The organized myofiber structure within the myocardium indicates its mechanical anisotropy. By projecting the MR Elastography (MRE) stiffness matrix along either the myocardial fiber or sheet orientations determined by Diffusion Tensor Imaging (DTI), anisotropic MRE (aMRE) maps axial and transverse shear and Young's moduli into three tensile and six shear deformation modes. Ten healthy ex vivo swine hearts were imaged three times at 3T using MRE and DTI sequences. aMRE results showed a within-subject coefficient of variation at 19% for the fiber model and 28% for the sheet model across specimens and metrics, with coefficients lower than 15% for seven of the ten specimens across models. Repeatability coefficient of ±0.5 kPa for Young's moduli and ±0.17 kPa for shear's moduli, demonstrating repeatability within the 95% agreement limit. Isotropic MRE underestimated stiffnesses by 31% compared to aMRE, where anisotropic moduli aligned more closely with prior finite element studies and some mechanical loading tests. The myocardium's anisotropic elasticity reflects with its helicoidal myofiber microstructure, with mid-wall circumferential fibers requiring twice the force to deform as longitudinal fibers at the epicardium or endocardium. At the mid-wall, fiber model values were μax = 1.9 ± 0.1 kPa, μtra = 1.3 ± 0.1 kPa, Eax = 5.6 ± 0.4 kPa, and Etra = 3.8 ± 0.3 kPa. Identified deformation modes included: (FF), (NN), (FF or SS), (NN or SS), (SN or NS), (FN or FS), (SF or FS), and (SN or NF), where N is normal to both fiber (F) and sheet (S) orientations. By aligning elasticity matrices more accurately with myocardial architecture than isotropic MRE, aMRE was able to reliably measure shear and Young's moduli in ex vivo swine hearts. These mappings of deformation modes may bring myocardial stiffness assessment closer to clinical application, providing a foundation for a non-invasive methodology capable of true mechanical characterization of the cardiac wall using MR imaging. STATEMENT OF SIGNIFICANCE: The myocardium's anisotropic elasticity, due to its helicoidal myofiber structure, is revealed through anisotropic MR elastography, using fiber and sheet elastic models. Mid-wall circumferential fibers require twice the force to deform equally compared to epicardial or endocardial fibers. Characterizing shear and Young's moduli across cardiac modes offers noninvasive measures of ventricular compliance, comparable to pressure-volume relationships. This could enhance early diagnosis of "stiff heart syndrome" and clarify its underlying mechanisms. Additionally, it aids understanding of myocardial pathologies, including amyloidosis, hypertrophic and dilated cardiomyopathies, and ischemic damage. By characterizing tensile and shear interactions, it may inform diagnosis and treatment of conduction issues and arrhythmia, where tissue has lost its normal mechanical behavior, while patient-specific models could optimize surgical and therapeutic outcomes.

Tattoo-based electronics have emerged as a transformative platform for next-generation wearable bioelectronics. Unlike conventional wearable devices, which rely on substrates, tattoo electrodes are directly formed or transferred onto the skin or internal organs, ensuring superior comfort, breathability, and long-term usability. This intimate interface minimizes motion-induced artifacts and enables reliable biosignal acquisition across diverse physiological and anatomical regions. However, the absence of a supporting substrate imposes unique challenges in fabrication and material design. The fabrication processes must be tailored to accommodate direct skin application, and the selection of functional materials is more constrained. Materials must not only be biocompatible and flexible but also capable of maintaining performance under the dynamic conditions of the human body. This review presents a comprehensive overview of tattoo electrode technology, beginning with fabrication strategies, including direct and indirect patterning methods. We then discuss a range of materials, such as metallic networks, carbon-based materials, polymers, and materials recently being studied. Finally, we explore the diverse applications of tattoo electrodes in strain and electrophysiological sensing, temperature and humidity detection, biochemical monitoring, and energy harvesting and storage. Through this review, we aim to highlight the potential and future directions of tattoo-based electronic systems.

Heart failure (HF) mediated by cardiac fibrosis (CF) is characterized by an excessive accumulation of collagen-based extracellular matrix (ECM) in the myocardium. CF is a common pathophysiological condition in many heart diseases and can be distinctly categorized into two types: replacement and interstitial. In ischemic heart diseases, sudden loss of cardiomyocytes leads to the replacement of CF to prevent ventricular rupture. In contrast, excessive collagen deposition in the interstitial space between cardiomyocytes (often in response to pressure overload, chronic cardiac stress, hypertension, etc.) is termed interstitial CF. The progression of HF due to cardiac fibrosis is mainly driven by compromised diastolic function, resulting from increased stiffness of the heart wall muscle due to collagen-based scar formation. Increased myocardial stiffness is primarily catalyzed by the differential cross linking of deposited collagens forming the scar in the fibrotic heart. Although collagen deposition remained a hallmark of fibrosis, the pathophysiological progression due to biochemical alterations and mechanistic discrepancy of collagens across cardiac fibrosis subtypes remains elusive. With the advent of next-generation RNA sequencing and high-resolution mass spectrometry, mechanistic insights into collagen-mediated scar maturation have gained impetus. A deeper understanding of the spatiocellular transcriptional heterogeneity and site-specific collagen posttranslational modifications (PTMs) in maneuvering ECM remodeling is gaining attention. The unexplored mechanisms of posttranslational modifications and subsequent collagen cross linking in various cardiac fibrosis may provide the prime target for therapeutic interventions. This review comprehensively summarizes the detailed pattern, role, signaling, and mechanical contributions of different collagens and their PTMs, including cross-linking patterns as newer therapeutic regimens during cardiac fibrosis.

