Three-Dimensional Microfibrous Scaffold with Aligned Topography Produced via a Combination of Melt-Extrusion Additive Manufacturing and Porogen Leaching for In Vitro Skeletal Muscle Modeling.

Skeletal muscle tissue (SMT) has a highly hierarchical and anisotropic morphology, featuring aligned and parallel structures at multiple levels. Various factors, including trauma and disease conditions, can compromise the functionality of skeletal muscle. The in vitro modeling of SMT represents a useful tool for testing novel drugs and therapies. The successful replication of SMT native morphology demands scaffolds with an aligned anisotropic 3D architecture. In this work, a 3D PCL fibrous scaffold with aligned morphology was developed through the synergistic combination of Melt-Extrusion Additive Manufacturing (MEAM) and porogen leaching, utilizing PCL as the bulk material and PEG as the porogen. PCL/PEG blends with different polymer ratios (60/40, 50/50, 40/60) were produced and characterized through a DSC analysis. The MEAM process parameters and porogen leaching in bi-distilled water allowed for the development of a micrometric anisotropic fibrous structure with fiber diameters ranging from 10 to 100 µm, depending on PCL/PEG blend ratios. The fibrous scaffolds were coated with Gelatin type A to achieve a biomimetic coating for an in vitro cell culture and mechanically characterized via AFM. The 40/60 PCL/PEG scaffolds yielded the most homogeneous and smallest fibers and the greatest physiological stiffness. In vitro cell culture studies were performed by seeding C2C12 cells onto a selected scaffold, enabling their attachment, alignment, and myotube formation along the PCL fibers during a 14-day culture period. The resultant anisotropic scaffold morphology promoted SMT-like cell conformation, establishing a versatile platform for developing in vitro models of tissues with anisotropic morphology.

In Vitro Mechanical Stimulation to Reproduce the Pathological Hallmarks of Human Cardiac Fibrosis on a Beating Chip and Predict The Efficacy of Drugs and Advanced Therapies.

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients.

Biomimetic Electrospun Scaffold-Based In Vitro Model Resembling the Hallmarks of Human Myocardial Fibrotic Tissue.

Adverse remodeling post-myocardial infarction is hallmarked by the phenotypic change of cardiac fibroblasts (CFs) into myofibroblasts (MyoFs) and over-deposition of the fibrotic extracellular matrix (ECM) mainly composed by fibronectin and collagens, with the loss of tissue anisotropy and tissue stiffening. Reversing cardiac fibrosis represents a key challenge in cardiac regenerative medicine. Reliable in vitro models of human cardiac fibrotic tissue could be useful for preclinical testing of new advanced therapies, addressing the limited predictivity of traditional 2D cell cultures and animal in vivo models. In this work, we engineered a biomimetic in vitro model, reproducing the morphological, mechanical, and chemical cues of native cardiac fibrotic tissue. Polycaprolactone (PCL)-based scaffolds with randomly oriented fibers were fabricated by solution electrospinning technique, showing homogeneous nanofibers with an average size of 131 ± 39 nm. PCL scaffolds were then surface-functionalized with human type I collagen (C1) and fibronectin (F) by dihydroxyphenylalanine (DOPA)-mediated mussel-inspired approach (PCL/polyDOPA/C1F), in order to mimic fibrotic cardiac tissue-like ECM composition and support human CF culture. BCA assay confirmed the successful deposition of the biomimetic coating and its stability during 5 days of incubation in phosphate-buffered saline. Immunostaining for C1 and F demonstrated their homogeneous distribution in the coating. AFM mechanical characterization showed that PCL/polyDOPA/C1F scaffolds, in wet conditions, resembled fibrotic tissue stiffness with an average Young's modulus of about 50 kPa. PCL/polyDOPA/C1F membranes supported human CF (HCF) adhesion and proliferation. Immunostaining for α-SMA and quantification of α-SMA-positive cells showed HCF activation into MyoFs in the absence of a transforming growth factor ß (TGF-ß) profibrotic stimulus, suggesting the intrinsic ability of biomimetic PCL/polyDOPA/C1F scaffolds to sustain the development of cardiac fibrotic tissue. A proof-of-concept study making use of a commercially available antifibrotic drug confirmed the potentialities of the developed in vitro model for drug efficacy testing. In conclusion, the proposed model was able to replicate the main hallmarks of early-stage cardiac fibrosis, appearing as a promising tool for future preclinical testing of advanced regenerative therapies.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Thiol-Ene Photo-Click Hydrogels with Tunable Mechanical Properties Resulting from the Exposure of Different -Ene Moieties through a Green Chemistry.

