Characterisation of Matrix-Bound Nanovesicles (MBVs) Isolated from Decellularised Bovine Pericardium: New Frontiers in Regenerative Medicine.

Matrix-bound nanovesicles (MBVs) are a recently discovered type of extracellular vesicles (EVs), and they are characterised by a strong adhesion to extracellular matrix structural proteins (ECM) and ECM-derived biomaterials. MBVs contain a highly bioactive and tissue-specific cargo that recapitulates the biological activity of the source ECM. The rich content of MBVs has shown to be capable of potent cell signalling and of modulating the immune system, thus the raising interest for their application in regenerative medicine. Given the tissue-specificity and the youthfulness of research on MBVs, until now they have only been isolated from a few ECM sources. Therefore, the objective of this research was to isolate and identify the presence of MBVs in decellularised bovine pericardium ECM and to characterise their protein content, which is expected to play a major role in their biological potential. The results showed that nanovesicles, corresponding to the definition of recently described MBVs, could be isolated from decellularised bovine pericardium ECM. Moreover, these MBVs were composed of numerous proteins and cytokines, thus preserving a highly potential biological effect. Overall, this research shows that bovine pericardium MBVs show a rich and tissue-specific biological potential.

Cardiac Differentiation Promotes Focal Adhesions Assembly through Vinculin Recruitment.

Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context, forces generated by actomyosin networks and those transmitted through focal adhesion (FA) complexes represent the key regulators of cellular behaviors in terms of cytoskeleton dynamism, cell adhesion, migration, differentiation, and tissue organization. In this study, we investigated the involvement of FAs on cardiomyocyte differentiation. In particular, vinculin and focal adhesion kinase (FAK) family, which are known to be involved in cardiac differentiation, were studied. Results revealed that differentiation conditions induce an upregulation of both FAK-Tyr397 and vinculin, resulting also in the translocation to the cell membrane. Moreover, the role of mechanical stress in contractile phenotype expression was investigated by applying a uniaxial mechanical stretching (5% substrate deformation, 1 Hz frequency). Morphological evaluation revealed that the cell shape showed a spindle shape and reoriented following the stretching direction. Substrate deformation resulted also in modification of the length and the number of vinculin-positive FAs. We can, therefore, suggest that mechanotransductive pathways, activated through FAs, are highly involved in cardiomyocyte differentiation, thus confirming their role during cytoskeleton rearrangement and cardiac myofilament maturation.

Paracrine Shear-Stress-Dependent Signaling from Endothelial Cells Affects Downstream Endothelial Function and Inflammation.

Cardiovascular diseases (CVDs), mainly ischemic heart disease (IHD) and stroke, are the leading cause of global mortality and major contributors to disability worldwide. Despite their heterogeneity, almost all CVDs share a common feature: the endothelial dysfunction. This is defined as a loss of functionality in terms of anti-inflammatory, anti-thrombotic and vasodilatory abilities of endothelial cells (ECs). Endothelial function is greatly ensured by the mechanotransduction of shear forces, namely, endothelial wall shear stress (WSS). Low WSS is associated with endothelial dysfunction, representing the primary cause of atherosclerotic plaque formation and an important factor in plaque progression and remodeling. In this work, the role of factors released by ECs subjected to different magnitudes of shear stress driving the functionality of downstream endothelium has been evaluated. By means of a microfluidic system, HUVEC monolayers have been subjected to shear stress and the conditioned media collected to be used for the subsequent static culture. The results demonstrate that conditioned media retrieved from low shear stress experimental conditions (LSS-CM) induce the downregulation of endothelial nitric oxide synthase (eNOS) expression while upregulating peripheral blood mononuclear cell (PBMC) adhesion by means of higher levels of adhesion molecules such as E-selectin and ICAM-1. Moreover, LSS-CM demonstrated a significant angiogenic ability comparable to the inflammatory control media (TNFα-CM); thus, it is likely related to tissue suffering. We can therefore suggest that ECs stimulated at low shear stress (LSS) magnitudes are possibly involved in the paracrine induction of peripheral endothelial dysfunction, opening interesting insights into the pathogenetic mechanisms of coronary microvascular dysfunction.

Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium.

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.

Overview of natural hydrogels for regenerative medicine applications.

Hydrogels from different materials can be used in biomedical field as an innovative approach in regenerative medicine. Depending on the origin source, hydrogels can be synthetized through chemical and physical methods. Hydrogel can be characterized through several physical parameters, such as size, elastic modulus, swelling and degradation rate. Lately, research is focused on hydrogels derived from biologic materials. These hydrogels can be derived from protein polymers, such as collage, elastin, and polysaccharide polymers like glycosaminoglycans or alginate among others. Introduction of decellularized tissues into hydrogels synthesis displays several advantages compared to natural or synthetic based hydrogels. Preservation of natural molecules such as growth factors, glycans, bioactive cryptic peptides and natural proteins can promote cell growth, function, differentiation, angiogenesis, anti-angiogenesis, antimicrobial effects, and chemotactic effects. Versatility of hydrogels make possible multiple applications and combinations with several molecules on order to obtain the adequate characteristic for each scope. In this context, a lot of molecules such as cross link agents, drugs, grow factors or cells can be used. This review focuses on the recent progress of hydrogels synthesis and applications in order to classify the most recent and relevant matters in biomedical field.

