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Abstract Organ‐on‐a‐chip (OOC) models based on microfluidic technology are increasingly used to obtain mechanistic insight into (patho)physiological processes in humans, and they hold great promise for application in drug development and regenerative medicine. Despite significant progress in OOC development, several limitations of conventional microfluidic devices pose challenges. First, most microfluidic systems have rectangular cross sections and flat walls, and therefore tubular/ curved structures, like blood vessels and nephrons, are not well represented. Second, polymers used as base materials for microfluidic devices are much stiffer than in vivo extracellular matrix (ECM). Finally, in current cell seeding methods, challenges exist regarding precise control over cell seeding location, unreachable spaces due to flow resistances, and restricted dimensions/geometries. To address these limitations, an alternative cell seeding technique and a corresponding workflow is introduced to create circular cross‐sectioned tubular OOC models by pre‐wrapping cells around sacrificial fiber templates. As a proof of concept, a perfusable renal proximal tubule‐on‐a‐chip is demonstrated with a diameter as small as 50 µm, cellular tubular structures with branches and curvature, and a preliminary vascular‐renal tubule interaction model. The cell pre‐wrapping seeding technique promises to enable the construction of diverse physiological/pathological models, providing tubular OOC systems for mechanistic investigations and drug development.

The dynamic nature of the extracellular matrix (ECM), particularly its stiffness, plays a pivotal role in cellular behavior, especially after myocardial infarction (MI), where cardiac fibroblasts (cFbs) are key in ECM remodeling. This study explores the effects of dynamic stiffness changes on cFb activation and ECM production, addressing a gap in understanding the dynamics of ECM stiffness and their impact on cellular behavior. Utilizing gelatin methacrylate (GelMA) hydrogels, we developed a model to dynamically alter the stiffness of cFb environment through a two-step photocrosslinking process. By inducing a quiescent state in cFbs with a TGF-β inhibitor, we ensured the direct observation of cFbs-responses to the engineered mechanical environment. Our findings demonstrate that the mechanical history of substrates significantly influences cFb activation and ECM-related gene expression. Cells that were initially cultured for 24 h on the soft substrate remained more quiescent when the hydrogel was stiffened compared to cells cultured directly to a stiff static substrate. This underscores the importance of past mechanical history in cellular behavior. The present study offers new insights into the role of ECM stiffness changes in regulating cellular behavior, with significant implications for understanding tissue remodeling processes, such as in post-MI scenarios.

Abstract 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.

Abstract 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.

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.

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.

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.

A method is developed to engineer cerebral tissues with intact 3D neuronal networks, mimicking neuropathological signatures such as the propagation of epileptiform discharges, using a multi-chambered tissue culture chip.

Abstract 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.

Low total energy expenditure (TEE) has been a hypothesized risk factor for weight gain, but longitudinal repeatability of TEE is incompletely understood. Here the authors report that TEE is repeatable for adults, but not for children, and increases in TEE (adjusted for fat-free mass, fat mass, age and sex) are not associated with body composition changes in short-term longitudinal analyses.

The myocardium is a mechanically active tissue typified by anisotropy of the resident cells [cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)] and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding of how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a two-dimensional human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain—mimicking the local anisotropic deformation of beating myocardium—causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs was no contributing factor. Our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study, therefore, highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

Abstract Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.

Mechanical stimuli experienced by vascular smooth muscle cells (VSMCs) and mechanosensitive Notch signaling are important regulators of vascular growth and remodeling. However, the interplay between mechanical cues and Notch signaling, and its contribution to regulate the VSMC phenotype are still unclear. Here, we investigated the role of Notch signaling in regulating strain-mediated changes in VSMC phenotype. Synthetic and contractile VSMCs were cyclically stretched for 48 h to determine the temporal changes in phenotypic features. Different magnitudes of strain were applied to investigate its effect on Notch mechanosensitivity and the phenotypic regulation of VSMCs. In addition, Notch signaling was inhibited via DAPT treatment and activated with immobilized Jagged1 ligands to understand the role of Notch on strain-mediated phenotypic changes of VSMCs. Our data demonstrate that cyclic strain induces a decrease in Notch signaling along with a loss of VSMC contractile features. Accordingly, the activation of Notch signaling during cyclic stretching partially rescued the contractile features of VSMCs. These findings demonstrate that Notch signaling has an important role in regulating strain-mediated phenotypic switching of VSMCs.

van Haaften et al. show numerical-experimental approach to reconstruct the graft–host response. They interrogate the mechanoregulation in dialysis grafts to solve the disturbed shear stress problem, which can be a cause of neointimal hyperplasia in blood vessels and grafts.

