The future of valvular heart disease assessment and therapy.

Valvular heart disease (VHD) is becoming more prevalent in an ageing population, leading to challenges in diagnosis and management. This two-part Series offers a comprehensive review of changing concepts in VHD, covering diagnosis, intervention timing, novel management strategies, and the current state of research. The first paper highlights the remarkable progress made in imaging and transcatheter techniques, effectively addressing the treatment paradox wherein populations at the highest risk of VHD often receive the least treatment. These advances have attracted the attention of clinicians, researchers, engineers, device manufacturers, and investors, leading to the exploration and proposal of treatment approaches grounded in pathophysiology and multidisciplinary strategies for VHD management. This Series paper focuses on innovations involving computational, pharmacological, and bioengineering approaches that are transforming the diagnosis and management of patients with VHD. Artificial intelligence and digital methods are enhancing screening, diagnosis, and planning procedures, and the integration of imaging and clinical data is improving the classification of VHD severity. The emergence of artificial intelligence techniques, including so-called digital twins-eg, computer-generated replicas of the heart-is aiding the development of new strategies for enhanced risk stratification, prognostication, and individualised therapeutic targeting. Various new molecular targets and novel pharmacological strategies are being developed, including multiomics-ie, analytical methods used to integrate complex biological big data to find novel pathways to halt the progression of VHD. In addition, efforts have been undertaken to engineer heart valve tissue and provide a living valve conduit capable of growth and biological integration. Overall, these advances emphasise the importance of early detection, personalised management, and cutting-edge interventions to optimise outcomes amid the evolving landscape of VHD. Although several challenges must be overcome, these breakthroughs represent opportunities to advance patient-centred investigations.

Shear flow affects selective monocyte recruitment into MCP-1-loaded scaffolds.

Novel cardiovascular replacements are being developed by using degradable synthetic scaffolds, which function as a temporary guide to induce neotissue formation directly in situ. Priming of such scaffolds with fast-releasing monocyte chemoattractant protein-1 (MCP-1) was shown to improve the formation of functional neoarteries in rats. However, the underlying mechanism has not been clarified. Therefore, the goal of this study was to investigate the effect of a burst-release of MCP-1 from a synthetic scaffold on the local recruitment of circulating leucocytes under haemodynamic conditions. Herein, we hypothesized that MCP-1 initiates a desired healing cascade by recruiting favourable monocyte subpopulations into the implanted scaffold. Electrospun poly(ε-caprolactone) scaffolds were loaded with fibrin gel containing various doses of MCP-1 and exposed to a suspension of human peripheral blood mononuclear cells in static or dynamic conditions. In standard migration assay, a dose-dependent migration of specific CD14(+) monocyte subsets was observed, as measured by flow cytometry. In conditions of pulsatile flow, on the other hand, a marked increase in immediate monocyte recruitment was observed, but without evident selectivity in monocyte subsets. This suggests that the selectivity was dependent on the release kinetics of the MCP-1, as it was overruled by the effect of shear stress after the initial burst-release. Furthermore, these findings demonstrate that local recruitment of specific MCP-1-responsive monocytes is not the fundamental principle behind the improved neotissue formation observed in long-term in vivo studies, and mobilization of MCP-1-responsive cells from the bone marrow into the bloodstream is suggested to play a predominant role in vivo.

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.

Functional regeneration at the blood-biomaterial interface.

The use of cardiovascular implants is commonplace in clinical practice. However, reproducing the key bioactive and adaptive properties of native cardiovascular tissues with an artificial replacement is highly challenging. Exciting new treatment strategies are under development to regenerate (parts of) cardiovascular tissues directly in situ using immunomodulatory biomaterials. Direct exposure to the bloodstream and hemodynamic loads is a particular challenge, given the risk of thrombosis and adverse remodeling that it brings. However, the blood is also a source of (immune) cells and proteins that dominantly contribute to functional tissue regeneration. This review explores the potential of the blood as a source for the complete or partial in situ regeneration of cardiovascular tissues, with a particular focus on the endothelium, being the natural blood-tissue barrier. We pinpoint the current scientific challenges to enable rational engineering and testing of blood-contacting implants to leverage the regenerative potential of the blood.

Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

Calcification in Pulmonary Heart Valve Tissue Engineering: A Systematic Review and Meta-Analysis of Large-Animal Studies.