Myocardial work assessment has emerged as a promising tool for left ventricular (LV) performance evaluation. Existing non-invasive methods for assessing it rely on assumptions on LV pressure and geometry. Recently, shear wave elastography allowed to quantify changes in myocardial stiffness throughout the cardiac cycle. Based on Hooke's law, it becomes theoretically possible to calculate myocardial stress and work from myocardial stiffness and strain measurements. The main objective of this study is to demonstrate the feasibility of this comprehensive ultrasound approach and to compare myocardial work values between populations where variations are anticipated.

Children with hypertrophic cardiomyopathy (HCM), aortic stenosis (AS) and healthy volunteers (HV) were included in this study. Segment dimensions, strain, thickness, and segmental myocardial stiffness were assessed in the basal antero-septal segment throughout the cardiac cycle. One-beat segmental work, the stress-strain loop area, and contributive and dissipative work were compared between groups. Twenty HV (9.8 ± 5.3 years of age), 20 HCM (10.0 ± 6.1 years of age), and 5 AS (5.3 ± 4.3 years of age) subjects were included. One-beat segmental work was significantly higher in AS (272.0 ± 102.9 µJ/mm) and lower in HCM (38.2 ± 106.9 µJ/mm) compared with HV (131.1 ± 83.3 µJ/mm), P = 0.02 and P = 0.01, respectively. Desynchronized work was prevailing in HCM with dissipative work during systole measured at 17.3 ± 28.9 µJ/mm and contributive work during diastole measured at 15.3 ± 18.0 µJ/mm. The stress-strain loop area was higher in AS (95.2 ± 31.1 kPa%) and HV (66.2 ± 35.9 kPa%) than in HCM (5.8 ± 13.0 kPa%), P < 0.01.

Calculating segmental myocardial work based on myocardial stiffness and strain measurements is technically feasible. This approach overcomes the inherent limitations of current methods by introducing a direct quantitative measure of myocardial stress.

Cardiovascular patient-specific phantoms can improve patient care through testing and simulation. However, materials like silicone and 3D-printing polymers differ mechanically from biological tissues. Agilus30 Clear, the primary material for 3D-printed phantoms, is much stiffer, nearly isotropic, and lacks strain-hardening behavior. To overcome these challenges, a novel 3D voxel-printing approach may provide an effective solution.

This study aimed to explore the applicability of 3D voxel printing, assess how different parameters (strand structure, density, and orientation) affect mechanical properties, and compare them to established phantom materials and porcine cardiovascular tissues. Progressive uniaxial cyclic tension tests were performed across nine stages, varying strain rates and target strain levels, with elastic modulus calculated for comparison. The goal was to stepwise assess whether the overall material stiffness can be reduced, achieving anisotropy and replicating strain-hardening behavior.

In the first step, varying the strand density, the tested samples showed a 0%-60% strain modulus of elasticity of 0.215-0.278 N/mm2, representing a 4-5-fold reduction in elastic modulus compared to that of the base material, Agilus30 Clear. In the second step, varying the orientation of the structures had a significant influence on the elastic modulus, which was measured. The 0%-60% modulus of elasticity decreased to 0.161-0.192 N/mm2, displaying anisotropic material behavior. In the third step, two strand structures specifically designed to mimic fiber recruitment were tested. These resulted in slightly flatter (more linear) stress-strain curves compared to the non-linear strain-softening behavior observed in Agilus30 Clear. However, they still fell short of replicating the desired non-linear strain-hardening behavior characteristic of fiber recruitment in cardiovascular tissues.

The novel 3D voxel-printing material approach resulted in reduced elastic modulus, anisotropic behavior, and strain-hardening properties, providing a much closer representation of the mechanical behavior of porcine cardiovascular tissues compared to other available phantom materials. However, there is still significant potential for optimization through further exploration of fiber recruitment replication.

Since the turn of the millennium, computational modelling of biological systems has evolved remarkably and sees matured use spanning basic and clinical research. While the topic of the peri-millennial debate about the virtues and limitations of 'reductionism and integrationism' seems less controversial today, a new apparent dichotomy dominates discussions: mechanistic versus data-driven modelling. In light of this distinction, we provide an overview of recent achievements and new challenges with a focus on the cardiovascular system. Attention has shifted from generating a universal model of the human to either models of individual humans (digital twins) or entire cohorts of models representative of clinical populations to enable in silico clinical trials. Disease-specific parametrization, inter-individual and intra-individual variability, uncertainty quantification as well as interoperable, standardized and quality-controlled data are important issues today, which call for open tools, data and metadata standards, as well as strong community interactions. The quantitative, biophysical and highly controlled approach provided by in silico methods has become an integral part of physiological and medical research. In silico methods have the potential to accelerate future progress also in the fields of integrated multi-physics modelling, multi-scale models, virtual cohort studies and machine learning beyond what is feasible today. In fact, mechanistic and data-driven modelling can complement each other synergistically and fuel tomorrow's artificial intelligence applications to further our understanding of physiology and disease mechanisms, to generate new hypotheses and assess their plausibility, and thus to contribute to the evolution of preventive, diagnostic and therapeutic approaches.This article is part of the theme issue 'Science into the next millennium: 25 years on'.

