Various biophysical and biochemical factors are important for determining the fate of neural stem cells (NSCs). Among biophysical signals, topographical stimulation by micro/nanopatterns has been applied to control NSC differentiation. In this study, we developed a hierarchically patterned substrate (HPS) platform that can synergistically enhance the differentiation of human NSCs (hNSCs) by simultaneously providing microscale and nanoscale spatial controls to facilitate the alignment of the cytoskeleton and the formation of focal adhesions. The multiscale HPS was fabricated by combining microgroove patterns (groove size: 1.5 μm), prepared by a conventional photolithographic process, and nanopore patterns (pore diameter: 10 nm), prepared from cylinder-forming block copolymer thin films. The hNSCs grown on the HPS exhibited not only a highly aligned, elongated morphology, but also a greatly enhanced differentiation into neuronal and astrocyte lineages, compared to hNSCs on a flat substrate (FS) or single-type patterned substrates [microgroove patterned substrate (MPS) and nanopore patterned substrate (NPS)]. Interestingly, the application of the HPS directed hNSC differentiation toward neurons rather than astrocytes. The expression of focal adhesion proteins in hNSCs was also significantly increased on the HPS compared to the FS, MPS, and NPS, likely a result of the presence of more focal contact points provided by nanopore structures. Inhibition of both β1 integrin-mediated binding and the intracellular Rho-associated protein kinase pathway of hNSCs eliminated the beneficial effects of the HPS on focal adhesion formation and actin filament alignment, which subsequently reduced hNSC differentiation. More importantly, hNSCs on the HPS differentiated into functional neurons exhibiting sodium currents and action potentials. The multiscale, hierarchically patterned topography would be useful for the design of functional biomaterial scaffolds to potentiate NSC therapeutic efficacy.
The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.
Biophysical cues, such as topography, and electrical cues can provide external stimulation for the promotion of stem cell neurogenesis. Here, we demonstrate an electroconductive surface nanotopography for enhancing neuronal differentiation and the functional maturation of human neural stem cells (hNSCs). The electroconductive nanopatterned substrates were prepared by depositing a thin layer of titanium (Ti) with nanograting topographies (150 to 300 nm groove/ridge, the thickness of the groove - 150 μm) onto polymer surfaces. The Ti-coated nanopatterned substrate (TNS) induced cellular alignment along the groove pattern via contact guidance and promoted focal adhesion and cytoskeletal reorganization, which ultimately led to enhanced neuronal differentiation and maturation of hNSCs as indicated by significantly elevated neurite extension and the upregulated expression of the neuronal markers Tuj1 and NeuN compared with the Ti-coated flat substrate (TFS) and the nanopatterned substrate (NS) without Ti coating. Mechanosensitive cellular events, such as β1-integrin binding/clustering and myosin-actin interaction, and the Rho-associated protein kinase (ROCK) and mitogen-activated protein kinase/extracellular signal regulated kinase (MEK-ERK) pathways, were found to be associated with enhanced focal adhesion and neuronal differentiation of hNSCs by the TNS. Among the neuronal subtypes, differentiation into dopaminergic and glutamatergic neurons was promoted on the TNS. Importantly, the TNS increased the induction rate of neuron-like cells exhibiting electrophysiological properties from hNSCs. Finally, the application of pulsed electrical stimulation to the TNS further enhanced neuronal differentiation of hNSCs due probably to calcium channel activation, indicating a combined effect of topographical and electrical cues on stem cell neurogenesis, which postulates the novelty of our current study. The present work suggests that an electroconductive nanopatterned substrate can serve as an effective culture platform for deriving highly mature, functional neuronal lineage cells from stem cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.