Mechanical guidance of tissue morphogenesis is an emerging method of regenerative medicine that can be employed to steer functional kidney architecture for the purpose of bioartificial kidney design or renal tissue engineering strategies. In kidney morphogenesis, apical-basal polarization of renal epithelial cells is paramount for tubule formation and subsequent tissue functions like excretion and resorption. In kidney epithelium, polarization is initiated by integrin-mediated cell-matrix adhesion at the cell membrane. Cellular mechanobiology research has indicated that this integrin-mediated adhesion is responsive to matrix stiffness, raising the possibility to use matrix stiffness as a handle to steer cell polarization. Herein, we evaluate apical-basal polarization in response to 2D substates of different stiffness (1, 10, 50 kPa and glass) in Madin Darby Canine Kidney cells (MDCKs), a classic canine-derived cell model of epithelial polarization, and in tubuloid-derived cells, established from human primary cells derived from adult kidney tissue. Our results show that sub-physiological (1 kPa) substrate stiffness with low integrin-based adhesion induces polarization in MDCKs, while MDCKs on supraphysiological (>10 kPa) stiffness remain unpolarized. Inhibition of integrin, indeed, allows for polarization on the supraphysiological substrates, suggesting that increased cellular adhesion on stiff substrates opposes polarization. In contrast, tubuloid-derived cells do not establish apical-basal polarization on 2D substrates, irrespective of substrate stiffness, despite their ability to polarize in 3D environments. Further analysis implies that the 2D cultured tubuloid-derived cells have a diminished mechanosensitive capacity when presented with different substrate stiffnesses due to immature focal adhesions and the absence of a connection between focal adhesions and the cytoskeleton. Overall, this study demonstrates that apical-basal polarization is a complex process, where cell type, the extracellular environment, and both the mechanical and chemical aspects in cell-matrix interactions performed by integrins play a role.

A fundamental process in the development and progression of heart failure is fibrotic remodeling, characterized by excessive deposition of extracellular matrix proteins in response to injury. Currently, therapies that effectively target and reverse cardiac fibrosis are lacking, warranting novel therapeutic strategies and reliable methods to study their effect. Using a gelatin methacryloyl hydrogel, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and human fetal cardiac fibroblasts (hfCF), we developed a multi-cellular mechanically tunable 3D in vitro model of human cardiac fibrosis. This model was used to evaluate the effects of a promising anti-fibrotic drug—pirfenidone—and yields proof-of-concept of the drug testing potential of this platform. Our study demonstrates that pirfenidone has anti-fibrotic effects but does not reverse all TGF-β1 induced pro-fibrotic changes, which provides new insights into its mechanism of action.

Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease.Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity.Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Tissue homeostasis is perturbed by stressful events, which can lead to organ dysfunction and failure. This is particularly true for the heart, where injury resulting from myocardial infarction or ischemic heart disease can result in a cascading event ultimately ending with the loss of functional myocardial tissue and heart failure. To help reverse this loss of healthy contractile tissue, researchers have spent decades in the hopes of characterizing a cell source capable of regenerating the injured heart. Unfortunately, these strategies have proven to be ineffective. With the goal of truly understanding cardiac regeneration, researchers have focused on the innate regenerative abilities of zebrafish and neonatal mammals. This has led to the realization that although cells play an important role in the repair of the diseased myocardium, inducing cardiac regeneration may instead lie in the composition of the extra cellular milieu, specifically the extra cellular matrix. In this review we will briefly summarize the current knowledge regarding cell sources used for cardiac regenerative approaches, since these have been extensively reviewed elsewhere. More importantly, by revisiting innate cardiac regeneration observed in zebrafish and neonatal mammals, we will stress the importance the extra cellular matrix has on reactivating this potential in the adult myocardium. Finally, we will address how we can harness the ability of the extra cellular matrix to guide cardiac repair thereby setting the stage of next generation regenerative strategies.

