Chitosan Micro-Grooved Membranes with Increased Asymmetry for the Improvement of the Schwann Cell Response in Nerve Regeneration.

Peripheral nerve injuries are a common condition in which a nerve is damaged, affecting more than one million people every year. There are still no efficient therapeutic treatments for these injuries. Artificial scaffolds can offer new opportunities for nerve regeneration applications; in this framework, chitosan is emerging as a promising biomaterial. Here, we set up a simple and effective method for the production of micro-structured chitosan films by solvent casting, with high fidelity in the micro-pattern reproducibility. Three types of chitosan directional micro-grooved patterns, presenting different levels of symmetricity, were developed for application in nerve regenerative medicine: gratings (GR), isosceles triangles (ISO) and scalene triangles (SCA). The directional patterns were tested with a Schwann cell line. The most asymmetric topography (SCA), although it polarized the cell shaping less efficiently, promoted higher cell proliferation and a faster cell migration, both individually and collectively, with a higher directional persistence of motion. Overall, the use of micro-structured asymmetrical directional topographies may be exploited to enhance the nerve regeneration process mediated by chitosan scaffolds.

Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease.

Krabbe disease (KD) is a genetic disorder caused by the absence of the galactosylceramidase (GALC) functional enzyme. No cure is currently available. Here, we investigate the mechanotransduction process in primary fibroblasts collected from the twitcher mouse, a natural KD murine model. Thanks to mechanotransduction, cells can sense their environment and convert external mechanical stimuli into biochemical signals that result in intracellular changes. In GALC-deficient fibroblasts, we show that focal adhesions (FAs), the protein clusters necessary to adhere and migrate, are increased, and that single-cell migration and wound healing are impaired. We also investigate the involvement of the autophagic process in this framework. We show a dysregulation in the FA turnover: here, the treatment with the autophagy activator rapamycin boosts cell migration and improves the clearance of FAs in GALC-deficient fibroblasts. We propose mechanosensing impairment as a novel potential pathological mechanism in twitcher fibroblasts, and more in general in Krabbe disease.

Current treatment options and novel nanotechnology-driven enzyme replacement strategies for lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are a vast group of more than 50 clinically identified metabolic diseases. They are singly rare, but they affect collectively 1 on 5,000 live births. They result in most of the cases from an enzymatic defect within lysosomes, which causes the subsequent augmentation of unwanted substrates. This accumulation process leads to plenty of clinical signs, determined by the specific substrate and accumulation area. The majority of LSDs present a broad organ and tissue engagement. Brain, connective tissues, viscera and bones are usually afflicted. Among them, brain disease is markedly frequent (two-thirds of LSDs). The most clinically employed approach to treat LSDs is enzyme replacement therapy (ERT), which is practiced by administering systemically the missed or defective enzyme. It represents a healthful strategy for 11 LSDs at the moment, but it solves the pathology only in the case of Gaucher disease. This approach, in fact, is not efficacious in the case of LSDs that have an effect on the central nervous system (CNS) due to the existence of the blood-brain barrier (BBB). Additionally, ERT suffers from several other weak points, such as low penetration of the exogenously administered enzyme to poorly vascularized areas, the development of immunogenicity and infusion-associated reactions (IARs), and, last but not least, the very high cost and lifelong needed. To ameliorate these weaknesses lot of efforts have been recently spent around the development of innovative nanotechnology-driven ERT strategies. They may boost the power of ERT and minimize adverse reactions by loading enzymes into biodegradable nanomaterials. Enzyme encapsulation into biocompatible liposomes, micelles, and polymeric nanoparticles, for example, can protect enzymatic activity, eliminating immunologic reactions and premature enzyme degradation. It can also permit a controlled release of the payload, ameliorating pharmacokinetics and pharmacodynamics of the drug. Additionally, the potential to functionalize the surface of the nanocarrier with targeting agents (antibodies or peptides), could promote the passage through biological barriers. In this review we examined the clinically applied ERTs, highlighting limitations that do not allow to completely cure the specific LSD. Later, we critically consider the nanotechnology-based ERT strategies that have beenin-vitroand/orin-vivotested to improve ERT efficacy.

Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder.

PCDH19 epilepsy (DEE9) is an X-linked syndrome associated with cognitive and behavioral disturbances. Since heterozygous females are affected, while mutant males are spared, it is likely that DEE9 pathogenesis is related to disturbed cell-to-cell communication associated with mosaicism. However, the effects of mosaic PCDH19 expression on cortical networks are unknown. We mimicked the pathology of DEE9 by introducing a patch of mosaic protein expression in one hemisphere of the cortex of conditional PCDH19 knockout mice one day after birth. In the contralateral area, PCDH19 expression was unaffected, thus providing an internal control. In this model, we characterized the physiology of the disrupted network using local field recordings and two photon Ca2+ imaging in urethane anesthetized mice. We found transient episodes of hyperexcitability in the form of brief hypersynchronous spikes or bursts of field potential oscillations in the 9-25 Hz range. Furthermore, we observed a strong disruption of slow wave activity, a crucial component of NREM sleep. This phenotype was present also when PCDH19 loss occurred in adult mice, demonstrating that PCDH19 exerts a function on cortical circuitry outside of early development. Our results indicate that a focal mosaic mutation of PCDH19 disrupts cortical networks and broaden our understanding of DEE9.

Chitosan films for regenerative medicine: fabrication methods and mechanical characterization of nanostructured chitosan films.

Regenerative medicine is continuously facing new challenges and it is searching for new biocompatible, green/natural polymer materials, possibly biodegradable and non-immunogenic. Moreover, the critical importance of the nano/microstructuring of surfaces is overall accepted for their full biocompatibility and in vitro/in vivo performances. Chitosan is emerging as a promising biopolymer for tissue engineering and its application can be further improved by exploiting its nano/microstructuration. Here, we report the state of the art of chitosan films and scaffolds nano/micro-structuration. We show that it is possible to obtain, by solvent casting, chitosan thin films with good mechanical properties and to structure them at the microscale and even nanoscale level, with resolutions down to 100 nm.