In tissue engineering, it is well accepted that a scaffold surface has a decisive impact on cell behaviour. Here we focused on microglia-the resident immune cells of the central nervous system (CNS)-and on their response to poly(trimethylene carbonate-co-1-caprolactone) (P(TMC-CL)) fibrous and flat surfaces obtained by electrospinning and solvent cast, respectively. This study aims to provide cues for the design of instructive surfaces that can contribute to the challenging process of CNS regeneration. Cell morphology was evidently affected by the substrate, mirroring the surface main features. Cells cultured on flat substrates presented a round shape, while cells with elongated processes were observed on the electrospun fibres. A higher concentration of the pro-inflammatory cytokine tumour necrosis factor-a was detected in culture media from microglia on fibres. Still, astrogliosis is not exacerbated when astrocytes are cultured in the presence of microgliaconditioned media obtained from cultures in contact with either substrate. Furthermore, a significant percentage of microglia was found to participate in the process of myelin phagocytosis, with the formation of multinucleated giant cells being observed only on films. Altogether, the results presented suggest that microglia in contact with the tested substrates may contribute to the regeneration process, putting forward P(TMC-CL) substrates as supporting matrices for nerve regeneration.

Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease.

Mammalian central nervous system (CNS) neurons do not regenerate after injury due to the inhibitory environment formed by the glial scar, largely constituted by myelin debris. The use of biomaterials to bridge the lesion area and the creation of an environment favoring axonal regeneration is an appealing approach, currently under investigation. This work aimed at assessing the suitability of three candidate polymers – poly(ε-caprolactone), poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) (11∶89 mol%) and poly(trimethylene carbonate) - with the final goal of using these materials in the development of conduits to promote spinal cord regeneration. Poly(L-lysine) (PLL) coated polymeric films were tested for neuronal cell adhesion and neurite outgrowth. At similar PLL film area coverage conditions, neuronal polarization and axonal elongation was significantly higher on P(TMC-CL) films. Furthermore, cortical neurons cultured on P(TMC-CL) were able to extend neurites even when seeded onto myelin. This effect was found to be mediated by the glycogen synthase kinase 3β (GSK3β) signaling pathway with impact on the collapsin response mediator protein 4 (CRMP4), suggesting that besides surface topography, nanomechanical properties were implicated in this process. The obtained results indicate P(TMC-CL) as a promising material for CNS regenerative applications as it promotes axonal growth, overcoming myelin inhibition.

It is now widely accepted that a therapeutic strategy for spinal cord injury (SCI) demands a multi-target approach. Here we propose the use of an easily implantable bilayer polymeric patch based on poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) that combines physical guidance cues provided by electrospun aligned fibres and the delivery of ibuprofen, as a mean to reduce the inhibitory environment at the lesion site by taming RhoA activation. Bilayer patches comprised a solvent cast film onto which electrospun aligned fibres have been deposited. Both layers were loaded with ibuprofen. In vitro release (37°C, in phosphate buffered saline) of the drug from the loaded scaffolds under sink condition was found to occur in the first 24 h. The released ibuprofen was shown to retain its bioactivity, as indicated by the reduction of RhoA activation when the neuronal-like cell line ND7/23 was challenged with lysophosphatidic acid. Ibuprofen-loaded P(TMC-CL) bilayer scaffolds were successfully implanted in vivo in a dorsal hemisection rat SCI model mediating the reduction of RhoA activation after 5 days of implantation in comparison to plain P(TMC-CL) scaffolds. Immunohistochemical analysis of the tissue shows βIII tubulin positive cells close to the ibuprofen-loaded patches further supporting the use of this strategy in the context of regeneration after a lesion in the spinal cord.

Cells are continuously exposed to physical forces and the central nervous system (CNS) is no exception. Cells dynamically adapt their behavior and remodel the surrounding environment in response to forces. The importance of mechanotransduction in the CNS is illustrated by exploring its role in CNS pathology development and progression. The crosstalk between the biochemical and biophysical components of the extracellular matrix (ECM) are here described, considering the recent explosion of literature demonstrating the powerful influence of biophysical stimuli like density, rigidity and geometry of the ECM on cell behavior. This review aims at integrating mechanical properties into our understanding of the molecular basis of CNS disease. The mechanisms that mediate mechanotransduction events, like integrin, Rho/ROCK and matrix metalloproteinases signaling pathways are revised. Analysis of CNS pathologies in this context has revealed that a wide range of neurological diseases share as hallmarks alterations of the tissue mechanical properties. Therefore, it is our belief that the understanding of CNS mechanotransduction pathways may lead to the development of improved medical devices and diagnostic methods as well as new therapeutic targets and strategies for CNS repair.

Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.

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