Radiation-induced cardiovascular disease is a well-known complication of radiation exposure. Over the last few years, planning for deep space missions has increased interest in the effects of space radiation on the cardiovascular system, as an increasing number of astronauts will be exposed to space radiation for longer periods of time. Research has shown that exposure to different types of particles found in space radiation can lead to the development of diverse cardiovascular disease via fibrotic myocardial remodeling, accelerated atherosclerosis and microvascular damage. Several underlying mechanisms for radiation-induced cardiovascular disease have been identified, but many aspects of the pathophysiology remain unclear. Existing pharmacological compounds have been evaluated to protect the cardiovascular system from space radiation-induced damage, but currently no radioprotective compounds have been approved. This review critically analyzes the effects of space radiation on the cardiovascular system, the underlying mechanisms and potential countermeasures to space radiation-induced cardiovascular disease.

Synthetic vascular grafts to be applied as access grafts for hemodialysis often require anti-kinking properties. Previously, electrospun microporous vascular implants based on synthetic supramolecular materials have been shown to perform adequately as resorbable grafts due to the microstructures, thereby enabling attraction of endogenous cells and consecutive matrix production in situ. Here, we use supramolecular materials based on hydrogen bonding interactions between bisurea (BU) or 2-ureido-4[1H]-pyrimidinones (UPy) to produce microporous anti-kinking tubular structures by combining solution electrospinning with 3D printing. A custom-made rational axis for 3D printing was developed to produce controlled tubular structures with freedom in design in order to print complex tubular architectures without supporting structures. Two different tubular grafts were developed, both composed of a three-layered design with a 3D printed spiral sandwiched in between luminal and adventitial electrospun layers. One tubular scaffold was composed of BU-polycarbonate electrospun layers with 3D printed polycaprolactone (PCL) strands in between for dimensional stability, and the other graft fully consisted of supramolecular polymers, using chain-extended UPy-PCL as electrospun layers and a bifunctional UPy-PCL for 3D printing. Both grafts, with a 3D printed spiral, demonstrated a reproducible dimensional stability and anti-kinking behavior under bending stresses.

Abstract There is a persistent and growing clinical need for readily-available substitutes for heart valves and small-diameter blood vessels. In situ tissue engineering is emerging as a disruptive new technology, providing ready-to-use biodegradable, cell-free constructs which are designed to induce regeneration upon implantation, directly in the functional site. The induced regenerative process hinges around the host response to the implanted biomaterial and the interplay between immune cells, stem/progenitor cell and tissue cells in the microenvironment provided by the scaffold in the hemodynamic environment. Recapitulating the complex tissue microstructure and function of cardiovascular tissues is a highly challenging target. Therein the scaffold plays an instructive role, providing the microenvironment that attracts and harbors host cells, modulating the inflammatory response, and acting as a temporal roadmap for new tissue to be formed. Moreover, the biomechanical loads imposed by the hemodynamic environment play a pivotal role. Here, we provide a multidisciplinary view on in situ cardiovascular tissue engineering using synthetic scaffolds; starting from the state-of-the art, the principles of the biomaterial-driven host response and wound healing and the cellular players involved, toward the impact of the biomechanical, physical, and biochemical microenvironmental cues that are given by the scaffold design. To conclude, we pinpoint and further address the main current challenges for in situ cardiovascular regeneration, namely the achievement of tissue homeostasis, the development of predictive models for long-term performances of the implanted grafts, and the necessity for stratification for successful clinical translation.

Human epicardium-derived cells (hEPDCs) transplanted in the NOD-SCID mouse heart after myocardial infarction (MI) are known to improve cardiac function, most likely orchestrated by paracrine mechanisms that limit adverse remodeling. It is not yet known, however, if hEPDCs contribute to preservation of cardiac function via the secretion of matrix proteins and/or matrix proteases to reduce scar formation. This study describes the ability of hEPDCs to produce human collagen type I after transplantation into the infarct border zone, thereby creating their own extracellular environment. As the in vivo environment is too complex to investigate the mechanisms involved, we use an in vitro set-up, mimicking biophysical and biochemical cues from the myocardial tissue to unravel hEPDC-induced matrix remodeling. The in vivo contribution of hEPDCs to the cardiac extracellular matrix (ECM) was assessed in a historical dataset of the NOD-SCID murine model of experimentally induced MI and cell transplantation. Analysis showed that within 48 h after transplantation, hEPDCs produce human collagen type I. The build-up of the human collagen microenvironment was reversed within 6 weeks. To understand the hEPDCs response to the pathologic cardiac microenvironment, we studied the influence of cyclic straining and/or transforming growth beta (TGFβ) signaling in vitro. We revealed that 48 h of cyclic straining induced collagen type I production via the TGFβ/ALK5 signaling pathway. The in vitro approach enables further unraveling of the hEPDCs ability to secrete matrix proteins and matrix proteases and the potential to create and remodel the cardiac matrix in response to injury.

