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.

Development of a hyaluronic acid-collagen bioink for shear-induced fibers and cells alignment.

Human tissues are characterized by complex composition and cellular and extracellular matrix (ECM) organization at microscopic level. In most of human tissues, cells and ECM show an anisotropic arrangement, which confers them specific properties.In vitro, the ability to closely mimic this complexity is limited. However, in the last years, extrusion bioprinting showed a certain potential for aligning cells and biomolecules, due to the application of shear stress during the bio-fabrication process. In this work, we propose a strategy to combine collagen (col) with tyramine-modified hyaluronic acid (THA) to obtain a printable col-THA bioink for extrusion bioprinting, solely-based on natural-derived components. Collagen fibers formation within the hybrid hydrogel, as well as collagen distribution and spatial organization before and after printing, were studied. For the validation of the biological outcome, fibroblasts were selected as cellular model and embedded in the col-THA matrix. Cell metabolic activity and cell viability, as well as cell distribution and alignment, were studied in the bioink before and after bioprinting. Results demonstrated successful collagen fibers formation within the bioink, as well as collagen anisotropic alignment along the printing direction. Furthermore, results revealed suitable biological properties, with a slightly reduced metabolic activity at day 1, fully recovered within the first 3 d post-cell embedding. Finally, results showed fibroblasts elongation and alignment along the bioprinting direction. Altogether, results validated the potential to obtain collagen-based bioprinted constructs, with both cellular and ECM anisotropy, without detrimental effects of the fabrication process on the biological outcome. This bioink can be potentially used for a wide range of applications in tissue engineering and regenerative medicine in which anisotropy is required.

Electrochemical and in vitro biological behaviors of a Ti-Mo-Fe alloy specifically designed for stent applications.

There is a deep interest in developing new Ni-free Ti-based alloys to replace 316 L stainless steel and Co-Cr alloys for endovascular stent application, mainly because the release of Ni can generate toxicity and allergenicity. Interactions of Ti alloy biomaterials with bone cells and tissues have been widely investigated and reported, while interactions with vascular cells and tissues, such as endothelial cells (ECs) and smooth muscle cells (SMCs), are scarce. Therefore, this study focused on the relationship among the surface finishing features, corrosion behavior and in vitro biological performances regarding human ECs, SMCs and blood of a newly developed Ti-8Mo-2Fe (TMF) alloy, specifically designed for balloon-expandable stent applications. The alloy performances were compared to those of 316 L and pure Ti, prepared with the same surface finishing techniques, which are mechanical polishing and electropolishing. Surface properties were investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA) and x-ray photoelectron spectroscopy (XPS). The corrosion behavior was assessed with potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) tests in phosphate buffered saline (PBS) solution. No significant differences were observed regarding the corrosion rate measured with PDP analyses, which was of the order of 2 × 10-4 mm/y for all the studied materials. Moreover, similarly to pure Ti, TMF exhibited an advantage over 316 L for biomedical applications, namely remarkable resistance to pitting corrosion up to high potentials. The results evidenced a good cytocompatibility and hemocompatibility, making this group of alloy a potential candidate for cardiovascular implants. In fact, both ECs and SMCs proliferated on TMF surfaces showing a 7-day viability similar to that of pure Ti. Regarding hemocompatibility, TMF did not cause hemolysis, and blood coagulation was delayed on its surface in comparison to pure Ti. When compared to 316 L, TMF showed similar hemocompatibility.

Prolonged Antibacterial Activity in Tannic Acid-Iron Complexed Chitosan Films for Medical Device Applications.

Healthcare-associated infections (HAIs) represent a global burden, leading to significant mortality and generating financial costs. One important cause of HAIs is the microbiological contamination of implantable medical devices. In this context, a novel antimicrobial drug-eluting system, based on chitosan and loaded with gentamicin, a broad-spectrum antibiotic, was developed. The effects of the addition of tannic acid and different FeSO4 concentrations on the loaded antibiotic release were evaluated. The properties of the films were assessed in terms of thickness, swelling, mass loss and wettability. The films' surface composition was characterized by X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy. The antibiotic release in phosphate buffer saline was quantified by high-performance liquid chromatography-mass spectrometry, and the antibacterial activity was evaluated. Hemolysis and cytotoxicity were also assessed. The results showed that the addition of tannic acid and iron decreased the swelling degree and degradation due to strong interactions between the different components, thus impacting gentamicin release for up to 35 days. In conclusion, this study presents a novel strategy to produce low-cost and biocompatible antimicrobial drug-eluting systems with sustained and prolonged antibacterial activity over more than a month.