Focal Adhesion's Role in Cardiomyocytes Function: From Cardiomyogenesis to Mechanotransduction.

Mechanotransduction refers to the ability of cells to sense mechanical stimuli and convert them into biochemical signals. In this context, the key players are focal adhesions (FAs): multiprotein complexes that link intracellular actin bundles and the extracellular matrix (ECM). FAs are involved in cellular adhesion, growth, differentiation, gene expression, migration, communication, force transmission, and contractility. Focal adhesion signaling molecules, including Focal Adhesion Kinase (FAK), integrins, vinculin, and paxillin, also play pivotal roles in cardiomyogenesis, impacting cell proliferation and heart tube looping. In fact, cardiomyocytes sense ECM stiffness through integrins, modulating signaling pathways like PI3K/AKT and Wnt/ß-catenin. Moreover, FAK/Src complex activation mediates cardiac hypertrophic growth and survival signaling in response to mechanical loads. This review provides an overview of the molecular and mechanical mechanisms underlying the crosstalk between FAs and cardiac differentiation, as well as the role of FA-mediated mechanotransduction in guiding cardiac muscle responses to mechanical stimuli.

Biological Materials for Tissue-Engineered Vascular Grafts: Overview of Recent Advancements.

The clinical demand for tissue-engineered vascular grafts is still rising, and there are many challenges that need to be overcome, in particular, to obtain functional small-diameter grafts. The many advances made in cell culture, biomaterials, manufacturing techniques, and tissue engineering methods have led to various promising solutions for vascular graft production, with available options able to recapitulate both biological and mechanical properties of native blood vessels. Due to the rising interest in materials with bioactive potentials, materials from natural sources have also recently gained more attention for vascular tissue engineering, and new strategies have been developed to solve the disadvantages related to their use. In this review, the progress made in tissue-engineered vascular graft production is discussed. We highlight, in particular, the use of natural materials as scaffolds for vascular tissue engineering.

Polymeric reinforcements for cellularized collagen-based vascular wall models: influence of the scaffold architecture on the mechanical and biological properties.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold's architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds' architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01 kPa, 100.59 ± 31.15 kPa and 245.78 ± 33.54 kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69 kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5-15] kPa for COL, [80-350] kPa for SES, [20-70] kPa for 3DP and [100-190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes' architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds' architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

Regenerative Potential of A Bovine ECM-Derived Hydrogel for Biomedical Applications.

Recent advancements in regenerative medicine have enhanced the development of biomaterials as multi-functional dressings, capable of accelerating wound healing and addressing the challenge of chronic wounds. Hydrogels obtained from decellularized tissues have a complex composition, comparable to the native extracellular environment, showing highly interesting characteristics for wound healing applications. In this study, a bovine pericardium decellularized extracellular matrix (dECM) hydrogel was characterized in terms of macromolecules content, and its immunomodulatory, angiogenic and wound healing potential has been evaluated. The polarization profile of human monocytes-derived macrophages seeded on dECM hydrogel was assessed by RT-qPCR. Angiogenic markers expression has been evaluated by Western blot and antibody array on cell lysates derived from endothelial cells cultured on dECM hydrogel, and a murine in vivo model of hindlimb ischemia was used to evaluate the angiogenic potential. Fibroblast migration was assessed by a transwell migration assay, and an in vivo murine wound healing model treated with dECM hydrogels was also used. The results showed a complex composition, of which the major component is collagen type I. The dECM hydrogel is biocompatible, able to drive M2 phenotype polarization, stimulate the expression of angiogenic markers in vitro, and prevent loss of functionality in hindlimb ischemia model. Furthermore, it drives fibroblast migration and shows ability to facilitate wound closure in vivo, demonstrating its great potential for regenerative applications.

Angiogenic Potential in Biological Hydrogels.

Hydrogels are three-dimensional (3D) materials able to absorb and retain water in large amounts while maintaining their structural stability. Due to their considerable biocompatibility and similarity with the body's tissues, hydrogels are one of the most promising groups of biomaterials. The main application of these hydrogels is in regenerative medicine, in which they allow the formation of an environment suitable for cell differentiation and growth. Deriving from these hydrogels, it is, therefore, possible to obtain bioactive materials that can regenerate tissues. Because vessels guarantee the right amount of oxygen and nutrients but also assure the elimination of waste products, angiogenesis is one of the processes at the base of the regeneration of a tissue. On the other hand, it is a very complex mechanism and the parameters to consider are several. Indeed, the factors and the cells involved in this process are numerous and, for this reason, it has been a challenge to recreate a biomaterial able to adequately sustain the angiogenic process. However, in this review the focal point is the application of natural hydrogels in angiogenesis enhancing and their potential to guide this process.