Temperature and light responsiveness are widely exploited stimuli to tune the physico-chemical properties of double network hydrogels. In this work, new amphiphilic poly(ether urethane)s bearing photo-sensitive moieties (i.e., thiol, acrylate and norbornene functionalities) were engineered by exploiting the versatility of poly(urethane) chemistry and carbodiimide-mediated green functionalization procedures. Polymers were synthesized according to optimized protocols maximizing photo-sensitive group grafting while preserving their functionality (approx. 1.0 × 1019, 2.6 × 1019 and 8.1 × 1017 thiol, acrylate and norbornene groups/gpolymer), and exploited to prepare thermo- and Vis-light-responsive thiol-ene photo-click hydrogels (18% w/v, 1:1 thiol:ene molar ratio). Green light-induced photo-curing allowed the achievement of a much more developed gel state with improved resistance to deformation (ca. 60% increase in critical deformation, γL). Triethanolamine addition as co-initiator to thiol-acrylate hydrogels improved the photo-click reaction (i.e., achievement of a better-developed gel state). Differently, L-tyrosine addition to thiol-norbornene solutions slightly hindered cross-linking, resulting in less developed gels with worse mechanical performances (~62% γL decrease). In their optimized composition, thiol-norbornene formulations resulted in prevalent elastic behavior at lower frequency compared to thiol-acrylate gels due to the formation of purely bio-orthogonal instead of heterogeneous gel networks. Our findings highlight that exploiting the same thiol-ene photo-click chemistry, a fine tuning of the gel properties is possible by reacting specific functional groups.

Biomaterials-Enhanced Intranasal Delivery of Drugs as a Direct Route for Brain Targeting.

Intranasal (IN) drug delivery is a non-invasive and effective route for the administration of drugs to the brain at pharmacologically relevant concentrations, bypassing the blood-brain barrier (BBB) and minimizing adverse side effects. IN drug delivery can be particularly promising for the treatment of neurodegenerative diseases. The drug delivery mechanism involves the initial drug penetration through the nasal epithelial barrier, followed by drug diffusion in the perivascular or perineural spaces along the olfactory or trigeminal nerves, and final extracellular diffusion throughout the brain. A part of the drug may be lost by drainage through the lymphatic system, while a part may even enter the systemic circulation and reach the brain by crossing the BBB. Alternatively, drugs can be directly transported to the brain by axons of the olfactory nerve. To improve the effectiveness of drug delivery to the brain by the IN route, various types of nanocarriers and hydrogels and their combinations have been proposed. This review paper analyzes the main biomaterials-based strategies to enhance IN drug delivery to the brain, outlining unsolved challenges and proposing ways to address them.

Design of Injectable Bioartificial Hydrogels by Green Chemistry for Mini-Invasive Applications in the Biomedical or Aesthetic Medicine Fields.

Bioartificial hydrogels are hydrophilic systems extensively studied for regenerative medicine due to the synergic combination of features of synthetic and natural polymers. Injectability is another crucial property for hydrogel mini-invasive administration. This work aimed at engineering injectable bioartificial in situ cross-linkable hydrogels by implementing green and eco-friendly approaches. Specifically, the versatile poly(ether urethane) (PEU) chemistry was exploited for the development of an amphiphilic PEU, while hyaluronic acid was selected as natural component. Both polymers were functionalized to expose thiol and catechol groups through green water-based carbodiimide-mediated grafting reactions. Functionalization was optimized to maximize grafting yield while preserving group functionality. Then, polymer miscibility was studied at the macro-, micro-, and nano-scale, suggesting the formation of hydrogen bonds among polymeric chains. All hydrogels could be injected through G21 and G18 needles in a wide temperature range (4-25 °C) and underwent sol-to-gel transition at 37 °C. The addition of an oxidizing agent to polymer solutions did not improve the gelation kinetics, while it negatively affected hydrogel stability in an aqueous environment, suggesting the occurrence of oxidation-triggered polymer degradation. In the future, the bioartificial hydrogels developed herein could find application in the biomedical and aesthetic medicine fields as injectable formulations for therapeutic agent delivery.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

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.