Anthocyanins Formulated with Carboxymethyl Starch for Gastric and Intestinal Delivery.

Anthocyanins obtained from jambolan have been used as active agents in different carboxymethyl starch-based tablet formulations and their release profiles evaluated in simulated gastric fluids (SGF) and simulated intestinal (SIF) fluids. Structural analysis highlighted a strong interaction between anthocyanins and carboxymethyl starch, evidenced by scanning electron microscopy and infrared analysis. Tablet dissolution behavior varied according to the pH of the media, being controlled by the swelling and/or erosion of the polymeric matrix. Various formulations for immediate, fast, and sustained release of anthocyanins for 30 min, 2 h and 12 h of dissolution have been developed. It was found that monolithic carboxymethyl starch tablets loaded with powdered jambolan extract efficiently afforded the complete delivery (100% of anthocyanins) to different sites of the simulated gastrointestinal tract and ensured the stability of these pigments, which maintained their antioxidant activity.

Quercetin-Crosslinked Chitosan Films for Controlled Release of Antimicrobial Drugs.

Natural polymer-based films, due to their favorable biological and mechanical properties, have demonstrated great potential as coatings for biomedical applications. Among them, chitosan films have been widely studied both as coating materials and as controlled drug release systems. Crosslinkers are often used to tune chitosan's crosslinking degree and thus to control the drug release kinetics. For this purpose, quercetin, a plant-derived natural polyphenol, has gained attention as a crosslinker, mainly for its intrinsic anti-inflammatory, antioxidant, and antibacterial features. In this study, chitosan films crosslinked with three different concentrations of quercetin (10, 20, and 30% w/w) have been used as controlled release systems for the delivery of the antibacterial drug trimethoprim (TMP, 10% w/w). Physicochemical and antimicrobial properties were investigated. Surface wettability and composition of the films were assessed by contact angle measurements, X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR), respectively. The release kinetic of TMP in phosphate-buffered saline (PBS) and 2-(N-morpholino) ethanesulfonic acid (MES) was studied over time. Finally, antibacterial properties were assessed on E. coli and S. aureus through Kirby-Bauer disc diffusion and micro-dilution broth assays. Results show that quercetin, at the tested concentrations, clearly increases the crosslinking degree in a dose-dependent manner, thus influencing the release kinetic of the loaded TMP while maintaining its bactericidal effects. In conclusion, this work demonstrates that quercetin-crosslinked chitosan films represent a promising strategy for the design of antibiotic-releasing coatings for biomedical applications.

Effect of silver in thermal treatments of Fe-Mn-C degradable metals: Implications for stent processing.

Twinning-induced plasticity (TWIP) steels are considered excellent materials for manufacturing products requiring extremely high mechanical properties for various applications including thin medical devices, such as biodegradable intravascular stents. It is also proven that the addition of Ag can guarantee an appropriate degradation while implanted in human body without affecting its bioactive properties. In order to develop an optimized manufacturing process for thin stents, the effect of Ag on the recrystallization behavior of TWIP steels needs to be elucidated. This is of major importance since manufacturing stents involves several intermediate recrystallization annealing treatments. In this work, the recrystallization mechanism of two Fe-Mn-C steels with and without Ag was thoroughly investigated by microstructural and mechanical analyses. It was observed that Ag promoted a finer microstructure with a different texture evolution, while the recrystallization kinetics resulted unaffected. The presence of Ag also reduced the effectiveness of the recrystallization treatment. This behavior was attributed to the presence of Ag-rich second phase particles, precipitation of carbides and to the preferential development of grains possessing a {111} orientation upon thermal treatment. The prominence of {111} grains can also give rise to premature twinning, explaining the role of Ag in reducing the ductility of TWIP steels already observed in other works. Furthermore, in vitro biological performances were unaffected by Ag. These findings could allow the design of efficient treatments for supporting the transformation of Fe-Mn-C steels alloyed with Ag into commercial products.

Tubular Bioartificial Organs: From Physiological Requirements to Fabrication Processes and Resulting Properties. A Critical Review.

In this featured review manuscript, the aim is to present a critical survey on the processes available for fabricating bioartificial organs (BAOs). The focus will be on hollow tubular organs for the transport of anabolites and catabolites, i.e., vessels, trachea, esophagus, ureter and urethra, and intestine. First, the anatomic hierarchical structures of tubular organs, as well as their principal physiological functions, will be presented, as this constitutes the mandatory requirements for effectively designing and developing physiologically relevant BAOs. Second, 3D bioprinting, solution electrospinning, and melt electrowriting will be introduced, together with their capacity to match the requirements imposed by designing scaffolds compatible with the anatomical and physiologically relevant environment. Finally, the intrinsic correlation between processes, materials, and cells will be critically discussed, and directives defining the strengths, weaknesses, and opportunities offered by each process will be proposed for assisting bioengineers in the selection of the appropriate process for the target BAO and its specific required functions.

