Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barré syndrome in children) and analgesic drug screening.

Controlling the Outgrowth and Functions of Neural Stem Cells: The Effect of Surface Topography.

Neural stem cells (NSCs) are self-renewing cells that generate the major cell types of the central nervous system, namely neurons, astrocytes and oligodendrocytes, during embryonic development and in the adult brain. NSCs reside in a complex niche where they are exposed to a plethora of signals, including both soluble and physical signals such as compressive and shear stresses, but also discontinuities and differences in morphology of the extracellular environment, termed as topographical features. Different approaches that incorporate artificial micro- and nano-scale surface topographical features have been developed aiming to recapitulate the in vivo NSC niche discontinuities and features, particularly for in vitro studies. The present review article aims at reviewing the existing body of literature on the use of artificial micro- and nano-topographical features to control NSCs orientation and differentiation into neuronal and/or neuroglial lineage. The different approaches on the study of the underlying mechanism of the topography-guided NSC responses are additionally revised and discussed.

Engineering Cell Adhesion and Orientation via Ultrafast Laser Fabricated Microstructured Substrates.

Cell responses depend on the stimuli received by the surrounding extracellular environment, which provides the cues required for adhesion, orientation, proliferation, and differentiation at the micro and the nano scales. In this study, discontinuous microcones on silicon (Si) and continuous microgrooves on polyethylene terephthalate (PET) substrates were fabricated via ultrashort pulsed laser irradiation at various fluences, resulting in microstructures with different magnitudes of roughness and varying geometrical characteristics. The topographical models attained were specifically developed to imitate the guidance and alignment of Schwann cells for the oriented axonal regrowth that occurs in nerve regeneration. At the same time, positive replicas of the silicon microstructures were successfully reproduced via soft lithography on the biodegradable polymer poly(lactide-co-glycolide) (PLGA). The anisotropic continuous (PET) and discontinuous (PLGA replicas) microstructured polymeric substrates were assessed in terms of their influence on Schwann cell responses. It is shown that the micropatterned substrates enable control over cellular adhesion, proliferation, and orientation, and are thus useful to engineer cell alignment in vitro. This property is potentially useful in the fields of neural tissue engineering and for dynamic microenvironment systems that simulate in vivo conditions.

Amniotic Membrane-Derived Multichannel Hydrogels for Neural Tissue Repair.

In the pursuit of advancing neural tissue regeneration, biomaterial scaffolds have emerged as promising candidates, offering potential solutions for nerve disruptions. Among these scaffolds, multichannel hydrogels, characterized by meticulously designed micrometer-scale channels, stand out as instrumental tools for guiding axonal growth and facilitating cellular interactions. This study explores the innovative application of human amniotic membranes modified with methacryloyl domains (AMMA) in neural stem cell (NSC) culture. AMMA hydrogels, possessing a tailored softness resembling the physiological environment, are prepared in the format of multichannel scaffolds to simulate native-like microarchitecture of nerve tracts. Preliminary experiments on AMMA hydrogel films showcase their potential for neural applications, demonstrating robust adhesion, proliferation, and differentiation of NSCs without the need for additional coatings. Transitioning into the 3D realm, the multichannel architecture fosters intricate neuronal networks guiding neurite extension longitudinally. Furthermore, the presence of synaptic vesicles within the cellular arrays suggests the establishment of functional synaptic connections, underscoring the physiological relevance of the developed neuronal networks. This work contributes to the ongoing efforts to find ethical, clinically translatable, and functionally relevant approaches for regenerative neuroscience.

Microfluidic Systems for Neural Cell Studies.

Whereas the axons of the peripheral nervous system (PNS) spontaneously regenerate after an injury, the occurring regeneration is rarely successful because axons are usually directed by inappropriate cues. Therefore, finding successful ways to guide neurite outgrowth, in vitro, is essential for neurogenesis. Microfluidic systems reflect more appropriately the in vivo environment of cells in tissues such as the normal fluid flow within the body, consistent nutrient delivery, effective waste removal, and mechanical stimulation due to fluid shear forces. At the same time, it has been well reported that topography affects neuronal outgrowth, orientation, and differentiation. In this review, we demonstrate how topography and microfluidic flow affect neuronal behavior, either separately or in synergy, and highlight the efficacy of microfluidic systems in promoting neuronal outgrowth.