Tissue-engineered heart valves (TEHVs) are emerging alternatives to current valve prostheses and prospectively a lifelong replacement. Calcification, a pathological complication for biological protheses, has been reported in preclinical TEHV studies. Systematic analysis of its occurrence is missing. This review aims to: 1) systematically review reported calcification of pulmonary TEHVs in large-animal studies; and 2) analyze the influence of engineering methodology (choice of scaffold material, cell preseeding) and animal model (animal species and age) on calcification. Baseline analysis included 80 studies, of which 41 studies containing 108 experimental groups were included in meta-analysis. Inclusion was low because only 55% of studies reported on calcification. Meta-analysis showed an overall average calcification event rate of 35% (95% CI: 28%-43%). Calcification was more prominent (P = 0.023) in the arterial conduit region (34%; 95% CI: 26%-43%) than in the valve leaflets (21%; 95% CI: 17%-27%), and was mostly (42% in leaflets, 60% in conduits) present in a mild form. Time-analysis showed an initial surge within 1 month after implantation, decreased calcification between 1 and 3 months, and then progression over time. There were no significant differences in degree of calcification between TEHV strategy nor animal models. Much variability between individual studies was observed in degree of calcification as well as quality of analysis and reporting thereof, hampering adequate comparisons between studies. These findings underline the need for improved analysis and better reporting standards of calcification in TEHVs. It also necessitates control-based research to further enlighten the risk of calcification for tissue-engineered transplants compared to current options. This can bring the field of heart valve tissue engineering forward toward safe clinical use.

Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem-Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel.

Introduction: Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human-induced pluripotent stem-cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of three-dimensional (3D) substrate properties under cyclic strain on phenotypic adaptation in hiPSC-derived VSMCs. Here, we aim to investigate the impact of intrinsic substrate properties, such as the hydrogel's elastic modulus and cross-linking density in a 3D static and dynamic environment, on the phenotypical adaptation of human mural cells derived from hiPSC-derived organoids (ODMCs), compared to aortic VSMCs. Methods and results: ODMCs were cultured in two-dimensional (2D) conditions with synthetic or contractile differentiation medium or in 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate Young's modulus and cross-linking density. Cells in 3D substrates were exposed to cyclic, unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity similar to primary human VSMCs. In static 3D culture, a substrate with a higher Young's modulus and cross-linking density promoted a contractile phenotype in ODMCs and VSMCs. Dynamic stimulation in the 3D substrate promoted a switch toward a contractile phenotype in both cell types. Conclusion: Our study demonstrates phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human VSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modeling and regenerative strategies.

3D Human iPSC Blood Vessel Organoids as a Source of Flow-Adaptive Vascular Cells for Creating a Human-Relevant 3D-Scaffold Based Macrovessel Model.

3D-scaffold based in vitro human tissue models accelerate disease studies and screening of pharmaceutics while improving the clinical translation of findings. Here is reported the use of human induced pluripotent stem cell (hiPSC)-derived vascular organoid cells as a new cell source for the creation of an electrospun polycaprolactone-bisurea (PCL-BU) 3D-scaffold-based, perfused human macrovessel model. A separation protocol is developed to obtain monocultures of organoid-derived endothelial cells (ODECs) and mural cells (ODMCs) from hiPSC vascular organoids. Shear stress responses of ODECs versus HUVECs and barrier function (by trans endothelial electrical resistance) are measured. PCL-BU scaffolds are seeded with ODECs and ODMCs, and tissue organization and flow adaptation are evaluated in a perfused bioreactor system. ODECs and ODMCs harvested from vascular organoids can be cryopreserved and expanded without loss of cell purity and proliferative capacity. ODECs are shear stress responsive and establish a functional barrier that self-restores after the thrombin challenge. Static bioreactor culture of ODECs/ODMCs seeded scaffolds results in a biomimetic vascular bi-layer hierarchy, which is preserved under laminar flow similar to scaffolds seeded with primary vascular cells. HiPSC-derived vascular organoids can be used as a source of functional, flow-adaptive vascular cells for the creation of 3D-scaffold based human macrovascular models.

Evaluation of pliable bioresorbable, elastomeric aortic valve prostheses in sheep during 12 months post implantation.