Development of photo-crosslinkable collagen hydrogel building blocks for vascular tissue engineering applications: A superior alternative to methacrylated gelatin?

The present work targets the development of collagen-based hydrogel precursors, functionalized with photo-crosslinkable methacrylamide moieties (COL-MA), for vascular tissue engineering (vTE) applications. The developed materials were physico-chemically characterized in terms of crosslinking kinetics, degree of modification/conversion, swelling behavior, mechanical properties and in vitro cytocompatibility. The collagen derivatives were benchmarked to methacrylamide-modified gelatin (GEL-MA), due to its proven track record in the field of tissue engineering. To the best of our knowledge, this is the first paper in its kind comparing these two methacrylated biopolymers for vTE applications. For both gelatin and collagen, two derivatives with varying degrees of substitutions (DS) were developed by altering the added amount of methacrylic anhydride (MeAnH). This led to photo-crosslinkable derivatives with a DS of 74 and 96% for collagen, and a DS of 73 and 99% for gelatin. The developed derivatives showed high gel fractions (i.e. 74% and 84%, for the gelatin derivatives; 87 and 83%, for the collagen derivatives) and an excellent crosslinking efficiency. Furthermore, the results indicated that the functionalization of collagen led to hydrogels with tunable mechanical properties (i.e. storage moduli of [4.8-9.4 kPa] for the developed COL-MAs versus [3.9-8.4 kPa] for the developed GEL-MAs) along with superior cell-biomaterial interactions when compared to GEL-MA. Moreover, the developed photo-crosslinkable collagens showed superior mechanical properties compared to extracted native collagen. Therefore, the developed photo-crosslinkable collagens demonstrate great potential as biomaterials for vTE applications.

A Novel Strategy to Coat Dopamine-Functionalized Titanium Surfaces With Agarose-Based Hydrogels for the Controlled Release of Gentamicin.

Introduction: The use of spinal implants for the treatment of back disorders is largely affected by the insurgence of infections at the implantation site. Antibacterial coatings have been proposed as a viable solution to limit such infections. However, despite being effective at short-term, conventional coatings lack the ability to prevent infections at medium and long-term. Hydrogel-based drug delivery systems may represent a solution controlling the release of the loaded antibacterial agents while improving cell integration. Agarose, in particular, is a biocompatible natural polysaccharide known to improve cell growth and already used in drug delivery system formulations. In this study, an agarose hydrogel-based coating has been developed for the controlled release of gentamicin (GS). Methods: Sand blasted Ti6Al4V discs were grafted with dopamine (DOPA) solution. After, GS loaded agarose hydrogels have been produced and additioned with tannic acid (TA) and calcium chloride (CaCl2) as crosslinkers. The different GS-loaded hydrogel formulations were deposited on Ti6Al4V-DOPA surfaces, and allowed to react under UV irradiation. Surface topography, wettability and composition have been analyzed with profilometry, static contact angle measurement, XPS and FTIR spectroscopy analyses. GS release was performed under pseudo-physiological conditions up to 28 days and the released GS was quantified using a specific ELISA test. The cytotoxicity of the produced coatings against human cells have been tested, along with their antibacterial activity against S. aureus bacteria. Results: A homogeneous coating was obtained with all the hydrogel formulations. Moreover, the coatings presented a hydrophilic behavior and micro-scale surface roughness. The addition of TA in the hydrogel formulations showed an increase in the release time compared to the normal GS-agarose hydrogels. Moreover, the GS released from these gels was able to significantly inhibit S. aureus growth compared to the GS-agarose hydrogels. The addition of CaCl2 to the gel formulation was able to significantly decrease cytotoxicity of the TA-modified hydrogels. Conclusions: Due to their surface properties, low cytotoxicity and high antibacterial effects, the hereby proposed gentamicin-loaded agarose-hydrogels provide new insight, and represent a promising approach for the surface modification of spinal implants, greatly impacting their application in the orthopedic surgical scenario.

Development of Multifunctional Materials Based on Poly(ether ether ketone) with Improved Biological Performances for Dental Applications.

