Advantageous environment of micro-patterned, high-density complementary metal-oxide-semiconductor electrode array for spiral ganglion neurons cultured in vitro.

This study investigated micro-patterned, high-density complementary metal-oxide-semiconductor (CMOS) electrode array to be used as biologically permissive environment for organization, guidance and electrical stimulation of spiral ganglion neurons (SGN). SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured 1, 4 and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry as well as the contact of a neuronal soma and neurites with the micro-electrodes. Our findings indicate that topographical environment of CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation and alignment of SGNs in vitro compared to control. Smaller spacing (0.8-1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. We found morphological basis for positioning of the micro-electrodes on the chip that was appropriate for direct contact of SGNs with them. This configuration allowed CMOS electrode array to electrically stimulate the SGN whose responses were observed with live Fluo 4 calcium imaging.

Differential neuronal and glial behavior on flat and micro patterned chitosan films.

Chitosan is a biodegradable natural polysaccharide that has been widely studied for regenerative purposes in the central nervous system. In this study we assessed the in vitro glial and neuronal cells response to chitosan either flat or patterned with grooves in the micrometric range. Chitosan demonstrated to be a good substrate for the attachment and growth of both neurons and glial cells. Chitosan micropatterns promoted glial cell maturation, suggesting astroglial activation. Nevertheless, those mature/reactive glial cells were permissive for axonal growth. Axons aligned and organized along the patterned grooves and the size of the linear topographic patterns is also affecting neurite and cell response. Patterns with 10µm width induced fasciculation of axons, which can be useful for CNS tissue engineering substrates when precise orientation of the axonal outgrowth is desired.

An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA.

BACKGROUND: Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-µm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. NEW METHOD: Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. RESULTS: Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. COMPARISON WITH EXISTING METHODS: The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. CONCLUSIONS: The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications.

Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA.

Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to de-differentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 µm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.

Silk fibroin matrices for the controlled release of nerve growth factor (NGF).

Nerve conduits (NC) for peripheral nerve repair should guide the sprouting axons and physically protect the axonal cone from any damage. The NC should also degrade after completion of its function to obviate the need of subsequent explanation and should optionally be suitable for controlled drug release of embedded growth factors to enhance nerve regeneration. Silk fibroin (SF) is a biocompatible and slowly biodegradable biomaterial with excellent mechanical properties that could meet the above stated requirements. SF material (films) supported the adherence and metabolic activity of PC12 cells, and, in combination with nerve growth factor (NGF), supported neurite outgrowth during PC12 cell differentiation. NGF-loaded SF-NC were prepared from aqueous solutions of NGF and SF (20%, w/w), which were air-dried or freeze-dried (freezing at -20 or -196 degrees C) in suitable molds. NGF release from the three differently prepared SF-NC was prolonged over at least 3 weeks, but the total amount released depended on the drying procedure of the NC. The potency of released NGF was retained within all formulations. Control experiments with differently dried NGF-lactose solutions did not evidence marked protein aggregation (SEC, HPLC), loss of ELISA-reactivity or PC12 cell bioactivity. This study encourages the further exploitation of SF-NC for growth factor delivery and evaluation in peripheral nerve repair.