Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Off-the-Shelf Synthetic Biodegradable Grafts Transform In Situ into a Living Arteriovenous Fistula in a Large Animal Model.

Current vascular access options require frequent interventions. In situ tissue engineering (TE) may overcome these limitations by combining the initial success of synthetic grafts with long-term advantages of autologous vessels by using biodegradable grafts that transform into autologous vascular tissue at the site of implantation. Scaffolds (6 mm-Ø) made of supramolecular polycarbonate-bisurea (PC-BU), with a polycaprolactone (PCL) anti-kinking-coil, are implanted between the carotid artery and jugular vein in goats. A subset is bio-functionalized using bisurea-modified-Stromal cell-derived factor-1α (SDF1α) derived peptides and ePTFE grafts as controls. Grafts are explanted after 1 and 3 months, and evaluated for material degradation, tissue formation, compliance, and patency. At 3 months, the scaffold is resorbed and replaced by vascular neo-tissue, including elastin, contractile markers, and endothelial lining. No dilations, ruptures, or aneurysms are observed and grafts are successfully cannulated at termination. SDF-1α-peptide-biofunctionalization does not influence outcomes. Patency is lower in TE grafts (50%) compared to controls (100% patency), predominantly caused by intimal hyperplasia. Rapid remodeling of a synthetic, biodegradable vascular scaffold into a living, compliant arteriovenous fistula is demonstrated in a large animal model. Despite lower patency compared to ePTFE, transformation into autologous and compliant living tissue with self-healing capacity may have long-term advantages.

Balancing Scaffold Degradation and Neo-Tissue Formation in In Situ Tissue Engineered Vascular Grafts.

An essential aspect of cardiovascular in situ tissue engineering (TE) is to ensure balance between scaffold degradation and neo-tissue formation. We evaluated the rate of degradation and neo-tissue formation of three electrospun supramolecular bisurea-based biodegradable scaffolds that differ in their soft-block backbone compositions only. Scaffolds were implanted as interposition grafts in the abdominal aorta in rats, and evaluated at different time points (t = 1, 6, 12, 24, and 40 weeks) on function, tissue formation, strength, and scaffold degradation. The fully carbonate-based biomaterial showed minor degradation after 40 weeks in vivo, whereas the other two ester-containing biomaterials showed (near) complete degradation within 6-12 weeks. Local dilatation was only observed in these faster degrading scaffolds. All materials showed to some extent mineralization, at early as well as late time points. Histological evaluation showed equal and non-native-like neo-tissue formation after total degradation. The fully carbonate-based scaffolds lagged in neo-tissue formation, presumably as its degradation was (far from) complete at 40 weeks. A significant difference in vessel wall contrast enhancement was observed by magnetic resonance imaging between grafts with total compared with minimal-degraded scaffolds.

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.

SFAlab: image-based quantification of mechano-active ventral actin stress fibers in adherent cells.

Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.

Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells.

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

Hydrogels for Cardiac Restorative Support: Relevance of Gelation Mechanisms for Prospective Clinical Use.

PURPOSE OF REVIEW: Cardiac tissue regenerative strategies have gained much traction over the years, in particular those utilizing hydrogels. With our review, and with special focus on supporting post-myocardial infarcted tissue, we aim to provide insights in determining crucial design considerations of a hydrogel and the implications these could have for future clinical use. RECENT FINDINGS: To date, two hydrogel delivery strategies are being explored, cardiac injection or patch, to treat myocardial infarction. Recent advances have demonstrated that the mechanism by which a hydrogel is gelated (i.e., physically or chemically cross-linked) not only impacts the biocompatibility, mechanical properties, and chemical structure, but also the route of delivery of the hydrogel and thus its effect on cardiac repair. With regard to cardiac regeneration, various hydrogels have been developed with the ability to function as a delivery system for therapeutic strategies (e.g., drug and stem cells treatments), as well as a scaffold to guide cardiac tissue regeneration following myocardial infarction. However, these developments remain within the experimental and pre-clinical realm and have yet to transition towards the clinical setting.

Shape-Morphing Photoresponsive Hydrogels Reveal Dynamic Topographical Conditioning of Fibroblasts.