Development of an Innovative Soft Piezoresistive Biomaterial Based on the Interconnection of Elastomeric PDMS Networks and Electrically-Conductive PEDOT:PSS Sponges.

A deeply interconnected flexible transducer of polydimethylsiloxane (PDMS) and poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) was obtained as a material for the application of soft robotics. Firstly, transducers were developed by crosslinking PEDOT:PSS with 3-glycidyloxypropryl-trimethoxysilane (GPTMS) (1, 2 and 3% v/v) and using freeze-drying to obtain porous sponges. The PEDOT:PSS sponges were morphologically characterized, showing porosities mainly between 200 and 600 µm2; such surface area dimensions tend to decrease with increasing degrees of crosslinking. A stability test confirmed a good endurance for up to 28 days for the higher concentrations of the crosslinker tested. Consecutively, the sponges were electromechanically characterized, showing a repeatable and linear resistance variation by the pressure triggers within the limits of their working range (∆RR0 max = 80% for 1-2% v/v of GPTMS). The sponges containing 1% v/v of GPTMS were intertwined with a silicon elastomer to increase their elasticity and water stability. The flexible transducer obtained with this method exhibited moderately lower sensibility and repeatability than the PEDOT:PSS sponges, but the piezoresistive response remained stable under mechanical compression. Furthermore, the transducer displayed a linear behavior when stressed within the limits of its working range. Therefore, it is still valid for pressure sensing and contact detection applications. Lastly, the flexible transducer was submitted to preliminary biological tests that indicate a potential for safe, in vivo sensing applications.

Tissue-Engineered Models of the Human Brain: State-of-the-Art Analysis and Challenges.

Neurological disorders affect billions of people across the world, making the discovery of effective treatments an important challenge. The evaluation of drug efficacy is further complicated because of the lack of in vitro models able to reproduce the complexity of the human brain structure and functions. Some limitations of 2D preclinical models of the human brain have been overcome by the use of 3D cultures such as cell spheroids, organoids and organs-on-chip. However, one of the most promising approaches for mimicking not only cell structure, but also brain architecture, is currently represented by tissue-engineered brain models. Both conventional (particularly electrospinning and salt leaching) and unconventional (particularly bioprinting) techniques have been exploited, making use of natural polymers or combinations between natural and synthetic polymers. Moreover, the use of induced pluripotent stem cells (iPSCs) has allowed the co-culture of different human brain cells (neurons, astrocytes, oligodendrocytes, microglia), helping towards approaching the central nervous system complexity. In this review article, we explain the importance of in vitro brain modeling, and present the main in vitro brain models developed to date, with a special focus on the most recent advancements in tissue-engineered brain models making use of iPSCs. Finally, we critically discuss achievements, main challenges and future perspectives.

Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through microRNAs (miRNAs) is a new emerging strategy for myocardial regeneration after ischemic heart disease. Previous studies have reported that murine fibroblasts can be directly reprogrammed into iCMs by transient transfection with four miRNAs (miRs-1, 133, 208 and 499 - termed "miRcombo"). While advancement in the knowledge of direct cell reprogramming molecular mechanism is in progress, it is important to investigate if this strategy may be translated to humans. Recently, we demonstrated that miRcombo transfection is able to induce direct reprogramming of adult human cardiac fibroblasts (AHCFs) into iCMs. Although additional studies are needed to achieve iCM maturation, our early findings pave the way toward new therapeutic strategies for cardiac regeneration in humans. This chapter describes methods for inducing direct reprogramming of AHCFs into iCMs through miRcombo transient transfection, showing experiments to perform for assessing iCM generation.

Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming.

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial DharmaFECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA downregulation. DE-DOPE/miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo.

Electroconductive Photo-Curable PEGDA-Gelatin/PEDOT:PSS Hydrogels for Prospective Cardiac Tissue Engineering Application.

