Spatial regulation of substrate adhesion directs fibroblast morphotype and phenotype.

The switching of the fibroblast phenotype to myofibroblast is a hallmark of a wide variety of tissue pathologies. This phenotypical switch is known to be influenced not only by humoral factors such as TGF-ß, but also by mechanical and physical cues in the cellular environment, and is accompanied by distinctive changes in cell morphology. However, the causative link between these cues, the concomitant morphological changes, and the resulting phenotypic switch remain elusive. Here, we use protein micropatterning to spatially control dermal fibroblast adhesion without invoking exogenous mechanical changes and demonstrate that varying the spatial configuration of focal adhesions (FAs) is sufficient to direct fibroblast phenotype. We further developed an automated morphometry analysis pipeline, which revealed FA eccentricity as the primary determinant of cell-state positioning along the spectrum of fibroblast phenotype. Moreover, linear fibronectin patterns that constrain the FAs were found to promote a further phenotype transition, characterized by dispersed expression of alpha-smooth muscle actin, pointing to an interesting possibility of controlling fibroblast phenotype beyond the canonical fibroblast-myofibroblast axis. Together, our study reveals that the spatial configuration of adhesion to the cellular microenvironment is a key factor governing fibroblast morphotype and phenotype, shedding new light on fibroblast phenotype regulation.

GelMA hydrogel dual photo-crosslinking to dynamically modulate ECM stiffness.

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-ß 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.

Steering cell orientation through light-based spatiotemporal modulation of the mechanical environment.

The anisotropic organization of cells and the extracellular matrix (ECM) is essential for the physiological function of numerous biological tissues, including the myocardium. This organization changes gradually in space and time, during disease progression such as myocardial infarction. The role of mechanical stimuli has been demonstrated to be essential in obtaining, maintaining and de-railing this organization, but the underlying mechanisms are scarcely known. To enable the study of the mechanobiological mechanisms involved,in vitrotechniques able to spatiotemporally control the multiscale tissue mechanical environment are thus necessary. Here, by using light-sensitive materials combined with light-illumination techniques, we fabricated 2D and 3Din vitromodel systems exposing cells to multiscale, spatiotemporally resolved stiffness anisotropies. Specifically, spatial stiffness anisotropies spanning from micron-sized (cellular) to millimeter-sized (tissue) were achieved. Moreover, the light-sensitive materials allowed to introduce the stiffness anisotropies at defined timepoints (hours) after cell seeding, facilitating the study of their temporal effects on cell and tissue orientation. The systems were tested using cardiac fibroblasts (cFBs), which are known to be crucial for the remodeling of anisotropic cardiac tissue. We observed that 2D stiffness micropatterns induced cFBs anisotropic alignment, independent of the stimulus timing, but dependent on the micropattern spacing. cFBs exhibited organized alignment also in response to 3D stiffness macropatterns, dependent on the stimulus timing and temporally followed by (slower) ECM co-alignment. In conclusion, the developed model systems allow improved fundamental understanding of the underlying mechanobiological factors that steer cell and ECM orientation, such as stiffness guidance and boundary constraints.

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

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

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

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

Comparison of a human acellular dermal matrix and a polypropylene mesh for pelvic floor reconstruction: a randomized trial study in a rabbit model.

Non-absorbable polypropylene (PP) meshes have been widely used in surgical reconstruction of the pelvic floor disorders. However, they are associated with serious complications. Human acellular dermal matrices (hADM) have demonstrated safety and efficacy in reconstructive medicine, but their suitability and efficacy at vaginal level is not known. This study compares the biological performance of PP mesh and a newly developed hADM. 20 rabbits were randomized to receive the hADM graft or the PP mesh. Grafts were surgically implanted in the abdominal wall and vagina. After 180 days, grafts were explanted and evaluated. The vaginal mesh extrusion rate was higher in the PP group (33% vs. 0%, p = 0.015). Full integration of the vaginal grafts was more frequent in the hADM group, where 35% of the grafts were difficult to recognize. In the PP group, the vaginal mesh was identified in 100% of the animals (p = 0.014). In PP group, the infiltrates had a focal distribution and were mostly located in the internal part of the epithelium, while in the hADM group, the infiltrates had a diffuse distribution. Additionally, the hADM group also presented more B-lymphocytes and less T-lymphocytes. Biomechanical analysis showed that hADM had lower resistance to stress. Moreover, PP mesh stiffness and elasticity were higher. Then, hADM is associated with fewer clinical complications, as well as better tissue integration. However, it shows greater incorporation into the surrounding native tissue, especially in the vaginal location, undergoing a reduction in its biomechanical properties 6 months after implantation.

Long-Term Strenuous Exercise Promotes Vascular Injury by Selectively Damaging the Tunica Media: Experimental Evidence.