Cardiac fibroblasts play a key role in chronic heart failure. The conversion from cardiac fibroblast to myofibroblast as a result of cardiac injury, will lead to excessive matrix deposition and a perpetuation of pro-fibrotic signaling. Cardiac cell therapy for chronic heart failure may be able to target fibroblast behavior in a paracrine fashion. However, no reliable human fibrotic tissue model exists to evaluate this potential effect of cardiac cell therapy. Using a gelatin methacryloyl hydrogel and human fetal cardiac fibroblasts (hfCF), we created a 3D in vitro model of human cardiac fibrosis. This model was used to study the possibility to modulate cellular fibrotic responses. Our approach demonstrated paracrine inhibitory effects of cardiac progenitor cells (CPC) on both cardiac fibroblast activation and collagen synthesis in vitro and revealed that continuous cross-talk between hfCF and CPC seems to be indispensable for the observed anti-fibrotic effect.

In situ tissue engineering is a technology in which non-cellular biomaterial scaffolds are implanted in order to induce local regeneration of replaced or damaged tissues. Degradable synthetic electrospun scaffolds are a versatile and promising class of biomaterials for various in situ tissue engineering applications, such as cardiovascular replacements. Functional in situ tissue regeneration depends on the balance between endogenous neo-tissue formation and scaffold degradation. Both these processes are driven by macrophages. Upon invasion into a scaffold, macrophages secrete reactive oxygen species (ROS) and hydrolytic enzymes, contributing to oxidative and enzymatic biomaterial degradation, respectively. This study aims to elucidate the effect of scaffold microarchitecture, i.e., μm-range fiber diameter and fiber alignment, on early macrophage-driven scaffold degradation. Electrospun poly-ε-caprolactone-bisurea (PCL-BU) scaffolds with either 2 or 6 μm (Ø) isotropic or anisotropic fibers were seeded with THP-1 derived human macrophages and cultured in vitro for 4 or 8 days. Our results revealed that macroph age-induced oxidative degradation in particular was dependent on scaffold microarchitecture, with the highest level of ROS-induced lipid peroxidation, NADPH oxidase gene expression and degradation in the 6 μm Ø anisotropic group. Whereas, biochemically polarized macrophages demonstrated a phenotype-specific degradative potential, the observed differences in macrophage degradative potential instigated by the scaffold microarchitecture could not be attributed to either distinct M1 or M2 polarization. This suggests that the scaffold microarchitecture uniquely affects macrophage-driven degradation. These findings emphasize the importance of considering the scaffold microarchitecture in the design of scaffolds for in situ tissue engineering applications and the tailoring of degradation kinetics thereof.

Prestress is a phenomenon present in many cardiovascular tissues and has profound implications on their in vivo functionality. For instance, the in vivo mechanical properties are altered by the presence of prestress, and prestress also influences tissue growth and remodeling processes. The development of tissue prestress typically originates from complex growth and remodeling phenomena which yet remain to be elucidated. One particularly interesting mechanism in which prestress develops is by active traction forces generated by cells embedded in the tissue by means of their actin stress fibers. In order to understand how these traction forces influence tissue prestress, many have used microfabricated, high-throughput, micrometer scale setups to culture microtissues which actively generate prestress to specially designed cantilevers. By measuring the displacement of these cantilevers, the prestress response to all kinds of perturbations can be monitored. In the present study, such a microfabricated tissue gauge platform was combined with the commercially available Flexcell system to facilitate dynamic cyclic stretching of microtissues. First, the setup was validated to quantify the dynamic microtissue stretch applied during the experiments. Next, the microtissues were subjected to a dynamic loading regime for 24 h. After this interval, the prestress increased to levels over twice as high compared to static controls. The prestress in these tissues was completely abated when a ROCK-inhibitor was added, showing that the development of this prestress can be completely attributed to the cell-generated traction forces. Finally, after switching the microtissues back to static loading conditions, or when removing the ROCK-inhibitor, prestress magnitudes were restored to original values. These findings show that intrinsic cell-generated prestress is a highly controlled parameter, where the actin stress fibers serve as a mechanostat to regulate this prestress. Since almost all cardiovascular tissues are exposed to a dynamic loading regime, these findings have important implications for the mechanical testing of these tissues, or when designing cardiovascular tissue engineering therapies.