Pliable microfibrous, bioresorbable elastomeric heart valve prostheses are investigated in search of sustainable heart valve replacement. These cell-free implants recruit cells and trigger tissue formation on the valves in situ. Our aim is to investigate the behaviour of these heart valve prostheses when exposed to the high-pressure circulation. We conducted a 12-month follow-up study in sheep to evaluate the in vivo functionality and neo-tissue formation of these valves in the aortic position. All valves remained free from endocarditis, thrombotic complications and macroscopic calcifications. Cell colonisation in the leaflets was mainly restricted to the hinge area, while resorption of synthetic fibers was limited. Most valves were pliable and structurally intact (10/15), however, other valves (5/15) showed cusp thickening, retraction or holes in the leaflets. Further research is needed to assess whether in-situ heart valve tissue engineering in the aortic position is possible or whether non-resorbable synthetic pliable prostheses are preferred.

Donor Heterogeneity in the Human Macrophage Response to a Biomaterial Under Hyperglycemia In Vitro.

Macrophages have a commanding role in scaffold-driven in situ tissue regeneration. Depending on their polarization state, macrophages mediate the formation and remodeling of new tissue by secreting growth factors and cytokines. Therefore, successful outcomes of material-driven in situ tissue vascular tissue engineering depend largely on the immuno-regenerative potential of the recipient. A large cohort of patients requiring vascular replacements suffers from systemic multifactorial diseases, such as diabetes, which gives rise to a hyperglycemic and aggressive oxidative inflammatory environment that is hypothesized to hamper a well-balanced regenerative process. Here, we aimed at fundamentally exploring the effects of hyperglycemia, as one of the hallmarks of diabetes, on the macrophage response to three-dimensional (3D) electrospun synthetic biomaterials for in situ tissue engineering, in terms of inflammatory profile and tissue regenerative capacity. To simulate the early phases of the in situ regenerative cascade, we used a bottom-up in vitro approach. Primary human macrophages (n = 8 donors) were seeded in two-dimensional (2D) culture wells and polarized to pro-inflammatory M1 and anti-inflammatory M2 phenotype in normoglycemic (5.5 mM glucose), hyperglycemic (25 mM), and osmotic control (OC) conditions (5.5 mM glucose, 19.5 mM mannitol). Unpolarized macrophages and (myo)fibroblasts were seeded in mono- or co-culture in a 3D electrospun resorbable polycaprolactone bisurea scaffold and exposed to normoglycemic, hyperglycemic, and OC conditions. The results showed that macrophage polarization by biochemical stimuli was effective under all glycemic conditions and that the polarization states dictated expression of the receptors SCL2A1 (glucose transporter 1) and CD36 (fatty acid transporter). In 3D, the macrophage response to hyperglycemic conditions was strongly donor-dependent in terms of phenotype, cytokine secretion profile, and metabolic receptor expression. When co-cultured with (myo)fibroblasts, hyperglycemic conditions led to an increased expression of fibrogenic markers (ACTA2, COL1, COL3, IL-1ß). Together, these findings show that the hyperglycemic and hyperosmotic conditions may, indeed, influence the process of macrophage-driven in situ tissue engineering, and that the extent of this is likely to be patient-specific. Impact Statement Success or failure of cell-free bioresorbable in situ tissue-engineered vascular grafts hinges around the immuno-regenerative response of the recipient. Most patients requiring blood vessel replacements suffer from additional multifactorial diseases, such as diabetes, which may compromise their intrinsic regenerative potential. In this study, we used a bottom-up approach to study the effects of hyperglycemia, a hallmark of diabetes, on important phases in the in situ regenerative cascade, such as macrophage polarization and macrophage-myofibroblast crosstalk. The results demonstrate a relatively large donor-to-donor variation, which stresses the importance of taking scaffold-independent patient-specific factors into account when studying in situ biomaterial-driven tissue engineering.

Marker-Independent Monitoring of in vitro and in vivo Degradation of Supramolecular Polymers Applied in Cardiovascular in situ Tissue Engineering.