Matrix-derived biophysical cues are known to regulate the activation of fibroblasts and their subsequent transdifferentiation into myofibroblasts1-6, but whether modulation of these signals can suppress fibrosis in intact tissues remains unclear, particularly in the cardiovascular system7-10. Here we demonstrate across multiple scales that inhibition of matrix mechanosensing in persistently activated cardiac fibroblasts potentiates-in concert with soluble regulators of the TGFβ pathway-a robust transcriptomic, morphological and metabolic shift towards quiescence. By conducting a meta-analysis of public human and mouse single-cell sequencing datasets, we identify the focal-adhesion-associated tyrosine kinase SRC as a fibroblast-enriched mechanosensor that can be targeted selectively in stromal cells to mimic the effects of matrix softening in vivo. Pharmacological inhibition of SRC by saracatinib, coupled with TGFβ suppression, induces synergistic repression of key profibrotic gene programs in fibroblasts, characterized by a marked inhibition of the MRTF-SRF pathway, which is not seen after treatment with either drug alone. Importantly, the dual treatment alleviates contractile dysfunction in fibrotic engineered heart tissues and in a mouse model of heart failure. Our findings point to joint inhibition of SRC-mediated stromal mechanosensing and TGFβ signalling as a potential mechanotherapeutic strategy for treating cardiovascular fibrosis.

The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules. Here, we demonstrate an approach that combines lipid-enriched maturation medium with a high concentration of calcium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with advanced electrophysiological, structural and metabolic phenotypes. Systematic testing reveals that electrostimulation is the key driver of enhanced mitochondrial development and metabolic maturation and improved electrophysiological properties of iPSC-CMs. Increased calcium concentration strongly promotes electrophysiological maturation, while nanopatterning primarily facilitates sarcomere organisation with minor effect on electrophysiological properties. Transcriptome analysis reveals that activation of HMCES and TFAM targets contributes to mitochondrial development, whereas downregulation of MAPK/PI3K and SRF targets is associated with iPSC-CM polyploidy. These findings provide mechanistic insights into iPSC-CM maturation, paving the way for pharmacological responses that more closely resemble those of adult CMs.

Introduction: Myocardial infarction is a serious and potentially life-threatening condition that requires immediate medical intervention. The earlier help is provided, the less likely irreversible damage to the heart muscle will occur. Experimental investigation of myocardium behaviour is necessary for advanced numerical models to predict treatment outcomes. Methods: The study investigates the mechanical characteristics of the sheep heart's mid-wall, right and left ventricles using equi-biaxial mechanical testing. This method allows for studying the myocardium's behaviour in multiple directions, specifically analyzing the mechanical stiffness and strain energy. Thirteen (13) sheep hearts were collected from a local abattoir, and ten (10) of them were prepared and subjected to equi-biaxial mechanical tests under physiological conditions. This was to ensure that hearts were healthy to minimise the variability in mechanical properties of the myocardium. The study measured stress-strain relationships in both the longitudinal and circumferential directions for the right ventricle (RV), left ventricle (LV), and mid-wall septum (MDW). To minimize viscoelastic effects, the preconditioning protocol involved cyclic loading of 10 cycles before testing. Results and discussion: Results indicated distinct mechanical properties between the chambers, with the RV showing higher strain energy storage and compliance in the circumferential direction than the LV. To minimize viscoelastic effects, the preconditioning protocol involved cyclic loading of 10 cycles before testing. Stress-strain behaviour exhibited nonlinear characteristics, with variability between samples. Stored strain energy values of linear elastic region for left ventricle were 7.317 kJ and 6.67 kJ in longitudinal and circumferential directions, respectively. The elastic modulus was determined from the linear elastic region for each heart wall specifically, from 16% to 40% strain for LV, MDW, and RV. The toe region peak stresses were those corresponding to 16% strain for LV, MDW, and RV. The stresses at 40% strain were obtained from the closest strain level. Anisotropic effects of myocardium were exhibited. Thus, the study provides insights into the mechanical anisotropy of the myocardium and its relevance to ventricular function, offering important data for understanding heart tissue mechanics and modelling heart diseases.

Basic science research in cardiopulmonary resuscitation (CPR) is limited by challenges in obtaining haemodynamic data from models that simulate physiological processes. In this study, we assessed the morphology of the heart and lungs and calculated the ejection fractions of cardiac chambers during CPR using a virtual simulation.

A finite element model of a complete thorax, including internal organs, thoracic rib cage, spine, musculature, and a generic material representing soft tissues, was constructed from magentic resonance images of a man. Twelve chest compression simulations were performed with forces ranging from F = 50 to 600 N. During compression, lung and heart volumes were assessed, and the ejection fraction of each cardiac chamber was calculated.

In our numerical simulations a compression depth of 5.06 cm was reached with a force of 450 N. At this depth, the right and left ventricular ejection fractions were 34.0% and 14.4%, respectively, while the right and left atrial ejection fractions were 22.1% and 24.2%, respectively. The cross-sectional area of the outflow tract decreased by 27.5% and 15.6% in the right and left ventricles, respectively. Lung volumes decreased by 193 cm3 and 169 cm3 in the right and left lungs, respectively, representing 11.2% of the total lung volume.

The right ventricle exhibited the highest ejection fraction among the cardiac chambers, and the left atrium showed a higher ejection fraction than the left ventricle during CPR.

Myocardial infarction can cause irreversible damage to the heart muscle, which can lead to heart failure. The difficulty of the treatment mainly arises from the anisotropic behavior of the myocardium fibrous structure. Patches or cardiac restraint devices appear to be a promising approach to post-infarction treatment. In this study, we propose a new model-assisted method to design patterned membranes. The proposed approach combines computer experiments and statistical models to optimize the design parameters and to meet the requirement for the post-infarction treatment. Finite element model, global sensitivity analysis, random forest model and response surface model are the key components of the strategy implemented in this study, which is applied to design a real membrane. The metamodel-based design method is able to estimate the equivalent Young's modulus of the membrane in a few seconds and optimization results have been validated a posteriori by laboratory measurements. This solution opens up new prospects for the design of customized membranes with technical specifications tailored to each patient.