Electroconductive hydrogels (ECHs) have attracted interest for tissue engineering applications due to their ability to promote the regeneration of electroactive tissues. Hence, ECHs with tunable electrical and mechanical properties, bioactivity, biocompatibility and biodegradability are demanded. In this work, ECHs based on photo-crosslinked blends of polyethylene glycol diacrylate (PEGDA) and gelatin with different PEGDA:gelatin ratios (1:1, 1.5:1 and 2:1 wt./wt.), and containing poly (3,4-ethylenedioxythiophene):poly (styrene sulfonate) (PEDOT:PSS) (0.0, 0.1, 0,3 and 0.5% w/v%) were prepared. Main novelty was the use of gelatin as bioactive component and co-initiator in the photo-crosslinking process, leading to its successful incorporation in the hydrogel network. Physical properties could be modulated by the initial PEGDA:gelatin weight ratio. Pristine hydrogels with increasing PEGDA:gelatin ratio showed: (i) an increasing compressive elastic modulus from 5 to 28 kPa; (ii) a decreasing weight loss from 62% to 43% after 2 weeks incubation in phosphate buffered saline at 37°C; (iii) reduced crosslinking time; (iv) higher crosslinking density and (v) lower water absorption. The addition of PEDOT:PSS in the hydrogels reduced photo-crosslinking time (from 60 to 10 s) increasing their surface and bulk electrical properties. Finally, in vitro tests with human cardiac fibroblasts showed that hydrogels were cytocompatible and samples with 1.5:1 initial PEGDA:gelatin ratio promoted the highest cell adhesion at 24 h. Results from this work suggested the potential of electroconductive photo-curable PEGDA-gelatin/PEDOT:PSS hydrogels for prospective cardiac tissue engineering applications.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Cardiac Tissue-like 3D Microenvironment Enhances Route towards Human Fibroblast Direct Reprogramming into Induced Cardiomyocytes by microRNAs.

The restoration of cardiac functionality after myocardial infarction represents a major clinical challenge. Recently, we found that transient transfection with microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocytes (iCMs) in vitro. However, achieving efficient direct reprogramming still remains a challenge. The aim of this study was to investigate the influence of cardiac tissue-like biochemical and biophysical stimuli on direct reprogramming efficiency. Biomatrix (BM), a cardiac-like extracellular matrix (ECM), was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. In a set of experiments, AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of uncoated and BM-coated polystyrene (PS) dishes and fibrin hydrogels (2D hydrogel) or embedded into 3D fibrin hydrogels (3D hydrogel). Cell culturing on BM-coated PS dishes and in 3D hydrogels significantly improved direct reprogramming outcomes. Biochemical and biophysical cues were then combined in 3D fibrin hydrogels containing BM (3D BM hydrogel), resulting in a synergistic effect, triggering increased CM gene and cardiac troponin T expression in miRcombo-transfected AHCFs. Hence, biomimetic 3D culture environments may improve direct reprogramming of miRcombo-transfected AHCFs into iCMs, deserving further study.

In situ Forming Hyperbranched PEG-Thiolated Hyaluronic Acid Hydrogels With Honey-Mimetic Antibacterial Properties.

The rapidly increasing resistance of bacteria to currently approved antibiotic drugs makes surgical interventions and the treatment of bacterial infections increasingly difficult. In recent years, complementary strategies to classical antibiotic therapy have, therefore, gained importance. One of these strategies is the use of medicinal honey in the treatment of bacterially colonized wounds. One of the several bactericidal effects of honey is based on the in situ generation of hydrogen peroxide through the activity of the enzyme glucose oxidase. The strategy underlying this work is to mimic this antibacterial redox effect of honey in an injectable, biocompatible, and rapidly forming hydrogel. The hydrogel was obtained by thiol-ene click reaction between hyperbranched polyethylene glycol diacrylate (HB PEGDA), synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiolated hyaluronic acid (HA-SH). After mixing 500 µL HB PEGDA (10%, w/w) and 500 µL HA-SH (1%, w/w) solutions, hydrogels formed in ∼60 s (HB PEGDA/HA-SH 10.0-1.0), as assessed by the tube inverting test. The HB PEGDA/HA-SH 10.0-1.0 hydrogel (200 µL) was resistant to in vitro dissolution in water for at least 64 days, absorbing up to 130 wt% of water. Varying glucose oxidase (GO) amounts (0-500 U/L) and constant glucose content (2.5 wt%) were loaded into HB PEGDA and HA-SH solutions, respectively, before hydrogel formation. Then, the release of H2O2 was evaluated through a colorimetric pertitanic acid assay. The GO content of 250 U/L was selected, allowing the formation of 10.8 ± 1.4 mmol H2O2/L hydrogel in 24 h, under static conditions. The cytocompatibility of HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with different GO activities (≤ 500 U/L) at a constant glucose amount (2.5 wt%) was investigated by in vitro assays at 24 h with L929 and HaCaT cell lines, according to DIN EN ISO 10993-5. The tests showed cytocompatibility for GO enzyme activity up to 250 U/L for both cell lines. The antibacterial activity of HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with increasing amounts of GO was demonstrated against various gram-positive bacteria (S. aureus and S. epidermidis), antibiotic-resistant gram-positive bacteria (MRSA and MRSE), gram-negative bacteria (P. aeruginosa, E. coli, and A. baumanii), and antibiotic-resistant gram-negative strains (P. aeruginosa and E. coli) using agar diffusion tests. For all gram-positive bacterial strains, increasing efficacy was measured with increasing GO activity. For the two P. aeruginosa strains, efficacy was shown only from an enzyme activity of 125 U/L and for E. coli and A. baumanii, efficacy was shown only from 250 U/L enzyme activity. HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with ≤250 U/L GO and 2.5 wt% glucose are promising formulations due to their fast-forming properties, cytocompatibility, and ability to produce antibacterial H2O2, warranting future investigations for bacterial infection treatment, such as wound care.

