Search

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduits have been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfated polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 μm convex lenses, 2 μm gratings, and 10 μm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0% to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 μm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration., (Creative Commons Attribution license.)

Topography mediated contact guidance affects multiple cell behaviors such as establishment of cellular morphology and migration. The direction of cell migration is associated with the establishment of cell polarity, which also affects the primary cilia in migrating cells. POPX2, a partner of PIX2, is involved in pathways essential to primary cilium formation, while over-expression of POPX2 has been reported to cause a loss of cell polarity during migration. This study aims to examine how topographical cues direct morphological changes, and how topography affects the process of cellular migration and primary cilium architecture, in the context of POPX2 over-expression. Thus, the effect of anisotropic topography, 2 μm grating pattern on tissue-culture polystyrene, was used as a contact guidance cue to investigate the migration and cell polarity of POPX2 overexpressing cells, in comparison to control NIH3T3 fibroblast cells. We report that POPX2 overexpressing NIH3T3 cells were more sensitive to surface topographical cues as the cells became more elongated. In addition, these cues also affected focal adhesion alignment of POPX2 overexpressing cells. Cell migration was further studied using wound closure assays, in which the 2 μm gratings were designed to be either perpendicular or parallel to wound-induced cell migration direction, which would be agonistic or antagonistic to cell migration, respectively. We observed that both POPX2 overexpressing cells' migration direction and migration rate were more significantly influenced by gratings direction compared to control NIH3T3 cells. The migration paths of POPX2 overexpressing cells become more direct in the presence of anisotropic topographical cues. Further, cilia and centrosome alignment, which is important in cell migration, was also affected by the direction of gratings during this migration process. Collectively, enhancement of NIH3T3 cell sensitivity towards surface topography through POPX2 overexpression might reflect one of the mechanisms that combine biochemical and mechanical cues for directional cell migration.

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol-gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

Studies of electrical stimulation therapies for the treatment of neurological disorders, such as deep brain stimulation, have almost exclusively been performed using animal-models. However, because animal-models can only approximate human brain disorders, these studies should be supplemented with an in vitro human cell-culture based model to substantiate the results of animal-based studies and further investigate therapeutic benefit in humans. This study presents a novel approach to analyze the effect of electrical stimulation on the neurogenesis of patient-induced pluripotent stem cell (iPSC) derived neural progenitor cell (NPC) lines, in vitro using a 3D graphene scaffold system. The iPSC-derived hNPCs used to demonstrate the system were collected from patients with Rett syndrome, a debilitating neurodevelopmental disorder. The graphene scaffold readily supported both the wild-type and Rett NPCs. Electrical stimulation parameters were optimized to accommodate both wild-type and Rett cells. Increased cell maturation and improvements in cell morphology of the Rett cells was observed after electrical stimulation. The results of the pilot study of electrical stimulation to enhance Rett NPCs neurogenesis were promising and support further investigation of the therapy. Overall, this system provides a valuable tool to study electrical stimulation as a potential therapy for neurological disorders using patient-specific cells.