Laser-Structured Si and PLGA Inhibit the Neuro2a Differentiation in Mono- and Co-Culture with Glia.

BACKGROUND: The first step towards a successful neural tissue engineering therapy is the development of an appropriate scaffold and the in vitro study of the cellular response onto it. METHODS: Here, we fabricated nano- and micro- patterned Si surfaces via direct ultrafast laser irradiation, as well as their replicas in the biodegradable poly(lactide-co-glycolide), in order to use them as culture substrates for neuronal cells. The differentiation of neuro2a cells on the Si platforms and their replicas was studied both in a mono-culture and in a co-culture with glial cells (Schwann-SW10). RESULTS: It was found that the substrate's roughness inhibits the differentiation of the neuronal cells even in the presence of the differentiation medium, and the higher the roughness is, the more the differentiation gets limited. CONCLUSION: Our results highlight the importance of the substrate's topography for the controlled growth and differentiation of the neuronal cells and their further study via protein screening methods could shed light on the factors that lead to limited differentiation; thus, contributing to the long standing request for culture substrates that induce cells to differentiate.

Interfacing reduced graphene oxide with an adipose-derived extracellular matrix as a regulating milieu for neural tissue engineering.

Enthralling evidence of the potential of graphene-based materials for neural tissue engineering is motivating the development of scaffolds using various structures related to graphene such as graphene oxide (GO) or its reduced form. Here, we investigated a strategy based on reduced graphene oxide (rGO) combined with a decellularized extracellular matrix from adipose tissue (adECM), which is still unexplored for neural repair and regeneration. Scaffolds containing up to 50 wt% rGO relative to adECM were prepared by thermally induced phase separation assisted by carbodiimide (EDC) crosslinking. Using partially reduced GO enables fine-tuning of the structural interaction between rGO and adECM. As the concentration of rGO increased, non-covalent bonding gradually prevailed over EDC-induced covalent conjugation with the adECM. Edge-to-edge aggregation of rGO favours adECM to act as a biomolecular physical crosslinker to rGO, leading to the softening of the scaffolds. The unique biochemistry of adECM allows neural stem cells to adhere and grow. Importantly, high rGO concentrations directly control cell fate by inducing the differentiation of both NE-4C cells and embryonic neural progenitor cells into neurons. Furthermore, primary astrocyte fate is also modulated as increasing rGO boosts the expression of reactivity markers while unaltering the expression of scar-forming ones.

Development of an Oriented Co-Culture System Using 3D Scaffolds Fabricated via Non-Linear Lithography.

Damage in the Peripheral Nervous System (PNS) is related to numerous neurodegenerative diseases and has consequently drawn the attention of Tissue Engineering (TE), which is considered a promising alternative to already established methods such as surgery and autografts. TE focuses on the design, optimization, and use of scaffolds in vitro and in vivo. In this work, the authors used a novel scaffold geometry fabricated via Multiphoton Lithography (MPL), a commonly used fabrication method, for the mono- and co-cultures of glial Schwann (SW10) and neuronal Neuro-2a (N2a) cells. Both cell types have already been used for the study of various neurodegenerative diseases. However, their focus has been on only one of the cell types at a time, with studies regarding their co-culture only recently documented. Here, the suitability of the fabricated scaffolds has been explored and the effects of topography on SW10 and N2a behavior have been investigated. Our findings demonstrate that scaffold co-culture systems favor the presence of neurites compared to mono-cultures at 21 days (31.4 ± 5.5% and 15.4 ± 5.4%, respectively), while there is also a significant decrease in long neurites in the mono-culture over time (45.3 ± 15.9% at 7 days versus 15.4 ± 5.4% at 21 days). It has been shown that the scaffolds can successfully manipulate cell growth, elongation, and morphology, and these results can form a basis for the development of an experimental model for the study of PNS-related diseases and understanding of key cell functions such as myelination.

Fabrication of Biomimetic 2D Nanostructures through Irradiation of Stainless Steel Surfaces with Double Femtosecond Pulses.