Geometrical cues provided by the intrinsic architecture of tissues and implanted biomaterials have a high relevance in controlling cellular behavior. Knowledge of how cells sense and subsequently respond to complex geometrical cues of various sizes and origins is needed to understand the role of the architecture of the extracellular environment as a cell-instructive parameter. This is of particular interest in the field of tissue engineering, where the success of scaffold-guided tissue regeneration largely depends on the formation of new tissue in a native-like organization in order to ensure proper tissue function. A well-considered internal scaffold design (i.e., the inner architecture of the porous structure) can largely contribute to the desired cell and tissue organization. Advances in scaffold production techniques for tissue engineering purposes in the last years have provided the possibility to accurately create scaffolds with defined macroscale external and microscale internal architectures. Using the knowledge of how cells sense geometrical cues of different size ranges can drive the rational design of scaffolds that control cellular and tissue architecture. This concise review addresses the recently gained knowledge of the sensory mechanisms of cells towards geometrical cues of different sizes (from the nanometer to millimeter scale) and points out how this insight can contribute to informed architectural scaffold designs.

In situ heart valve tissue engineering using cell-free synthetic, biodegradable scaffolds is under development as a clinically attractive approach to create living valves right inside the heart of a patient. In this approach, a valve-shaped porous scaffold “implant” is rapidly populated by endogenous cells that initiate neo-tissue formation in pace with scaffold degradation. While this may constitute a cost-effective procedure, compatible with regulatory and clinical standards worldwide, the new technology heavily relies on the development of advanced biomaterials, the processing thereof into (minimally invasive deliverable) scaffolds, and the interaction of such materials with endogenous cells and neo-tissue under hemodynamic conditions. Despite the first positive preclinical results and the initiation of a small-scale clinical trial by commercial parties, in situ tissue formation is not well understood. In addition, it remains to be determined whether the resulting neo-tissue can grow with the body and preserves functional homeostasis throughout life. More important yet, it is still unknown if and how in situ tissue formation can be controlled under conditions of genetic or acquired disease. Here, we discuss the recent advances of material-based in situ heart valve tissue engineering and highlight the most critical issues that remain before clinical application can be expected. We argue that a combination of basic science – unveiling the mechanisms of the human body to respond to the implanted biomaterial under (patho)physiological conditions – and technological advancements – relating to the development of next generation materials and the prediction of in situ tissue growth and adaptation – is essential to take the next step towards a realistic and rewarding translation of in situ heart valve tissue engineering.

Background Valvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with 90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2‐dimensional in vitro culture and their utility in studying valve pathobiology. Methods and Results Within 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α‐smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation. Conclusions The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.

The microenvironment plays a crucial role in the behavior of stem and progenitor cells. In the heart, cardiac progenitor cells (CPCs) reside in specific niches, characterized by key components that are altered in response to a myocardial infarction. To date, there is a lack of knowledge on these niches and on the CPC interplay with the niche components. Insight into these complex interactions and into the influence of microenvironmental factors on CPCs can be used to promote the regenerative potential of these cells. In this review, we discuss cardiac resident progenitor cells and their regenerative potential and provide an overview of the interactions of CPCs with the key elements of their niche. We focus on the interaction between CPCs and supporting cells, extracellular matrix, mechanical stimuli, and soluble factors. Finally, we describe novel approaches to modulate the CPC niche that can represent the next step in recreating an optimal CPC microenvironment and thereby improve their regeneration capacity.

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.

In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.

The paradigm of regenerative medicine has recently shifted from in vitro to in situ tissue engineering: implanting a cell-free, biodegradable, off-the-shelf available scaffold and inducing the development of functional tissue by utilizing the regenerative potential of the body itself. This approach offers a prospect of not only alleviating the clinical demand for autologous vessels but also circumventing the current challenges with synthetic grafts. In order to move towards a hypothesis-driven engineering approach, we review three crucial aspects that need to be taken into account when regenerating vessels: (1) the structure-function relation for attaining mechanical homeostasis of vascular tissues, (2) the environmental cues governing cell function, and (3) the available experimental platforms to test instructive scaffolds for in situ tissue engineering. The understanding of cellular responses to environmental cues leads to the development of computational models to predict tissue formation and maturation, which are validated using experimental platforms recapitulating the (patho)physiological micro-environment. With the current advances, a progressive shift is anticipated towards a rational and effective approach of building instructive scaffolds for in situ vascular tissue regeneration.