The heart, with its complex structural and functional characteristics, plays a critical role in sustaining life by pumping blood throughout the entire body to supply nutrients and oxygen. Engineered heart tissues have been introduced to reproduce heart functions to understand the pathophysiological properties of the heart and to test and develop potential therapeutics. Although numerous studies have been conducted in various fields to increase the functionality of heart tissue to be similar to reality, there are still many difficulties in reproducing the blood-pumping function of the heart. In this review, we discuss advancements in cells, biomaterials, and biofabrication in cardiac tissue engineering to achieve cardiac models that closely mimic the pumping function. Moreover, we provide insight into future directions by proposing future perspectives to overcome remaining challenges, such as scaling up and biomimetic patterning of blood vessels and nerves through bioprinting.

The use of a video method based on the Digital Image Correlation (DIC) algorithm from experimental mechanics to estimate the displacements, strain field, and sarcolemma length in a beating single-cell cardiomyocyte is proposed in this work. The obtained deformation is then correlated with the calcium signal, from calcium imaging where fluorescent dyes sensitive to calcium Ca2+ are used. Our proposed video-based method for simultaneous contraction and intracellular calcium analysis results in a low-cost, non-invasive, and label-free method. This technique has shown great advantages in long-term observations because this type of intervention-free measurement neutralizes the possible alteration in the beating cardiomyocyte introduced by other techniques for measuring cell contractility (e.g., Traction Force Microscopy, Atomic Force Microscopy, Microfabrication or Optical tweezers). Three tests were performed with synthetically augmented data from cardiomyocyte images to validate the robustness of the algorithm. First, a simulated rigid translation of a referenced image is applied, then a rotation, and finally a controlled longitudinal deformation of the referenced image, thus simulating a native realistic deformation. Finally, the proposed framework is evaluated with real experimental data. To validate contraction induced by intracellular calcium concentration, this signal is correlated with a new deformation measure proposed in this article, which is independent of cell orientation in the imaging setup. Finally, based on the displacements obtained by the DIC algorithm, the change in sarcolemma length in a contracting cardiomyocyte is calculated and its temporal correlation with the calcium signal is obtained.

Mechanical metamaterials are rationally designed structures engineered to exhibit extraordinary properties, often surpassing those of their constituent materials. The geometry of metamaterials' building blocks, referred to as unit cells, plays an essential role in determining their macroscopic mechanical behavior. Due to their hierarchical design and remarkable properties, metamaterials hold significant potential for tissue engineering; however their implementation in the field remains limited. The major challenge hindering the broader use of metamaterials lies in the complexity of unit cell design and fabrication. To address this gap, a comprehensive guide is presented detailing the design principles of well-established metamaterials. The essential unit cell geometric parameters and design constraints, as well as their influence on mechanical behavior, are summarized highlighting essential points for effective fabrication. Moreover, the potential integration of artificial intelligence techniques is explored in meta-biomaterial design for patient- and application-specific design. Furthermore, a comprehensive overview of current applications of mechanical metamaterials is provided in tissue engineering, categorized by tissue type, thereby showcasing the versatility of different designs in matching the mechanical properties of the target tissue. This review aims to provide a valuable resource for tissue engineering researchers and aid in the broader use of metamaterials in the field.

Cardiac tissue engineering has been rapidly evolving with diverse applications, ranging from the repair of fibrotic tissue caused by "adverse remodeling," to the replacement of specific segments of heart tissue, and ultimately to the creation of a whole heart. The repair or replacement of cardiac tissue often involves the development of tissue scaffolds or constructs and the subsequent assessment of their performance and functionality. For this, the design and/or selection of biomaterials, and cell types, scaffold fabrication, and in vitro characterizations are the first starting points, yet critical, to ensure success in subsequent implantation in vivo. This highlights the importance of scaffold fabrication and in vitro experiments/characterization with protocols for cardiac tissue engineering. Yet, a comprehensive and critical review of these has not been established and documented. As inspired, herein, the latest development and advances in scaffold fabrication and in vitro characterization for cardiac tissue engineering are critically reviewed, with focus on biomaterials, cell types, additive manufacturing techniques for scaffold fabrication, and common in vitro characterization techniques or methods. This article would be of benefit to the ones who are working on cardiac tissue engineering by providing insights into the scaffold fabrication and in vitro investigations.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28 days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.

Without intervention, cardiac arrhythmias pose a risk of fatality. However, timely intervention can be challenging in environments where transporting a large, heavy defibrillator is impractical, or emergency surgery to implant cardiac stimulation devices is not feasible. Here, we introduce an injectable cardiac stimulator, a syringe loaded with a nanoparticle solution comprising a conductive polymer and a monomer that, upon injection, forms a conductive structure around the heart for cardiac stimulation. Following treatment, the electrode is cleared from the body, eliminating the need for surgical extraction. The mixture adheres to the beating heart in vivo without disrupting its normal rhythm. The electrofunctionalized injectable cardiac stimulator demonstrates a tissue-compatible Young's modulus of 21 kPa and a high conductivity of 55 S/cm. The injected electrode facilitates electrocardiogram measurements, regulates heartbeat in vivo, and rectifies arrhythmia. Conductive functionality is maintained for five consecutive days, and no toxicity is observed at the organism, organ, or cellular levels.