Femtosecond laser induced changes on the topography of stainless steel with double pulses is investigated to reveal the role of parameters such as the fluence, the energy dose and the interpulse delay on the features of the produced patterns. Our results indicate that short pulse separation (Δτ = 5 ps) favors the formation of 2D Low Spatially Frequency Laser Induced Periodic Surface Structures (LSFL) while longer interpulse delays (Δτ = 20 ps) lead to 2D High Spatially Frequency LIPSS (HSFL). The detailed investigation is complemented with an analysis of the produced surface patterns and characterization of their wetting and cell-adhesion properties. A correlation between the surface roughness and the contact angle is presented which confirms that topographies of variable roughness and complexity exhibit different wetting properties. Furthermore, our analysis indicates that patterns with different spatial characteristics demonstrate variable cell adhesion response which suggests that the methodology can be used as a strategy towards the fabrication of tailored surfaces for the development of functional implants.

How the Physicochemical Properties of Manufactured Nanomaterials Affect Their Performance in Dispersion and Their Applications in Biomedicine: A Review.

The growth in novel synthesis methods and in the range of possible applications has led to the development of a large variety of manufactured nanomaterials (MNMs), which can, in principle, come into close contact with humans and be dispersed in the environment. The nanomaterials interact with the surrounding environment, this being either the proteins and/or cells in a biological medium or the matrix constituent in a dispersion or composite, and an interface is formed whose properties depend on the physicochemical interactions and on colloidal forces. The development of predictive relationships between the characteristics of individual MNMs and their potential practical use critically depends on how the key parameters of MNMs, such as the size, shape, surface chemistry, surface charge, surface coating, etc., affect the behavior in a test medium. This relationship between the biophysicochemical properties of the MNMs and their practical use is defined as their functionality; understanding this relationship is very important for the safe use of these nanomaterials. In this mini review, we attempt to identify the key parameters of nanomaterials and establish a relationship between these and the main MNM functionalities, which would play an important role in the safe design of MNMs; thus, reducing the possible health and environmental risks early on in the innovation process, when the functionality of a nanomaterial and its toxicity/safety will be taken into account in an integrated way. This review aims to contribute to a decision tree strategy for the optimum design of safe nanomaterials, by going beyond the compromise between functionality and safety.

The role of mechanobiology on the Schwann cell response: A tissue engineering perspective.

Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS), do not only form myelin sheaths thereby insulating the electrical signal propagated by the axons, but also play an essential role in the regeneration of injured axons. SCs are inextricably connected with their extracellular environment and the mechanical stimuli that are received determine their response during development, myelination and injuries. To this end, the mechanobiological response of SCs is being actively researched, as it can determine the suitability of fabricated scaffolds for tissue engineering and regenerative medicine applications. There is growing evidence that SCs are sensitive to changes in the mechanical properties of the surrounding environment (such as the type of material, its elasticity and stiffness), different topographical features provided by the environment, as well as shear stress. In this review, we explore how different mechanical stimuli affect SC behaviour and highlight the importance of exploring many different avenues when designing scaffolds for the repair of PNS injuries.

Following the course of pre-implantation embryo patterning by non-linear microscopy.

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

Combined effect of shear stress and laser-patterned topography on Schwann cell outgrowth: synergistic or antagonistic?

Although the peripheral nervous system exhibits a higher rate of regeneration than that of the central nervous system through a spontaneous regeneration after injury, the functional recovery is fairly infrequent and misdirected. Thus, the development of successful methods to guide neuronal outgrowth, in vitro, is of great importance. In this study, a precise flow controlled microfluidic system with specific custom-designed chambers, incorporating laser-microstructured polyethylene terephthalate (PET) substrates comprising microgrooves, was fabricated to assess the combined effect of shear stress and topography on Schwann cells' behavior. The microgrooves were positioned either parallel or perpendicular to the direction of the flow inside the chambers. Additionally, the cell culture results were combined with computational flow simulations to calculate accurately the shear stress values. Our results demonstrated that wall shear stress gradients may be acting either synergistically or antagonistically depending on the substrate groove orientation relative to the flow direction. The ability to control cell alignment in vitro could potentially be used in the fields of neural tissue engineering and regenerative medicine.