Moderate exercise has well-founded benefits in cardiovascular health. However, increasing, yet controversial, evidence suggests that extremely trained athletes may not be protected from cardiovascular events as much as moderately trained individuals. In our rodent model, intensive but not moderate training promoted aorta and carotid stiffening and elastic lamina ruptures, tunica media thickening of intramyocardial arteries, and an imbalance between vasoconstrictor and relaxation agents. An up-regulation of angiotensin-converter enzyme, miR-212, miR-132, and miR-146b might account for this deleterious remodeling. Most changes remained after a 4-week detraining. In conclusion, our results suggest that intensive training blunts the benefits of moderate exercise.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions.

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

Measuring ventral nerve cord stiffness in live flat-dissected Drosophila embryos by atomic force microscopy.

Drosophila is an amenable system for addressing the mechanics of morphogenesis. We describe a workflow for characterizing the mechanical properties of its ventral nerve cord (VNC), at different developmental stages, in live, flat-dissected embryos employing atomic force microscopy (AFM). AFM is performed with spherical probes, and stiffness (Young's modulus) is calculated by fitting force curves with Hertz's contact model. For complete details on the use and execution of this protocol, please refer to Karkali et al. (2022).

In Vitro Methods to Model Cardiac Mechanobiology in Health and Disease.

In vitro cardiac modeling has taken great strides in the past decade. While most cell and engineered tissue models have focused on cell and tissue contractile function as readouts, mechanobiological cues from the cell environment that affect this function, such as matrix stiffness or organization, are less well explored. In this study, we review two-dimensional (2D) and three-dimensional (3D) models of cardiac function that allow for systematic manipulation or precise control of mechanobiological cues under simulated (patho)physiological conditions while acquiring multiple readouts of cell and tissue function. We summarize the cell types used in these models and highlight the importance of linking 2D and 3D models to address the multiscale organization and mechanical behavior. Finally, we provide directions on how to advance in vitro modeling for cardiac mechanobiology using next generation hydrogels that mimic mechanical and structural environmental features at different length scales and diseased cell types, along with the development of new tissue fabrication and readout techniques. Impact statement Understanding the impact of mechanobiology in cardiac (patho)physiology is essential for developing effective tissue regeneration and drug discovery strategies and requires detailed cause-effect studies. The development of three-dimensional in vitro models allows for such studies with high experimental control, while integrating knowledge from complementary cell culture models and in vivo studies for this purpose. Complemented by the use of human-induced pluripotent stem cells, with or without predisposed genetic diseases, these in vitro models will offer promising outlooks to delineate the impact of mechanobiological cues on human cardiac (patho)physiology in a dish.

Dopamine D1 receptor stimulates cathepsin K-dependent degradation and resorption of collagen I in lung fibroblasts.

Matrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution.This article has an associated First Person interview with the first author of the paper.

First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft.

BACKGROUND: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. METHODS: The PeriCord is a clinical-size (12-16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. FINDINGS: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. INTERPRETATION: This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation. FUNDING: La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, "La Caixa" Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.

Biomechanical Response of Lung Epithelial Cells to Iron Oxide and Titanium Dioxide Nanoparticles.

Increasing evidence shows that lungs can be damaged by inhalation of nanoparticles (NPs) at environmental and occupational settings. Recent findings have associated the exposure to iron oxide (Fe2O3) and titanium dioxide (TiO2) - NPs widely used in biomedical and clinical research - with pulmonary oxidative stress and inflammation. Although changes on cellular mechanics could contribute to pulmonary inflammation, there is no information regarding the effects of Fe2O3 and TiO2 on alveolar epithelial cell biomechanics. The aim was to investigate the NPs-induced biomechanical effects in terms of cell stiffness and traction forces exerted by human alveolar epithelial cells. Cell Young's modulus (E) measured by atomic force microscopy in alveolar epithelial cells significantly decreased after exposure to Fe2O3 and TiO2 (∼28 and ∼25%, respectively) compared to control conditions. Moreover, both NPs induced a similar reduction in the traction forces exerted by the alveolar epithelial cells in comparison to the control conditions. Accordingly, immunofluorescence images revealed a reduction of actomyosin stress fibers in response to the exposure to NPs. However, no inflammatory response was detected. In conclusion, an acute exposure of epithelial pulmonary cells to Fe2O3 and TiO2 NPs, which was mild since it was non-cytotoxic and did not induce inflammation, modified cell biomechanical properties which could be translated into damage of the epithelial barrier integrity, suggesting that mild environmental inhalation of Fe2O3 and TiO2 NPs could not be innocuous.

Proteomics Analysis of Extracellular Matrix Remodeling During Zebrafish Heart Regeneration.

Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days postamputation) time point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.

Nonlinear elasticity of the lung extracellular microenvironment is regulated by macroscale tissue strain.