This study investigates how dynamic fluctuations in matrix stiffness affect the behavior of cardiac fibroblasts (CFs) within a three-dimensional (3D) hydrogel environment. Using hybrid hydrogels with tunable stiffness, we created an in vitro model to mimic the varying stiffness of the cardiac microenvironment. By manipulating hydrogel stiffness, we examined CF responses, particularly the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation. Our findings reveal that increased matrix stiffness promotes the differentiation of CFs into myofibroblasts, while matrix softening reverses this process. Additionally, we identified the role of focal adhesions and integrin β1 in mediating stiffness-induced phenotypic switching. This study provides significant insights into the mechanobiology of cardiac fibrosis and suggests that modulating matrix stiffness could be a potential therapeutic strategy for treating cardiovascular diseases.

Global longitudinal active strain energy density (GLASED) is an innovative method for assessing myocardial function and quantifies the work performed per unit volume of the left ventricular myocardium. The GLASED, measured using MRI, is the best prognostic marker currently available. This study aimed to evaluate the feasibility of measuring the GLASED using echocardiography and to investigate potential differences in the GLASED among athletes based on age and sex.

An echocardiographic study was conducted with male controls, male and female young athletes, and male and female veteran athletes. GLASED was calculated from the myocardial stress and strain.

The mean age (in years) of the young athletes was 21.6 for males and 21.4 for females, while the mean age of the veteran athletes was 53.5 for males and 54.2 for females. GLASED was found to be highest in young male athletes (2.40 kJ/m3) and lowest in female veterans (1.96 kJ/m3). Veteran males exhibited lower values (1.96 kJ/m3) than young male athletes did (P < 0.001). Young females demonstrated greater GLASED (2.28 kJ/m3) than did veteran females (P < 0.01). However, no significant difference in the GLASED was observed between male and female veterans.

Our findings demonstrated the feasibility of measuring GLASED using echocardiography. GLASED values were greater in young male athletes than in female athletes and decreased with age, suggesting possible physiological differences in their myocardium. The sex-related differences observed in GLASED values among young athletes were no longer present in veteran athletes. We postulate that measuring the GLASED may serve as a useful additional screening tool for cardiac diseases in athletes, particularly for those with borderline phenotypes of hypertrophic and dilated cardiomyopathies.

Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ1) followed by a slower one (τ2). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ1 with respect to τ2. Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis.

In accordance with the World Health Organization data, cancer remains at the forefront of fatal diseases. An upward trend in cancer incidence and mortality has been observed globally, emphasizing that efforts in developing detection and treatment methods should continue. The diagnostic path typically begins with learning the medical history of a patient; this is followed by basic blood tests and imaging tests to indicate where cancer may be located to schedule a needle biopsy. Prompt initiation of diagnosis is crucial since delayed cancer detection entails higher costs of treatment and hospitalization. Thus, there is a need for novel cancer detection methods such as liquid biopsy, elastography, synthetic biosensors, fluorescence imaging, and reflectance confocal microscopy. Conventional therapeutic methods, although still common in clinical practice, pose many limitations and are unsatisfactory. Nowadays, there is a dynamic advancement of clinical research and the development of more precise and effective methods such as oncolytic virotherapy, exosome-based therapy, nanotechnology, dendritic cells, chimeric antigen receptors, immune checkpoint inhibitors, natural product-based therapy, tumor-treating fields, and photodynamic therapy. The present paper compares available data on conventional and modern methods of cancer detection and therapy to facilitate an understanding of this rapidly advancing field and its future directions. As evidenced, modern methods are not without drawbacks; there is still a need to develop new detection strategies and therapeutic approaches to improve sensitivity, specificity, safety, and efficacy. Nevertheless, an appropriate route has been taken, as confirmed by the approval of some modern methods by the Food and Drug Administration.

Human cardiac fibrotic tissues are characterized by a higher stiffness relative to healthy cardiac tissues. These tissues are unable to spontaneously contract and are subjected to passive mechanical stimulation during heart functionality. Moreover, scaffolds that can recapitulate the in vivo mechanical properties of the cardiac fibrotic tissues are lacking. Herein, this study aimed to design and fabricate mechanically stretchable bioartificial scaffolds with biomimetic composition and stiffness comparable to human cardiac fibrotic tissues. Poly(ε-caprolactone) (PCL) scaffolds with a stretchable mesh architecture were initially designed through structural and finite element method (FEM) analyses and subsequently fabricated by melt extrusion additive manufacturing (MEX). Scaffolds were surface functionalized by 3,4-dihydroxy-DL-phenylalanine (DOPA) polymerization (polyDOPA) to improve their interaction with natural polymers. Scaffold pores were then filled with different concentrations (5%, 7%, and 10% w/v) of gelatin methacryloyl (GelMA) hydrogels to support in vitro human cardiac fibroblasts (HCFs) 3D culture, thereby producing bioartificial PCL/GelMA scaffolds. Uniaxial tensile mechanical tests in static and dynamic conditions (1 Hz; 22% maximum strain) demonstrated that the bioartificial scaffolds had in vivo-like stretchability and similar stiffness to that of pathological cardiac tissue (tailored as a function of the number of PCL scaffold layers and GelMA hydrogel concentration). In vitro cell tests on bioartificial scaffolds using HCF-embedded GelMA hydrogels under static conditions displayed increasing cell viability, spreading, and cytoskeleton organization with decreasing GelMA hydrogel concentration. Moreover, α-smooth muscle actin (α-SMA)-positive cells were detected after 7 days of culture in static conditions followed by another 7 days of culture in dynamic conditions and not in HCF-loaded scaffolds cultured in static conditions for 14 days. These results suggested that in vitro culture under cyclic mechanical stimulations could induce an HCF phenotypic switch into myofibroblasts.