The extracellular environment defines a physical boundary condition with which cells interact. However, to date, cell response to geometrical environmental cues is largely studied in static settings, which fails to capture the spatiotemporally varying cues cells receive in native tissues. Here, a photoresponsive spiropyran-based hydrogel is presented as a dynamic, cell-compatible, and reconfigurable substrate. Local stimulation with blue light (455 nm) alters hydrogel swelling, resulting in on-demand reversible micrometer-scale changes in surface topography within 15 min, allowing investigation into cell response to controlled geometry actuations. At short term (1 h after actuation), fibroblasts respond to multiple rounds of recurring topographical changes by reorganizing their nucleus and focal adhesions (FA). FAs form primarily at the dynamic regions of the hydrogel; however, this propensity is abolished when the topography is reconfigured from grooves to pits, demonstrating that topographical changes dynamically condition fibroblasts. Further, this dynamic conditioning is found to be associated with long-term (72 h) maintenance of focal adhesions and epigenetic modifications. Overall, this study offers a new approach to dissect the dynamic interplay between cells and their microenvironment and shines a new light on the cell's ability to adapt to topographical changes through FA-based mechanotransduction.

How Smart are Smart Materials? A Conceptual and Ethical Analysis of Smart Lifelike Materials for the Design of Regenerative Valve Implants.

It may soon become possible not just to replace, but to re-grow healthy tissues after injury or disease, because of innovations in the field of Regenerative Medicine. One particularly promising innovation is a regenerative valve implant to treat people with heart valve disease. These implants are fabricated from so-called 'smart', 'lifelike' materials. Implanted inside a heart, these implants stimulate re-growth of a healthy, living heart valve. While the technological development advances, the ethical implications of this new technology are still unclear and a clear conceptual understanding of the notions 'smart' and 'lifelike' is currently lacking. In this paper, we explore the conceptual and ethical implications of the development of smart lifelike materials for the design of regenerative implants, by analysing heart valve implants as a showcase. In our conceptual analysis, we show that the materials are considered 'smart' because they can communicate with human tissues, and 'lifelike' because they are structurally similar to these tissues. This shows that regenerative valve implants become intimately integrated in the living tissues of the human body. As such, they manifest the ontological entanglement of body and technology. In our ethical analysis, we argue this is ethically significant in at least two ways: It exacerbates the irreversibility of the implantation procedure, and it might affect the embodied experience of the implant recipient. With our conceptual and ethical analysis, we aim to contribute to responsible development of smart lifelike materials and regenerative implants.

Greater male variability in daily energy expenditure develops through puberty.

There is considerably greater variation in metabolic rates between men than between women, in terms of basal, activity and total (daily) energy expenditure (EE). One possible explanation is that EE is associated with male sexual characteristics (which are known to vary more than other traits) such as musculature and athletic capacity. Such traits might be predicted to be most prominent during periods of adolescence and young adulthood, when sexual behaviour develops and peaks. We tested this hypothesis on a large dataset by comparing the amount of male variation and female variation in total EE, activity EE and basal EE, at different life stages, along with several morphological traits: height, fat free mass and fat mass. Total EE, and to some degree also activity EE, exhibit considerable greater male variation (GMV) in young adults, and then a decreasing GMV in progressively older individuals. Arguably, basal EE, and also morphometrics, do not exhibit this pattern. These findings suggest that single male sexual characteristics may not exhibit peak GMV in young adulthood, however total and perhaps also activity EE, associated with many morphological and physiological traits combined, do exhibit GMV most prominently during the reproductive life stages.

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.

Cyclic strain has antifibrotic effects on the human cardiac fibroblast transcriptome in a human cardiac fibrosis-on-a-chip platform.