The extracellular matrix (ECM) of the lung provides physical support and key mechanical signals to pulmonary cells. Although lung ECM is continuously subjected to different stretch levels, detailed mechanics of the ECM at the scale of the cell is poorly understood. Here, we developed a new polydimethylsiloxane (PDMS) chip to probe nonlinear mechanics of tissue samples with atomic force microscopy (AFM). Using this chip, we performed AFM measurements in decellularized rat lung slices at controlled stretch levels. The AFM revealed highly nonlinear ECM elasticity with the microscale stiffness increasing with tissue strain. To correlate micro- and macroscale ECM mechanics, we also assessed macromechanics of decellularized rat lung strips under uniaxial tensile testing. The lung strips exhibited exponential macromechanical behavior but with stiffness values one order of magnitude lower than at the microscale. To interpret the relationship between micro- and macromechanical properties, we carried out a finite element (FE) analysis which revealed that the stiffness of the alveolar cell microenvironment is regulated by the global strain of the lung scaffold. The FE modeling also indicates that the scale dependence of stiffness is mainly due to the porous architecture of the lung parenchyma. We conclude that changes in tissue strain during breathing result in marked changes in the ECM stiffness sensed by alveolar cells providing tissue-specific mechanical signals to the cells. STATEMENT OF SIGNIFICANCE: The micromechanical properties of the extracellular matrix (ECM) are a major determinant of cell behavior. The ECM is exposed to mechanical stretching in the lung and other organs during physiological function. Therefore, a thorough knowledge of the nonlinear micromechanical properties of the ECM at the length scale that cells probe is required to advance our understanding of cell-matrix interplay. We designed a novel PDMS chip to perform atomic force microscopy measurements of ECM micromechanics on decellularized rat lung slices at different macroscopic strain levels. For the first time, our results reveal that the microscale stiffness of lung ECM markedly increases with macroscopic tissue strain. Therefore, changes in tissue strain during breathing result in variations in ECM stiffness providing tissue-specific mechanical signals to lung cells.

Leaves of isoprene-emitting tobacco plants maintain PSII stability at high temperatures.

At high temperatures, isoprene-emitting plants display a higher photosynthetic rate and a lower nonphotochemical quenching (NPQ) compared with nonemitting plants. The mechanism of this phenomenon, which may be very important under current climate warming, is still elusive. NPQ was dissected into its components, and chlorophyll fluorescence lifetime imaging microscopy (FLIM) was used to analyse the dynamics of excited chlorophyll relaxation in isoprene-emitting and nonemitting plants. Thylakoid membrane stiffness was also measured using atomic force microscope (AFM) to identify a possible mode of action of isoprene in improving photochemical efficiency and photosynthetic stability. We show that, when compared with nonemitters, isoprene-emitting tobacco plants exposed at high temperatures display a reduced increase of the NPQ energy-dependent component (qE) and stable (1) chlorophyll fluorescence lifetime; (2) amplitude of the fluorescence decay components; and (3) thylakoid membrane stiffness. Our study shows for the first time that isoprene maintains PSII stability at high temperatures by preventing the modifications of the surrounding environment, namely providing a more steady and homogeneous distribution of the light-absorbing centres and a stable thylakoid membrane stiffness. Isoprene photoprotects leaves with a mechanism alternative to NPQ, enabling plants to maintain a high photosynthetic rate at rising temperatures.

Passive Stiffness of Left Ventricular Myocardial Tissue Is Reduced by Ovariectomy in a Post-menopause Mouse Model.

Background: Heart failure (HF) - a very prevalent disease with high morbidity and mortality - usually presents with diastolic dysfunction. Although post-menopause women are at increased risk of HF and diastolic dysfunction, poor attention has been paid to clinically and experimentally investigate this group of patients. Specifically, whether myocardial stiffness is affected by menopause is unknown. Aim: To investigate whether loss of female sexual hormones modifies the Young's modulus (E) of left ventricular (LV) myocardial tissue in a mouse model of menopause induced by ovariectomy (OVX). Methods: After 6 months of bilateral OVX, eight mice were sacrificed, fresh LV myocardial strips were prepared (∼8 × 1 × 1 mm), and their passive stress-stretch relationship was measured. E was computed by exponential fitting of the stress-stretch relationship. Subsequently, to assess the relative role of cellular and extracellular matrix components in determining OVX-induced changes in E, the tissues strips were decellularized and subjected to the same stretching protocol to measure E. A control group of eight sham-OVX mice was simultaneously studied. Results: E (kPa; m ± SE) in OVX mice was ∼twofold lower than in controls (11.7 ± 1.8 and 22.1 ± 4.4, respectively; p < 0.05). No significant difference between groups was found in E of the decellularized tissue (31.4 ± 12.05 and 40.9 ± 11.5, respectively; p = 0.58). Conclusion: Loss of female sexual hormones in an OVX model induces a reduction in the passive stiffness of myocardial tissue, suggesting that active relaxation should play a counterbalancing role in diastolic dysfunction in post-menopausal women with HF.