Bioengineering Methods in MicroRNA-Mediated Direct Reprogramming of Fibroblasts Into Cardiomyocytes.

Ischemic heart disease is the major cause of mortality worldwide. Despite the most recent pharmacological progresses, cardiac regeneration is yet not possible, and heart transplantation is the only therapeutic option for end-stage heart failure. Traditional cardiac regenerative medicine approaches, such as cell therapies and tissue engineering, have failed in the obtainment of human functional cardiac tissue, mainly due to unavailability of high quantities of autologous functional cardiomyocytes (CMs), low grafting efficiency, and/or arrhythmic events. Direct reprogramming (DR) of fibroblasts into induced CMs (iCMs) has emerged as a new promising approach for myocardial regeneration by in situ transdifferentiation or providing additional CM source for cell therapy. Among available DR methods, non-viral transfection with microRNAs (miRcombo: miR-1, miR-133, miR-208, and miR-499) appears promising for future clinical translation. MiRcombo transfection of fibroblasts could be significantly improved by the development of safe nanocarriers, efficiently delivering their cargo to target cells at the required stoichiometric ratio and overall dose in due times. Newly designed in vitro 3D culture microenvironments, providing biomimetic biophysical and biochemical stimuli to miRcombo-transfected cells, significantly increase the yield of fibroblast transdifferentiation into iCMs, enhancing CM gene expression. Epigenetic regulation of gene expression programs, critical to cell lineage commitment, can also be promoted by the administration of specific anti-inflammatory and anti-fibrotic soluble factors, helping in suppressing fibroblast signature. The aim of this mini-review is to introduce the readers to a relatively unknown field of cardiac research integrating bioengineering tools as relevant for the progress of miRNA-mediated cardiac DR.

Dual stimuli-responsive polyurethane-based hydrogels as smart drug delivery carriers for the advanced treatment of chronic skin wounds.

The design of multi-stimuli-responsive vehicles for the controlled and localized release of drugs is a challenging issue increasingly catching the attention of many research groups working on the advanced treatment of hard-to-close wounds. In this work, a thermo- and pH-responsive hydrogel (P-CHP407) was prepared from an ad hoc synthesized amphiphilic poly(ether urethane) (CHP407) exposing a significant amount of -COOH groups (8.8 ± 0.9 nmol/gpolymer). The exposure of acid moieties in P-CHP407 hydrogel led to slightly lower initial gelation temperature (12.1 °C vs. 14.6 °C, respectively) and gelation rate than CHP407 hydrogel, as rheologically assessed. Nanoscale hydrogel characterization by Low Field NMR (LF-NMR) spectroscopy suggested that the presence of carboxylic groups in P-CHP407 caused the formation of bigger micelles with a thicker hydrated shell than CHP407 hydrogels, as further proved by Dynamic Light Scattering analyses. In addition, P-CHP407 hydrogel showed improved capability to change its internal pH compared to CHP407 one when incubated with an alkaline buffer (pH 8) (e.g., pHchange_5min = 3.76 and 1.32, respectively). Moreover, LF-NMR characterization suggested a stronger alkaline-pH-induced interaction of water molecules with micelles exposing -COOH groups. Lastly, the hydrogels were found biocompatible according to ISO 10993 and able to load and release Ibuprofen: delivery kinetics of Ibuprofen was enhanced by P-CHP407 hydrogels at alkaline pH, suggesting their potential use as smart delivery systems in the treatment of chronic infected wounds.