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.

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 this work, we investigated, for the first time, the possibility of developing scaffolds for bone tissue engineering through three-dimensional (3D) melt-extrusion printing of medium chain length polyhydroxyalkanoate (mcl-PHA) (i.e., poly(3-hydroxyoctanoate-co-hydroxydecanoate-co-hydroxydodecanoate), P(3HO-co-3HD-co-3HDD)). The process parameters were successfully optimized to produce well-defined and reproducible 3D P(3HO-co-3HD-co-3HDD) scaffolds, showing high cell viability (100%) toward both undifferentiated and differentiated MC3T3-E1 cells. To introduce antibacterial features in the developed scaffolds, two strategies were investigated. For the first strategy, P(3HO-co-3HD-co-3HDD) was combined with PHAs containing thioester groups in their side chains (i.e., PHACOS), inherently antibacterial PHAs. The 3D blend scaffolds were able to induce a 70% reduction of Staphylococcus aureus 6538P cells by direct contact testing, confirming their antibacterial properties. Additionally, the scaffolds were able to support the growth of MC3T3-E1 cells, showing the potential for bone regeneration. For the second strategy, composite materials were produced by the combination of P(3HO-co-3HD-co-HDD) with a novel antibacterial hydroxyapatite doped with selenium and strontium ions (Se-Sr-HA). The composite material with 10 wt % Se-Sr-HA as a filler showed high antibacterial activity against both Gram-positive (S. aureus 6538P) and Gram-negative bacteria (Escherichia coli 8739), through a dual mechanism: by direct contact (inducing 80% reduction of both bacterial strains) and through the release of active ions (leading to a 54% bacterial cell count reduction for S. aureus 6538P and 30% for E. coli 8739 after 24 h). Moreover, the composite scaffolds showed high viability of MC3T3-E1 cells through both indirect and direct testing, showing promising results for their application in bone tissue engineering.

Growing evidence has shown that cells respond to the viscoelastic properties of the extracellular matrix (ECM), particularly its stress-relaxation, which influences their spreading, proliferation, and remodeling. Since cardiac tissue viscoelasticity plays a key role in modulating cellular mechanosensing, the development of biomimetic viscoelastic hydrogels is highly needed in cardiac tissue engineering (CTE). This work presents bio-orthogonal double cross-linked alginate-gelatin hydrogels with tunable viscoelasticity, designed to replicate the dynamic mechanical properties of cardiac ECM. Alginate and gelatin were functionalized with azide groups and cross-linked by a 4-arm-dibenzocyclooctyne (DBCO) crosslinker using strain-promoted azide-alkyne cycloaddition (SPAAC) with 0.5:1 (AG_Click(R0.5)) and 1:1 (AG_Click(R1)) DBCO:azide molar ratios. Calcium ions were also introduced to obtain double cross-linked hydrogels (AG_DC(R0.5) and AG_DC(R1)). Rheology showed that hydrogels exhibited tunable stiffness and stress relaxation, closely mimicking the properties of native cardiac tissue. The behavior of human cardiac fibroblasts (HCFs), seeded on hydrogels, was analyzed. When compared to purely elastic polyacrylamide (pAAm) hydrogels with comparable stiffness, soft stress-relaxing hydrogels (AG_Click(R0.5) and AG_DC(R0.5)) were found to promote cell spreading area, while stiffer stress-relaxing hydrogels (AG_Click(R1) and AG_DC(R1)) enhanced asymmetric cell elongation, reflecting substrate-mediated mechanosensing. Additionally, HCFs showed high viability when cultured in 3D hydrogels over 7 days. Overall, rapid gelation, biocompatibility, and tunable viscoelastic properties of bio-orthogonal double cross-linked alginate-gelatin hydrogels support their use as injectable formulations or engineered cardiac tissues for CTE.

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.

Replicating skeletal muscle architecture remains challenging in 3D bioprinting, as conventional bioinks lack multiscale directional cues. Herein, we propose a next-generation fibrous bioink composed of fragmented electrospun gelatin fibers (f-GFs), uniformly embedded in an alginate/gelatin hydrogel matrix (f-ALG/Gel). Upon micro-extrusion bioprinting, shear-induced f-GF alignment enabled the fabrication of microfilament-based scaffolds with intrinsic anisotropy. The resulting constructs exhibited high shape fidelity, viscoelastic properties, and physiologically relevant stiffness (Young’s modulus: 16.1 ± 1.7 kPa). In vitro studies using C2C12 murine myoblasts demonstrated that the embedded f-GFs provided strong topographical guidance, enhancing cell alignment and myogenesis. After 14 days of culture, the f-ALG/Gel scaffolds supported a 2.5-fold increase in myotube fusion index and length, alongside reduced angular dispersion. These effects were achieved without the need for biochemical induction with a differentiation medium, underscoring the key role of structural cues at the micro- and nanoscale on C2C12 differentiation and maturation. In conclusion, this work proposed a scalable, cell-compatible strategy to recapitulate the hierarchical organization of skeletal muscle tissue within 3D printed constructs. The platform holds broad potential for applications in regenerative medicine, skeletal muscle tissue modelling and the engineering of cultured meat.