Cardiovascular diseases are a leading cause of mortality and pose a significant burden on healthcare systems worldwide. Despite remarkable progress in medical research, the development of effective cardiovascular drugs has been hindered by high failure rates and escalating costs. One contributing factor is the limited availability of mature cardiomyocytes (CMs) for accurate disease modeling and drug screening. Human induced pluripotent stem cell-derived CMs offer a promising source of CMs; however, their immature phenotype presents challenges in translational applications. This review focuses on the road to achieving mature CMs by summarizing the major differences between immature and mature CMs, discussing the importance of adult-like CMs for drug discovery, highlighting the limitations of current strategies, and exploring potential solutions using electro-mechano active polymer-based scaffolds based on conductive polymers. However, critical considerations such as the trade-off between 3D systems and nutrient exchange, biocompatibility, degradation, cell adhesion, longevity, and integration into wider systems must be carefully evaluated. Continued advancements in these areas will contribute to a better understanding of cardiac diseases, improved drug discovery, and the development of personalized treatment strategies for patients with cardiovascular disorders.

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

Myocardial infarction (MI) dramatically changes the mechanical stress, which is intensified by the fibrotic remodeling. Integrins, especially the αV subunit, mediate mechanical signal and mechanoparacrine of transforming growth factor β1 (TGF-β1) in various organ fibrosis by activating CFs into myofibroblasts (MFBs). We investigated a possible role of integrin αV mediated mechanoparacrine of TGF-β1 in MFBs activation for fibrous reparation in mice with MI.

Heart samples from MI, sham, or MI plus cilengitide (14 mg/kg, specific integrin αV inhibitor) treated mice, underwent functional and morphological assessments by echocardiography, and histochemistry on 7, 14 and 28 days post-surgery. The mechanical and ultrastructural changes of the fibrous scar were further evaluated by atomic mechanics microscope (AFM), immunofluorescence, second harmonic generation (SHG) imaging, polarized light and scanning electron microscope, respectively. Hydroxyproline assay was used for total collagen content, and western blot for protein expression profile examination. Fibroblast bioactivities, including cell shape, number, Smad2/3 signal and expression of extracellular matrix (ECM) related proteins, were further evaluated by microscopic observation and immunofluorescence in polyacrylamide (PA) hydrogel with adjustable stiffness, which was re-explored in fibroblast cultured on stiff matrix after silencing of integrin αV. The content of total and free TGF-β1 was tested by enzyme-linked immunosorbent assay (ELISA) in both infarcted tissue and cell samples.

Increased stiffness with heterogeneity synchronized with integrin αV and alpha smooth muscle actin (α-SMA) positive MFBs accumulation in those less mature fibrous areas. Cilengitide abruptly reduced collagen content and disrupted collagen alignment, which also decreased TGF-β1 bioavailability, Smad2/3 phosphorylation, and α-SMA expression in the fibrous area. Accordingly, fibroblast on stiff but not soft matrix exhibited obvious MFB phenotype, as evidenced by enlarged cell, hyperproliferation, well-developed α-SMA fibers, and elevated ECM related proteins, while silencing of integrin αV almost abolished this switch via attenuating paracrine of TGF-β1 and nuclear translocation of Smad2/3.

This study illustrated that increased tissue stiffness activates CFs into MFBs by integrin αV mediated mechanoparacrine of TGF-β1, especially in immature scar area, which ultimately promotes fibrous scar maturation.

Background: Cardiovascular phantoms for patient education, pre-operative planning, surgical training, haemodynamic simulation, and device testing may help improve patient care. However, currently used materials may have different mechanical properties compared to biological tissue. Methods/Aim: The aim of this study was to investigate the mechanical properties of 3D-printing and silicone materials in comparison to biological cardiovascular tissues. Uniaxial cyclic tension testing was performed using dumbbell samples from porcine tissue (aorta, pulmonary artery, right and left ventricle). Flexible testing materials included 15 silicone (mixtures) and three 3D-printing materials. The modulus of elasticity was calculated for different deformation ranges. Results: The modulus of elasticity (0%-60%) for the aorta ranged from 0.16 to 0.18 N/mm2, for the pulmonary artery from 0.07 to 0.09 N/mm2, and for the right ventricle as well as the left ventricle short-axis from 0.1 to 0.16 N/mm2. For silicones the range of modulus of elasticity was 0.02-1.16 N/mm2, and for the 3D-printed materials from 0.85 to 1.02 N/mm2. The stress-strain curves of all tissues showed a non-linear behaviour in the cyclic tensile testing, with a distinct toe region, followed by exponential strain hardening behaviour towards the peak elongation. The vessel samples showed a more linear behaviour comparted to myocardial samples. The silicones and 3D printing materials exhibited near-linearity at higher strain ranges, with a decrease in stiffness following the initial deformation. All samples showed a deviation between the loading and unloading curves (hysteresis), and a reduction in peak force over the first few cycles (adaptation effect) at constant deformation. Conclusion: The modulus of elasticity of silicone mixtures is more in agreement to porcine cardiovascular tissues than 3D-printed materials. All synthetic materials showed an almost linear behaviour in the mechanical testing compared to the non-linear behaviour of the biological tissues, probably due to fibre recruitment mechanism in the latter.