In cardiac fibrosis, in response to stress or injury, cardiac fibroblasts deposit excessive amounts of collagens which contribute to the development of heart failure. The biochemical stimuli in this process have been extensively studied, but the influence of cyclic deformation on the fibrogenic behavior of cardiac fibroblasts in the ever-beating heart is not fully understood. In fact, most investigated mechanotransduction pathways in cardiac fibroblasts seem to ultimately have profibrotic effects, which leaves an important question in cardiac fibrosis research unanswered: how do cardiac fibroblasts stay quiescent in the ever-beating human heart? In this study, we developed a human cardiac fibrosis-on-a-chip platform and utilized it to investigate if and how cyclic strain affects fibrogenic signaling. The pneumatically actuated platform can expose engineered tissues to controlled strain magnitudes of 0-25% - which covers the entire physiological and pathological strain range in the human heart - and to biochemical stimuli and enables high-throughput screening of multiple samples. Microtissues of human fetal cardiac fibroblasts (hfCF) embedded in gelatin methacryloyl (GelMA) were 3D-cultured on this platform and exposed to strain conditions which mimic the healthy human heart. The results provide evidence of an antifibrotic effect of the applied strain conditions on cardiac fibroblast behavior, emphasizing the influence of biomechanical stimuli on the fibrogenic process and giving a detailed overview of the mechanosensitive pathways and genes involved, which can be used in the development of novel therapies against cardiac fibrosis.

The Importance of Effective Ligand Concentration to Direct Epithelial Cell Polarity in Dynamic Hydrogels.

Epithelial cysts and organoids are multicellular hollow structures formed by correctly polarized epithelial cells. Important in steering these cysts from single cells is the dynamic regulation of extracellular matrix presented ligands, and matrix dynamics. Here, control over the effective ligand concentration is introduced, decoupled from bulk and local mechanical properties, in synthetic dynamic supramolecular hydrogels formed through noncovalent crosslinking of supramolecular fibers. Control over the effective ligand concentration is realized by 1) keeping the ligand concentration constant, but changing the concentration of nonfunctionalized molecules or by 2) varying the ligand concentration, while keeping the concentration of non-functionalized molecules constant. The results show that in 2D, the effective ligand concentration within the supramolecular fibers rather than gel stiffness (from 0.1 to 8 kPa) regulates epithelial polarity. In 3D, increasing the effective ligand concentration from 0.5 × 10-3 to 2 × 10-3 m strengthens the effect of increased gel stiffness from 0.1 to 2 kPa, to synergistically yield more correctly polarized cysts. Through integrin manipulation, it is shown that epithelial polarity is regulated by tension-based homeostasis between cells and matrix. The results reveal the effective ligand concentration as influential factor in regulating epithelial polarity and provide insights on engineering of synthetic biomaterials for cell and organoid culture.

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.

The effect of chronic kidney disease on tissue formation of in situ tissue-engineered vascular grafts.

Vascular in situ tissue engineering encompasses a single-step approach with a wide adaptive potential and true off-the-shelf availability for vascular grafts. However, a synchronized balance between breakdown of the scaffold material and neo-tissue formation is essential. Chronic kidney disease (CKD) may influence this balance, lowering the usability of these grafts for vascular access in end-stage CKD patients on dialysis. We aimed to investigate the effects of CKD on in vivo scaffold breakdown and tissue formation in grafts made of electrospun, modular, supramolecular polycarbonate with ureido-pyrimidinone moieties (PC-UPy). We implanted PC-UPy aortic interposition grafts (n = 40) in a rat 5/6th nephrectomy model that mimics systemic conditions in human CKD patients. We studied patency, mechanical stability, extracellular matrix (ECM) components, total cellularity, vascular tissue formation, and vascular calcification in CKD and healthy rats at 2, 4, 8, and 12 weeks post-implantation. Our study shows successful in vivo application of a slow-degrading small-diameter vascular graft that supports adequate in situ vascular tissue formation. Despite systemic inflammation associated with CKD, no influence of CKD on patency (Sham: 95% vs CKD: 100%), mechanical stability, ECM formation (Sirius red+, Sham 16.5% vs CKD 25.0%-p:0.83), tissue composition, and immune cell infiltration was found. We did find a limited increase in vascular calcification at 12 weeks (Sham 0.08% vs CKD 0.80%-p:0.02) in grafts implanted in CKD animals. However, this was not associated with increased stiffness in the explants. Our findings suggest that disease-specific graft design may not be necessary for use in CKD patients on dialysis.

Methacrylated human recombinant collagen peptide as a hydrogel for manipulating and monitoring stiffness-related cardiac cell behavior.

Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14 days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.

Transiently heritable fates and quorum sensing drive early IFN-I response dynamics.

