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-Barre syndrome in children) and analgesic drug screening

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

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

<p>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</p&gt

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

In vitro investigation of the cellular mechanisms activated by GO and rGO in Mesenchymal Stwm Cells (MSCs)

Η Μηχανική Ιστών ενσωματώνει τη χρήση καινοτόμων βιοϋλικών στοχεύοντας στην επιδιόρθωση της λειτουργίας ή την ανακατασκευή ιστών/οργάνων. Τα υλικά με βάση το γραφένιο (GBMs) έχουν προσελκύσει τεράστιο ενδιαφέρον λόγω της μοναδικής δομής και των ιδιαίτερων ιδιοτήτων τους, ωστόσο, έχει εκφραστεί ανησυχία για τις πιθανές δυσμενείς επιπτώσεις τους. Ως εκ τούτου, είναι υψίστης σημασίας να πραγματοποιηθεί αξιολόγηση των αλληλεπιδράσεων κυττάρου-γραφενίου, καθώς και των υποκείμενων μηχανισμών, προκειμένου να διευκολυνθεί η σωστή ανάπτυξη και χρήση τους για βιοϊατρικές εφαρμογές. Σε αυτή τη διατριβή, μελετήσαμε δύο υλικά που έχουν ως βάση το γραφένιο – το οξείδιο γραφενίου (GO) και το ανηγμένο οξείδιο γραφενίου (rGO) - και την επίδρασή τους στα μεσεγχυματικά βλαστοκύτταρα (MSCs), μια πολυδύναμη κυτταρική σειρά που χρησιμοποιείται ευρέως σε εφαρμογές μηχανικής ιστών. Εστιάσαμε στο αν η παρουσία αυτών των δύο υλικών ενεργοποιεί την απόκριση του κυτταρικού οξειδωτικού στρες, ένα πανταχού παρόν φαινόμενο που σχετίζεται με διαταραχές στη φυσιολογική οξειδοαναγωγική κατάσταση των κυττάρων. Για τον προσδιορισμό της τοξικότητας του γραφενίου, πραγματοποιήθηκαν δοκιμασίες κυτταροτοξικότητας και πολλαπλασιασμού σε κυτταροκαλλιέργειες για διαφορετικές συγκεντρώσεις GO και rGO. Ο εντοπισμός των σημαντικών μεταγραφικών παραγόντων που σχετίζονται με την απόκριση του οξειδωτικού στρες αξιολογήθηκε μέσω ανοσοφθορισμού χρώσης και συνεστιακής μικροσκοπίας. Τα πρότυπα έκφρασης των γονιδίων που κωδικοποιούν πρωτεΐνες των συστημάτων κυτταρικής αντιοξειδωτικής δράσης - γλουταροξίνης και θειορεδοξίνης - μελετήθηκαν επίσης και αναλύθηκαν μέσω της ποσοτικής αλυσιδωτής αντίδρασης της πολυμεράσης με αντίστροφη μεταγραφάση (q RT-PCR), για να ανιχνευθούν πιθανές εναλλαγές στην έκφραση του mRNA που προκαλούνται από την έκθεση των κυττάρων σε GO και rGO. Στην παρούσα εργασία, μπορέσαμε να προσδιορίσουμε ότι τόσο το GO όσο και το rGO επηρεάζουν τις κυτταρικές αποκρίσεις συναρτήσει της συγκέντρωσης/δόσης του υλικού.Tissue Engineering embraces the employment of novel biomaterials in order to realize functional tissue/organ repair or reconstruction. Graphene-based materials (GBMs) have attracted enormous interest due to their unique structure and properties, however, concern has been raised about their potential adverse effects. Therefore, it is of utmost importance to evaluate the cell-graphene interactions, as well as the underlying mechanisms in order to facilitate their proper development and use for biomedical applications. In this thesis, we have studied two GBMs – graphene oxide (GO) and reduced graphene oxide (rGO) – and their effect on mesenchymal stem cells (MSCs), a pluripotent cell line extensively used in tissue engineering applications. We focused on whether the presence of these two materials activates the cellular oxidative stress response, a ubiquitous phenomenon related with disturbances in the normal redox state of cells. To determine the toxicity of graphene, cytotoxicity and proliferation assays were performed in cell cultures for different concentrations of GO and rGO. The localization of key transcription factors relating to the oxidative stress response was evaluated through immunofluorescent staining and confocal microscopy. The expression patterns of genes encoding for proteins of the glutaredoxin and thioredoxin cellular detoxification systems were also studied and analysed via quantitative RT-PCR, to detect potential alternations in mRNA expression caused by GO and rGO exposure. Through this work, we were able to determine that both GO and rGO affect cellular responses in a dose-dependent manner

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

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

Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers

The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity. © 2011 IOP Publishing Ltd