Biomimetic materials for novel cardiac regeneration approaches

The quest for novel biomaterials to promote cell structural and functional maturation for cardiac tissue regeneration has emphasized a need to create microenvironments with physiological features. Substrate stiffness constitutes a structural property of crucial importance in the field of tissue engineering and many studies have shown how cardiac cells sense the rigidity of the substrate on which they grow. In this work, we focused on the relevance of substrates mimicking cardiac extracellular matrix (cECM) rigidity for the understanding of the complex interplay between the extracellular and intracellular compartments. Among the most promising biomaterials, Liquid Crystalline Elastomers (LCEs) represent a novel class of polymers previously investigated both as artificial muscles for biomedical purposes and dynamic cell scaffolds. The development of new smart materials which can provide bioactive cues to control and regulate cell fate has been recently encouraged. Indeed, mechanical cues play a significant role in maintaining cell and tissues/organs functions and, in this respect, cell models and substrate stiffness appear as intriguing tools for the investigation of cECM-cell interactions both in physiological and pathological conditions. From the perspective of materials, we have explored the fabrication of biomimetic patterned substrates to direct human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) growth and evaluate their effect on cell functional properties. In the field of regenerative medicine, the advent of hiPSC-CMs has paved the way for a patient-specific therapy but the development of more mature hiPSC-CMs is still needed. Promising approaches that have begun to be investigated include long-term culture, mechanical loading, 3-dimensional tissue engineering and, above all, the use of dynamic scaffolds to boost cell maturation by giving a mechanical stimulus. Finally, with the aim of creating an effective dynamic cell substrate, we have introduced the design of the first prototype of LCE-based biomimetic contractile unit by optimizing a miniaturization of the mechanical device. The functional properties of the contractile apparatus have been investigated and then modulated to closely reproduce the features of native myocardium. Overall, in this work we have provided an overview of some functional aspects of biomaterials which are considered of key relevance in different biomedical fields to elucidate how recent advances may impact future tissue engineering applications

The Role of Crosslinker Molecular Structure on Mechanical and Light-Actuation Properties in Liquid Crystalline Networks

: Phase behavior modulation of liquid crystalline molecules can be addressed by structural modification at molecular level. Starting from a rigid rod-like core reduction of the symmetry or increase of the steric hindrance by different substituents generally reduces the clearing temperature. Similar approaches can be explored to modulate the properties of liquid crystalline networks (LCNs)-shape-changing materials employed as actuators in many fields. Depending on the application, the polymer properties have to be adjusted in terms of force developed under stimuli, kinetics of actuation, elasticity, and resistance to specific loads. In this work, the crosslinker modification at molecular level is explored towards the optimization of LCN properties as light-responsive artificial muscles. The synthesis and characterization of photopolymerizable crosslinkers, bearing different lateral groups on the aromatic core is reported. Such molecules are able to strongly modulate the material mechanical properties, such as kinetics and maximum tension under light actuation, opening up to interesting materials for biomedical applications

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions

: Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells

DataSheet1_Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.pdf

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).</p