Accelerated epithelial layer healing induced by tactile anisotropy in surface topography.

Mammalian cells respond to tactile cues from topographic elements presented by the substrate. Among these, anisotropic features distributed in an ordered manner give directionality. In the extracellular matrix, this ordering is embedded in a noisy environment altering the contact guidance effect. To date, it is unclear how cells respond to topographical signals in a noisy environment. Here, using rationally designed substrates, we report morphotaxis, a guidance mechanism enabling fibroblasts and epithelial cells to move along gradients of topographic order distortion. Isolated cells and cell ensembles perform morphotaxis in response to gradients of different strength and directionality, with mature epithelia integrating variations of topographic order over hundreds of micrometers. The level of topographic order controls cell cycle progression, locally delaying or promoting cell proliferation. In mature epithelia, the combination of morphotaxis and noise-dependent distributed proliferation provides a strategy to enhance wound healing as confirmed by a mathematical model capturing key elements of the process.

Neuronal contact guidance and YAP signaling on ultra-small nanogratings.

Contact interaction of neuronal cells with extracellular nanometric features can be exploited to investigate and modulate cellular responses. By exploiting nanogratings (NGs) with linewidth from 500 nm down to 100 nm, we here study neurite contact guidance along ultra-small directional topographies. The impact of NG lateral dimension on the neuronal morphotype, neurite alignment, focal adhesion (FA) development and YAP activation is investigated in nerve growth factor (NGF)-differentiating PC12 cells and in primary hippocampal neurons, by confocal and live-cell total internal reflection fluorescence (TIRF) microscopy, and at molecular level. We demonstrate that loss of neurite guidance occurs in NGs with periodicity below 400 nm and correlates with a loss of FA lateral constriction and spatial organization. We found that YAP intracellular localization is modulated by the presence of NGs, but it is not sensitive to their periodicity. Nocodazole, a drug that can increase cell contractility, is finally tested for rescuing neurite alignment showing mild ameliorative effects. Our results provide new indications for a rational design of biocompatible scaffolds for enhancing nerve-regeneration processes.

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons.

Background: Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods: Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results: We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber-binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions: We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

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.

Perfluoropolyether (PFPE) Intermediate Molds for High-Resolution Thermal Nanoimprint Lithography.

Among soft lithography techniques, Thermal Nanoimprint Lithography (NIL) is a high-throughput and low-cost process that can be applied to a broad range of thermoplastic materials. By simply applying the appropriate pressure and temperature combination, it is possible to transfer a pattern from a mold surface to the chosen material. Usually, high-resolution and large-area NIL molds are difficult to fabricate and expensive. Furthermore, they are typically made of silicon or other hard materials such as nickel or quartz for preserving their functionality. Nonetheless, after a large number of imprinting cycles, they undergo degradation and become unusable. In this paper, we introduce and characterize an innovative two-step NIL process based on the use of a perfluoropolyether (PFPE) intermediate mold to replicate sub-100 nm features from a silicon mold to the final thermoplastic material. We compare PFPE elastomeric molds with molds made of the standard polydimethylsiloxane (PDMS) elastomer, which demonstrates better resolution and fidelity of the replica process. By using PFPE intermediate molds, the nanostructured masters are preserved and the throughput of the process is significantly enhanced.

Hierarchical thermoplastic rippled nanostructures regulate Schwann cell adhesion, morphology and spatial organization.

Periodic ripples are a variety of anisotropic nanostructures that can be realized by ion beam irradiation on a wide range of solid surfaces. Only a few authors have investigated these surfaces for tuning the response of biological systems, probably because it is challenging to directly produce them in materials that well sustain long-term cellular cultures. Here, hierarchical rippled nanotopographies with a lateral periodicity of ∼300 nm are produced from a gold-irradiated germanium mold in polyethylene terephthalate (PET), a biocompatible polymer approved by the US Food and Drug Administration for clinical applications, by a novel three-step embossing process. The effects of nano-ripples on Schwann Cells (SCs) are studied in view of their possible use for nerve-repair applications. The data demonstrate that nano-ripples can enhance short-term SC adhesion and proliferation (3-24 h after seeding), drive their actin cytoskeleton spatial organization and sustain long-term cell growth. Notably, SCs are oriented perpendicularly with respect to the nanopattern lines. These results provide information about the possible use of hierarchical nano-rippled elements for nerve-regeneration protocols.

Novel Light-Responsive Biocompatible Hydrogels Produced by Initiated Chemical Vapor Deposition.

A novel multiresponsive hydrogel has been synthesized by initiated chemical vapor deposition (iCVD). Hydrogels are known for their dynamic swelling response to aqueous environments. A chemical functionalization of the hydrogel surface was performed to add other stimuli-responsive functionalities and obtain a smart material that responds to two stimuli: light irradiation and exposure to aqueous environment. Modifying the hydrogel surface with solution-based methods is often problematic because of the damages caused by the permeation of solvents in the hydrogel. This issue is completely bypassed by the use of solvent-free techniques. Cross-linked polymers of 2-hydroxyethyl methacrylate (HEMA) were functionalized with azobenzene groups, as confirmed by IR spectroscopy and X-ray photoelectron spectroscopy (XPS). Through photoisomerization of the azobenzene, the polarity within the hydrogel is modified and as a consequence the affinity to water. Light irradiation modifies the degree of swelling within thin hydrogel films from 13% before exposure to UV light to 25% after exposure. The possibility of controlling the degree and rate of swelling by light irradiation was never reported before on these time scales and can have exceptional implications for light-induced drug delivery or light-controlled microfluidic systems. The light-responsive hydrogels showed also biocompatibility, which makes them suitable for a great variety of applications as biomaterials.

Organosilicon phantom for photoacoustic imaging.

Photoacoustic imaging is an emerging technique. Although commercially available photoacoustic imaging systems currently exist, the technology is still in its infancy. Therefore, the design of stable phantoms is essential to achieve semiquantitative evaluation of the performance of a photoacoustic system and can help optimize the properties of contrast agents. We designed and developed a polydimethylsiloxane (PDMS) phantom with exceptionally fine geometry; the phantom was tested using photoacoustic experiments loaded with the standard indocyanine green dye and compared to an agar phantom pattern through polyethylene glycol-gold nanorods. The linearity of the photoacoustic signal with the nanoparticle number was assessed. The signal-tonoiseratio and contrast were employed as image quality parameters, and enhancements of up to 50 and up to 300%, respectively, were measured with the PDMS phantom with respect to the agar one. A tissue-mimicking (TM)-PDMS was prepared by adding TiO2 and India ink; photoacoustic tests were performed in order to compare the signal generated by the TM-PDMS and the biological tissue. The PDMS phantom can become a particularly promising tool in the field of photoacoustics for the evaluation of the performance of a PA system and as a model of the structure of vascularized soft tissues.