Intermittent Hypoxia Mimicking Sleep Apnea Increases Passive Stiffness of Myocardial Extracellular Matrix. A Multiscale Study.

Background: Tissue hypoxia-reoxygenation characterizes obstructive sleep apnea (OSA), a very prevalent respiratory disease associated with increased cardiovascular morbidity and mortality. Experimental studies indicate that intermittent hypoxia (IH) mimicking OSA induces oxidative stress and inflammation in heart tissue at the cell and molecular levels. However, it remains unclear whether IH modifies the passive stiffness of the cardiac tissue extracellular matrix (ECM). Aim: To investigate multiscale changes of stiffness induced by chronic IH in the ECM of left ventricular (LV) myocardium in a murine model of OSA. Methods: Two-month and 18-month old mice (N = 10 each) were subjected to IH (20% O2 40 s-6% O2 20 s) for 6 weeks (6 h/day). Corresponding control groups for each age were kept under normoxia. Fresh LV myocardial strips (∼7 mm × 1 mm × 1 mm) were prepared, and their ECM was obtained by decellularization. Myocardium ECM macroscale mechanics were measured by performing uniaxial stress-strain tensile tests. Strip macroscale stiffness was assessed as the stress value (σ) measured at 0.2 strain and Young's modulus (EM) computed at 0.2 strain by fitting Fung's constitutive model to the stress-strain relationship. ECM stiffness was characterized at the microscale as the Young's modulus (Em) measured in decellularized tissue slices (∼12 µm tick) by atomic force microscopy. Results: Intermittent hypoxia induced a ∼1.5-fold increase in σ (p < 0.001) and a ∼2.5-fold increase in EM (p < 0.001) of young mice as compared with normoxic controls. In contrast, no significant differences emerged in Em among IH-exposed and normoxic mice. Moreover, the mechanical effects of IH on myocardial ECM were similar in young and aged mice. Conclusion: The marked IH-induced increases in macroscale stiffness of LV myocardium ECM suggests that the ECM plays a role in the cardiac dysfunction induced by OSA. Furthermore, absence of any significant effects of IH on the microscale ECM stiffness suggests that the significant increases in macroscale stiffening are primarily mediated by 3D structural ECM remodeling.

The local microenvironment limits the regenerative potential of the mouse neonatal heart.

Neonatal mice have been shown to regenerate their hearts during a transient window of time of approximately 1 week after birth. However, experimental evidence for this phenomenon is not undisputed, because several laboratories have been unable to detect neonatal heart regeneration. We first confirmed that 1-day-old neonatal mice are indeed able to mount a robust regenerative response after heart amputation. We then found that this regenerative ability sharply declines within 48 hours, with hearts of 2-day-old mice responding to amputation with fibrosis, rather than regeneration. By comparing the global transcriptomes of 1- and 2-day-old mouse hearts, we found that most differentially expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate. Testing this hypothesis by pharmacologically decreasing the stiffness of the extracellular matrix in 3-day-old mice, we found that decreased matrix stiffness rescued the ability of mice to regenerate heart tissue after apical resection. Together, our results identify an unexpectedly restricted time window of regenerative competence in the mouse neonatal heart and open new avenues for promoting cardiac regeneration by local modification of the extracellular matrix stiffness.

Head-to-head comparison of two engineered cardiac grafts for myocardial repair: From scaffold characterization to pre-clinical testing.

Cardiac tissue engineering, which combines cells and supportive scaffolds, is an emerging treatment for restoring cardiac function after myocardial infarction (MI), although, the optimal construct remains a challenge. We developed two engineered cardiac grafts, based on decellularized scaffolds from myocardial and pericardial tissues and repopulated them with adipose tissue mesenchymal stem cells (ATMSCs). The structure, macromechanical and micromechanical scaffold properties were preserved upon the decellularization and recellularization processes, except for recellularized myocardium micromechanics that was ∼2-fold stiffer than native tissue and decellularized scaffolds. Proteome characterization of the two acellular matrices showed enrichment of matrisome proteins and major cardiac extracellular matrix components, considerably higher for the recellularized pericardium. Moreover, the pericardial scaffold demonstrated better cell penetrance and retention, as well as a bigger pore size. Both engineered cardiac grafts were further evaluated in pre-clinical MI swine models. Forty days after graft implantation, swine treated with the engineered cardiac grafts showed significant ventricular function recovery. Irrespective of the scaffold origin or cell recolonization, all scaffolds integrated with the underlying myocardium and showed signs of neovascularization and nerve sprouting. Collectively, engineered cardiac grafts -with pericardial or myocardial scaffolds- were effective in restoring cardiac function post-MI, and pericardial scaffolds showed better structural integrity and recolonization capability.