The main target for the future of materials in dentistry aims to develop dental implants that will have optimal integration with the surrounding tissues, while preventing or avoiding bacterial infections. In this project, poly(ether ether ketone) (PEEK), known for its suitable biocompa-tibility and mechanical properties for dental applications, was loaded with 1, 3, and 5 wt.% ZnO nanoparticles to provide antibacterial properties and improve interaction with cells. Sample cha-racterization by X-ray diffraction (XRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) as well as mechanical properties showed the presence of the nanoparticles and their effect in PEEK matrices, preserving their relevant properties for dental applications. Al-though, the incorporation of ZnO nanoparticles did not improve the mechanical properties and a slight decrease in the thermal stability of the materials was observed. Hemocompatibility and osteoblasts-like cell viability tests showed improved biological performances when ZnO was present, demonstrating high potential for dental implant applications.

Synthesis and characterization of a polymeric network made of polyethylene glycol and chitosan as a treatment with antibacterial properties for skin wounds.

Polyethylene glycol has been widely investigated for wound healing and dressing applications. Despite its advantages (i.e. great biocompatibility), polyethylene glycol lacks antibacterial activity. For this reason, semi-interpenetrated polymeric networks were prepared by combining a chemically cross-linked polyethylene glycol network with chitosan. The aim of this work was to identify the best amount of chitosan able to improve the antibacterial properties against Staphylococcus aureus. Briefly, the networks were synthesized by a sequential method, adding chitosan in different proportion to the polyethylene glycol. The antibacterial activity was tested following the MGA 0100 of the Pharmacopeia of the United States of Mexico. Fourier-transform infrared with attenuated total reflection spectroscopy, scanning electron microscopy and swelling behavior PBS at 37° C and room temperature were also performed to characterize the polymeric networks. The results showed that PC-2% was able to inhibit the bacterial growth of Staphylococcus aureus even more than Fosfomycin antibiotic. The networks showed cylindrical pores of different sizes (50-100 µm). The maximum swelling of all the networks was achieved in PBS at 37°C (>315%). Free hemoglobin and hemolysis assays were also evaluated to know the compatibility with erythrocytes. Human dermal fibroblasts were used to evaluate direct cytotoxicity. Therefore, the produced gels exerted interesting antibacterial activity and showed good biocompatibility properties.

Collagen-Based Tissue Engineering Strategies for Vascular Medicine.

Cardiovascular diseases (CVDs) account for the 31% of total death per year, making them the first cause of death in the world. Atherosclerosis is at the root of the most life-threatening CVDs. Vascular bypass/replacement surgery is the primary therapy for patients with atherosclerosis. The use of polymeric grafts for this application is still burdened by high-rate failure, mostly caused by thrombosis and neointima hyperplasia at the implantation site. As a solution for these problems, the fast re-establishment of a functional endothelial cell (EC) layer has been proposed, representing a strategy of crucial importance to reduce these adverse outcomes. Implant modifications using molecules and growth factors with the aim of speeding up the re-endothelialization process has been proposed over the last years. Collagen, by virtue of several favorable properties, has been widely studied for its application in vascular graft enrichment, mainly as a coating for vascular graft luminal surface and as a drug delivery system for the release of pro-endothelialization factors. Collagen coatings provide receptor-ligand binding sites for ECs on the graft surface and, at the same time, act as biological sealants, effectively reducing graft porosity. The development of collagen-based drug delivery systems, in which small-molecule and protein-based drugs are immobilized within a collagen scaffold in order to control their release for biomedical applications, has been widely explored. These systems help in protecting the biological activity of the loaded molecules while slowing their diffusion from collagen scaffolds, providing optimal effects on the targeted vascular cells. Moreover, collagen-based vascular tissue engineering substitutes, despite not showing yet optimal mechanical properties for their use in the therapy, have shown a high potential as physiologically relevant models for the study of cardiovascular therapeutic drugs and diseases. In this review, the current state of the art about the use of collagen-based strategies, mainly as a coating material for the functionalization of vascular graft luminal surface, as a drug delivery system for the release of pro-endothelialization factors, and as physiologically relevant in vitro vascular models, and the future trend in this field of research will be presented and discussed.

Heparin-Modified Collagen Gels for Controlled Release of Pleiotrophin: Potential for Vascular Applications.

