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In situ vascular tissue engineering aims to regenerate vessels "at the target site" using synthetic scaffolds that are capable of inducing endogenous regeneration. Critical to the success of this approach is a fine balance between functional neo-tissue formation and scaffold degradation. Circulating immune cells are important regulators of this process as they drive the host response to the scaffold and they play a central role in scaffold resorption. Despite the progress made with synthetic scaffolds, little is known about the host response and neo-tissue development during and after scaffold resorption. In this study, we designed a fast-degrading biodegradable supramolecular scaffold for arterial applications and evaluated this development in vivo. Bisurea-modified polycaprolactone (PCL2000-U4U) was electrospun in tubular scaffolds and shielded by non-degradable expanded polytetrafluoroethylene in order to restrict transmural and transanastomotic cell ingrowth. In addition, this shield prevented graft failure, permitting the study of neo-tissue and host response development after degradation. Scaffolds were implanted in 60 healthy male Lewis rats as an interposition graft into the abdominal aorta and explanted at different time points up to 56 days after implantation to monitor sequential cell infiltration, differentiation, and tissue formation in the scaffold. Endogenous tissue formation started with an acute immune response, followed by a dominant presence of pro-inflammatory macrophages during the first 28 days. Next, a shift towards tissue-producing cells was observed, with a striking increase in α-Smooth Muscle Actin-positive cells and extracellular matrix by day 56. At that time, the scaffold was resorbed and immune markers were low. These results suggest that neo-tissue formation was still in progress, while the host response became quiescent, favoring a regenerative tissue outcome. Future studies should confirm long-term tissue homeostasis, but require the strengthening of the supramolecular scaffold if a non-shielded model will be used. [ABSTRACT FROM AUTHOR]

It is estimated that the number of patients requiring heart valve replacement will triple over the next five decades. None of the current replacement valves can fully restore native valve function because they lack growth and remodeling capabilities. Heart valve tissue engineering is a promising technology to overcome these limitations. Various approaches are being employed, either aimed at development of the valve substitute in vitro or at the use of the regenerative potential of the body (in situ) for the tissue culture phase. This review provides an overview of the progress within both the in vitro and in situ tissue engineering approaches for trileaflet heart valve tissue engineering. Current challenges with these approaches are discussed, focusing in particular on the use of synthetic scaffold materials. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: This research was financially supported by the Gravitation Program “Materials Driven Regeneration,” funded by the Netherlands Organization for Scientific Research (024.003.013), and the More Knowledge with Fewer Animals program by ZonMw (Practice in Synthesis of Evidence grant 114024119). Dr Akiva has received support from the Netherlands Organization for Scientific Research through a VENI grant (VI.Veni.192.094). Dr Bouten is a shareholder of Xeltis BV. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

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., (© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

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-1β). 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.

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.

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.

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., Competing Interests: This research forms part of the iValve-II project, powered by Health∼Holland, top sector Life Sciences and Health (Grant number TTTI1403B), supported by the Dutch Ministry of Economic Affairs. This work was supported by the Netherlands Cardiovascular Research Initiative (CVON 2012-01): The Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, the Royal Netherlands Academy of Sciences, and by the Gravitation Program “Materials Driven Regeneration,” funded by the Netherlands Organization for Scientific Research (024.003.013). Dr. Aikawa is supported by grants from the National Institutes of Health (R01 HL114805, R01 HL136431, R01 HL147095). Dr. Bouten is a shareholder of Xeltis BV. Drs. Tristan and Mes are employees of Suprapolix BV. Dr. Brugmans is an employee of Xeltis BV. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2020 The Authors.)