The equilibrium between scaffold degradation and neotissue formation, is highly essential for in situ tissue engineering. Herein, biodegradable grafts function as temporal roadmap to guide regeneration. The ability to monitor and understand the dynamics of degradation and tissue deposition in in situ cardiovascular graft materials is therefore of great value to accelerate the implementation of safe and sustainable tissue-engineered vascular grafts (TEVGs) as a substitute for conventional prosthetic grafts. In this study, we investigated the potential of Raman microspectroscopy and Raman imaging to monitor degradation kinetics of supramolecular polymers, which are employed as degradable scaffolds in in situ tissue engineering. Raman imaging was applied on in vitro degraded polymers, investigating two different polymer materials, subjected to oxidative and enzymatically-induced degradation. Furthermore, the method was transferred to analyze in vivo degradation of tissue-engineered carotid grafts after 6 and 12 months in a sheep model. Multivariate data analysis allowed to trace degradation and to compare the data from in vitro and in vivo degradation, indicating similar molecular observations in spectral signatures between implants and oxidative in vitro degradation. In vivo degradation appeared to be dominated by oxidative pathways. Furthermore, information on collagen deposition and composition could simultaneously be obtained from the same image scans. Our results demonstrate the sensitivity of Raman microspectroscopy to determine degradation stages and the assigned molecular changes non-destructively, encouraging future exploration of this techniques for time-resolved quality assessment of in situ tissue engineering processes.

Animal studies for the evaluation of in situ tissue-engineered vascular grafts - a systematic review, evidence map, and meta-analysis.

Vascular in situ tissue engineering (TE) is an approach that uses bioresorbable grafts to induce endogenous regeneration of damaged blood vessels. The evaluation of newly developed in situ TE vascular grafts heavily relies on animal experiments. However, no standard for in vivo models or study design has been defined, hampering inter-study comparisons and translational efficiency. To provide input for formulating such standard, the goal of this study was to map all animal experiments for vascular in situ TE using off-the-shelf available, resorbable synthetic vascular grafts. A literature search (PubMed, Embase) yielded 15,896 studies, of which 182 studies met the inclusion criteria (n = 5,101 animals). The reports displayed a wide variety of study designs, animal models, and biomaterials. Meta-analysis on graft patency with subgroup analysis for species, age, sex, implantation site, and follow-up time demonstrated model-specific variations. This study identifies possibilities for improved design and reporting of animal experiments to increase translational value.

Inconsistency in Graft Outcome of Bilayered Bioresorbable Supramolecular Arterial Scaffolds in Rats.

There is a continuous search for the ideal bioresorbable material to develop scaffolds for in situ vascular tissue engineering. As these scaffolds are exposed to the harsh hemodynamic environment during the entire transformation process from scaffold to neotissue, it is of crucial importance to maintain mechanical integrity and stability at all times. Bilayered scaffolds made of supramolecular polycarbonate-ester-bisurea were manufactured using dual electrospinning. These scaffolds contained a porous inner layer to allow for cellular infiltration and a dense outer layer to provide strength. Scaffolds (n = 21) were implanted as an interposition graft into the abdominal aorta of male Lewis rats and explanted after 1, 3, and 5 months in vivo to assess mechanical functionality and neotissue formation upon scaffold resorption. Results demonstrated conflicting graft outcomes despite homogeneity in the experimental group and scaffold production. Most grafts exhibited adverse remodeling, resulting in aneurysmal dilatation and calcification. However, a few grafts did not demonstrate such features, but instead were characterized by graft extension and smooth muscle cell proliferation in the absence of endothelium, while remaining patent throughout the study. We conclude that it remains extremely difficult to anticipate graft development and performance in vivo. Next to rational mechanical design and good performance in vitro, a thorough understanding of the mechanobiological mechanisms governing scaffold-driven arterial regeneration as well as potential influences of surgical procedures is warranted to further optimize scaffold designs. Careful analysis of the differences between preclinical successes and failures, as is done in this study, may provide initial handles for scaffold optimization and standardized surgical procedures to improve graft performance in vivo. Impact statement In situ vascular tissue engineering using cell-free bioresorbable scaffolds is investigated as an off-the-shelf option to grow small caliber arteries inside the body. In this study, we developed a bilayered electrospun supramolecular scaffold with a dense outer layer to provide mechanical integrity and a porous inner layer for cell recruitment and tissue formation. Despite homogenous scaffold properties and mechanical performance in vitro, in vivo testing as rat aorta interposition grafts revealed distinct graft outcomes, ranging from aneurysms to functional arteries. Careful analysis of this variability provided valuable insights into materials-driven in situ artery formation relevant for scaffold design and implantation procedures.

Imparting Immunomodulatory Activity to Scaffolds via Biotin-Avidin Interactions.