A fast re-endothelialization, along with the inhibition of neointima hyperplasia, are crucial to reduce the failure of vascular bypass grafts. Implants modifications with molecules capable of speeding up the re-endothelialization process have been proposed over the last years. However, clinical trials of angiogenic factor delivery have been mostly disappointing, underscoring the need to investigate a wider array of angiogenic factors. In this work, a drug release system based on a type I collagen hydrogel has been proposed for the controlled release of Pleiotrophin (PTN), a cytokine known for its pro-angiogenetic effects. Heparin, in virtue of its ability to sequester, protect and release growth factors, has been used to better control the release of PTN. Performances of the PTN drug delivery system on endothelial (ECs) and smooth muscle cells (SMCs) have been investigated. Structural characterization (mechanical tests and immunofluorescent analyses of the collagen fibers) was performed on the gels to assess if heparin caused changes in their mechanical behavior. The release of PTN from the different gel formulations has been analyzed using a PTN-specific ELISA assay. Cell viability was evaluated with the Alamar Blue Cell Viability Assay on cells directly seeded on the gels (direct test) and on cells incubated with supernatant, containing the released PTN, obtained from the gels (indirect test). The effects of the different gels on the migration of both ECs and SMCs have been evaluated using a Transwell migration assay. Hemocompatibility of the gel has been assessed with a clotting/hemolysis test. Structural analyses showed that heparin did not change the structural behavior of the collagen gels. ELISA quantification demonstrated that heparin induced a constant release of PTN over time compared to other conditions. Both direct and indirect viability assays showed an increase in ECs viability while no effects were noted on SMCs. Cell migration results evidenced that the heparin/PTN-modified gels significantly increased ECs migration and decreased the SMCs one. Finally, heparin significantly increased the hemocompatibility of the collagen gels. In conclusion, the PTN-heparin-modified collagen here proposed can represent an added value for vascular medicine, able to ameliorate the biological performance, and integration of vascular grafts.

Pleiotrophin: Analysis of the endothelialisation potential.

PURPOSE: Endothelialisation of vascular substitutes, in fact, remains one of the most unsolved problems in cardiovascular diseases treatment. Stromal Derived Factor 1 (SDF-1) has been largely investigated as an endothelialisation promoter and Pleiotrophin is a promising alternative. Although it has been known to exert beneficial effects on different cell types, its potential as an inducer of proliferation and migration of endothelial cells was not investigated. Therefore, this work is aimed to compare the effects of Pleiotrophin on proliferation and migration of endothelial cells with respect to SDF-1. MATERIALS/METHODS: Endothelial cell line EA.hy926 was treated with Pleiotrophin (50 ng/ml) or SDF-1 (50 ng/ml). Cell viability was evaluated by MTT assay and migration assays were performed in Transwell chambers. Wound healing potential was evaluated by scratch wound assay. CXCR4, RPTP ß/ζ, PCNA and Rac1 expression was detected by Western Blot. RESULTS: Interestingly, Pleiotrophin significantly increased the viability of the treated endothelial cells with respects to SDF-1. The migratory ability of the endothelial cells was also improved in the presence of Pleiotrophin with reference to the SDF-1 treatment. Moreover, Western Blot analysis showed how the treatment with Pleiotrophin can induce an increase in the expression of RPTP ß/ζ, PCNA and Rac1 compared to SDF-1. CONCLUSION: Due to the significant effects exerted on viability, migration and repair ability of endothelial cells compared to SDF-1, Pleiotrophin can be considered as an interesting molecule to promote re-endothelialisation.

Decellularized biological matrices: an interesting approach for cardiovascular tissue repair and regeneration.

The repair and replacement of blood vessels is one of the most challenging topics for biomedical research. Autologous vessels are preferred as graft materials, but they still have many issues to overcome: for instance, they need multiple surgical procedures and often patients may not have healthy and surgically valuable arteries useful as an autograft. A tissue-engineering approach is widely desirable to generate biological vascular prostheses. Recently, decellularization of native tissue has gained significant attention in the biomedical research field. This method is used to obtain biological scaffolds that are expected to maintain the complex three-dimensional structure of the extracellular matrix, preserving the biomechanical properties of the native tissues. The decellularizing methods and the biomechanical characteristics of these products are presented in this review. Decellularization of biological matrices induces the loss of major histocompatibility complex (MHC), which is expected to promote an immunological response by the host. All the studies showed that decellularized biomaterials possess adequate properties for xenografting. Concerning their mechanical properties, several studies have demonstrated that, although chemical decellularization methods do not affect the scaffolds' mechanical properties, these materials can be modified through different treatments in order to provide the desired mechanical characteristics, depending on the specific application. A short overview of legislative issues concerning the use of decellularized substitutes and future perspectives in surgical applications is also presented. Copyright © 2015 John Wiley & Sons, Ltd.

Conditioned medium from human amniotic mesenchymal stromal cells limits infarct size and enhances angiogenesis.

The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.