made from human-cell-derived tissue-engineered extracellular matrices (hTEMs) demonstrated their recellularization capacity in vivo and may represent promising candidates to avoid antithrombotic therapy. To further enhance their hemocompatibility, we tested hTEMs pre-endothelialization potential using human-blood-derived endothelial-colony-forming cells (ECFCs) and umbilical vein cells (control), cultured under static and dynamic orbital conditions, with either FBS or hPL. ECFCs performance was assessed via scratch assay, thereby recapitulating the surface damages occurring in transcatheter valves during crimping procedures. Our study demonstrated: feasibility to form a confluent and functional endothelium on hTEMs with expression of endothelium-specific markers; ECFCs migration and confluency restoration after crimping tests; hPL-induced formation of neo-microvessel-like structures; feasibility to pre-endothelialize hTEMs-based TEHVs and ECFCs retention on their surface after crimping. Our findings may stimulate new avenues towards next-generation pre-endothelialized implants with enhanced hemocompatibility, being beneficial for selected high-risk patients.

Next-generation tissue-engineered heart valves with repair, remodelling and regeneration capacity.

Valvular heart disease is a major cause of morbidity and mortality worldwide. Surgical valve repair or replacement has been the standard of care for patients with valvular heart disease for many decades, but transcatheter heart valve therapy has revolutionized the field in the past 15 years. However, despite the tremendous technical evolution of transcatheter heart valves, to date, the clinically available heart valve prostheses for surgical and transcatheter replacement have considerable limitations. The design of next-generation tissue-engineered heart valves (TEHVs) with repair, remodelling and regenerative capacity can address these limitations, and TEHVs could become a promising therapeutic alternative for patients with valvular disease. In this Review, we present a comprehensive overview of current clinically adopted heart valve replacement options, with a focus on transcatheter prostheses. We discuss the various concepts of heart valve tissue engineering underlying the design of next-generation TEHVs, focusing on off-the-shelf technologies. We also summarize the latest preclinical and clinical evidence for the use of these TEHVs and describe the current scientific, regulatory and clinical challenges associated with the safe and broad clinical translation of this technology.

Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

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.

Tissue engineered heart valves for transcatheter aortic valve implantation: current state, challenges, and future developments.

INTRODUCTION: The establishment of transcatheter aortic valve implantation (TAVI) has revolutionized the treatment of severe aortic stenosis. However, with TAVI being approved for low-risk patients, valve durability is becoming of central importance. Here, we summarize how tissue engineered heart valves (TEHVs) may provide a clinically-relevant durable valve replacement compatible with TAVI. AREAS COVERED: Since its introduction, TAVI prostheses have advanced in design and development. However, TAVI bioprostheses are based on fixed xenogeneic materials prone to progressive degeneration. Transcatheter TEHVs may have the potential to overcome the drawbacks of current TAVI bioprostheses, with their remodeling, self-repair, and growth capacities. So far, performance and remodeling of transcatheter TEHV with in-situ regenerative potential were demonstrated in the low-pressure system, with acute performance proved in the systemic circulation. However, several challenges remain to be solved to ensure a safe clinical translation of TEHVs for TAVI approaches. EXPERT OPINION: With TAVI rapidly evolving, the establishment of long-term valve durability represents the top priority to reduce the rate of patient re-interventions, remove the associated risks and adverse events, and improve patients' life quality worldwide. With long-term performance and remodeling proved, TEHVs may represent the next-generation technology for a life-long TAVI prosthesis.

Differential Leaflet Remodeling of Bone Marrow Cell Pre-Seeded Versus Nonseeded Bioresorbable Transcatheter Pulmonary Valve Replacements.

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.

Human cell-derived tissue-engineered heart valve with integrated Valsalva sinuses: towards native-like transcatheter pulmonary valve replacements.

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 tissue-engineered 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.

Computational modeling guides tissue-engineered heart valve design for long-term in vivo performance in a translational sheep model.

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.

Development of an Off-the-Shelf Tissue-Engineered Sinus Valve for Transcatheter Pulmonary Valve Replacement: a Proof-of-Concept Study.

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 future of heart valve replacement: recent developments and translational challenges for heart valve tissue engineering.

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.

Off-the-shelf tissue engineered heart valves for in situ regeneration: current state, challenges and future directions.

INTRODUCTION: Transcatheter aortic valve replacement (TAVR) is continuously evolving and is expected to surpass surgical valve implantation in the near future. Combining durable valve substitutes with minimally invasive implantation techniques might increase the clinical relevance of this therapeutic option for younger patient populations. Tissue engineering offers the possibility to create tissue engineered heart valves (TEHVs) with regenerative and self-repair capacities which may overcome the pitfalls of current TAVR prostheses. AREAS COVERED: This review focuses on off-the-shelf TEHVs which rely on a clinically-relevant in situ tissue engineering approach and which have already advanced into preclinical or first-in-human investigation. EXPERT COMMENTARY: Among the off-the-shelf in situ TEHVs reported in literature, the vast majority covers pulmonary valve substitutes, and only few are combined with transcatheter implantation technologies. Hence, further innovations should include the development of transcatheter tissue engineered aortic valve substitutes, which would considerably increase the clinical relevance of such prostheses.