Ischemic heart disease is one of the leading causes of death worldwide. The efficient delivery of therapeutic growth factors could counteract the adverse prognosis of post-myocardial infarction (post-MI). In this study, a collagen hydrogel that is able to load and appropriately deliver pro-angiogenic stromal cell-derived factor 1 (SDF1) was physically coupled with a compact collagen membrane in order to provide the suture strength required for surgical implantation. This bilayer collagen-on-collagen scaffold (bCS) showed the suitable physicochemical properties that are needed for efficient implantation, and the scaffold was able to deliver therapeutic growth factors after MI. In vitro collagen matrix biodegradation led to a sustained SDF1 release and a lack of cytotoxicity in the relevant cell cultures. In vivo intervention in a rat subacute MI model resulted in the full integration of the scaffold into the heart after implantation and biocompatibility with the tissue, with a prevalence of anti-inflammatory and pro-angiogenic macrophages, as well as evidence of revascularization and improved cardiac function after 60 days. Moreover, the beneficial effect of the released SDF1 on heart remodeling was confirmed by a significant reduction in cardiac tissue stiffness. Our findings demonstrate that this multimodal scaffold is a desirable matrix that can be used as a drug delivery system and a scaffolding material to promote functional recovery after MI.

Cardiovascular diseases, including heart failure, pose significant challenges in medical practice, necessitating innovative approaches for cardiac repair and regeneration. Cardiac tissue engineering has emerged as a promising solution, aiming to develop functional and physiologically relevant cardiac tissue constructs. Replicating the native heart microenvironment, with its complex and dynamic milieu necessary for cardiac tissue growth and function, is crucial in tissue engineering. Biomimetic strategies that closely mimic the natural heart microenvironment have gained significant interest due to their potential to enhance synthetic cardiac tissue functionality and therapeutic applicability. Biomimetic approaches focus on mimicking biochemical cues, mechanical stimuli, coordinated electrical signaling, and cell-cell/cell-matrix interactions of cardiac tissue. By combining bioactive ligands, controlled delivery systems, appropriate biomaterial characteristics, electrical signals, and strategies to enhance cell interactions, biomimetic approaches provide a more physiologically relevant environment for tissue growth. The replication of the native cardiac microenvironment enables precise regulation of cellular responses, tissue remodeling, and the development of functional cardiac tissue constructs. Challenges and future directions include refining complex biochemical signaling networks, paracrine signaling, synchronized electrical networks, and cell-cell/cell-matrix interactions. Advancements in biomimetic approaches hold great promise for cardiovascular regenerative medicine, offering potential therapeutic strategies and revolutionizing cardiac disease modeling. These approaches contribute to the development of more effective treatments, personalized medicine, and improved patient outcomes. Ongoing research and innovation in biomimetic approaches have the potential to revolutionize regenerative medicine and cardiac disease modeling by replicating the native heart microenvironment, advancing functional cardiac tissue engineering, and improving patient outcomes.

Background: Engineering cardiac tissue that mimics the hierarchical structure of cardiac tissue remains challenging, raising the need for developing novel methods capable of creating structures with high complexity. Three-dimensional (3D)-printing techniques are among promising methods for engineering complex tissue constructs with high precision. By means of 3D printing, this study aims to develop cardiac constructs with a novel angular structure mimicking cardiac architecture from alginate (Alg) and gelatin (Gel) composite. The 3D-printing conditions were optimized and the structures were characterized in vitro, with human umbilical vein endothelial cells (HUVECs) and cardiomyocytes (H9c2 cells), for potential cardiac tissue engineering. Methods: We synthesized the composites of Alg and Gel with varying concentrations and examined their cytotoxicity with both H9c2 cells and HUVECs, as well as their printability for creating 3D structures of varying fibre orientations (angular design). The 3D-printed structures were characterized in terms of morphology by both scanning electron microscopy (SEM) and synchrotron radiation propagation-based imaging computed tomography (SR-PBI-CT), and elastic modulus, swelling percentage, and mass loss percentage as well. The cell viability studies were conducted via measuring the metabolic activity of the live cells with MTT assay and visualizing the cells with live/dead assay kit. Results: Among the examined composite groups of Alg and Gel, two combinations with ratios of 2 to 1 and 3 to 1 (termed as Alg2Gel1 and Alg3Gel1) showed the highest cell survival; they accordingly were used to fabricate two different structures: a novel angular and a conventional lattice structure. Scaffolds made of Alg3Gel1 showed higher elastic modulus, lower swelling percentage, less mass loss, and higher cell survival compared to that of Alg2Gel1. Although the viability of H9c2 cells and HUVECs on all scaffolds composed of Alg3Gel1 was above 99%, the group of the constructs with the angular design maintained significantly more viable cells compared to other investigated groups. Conclusion: The group of angular 3D-ptinted constructs has illustrated promising properties for cardiac tissue engineering by providing high cell viability for both endothelial and cardiac cells, high mechanical strength as well as appropriate swelling, and degradation properties during 21 days of incubation. Statement of Significance: 3D-printing is an emerging method to create complex constructs with high precision in a large scale. In this study, we have demonstrated that 3D-printing can be used to create compatible constructs from the composite of Alg and Gel with endothelial cells and cardiac cells. Also, we have demonstrated that these constructs are able to enhance the viability of cardiac and endothelial cells via creating a 3D structure mimicking the alignment and orientation of the fibers in the native heart.