Response of NIH 3T3 Fibroblast Cells on Laser-Induced Periodic Surface Structures on a 15×(Ti/Zr)/Si Multilayer System.

Ultrafast laser processing with the formation of periodic surface nanostructures on the 15×(Ti/Zr)/Si multilayers is studied in order to the improve cell response. A novel nanocomposite structure in the form of 15x(Ti/Zr)/Si multilayer thin films, with satisfying mechanical properties and moderate biocompatibility, was deposited by ion sputtering on an Si substrate. The multilayer 15×(Ti/Zr)/Si thin films were modified by femtosecond laser pulses in air to induce the following modifications: (i) mixing of components inside of the multilayer structures, (ii) the formation of an ultrathin oxide layer at the surfaces, and (iii) surface nano-texturing with the creation of laser-induced periodic surface structure (LIPSS). The focus of this study was an examination of the novel Ti/Zr multilayer thin films in order to create a surface texture with suitable composition and structure for cell integration. Using the SEM and confocal microscopies of the laser-modified Ti/Zr surfaces with seeded cell culture (NIH 3T3 fibroblasts), it was found that cell adhesion and growth depend on the surface composition and morphological patterns. These results indicated a good proliferation of cells after two and four days with some tendency of the cell orientation along the LIPSSs.

Cells on hierarchically-structured platforms hosting functionalized nanoparticles.

In this work, we report on a novel approach to develop hierarchically-structured cell culture platforms incorporating functionalized gold nanoparticles (AuNPs). In particular, the hierarchical substrates comprise primary pseudo-periodic arrays of silicon microcones combined with a secondary nanoscale pattern of homogeneously deposited AuNPs terminated with bio-functional moieties. AuNPs with various functionalities (i.e. oligopeptides, small molecules and oligomers) were successfully attached onto the microstructures. Experiments with PC12 cells on hierarchical substrates incorporating AuNPs carrying the RGD peptide showed an impressive growth and NGF-induced differentiation of the PC12 cells, compared to that on the NP-free, bare, micropatterned substrates. The exploitation of the developed methodology for the binding of AuNPs as carriers of specific bio-functional moieties onto micropatterned culture substrates for cell biology studies is envisaged.

Iron Oxide Colloidal Nanoclusters as Theranostic Vehicles and Their Interactions at the Cellular Level.

Advances in surfactant-assisted chemical approaches have led the way for the exploitation of nanoscale inorganic particles in medical diagnosis and treatment. In this field, magnetically-driven multimodal nanotools that perform both detection and therapy, well-designed in size, shape and composition, are highly advantageous. Such a theranostic material—which entails the controlled assembly of smaller (maghemite) nanocrystals in a secondary motif that is highly dispersible in aqueous media—is discussed here. These surface functionalized, pomegranate-like ferrimagnetic nanoclusters (40⁻85 nm) are made of nanocrystal subunits that show a remarkable magnetic resonance imaging contrast efficiency, which is better than that of the superparamagnetic contrast agent Endorem©. Going beyond this attribute and with their demonstrated low cytotoxicity in hand, we examine the critical interaction of such nanoprobes with cells at different physiological environments. The time-dependent in vivo scintigraphic imaging of mice experimental models, combined with a biodistribution study, revealed the accumulation of nanoclusters in the spleen and liver. Moreover, the in vitro proliferation of spleen cells and cytokine production witnessed a size-selective regulation of immune system cells, inferring that smaller clusters induce mainly inflammatory activities, while larger ones induce anti-inflammatory actions. The preliminary findings corroborate that the modular chemistry of magnetic iron oxide nanoclusters stimulates unexplored pathways that could be driven to alter their function in favor of healthcare.

Laser processing of protein films as a method for accomplishment of cell patterning at the microscale.

In this study, we propose a photostructuring approach for protein films based on a treatment with nanosecond pulses of a KrF excimer laser. As a model protein we used an amyloid fibril-forming protein. Laser treatment induced a foaming of the sample surface exhibiting an interconnected fibrous mesh with a high degree of control and precision. The surface foaming was well characterized by scanning electron microscopy, atomic force microscopy, laser induced fluorescence and contact angle measurements. The laser irradiated areas of the protein films acquired new morphological and physicochemical properties that could be exploited to fulfill unmet challenges in the tissue engineering field. In this context we subsequently evaluated the response of NIH/3T3 fibroblast cell line on the processed film. Our results show a strong and statistically significant preference for adhesion and proliferation of cells on the irradiated areas compared to the non-irradiated ones. We propose that this strategy can be followed to induce selective cell patterning on protein films at the microscale.