Type I interferon (IFN-I)-mediated antiviral responses are central to host defense against viral infections. Crucial is the tight and well-orchestrated control of cellular decision-making leading to the production of IFN-Is. Innovative single-cell approaches revealed that the initiation of IFN-I production is limited to only fractions of 1-3% of the total population, both found in vitro, in vivo, and across cell types, which were thought to be stochastically regulated. To challenge this dogma, we addressed the influence of various stochastic and deterministic host-intrinsic factors on dictating early IFN-I responses, using a murine fibroblast reporter model. Epigenetic drugs influenced the percentage of responding cells. Next, with the classical Luria-Delbrück fluctuation test, we provided evidence for transient heritability driving responder fates, which was verified with mathematical modeling. Finally, while studying varying cell densities, we substantiated an important role for cell density in dictating responsiveness, similar to the phenomenon of quorum sensing. Together, this systems immunology approach opens up new avenues to progress the fundamental understanding on cellular decision-making during early IFN-I responses, which can be translated to other (immune) signaling systems.

When we start to develop a cold, influenza or another viral infection, some of our cells produce signaling molecules known as type I interferons (or IFN-Is for short). These early IFN-I signals establish defenses against viruses in both infected and as yet uninfected cells. If the cells produce too much IFN-Is, however, it can result in uncontrolled inflammation that may harm the body and cause life threatening illness. Individual cells need to tightly control how much IFN-Is they produce and match this with the course of the viral infection. They also need to assess how much IFN-I their neighbors are producing and adjust their behavior accordingly. Cells have evolved a myriad of mechanisms to ensure the right amounts of IFN-Is are produced in different circumstances. Broadly, these mechanisms can be divided into two categories: stochastic regulation and deterministic regulation. Stochastic regulation occurs when individual cells receive the exact same information, but this leads to different outcomes, such as, different cells producing various quantities of IFN-Is. In contrast, deterministic regulation causes the same outcome in different cells independent on the information they receive. It was thought that stochastic regulation is the main driver of early IFN-1 responses, but recently a handful of studies have reported deterministic regulation being primarily responsible, instead. Here, Van Eyndhoven et al. explored the roles of both types of regulation in the early IFN-I responses of mouse cells. Van Eyndhoven et al. used genetic approaches and mathematical modelling to show that the fraction of cells that initiate early IFN-I responses can be considered deterministic. Moreover, this deterministic feature turned out to be heritable, such that the fate to produce IFN-I gets passed on for several generations of cells. Additionally, the experiments suggest that cell density, that is, how tightly packed together the cells are, plays an important role in controlling how many cells make IFN-I, with a lower cell density resulting in a higher fraction of cells producing IFN-Is. The findings of Van Eyndhoven et al. add to a growing body of evidence reporting heritable states that can guide decision-making in individual cells. Furthermore, it revises our view on how individual immune cells coordinate population-wide responses.

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.

Engineering Strategies to Move from Understanding to Steering Renal Tubulogenesis.

Rebuilding the kidney in the context of tissue engineering offers a major challenge as the organ is structurally complex and has a high variety of specific functions. Recreation of kidney function is inherently connected to the formation of tubules since the functional subunit of the kidney, the nephron, is based on tubular structures. In vivo, tubulogenesis culminates in a perfectly shaped, patterned, and functional renal tubule via different morphogenic processes that depend on delicately orchestrated chemical, physical, and mechanical interactions between cells and between cells and their microenvironment. This review summarizes the current understanding of the role of the microenvironment in the morphogenic processes involved in in vivo renal tubulogenesis. We highlight the current state-of-the-art of renal tubular engineering and provide a view on the design elements that can be extracted from these studies. Next, we discuss how computational modeling can aid in specifying and identifying design parameters and provide directions on how these design parameters can be incorporated in biomaterials for the purpose of engineering renal tubulogenesis. Finally, we propose that a step-by-step reciprocal interaction between understanding and engineering is necessary to effectively guide renal tubulogenesis. Impact statement Tubular tissue engineering lies at the foundation of regenerating kidney tissue function, as the functional subunit of the kidney, the nephron, is based on tubular structures. Guiding renal tubulogenesis toward functional renal tubules requires in-depth knowledge of the developmental processes that lead to the formation of native tubules as well as engineering approaches to steer these processes. In this study, we review the role of the microenvironment in the developmental processes that lead to functional renal tubules and give directions how this knowledge can be harnessed for biomaterial-based tubular engineering using computational models.