Biotin-avidin interactions have been explored for decades as a technique to functionalize biomaterials, as well as for in vivo targeting, but whether changes in these interactions can be leveraged for immunomodulation remain unknown. The goal of this study was to investigate how biotin density and avidin variant can be used to deliver the immunomodulatory cytokine, interleukin 4 (IL4), from a porous gelatin scaffold, Gelfoam, to primary human macrophages in vitro. Here, we demonstrate that the degree of scaffold biotinylation controlled the binding of two different avidin variants, streptavidin and CaptAvidin. Biotinylated scaffolds were also loaded with streptavidin and biotinylated IL4 under flow, suggesting a potential use for targeting this biomaterial in vivo. While biotin-avidin interactions did not appear to influence the protein release in this system, increasing degrees of biotinylation did lead to increased M2-like polarization of primary human macrophages over time in vitro, highlighting the capability to leverage biotin-avidin interactions to modulate the macrophage phenotype. These results demonstrate a versatile and modular strategy to impart immunomodulatory activity to biomaterials.

Distinct Effects of Heparin and Interleukin-4 Functionalization on Macrophage Polarization and In Situ Arterial Tissue Regeneration Using Resorbable Supramolecular Vascular Grafts in Rats.

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months). Routine analyses are augmented with Raman microspectroscopy, in order to acquire the local molecular fingerprints of the resorbing scaffold and developing tissue. Thrombosis is found not to be a confounding factor in any of the groups. Hep-only-functionalized grafts resulted in adverse tissue remodeling, with cases of local intimal hyperplasia. This is negated with the addition of IL-4, which promoted M2 macrophage polarization and more mature neotissue formation. This study shows that with bioactive functionalization, the early inflammatory response can be modulated and affect the composition of neotissue. Nevertheless, variability between graft outcomes is observed within each group, warranting further evaluation in light of clinical translation.

Human In Vitro Model Mimicking Material-Driven Vascular Regeneration Reveals How Cyclic Stretch and Shear Stress Differentially Modulate Inflammation and Matrix Deposition.

Resorbable synthetic scaffolds designed to regenerate living tissues and organs inside the body have emerged as a clinically attractive technology to replace diseased blood vessels. However, mismatches between scaffold design and in vivo hemodynamic loading (i.e., cyclic stretch and shear stress) can result in aberrant inflammation and adverse tissue remodeling, leading to premature graft failure. Yet, the underlying mechanisms remain elusive. Here, a human in vitro model is presented that mimics the transient local inflammatory and biomechanical environments that drive scaffold-guided tissue regeneration. The model is based on the coculture of human (myo)fibroblasts and macrophages in a bioreactor platform that decouples cyclic stretch and shear stress. Using a resorbable supramolecular elastomer as the scaffold material, it is revealed that cyclic stretch initially reduces proinflammatory cytokine secretion and, especially when combined with shear stress, stimulates IL-10 secretion. Moreover, cyclic stretch stimulates downstream (myo)fibroblast proliferation and matrix deposition. In turn, shear stress attenuates cyclic-stretch-induced matrix growth by enhancing MMP-1/TIMP-1-mediated collagen remodeling, and synergistically alters (myo)fibroblast phenotype when combined with cyclic stretch. The findings suggest that shear stress acts as a stabilizing factor in cyclic stretch-induced tissue formation and highlight the distinct roles of hemodynamic loads in the design of resorbable vascular grafts.

Transcatheter-Delivered Expandable Bioresorbable Polymeric Graft With Stenting Capacity Induces Vascular Regeneration.

As the next step in the translation of vascular tissue engineering, this study uniquely combines transcatheter delivery and in situ tissue regeneration using a novel bioresorbable electrospun polymer graft that can be implanted minimally invasively. Once delivered inside a small-diameter vessel, the electrospun microstructure supports the vessel wall, facilitates cellular infiltration, and guides organized tissue formation.

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach-the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis-are discussed in detail.

In Situ Remodeling Overrules Bioinspired Scaffold Architecture of Supramolecular Elastomeric Tissue-Engineered Heart Valves.

In situ tissue engineering that uses resorbable synthetic heart valve scaffolds is an affordable and practical approach for heart valve replacement; therefore, it is attractive for clinical use. This study showed no consistent collagen organization in the predefined direction of electrospun scaffolds made from a resorbable supramolecular elastomer with random or circumferentially aligned fibers, after 12 months of implantation in sheep. These unexpected findings and the observed intervalvular variability highlight the need for a mechanistic understanding of the long-term in situ remodeling processes in large animal models to improve predictability of outcome toward robust and safe clinical application.

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.