Impaired cardiac filling in response to increased passive myocardial stiffness contributes to the pathophysiology of heart failure. By leveraging cardiac MRI data and ventricular pressure measurements, we can estimate in vivo passive myocardial stiffness using personalized inverse finite element models. While it is well-known that this approach is subject to uncertainties, only few studies quantify the accuracy of these stiffness estimates. This lack of validation is, at least in part, due to the absence of ground truth in vivo passive myocardial stiffness values. Here, using 3D printing, we created soft, homogenous, isotropic, hyperelastic heart phantoms of varying geometry and stiffness and simulate diastolic filling by incorporating the phantoms into an MRI-compatible left ventricular inflation system. We estimate phantom stiffness from MRI and pressure data using inverse finite element analyses based on a Neo-Hookean model. We demonstrate that our identified softest and stiffest values of 215.7 and 512.3 kPa agree well with the ground truth of 226.2 and 526.4 kPa. Overall, our estimated stiffnesses revealed a good agreement with the ground truth ([Formula: see text] error) across all models. Our results suggest that MRI-driven computational constitutive modeling can accurately estimate synthetic heart material stiffnesses in the range of 200-500 kPa.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

This commentary constitutes the October edition of the 'Editors' roundup'-a multi-author omnibus of personal recommendations to interesting biophysics-related articles contributed by members of the editorial boards of leading international biophysics journals. The present commentary contains contributions from Progress in Biochemistry and Biophysics (an official journal of the Biophysical Society of China), European Biophysics Journal (the official journal of the European Biophysical Societies Association), Biophysical Reviews (the official IUPAB journal), and Biophysics (an official journal of the Russian Academy of Sciences). This edition of the Editors' Roundup also contains a new section from an editor at large who has provided selections from a number of journals on a single thematic topic.

Mitochondrial dysfunction of cardiomyocytes (CMs) has been identified as a significant pathogenesis of early myocardial infarction (MI). However, only a few agents or strategies have been developed to improve mitochondrial dysfunction for the effective MI treatment. Herein, a reactive oxygen species (ROS)-responsive PAMB-G-TK/4-arm-PEG-SG hydrogel is developed for localized drug-loaded liposome delivery. Notably, the liposomes contain both elamipretide (SS-31) and sphingosine-1-phosphate (S1P), where SS-31 acts as an inhibitor of mitochondrial oxidative damage and S1P as a signaling molecule for activating angiogenesis. Liposome-encapsulated PAMB-G-TK/4-arm-PEG-SG hydrogels demonstrate myocardium-like mechanical strength and electrical conductivity, and ROS-sensitive release of SS-31 and S1P-loaded liposomes. Further liposomal release of SS-31, which can target cytochrome c in the mitochondrial inner membrane of damaged CMs, inhibits pathological ROS production, improving mitochondrial dysfunction. Meanwhile, S1P released from the liposome induces endothelial cell angiogenesis by activating the S1PR1/PI3K/Akt pathway. In a rat MI model, the resulting liposomal composite hydrogel improves cardiac function by scavenging excess ROS, improving mitochondrial dysfunction, and promoting angiogenesis. This study reports for the first time a liposomal composite hydrogel that can directly target mitochondria of damaged CMs for a feedback-regulated release of encapsulated liposomes to consume the overproduced pathological ROS for improved CM activity and enhanced MI treatment.

Across differing spectrums of cardiac function and cardiac pathologies, there are strong associations between measures of cardiorespiratory fitness and burden of symptoms, quality of life, and prognosis. In this part 2 of a 4-part series, we contend that there is a strong association among physical activity, cardiorespiratory fitness, and cardiac function. We argue that a chronic lack of exercise is a major risk factor for heart failure with preserved ejection fraction in some patients. In support of this hypothesis, increasing physical activity is associated with greater cardiac mass, greater stroke volumes, greater cardiac output and peak oxygen consumption, and fewer clinical events. Conversely, physical inactivity results in cardiac atrophy, reduced output, reduced chamber size, and decreased ability to augment cardiac performance with exercise. Moreover, physical inactivity is a strong predictor of heart failure risk and death. In sum, exercise deficiency should be considered part of the broad heart failure with preserved ejection fraction phenotype.

Tissue engineering with human cardiac fibroblasts (CF) allows identifying novel mechanisms and anti-fibrotic drugs in the context of cardiac fibrosis. However, substantial knowledge on the influences of the used materials and tissue geometries on tissue properties and cell phenotypes is necessary to be able to choose an appropriate model for a specific research question. As there is a clear lack of information on how CF react to the mold architecture in engineered connective tissues (ECT), we first compared the effect of two mold geometries and materials with different hardnesses on the biomechanical properties of ECT. We could show that ECT, which formed around two distant poles (non-uniform model) were less stiff and more strain-resistant than ECT, which formed around a central rod (uniform model), independent of the materials used for poles and rods. Next, we investigated the cell state and could demonstrate that in the uniform versus non-uniform model, the embedded cells have a higher cell cycle activity and display a more pronounced myofibroblast phenotype. Differential gene expression analysis revealed that uniform ECT displayed a fibrosis-associated gene signature similar to the diseased heart. Furthermore, we were able to identify important relationships between cell and tissue characteristics, as well as between biomechanical tissue parameters by implementing cells from normal heart and end-stage heart failure explants from patients with ischemic or dilated cardiomyopathy. Finally, we show that the application of pro- and anti-fibrotic factors in the non-uniform and uniform model, respectively, is not sufficient to mimic the effect of the other geometry. Taken together, we demonstrate that modifying the mold geometry in tissue engineering with CF offers the possibility to compare different cellular phenotypes and biomechanical tissue properties.