In situ heart valve tissue engineering using a bioresorbable elastomeric implant - From material design to 12 months follow-up in sheep.

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.

Translational Challenges in Cardiovascular Tissue Engineering.

Valvular heart disease and congenital heart defects represent a major cause of death around the globe. Although current therapy strategies have rapidly evolved over the decades and are nowadays safe, effective, and applicable to many affected patients, the currently used artificial prostheses are still suboptimal. They do not promote regeneration, physiological remodeling, or growth (particularly important aspects for children) as their native counterparts. This results in the continuous degeneration and subsequent failure of these prostheses which is often associated with an increased morbidity and mortality as well as the need for multiple re-interventions. To overcome this problem, the concept of tissue engineering (TE) has been repeatedly suggested as a potential technology to enable native-like cardiovascular replacements with regenerative and growth capacities, suitable for young adults and children. However, despite promising data from pre-clinical and first clinical pilot trials, the translation and clinical relevance of such TE technologies is still very limited. The reasons that currently limit broad clinical adoption are multifaceted and comprise of scientific, clinical, logistical, technical, and regulatory challenges which need to be overcome. The aim of this review is to provide an overview about the translational problems and challenges in current TE approaches. It further suggests directions and potential solutions on how these issues may be efficiently addressed in the future to accelerate clinical translation. In addition, a particular focus is put on the current regulatory guidelines and the associated challenges for these promising TE technologies.

Heart Valve Replacements with Regenerative Capacity.

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.

Improved Geometry of Decellularized Tissue Engineered Heart Valves to Prevent Leaflet Retraction.

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.

Differential response of endothelial and endothelial colony forming cells on electrospun scaffolds with distinct microfiber diameters.

Electrospun scaffolds for in situ tissue engineering can be prepared with different fiber diameters to influence cell recruitment, adhesion, and differentiation. For cardiovascular applications, we investigated the impact of different fiber diameters (2, 5, 8, and 11 µm) in electrospun poly(ε-caprolactone) scaffolds on endothelial colony forming cells (ECFCs) in comparison to mature endothelial cells (HUVECs). In 2D cultures and on 2 µm fiber scaffolds, ECFC morphology and phenotype resemble those of HUVECs. When cultured on scaffolds with 5-11 µm fibers, a different behavior was detected. HUVECs developed a cytoskeleton organized circumferentially around the fibers, with collagen alignment in the same direction. ECFCs, instead, aligned the cytoskeleton along the scaffold fiber axis and deposited a homogeneous layer of collagen over the fibers; moreover, a subpopulation of ECFCs gained the αSMA marker. These results showed that ECFCs do not behave like mature endothelial cells in a 3D fibrous environment.

Matrix production and organization by endothelial colony forming cells in mechanically strained engineered tissue constructs.

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 ß1 (TGFß1). 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 TGFß1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFß1 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.

Polymer-based scaffold designs for in situ vascular tissue engineering: controlling recruitment and differentiation behavior of endothelial colony forming cells.

In situ vascular tissue engineering has been proposed as a promising approach to fulfill the need for small-diameter blood vessel substitutes. The approach comprises the use of a cell-free instructive scaffold to guide and control cell recruitment, differentiation, and tissue formation at the locus of implantation. Here we review the design parameters for such scaffolds, with special emphasis on differentiation of recruited ECFCs into the different lineages that constitute the vessel wall. Next to defining the target properties of the vessel, we concentrate on the target cell source, the ECFCs, and on the environmental control of the fate of these cells within the scaffold. The prospects of the approach are discussed in the light of current technical and biological hurdles.

Influence of substrate stiffness on circulating progenitor cell fate.

In situ vascular tissue engineering (TE) aims at regenerating vessels using implanted synthetic scaffolds. An envisioned strategy is to capture and differentiate progenitor cells from the bloodstream into the porous scaffold to initiate tissue formation. Among these cells are the endothelial colonies forming cells (ECFCs) that can differentiate into endothelial cells and transdifferentiate into smooth muscle cells under biochemical stimulation. The influence of mechanical stimulation is unknown, but relevant for in situ vascular TE because the cells perceive a change in mechanical environment when captured inside the scaffold, where they are shielded from blood flow induced shear stresses. Here we investigate the effects of substrate stiffness as one of the environmental mechanical cues to control ECFC fate within scaffolds. ECFCs were seeded on soft (3.58±0.90 kPa), intermediate (21.59±2.91 kPa), and stiff (93.75±18.36 kPa) fibronectin-coated polyacrylamide gels, as well as on glass controls, and compared to peripheral blood mononuclear cells (PBMC). Cell behavior was analyzed in terms of adhesion (vinculin staining), proliferation (BrdU), phenotype (CD31, αSMA staining, and flow cytometry), and collagen production (col I, III, and IV). While ECFCs adhesion and proliferation increased with substrate stiffness, no change in phenotype was observed. The cells produced no collagen type I, but abundant amounts of collagen type III and IV, albeit in a stiffness-dependent organization. PBMCs did not adhere to the gels, but they did adhere to glass, where they expressed CD31 and collagen type III. Addition mechanical cues, such as cyclic strains, should be studied to further investigate the effect of the mechanical environment on captured circulating cells for in situ TE purposes.