Modulation of Microglial Phenotype by Textured Nanofibrils

A key challenge in neuronal regeneration is the understanding and the control of the inflammatory response in pathological situations, in which microglia play a crucial role. Genetic findings have suggested that microglia are heterogeneous cells and are involved in almost every neurodegenerative disorder [1-4]. A comprehensive understanding how microglia adapt provides insight for therapeutic purposes. Nowadays it has been established that biomaterials can modulate cell morphology, migration and immune responses [5-9]. In an in vivo environment, the topological structure of the extracellular matrix (ECM) at the nanoscale provides a natural web of intricate nanofibers to support cells and guide their behavior [10,11]. Biomaterials with a topography close to the biological scale provide a substrate that does not require bioactive agents to modulate macrophage behavior [12,13]. Understanding and evaluating how the material surface regulates microglia behavior will provide cues for the design of instructive scaffolds and neuroinflammation models that can contribute to central nervous system (CNS) regeneration. In my Ph.D., I have addressed several aspects of the interaction between substrates and cells, such as neurons and microglia. This collaborative work has produced several papers which are included in my Ph.D. thesis. The main and most original contribution of my Ph.D. work is related to microglia and specifically to the modulatory effect of nanofibrils substrates on microglia properties. These are the main results of my thesis: 1) The nanofibril topography of modified bacterial cellulose (mBC) substrates induces ramified microglia with constantly extending and retracting processes, reminiscent of what is observed in vivo. 2) Microglia cultured on mBC substrates have a more negative resting membrane potential and increased inward rectifier K+ currents, which is similar to what is found in aged microglia. 3) Transcriptome analysis showed up-regulation of genes involved in the immune response and down-regulation of genes linked to cell adhesion and cell motion, which strengthened the aging-like phenotype. Moreover, we demonstrated that the elevated Kir2.1 channel expression accounts for the increased inward rectifier currents and membrane hyperpolarization. Meanwhile, the Arp2/3 complex activation is responsible for the modulation of microglia morphology and motility. 4) When stimulated by lipopolysaccharide, microglia cultured on both coverslip and mBC substrates displayed activated phenotype, with retracted processes, enlarged cell areas and meanwhile increased pro-inflammation mediator release. However, compared with coverslip substrates, microglia cultured on mBC substrates showed less cell area enlargement and pro-inflammation mediator secretion. For the interface between three-dimensional (3D) scaffolds and neurons: 5) We have demonstrated that hippocampal neurons cultured on 3D graphene foam (GF) scaffolds formed a neural network with higher firing frequency and synchronization compared with those cultured on flat 2D substrates. In addition, this 3D network had two synchronized regimes: a highly synchronized (HS) and a moderately synchronized (MS) regime, while the HS regime was never observed in 2D networks. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. 6) By growing carbon nanotubes (CNTs) in the pores of graphene foam (GF), we have obtained a fully interconnected graphene-carbon nanotube (GCNT) web. Dissociated cortical cells cultured inside the GCNT web form a functional 3D network exhibiting a spontaneous electrical activity that is closer to what is observed in vivo. By co-culturing and fluorescently labeling glioma and healthy cortical cells with different colors, a new in vitro model is obtained to investigate malignant glioma infiltration.