Self-Assembled Amyloid Peptides with Arg-Gly-Asp (RGD) Motifs As Scaffolds for Tissue Engineering.

Self-assembled peptides gain increasing interest as biocompatible and biodegradable scaffolds for tissue engineering. Rationally designed self-assembling building blocks that carry cell adhesion motifs such as Arg-Gly-Asp (RGD) are especially attractive. We have used a combination of theoretical and experimental approaches toward such rational designs, especially focusing on modular designs that consist of a central ultrashort amphiphilic motif derived from the adenovirus fiber shaft. In this study, we rationally designed RGDSGAITIGC, a bifunctional self-assembling amyloid peptide which encompasses cell adhesion and potential cysteine-mediated functionalization properties through the incorporation of an RGD sequence motif and a cysteine residue at the N- and C- terminal end, respectively. We performed replica exchange MD simulations that suggested that the key factor determining cell adhesion is the total solvent accessibility of the RGD motif and also that the C-terminal cysteine is adequately exposed. The designer peptides self-assembled into fibers that are structurally characterized with Transmission Electron Microscopy, Scanning Electron Microscopy and X-ray fiber diffraction. Furthermore, they supported cell adhesion and proliferation of a model cell line. We consider that the current bifunctional properties of the RGDSGAITIGC fibril-forming peptide can be exploited to fabricate novel biomaterials with promising biomedical applications. Such short self-assembling peptides that are amenable to computational design offer open-ended possibilities toward multifunctional tissue engineering scaffolds of the future.

Constitutive intracellular expression of human leukocyte antigen (HLA)-DO and HLA-DR but not HLA-DM in trophoblast cells.

The nonclassic human leukocyte antigen (HLA)-DM molecules have been proved to positively regulate antigen presentation in classic antigen-presenting cells, whereas in B lymphocytes HLA-DO have been identified as negative regulators of the process. The present report examines whether the negative expression of classic class II molecules in trophoblasts implies negative regulation by HLA-DO. It was revealed by immunofluorescence, confocal microscopy, and subcellular fractionation techniques that human trophoblasts, although not expressing any surface HLA-DR antigens, constitutively express intracellular HLA-DR, HLA-DO, and CD74, but not HLA-DM. Administration of interferon-gamma to the cell culture increased HLA-DR and CD74, induced HLA-DM, but did not alter the expression of HLA-DO and induced HLA-DR release from the cells. These results were confirmed by reverse transcriptase-polymerase chain reaction analysis except that HLA-DM mRNA was detected in control cells, indicating a posttranscriptional regulation. Under the same experimental conditions, human monocytes/macrophages were not expressing intracellular HLA-DO while exhibiting significant levels of HLA-DR, HLA-DM, and CD74. The results presented here reveal for the first time expression of HLA-DO in trophoblasts, which can be of great importance in maintaining the class II-negative state in these cells and consequently protecting the fetus from maternal immune attack.

Implantable vaccine development using in vitro antigen-pulsed macrophages absorbed on laser micro-structured Si scaffolds.

To overcome the limiting antigenic repertoire of protein sub-units and the side effects of adjuvants applied in second generation vaccines, the present work combined in vitro and in vivo manipulations to develop biomaterials allowing natural antigen-loading and presentation in vitro and further activation of the immune response in vivo. 3-dimensional laser micro-textured implantable Si-scaffolds supported mouse macrophage adherence, allowed natural seeding with human serum albumin (antigen) and specific antibody and inflammatory cytokine production in vitro. Implantation of Si-scaffolds loaded with antigen-activated macrophages induced an inflammatory reaction along with antigen-specific antibody production in vivo, which could be detected even 30 days post implantation. Analysis of implant histology using scanning electron microscopy showed that Si-scaffolds could be stable for a 6-month period. Such technology leads to personalized implantable vaccines, opening novel areas of research and treatment.