RESUMEN
Objective. Engineered nerve conduits must simultaneously enhance axon regeneration and orient axon extension to effectively restore function of severely injured peripheral nerves. The dental pulp contains a population of stem/progenitor cells that endogenously express neurotrophic factors (NTFs), growth factors known to induce axon repair. We have previously generated scaffold-free dental pulp stem/progenitor cell (DPSC) sheets comprising an aligned extracellular matrix (ECM). Through the intrinsic NTF expression of DPSCs and the topography of the aligned ECM, these sheets both induce and guide axon regeneration. Here, the capacity of bioactive conduits generated using these aligned DPSC sheets to restore function in critical-sized nerve injuries in rodents was evaluated.Approach. Scaffold-free nerve conduits were formed by culturing DPSCs on a substrate with aligned microgrooves, inducing the cells to align and deposit an aligned ECM. The sheets were then detached from the substrate and assembled into scaffold-free cylindrical tissues.Main results. In vitroanalyses confirmed that scaffold-free DPSC conduits maintained an aligned ECM and had uniformly distributed NTF expression. Implanting the aligned DPSC conduits across critical-sized defects in the buccal branch of rat facial nerves resulted in the regeneration of a fascicular nerve-like structure and myelinated axon extension across the injury site. Furthermore, compound muscle action potential and stimulated whisker movement measurements revealed that the DPSC conduit treatment promoted similar functional recovery compared to the clinical standard of care, autografts. Significance. This study demonstrates that scaffold-free aligned DPSC conduits supply trophic and guidance cues, key design elements needed to successfully promote and orient axon regeneration. Consequently, these conduits restore function in nerve injuries to similar levels as autograft treatments. These conduits offer a novel bioactive approach to nerve repair capable of improving clinical outcomes and patient quality of life.
Asunto(s)
Pulpa Dental , Matriz Extracelular , Regeneración Nerviosa , Células Madre , Ingeniería de Tejidos , Andamios del Tejido , Pulpa Dental/citología , Pulpa Dental/fisiología , Animales , Matriz Extracelular/fisiología , Regeneración Nerviosa/fisiología , Ratas , Andamios del Tejido/química , Células Madre/fisiología , Células Madre/citología , Ingeniería de Tejidos/métodos , Células Cultivadas , Ratas Sprague-Dawley , Nervio Facial/fisiología , Traumatismos del Nervio Facial/terapia , Masculino , HumanosRESUMEN
ConspectusNeural interface technologies enable bidirectional communication between the nervous system and external instrumentation. Advancements in neural interface devices not only open new frontiers for neuroscience research, but also hold great promise for clinical diagnosis, therapy, and rehabilitation for various neurological disorders. However, the performance of current neural electrode devices, often termed neural probes, is far from satisfactory. Glial scarring, neuronal degeneration, and electrode degradation eventually cause the devices to lose their connection with the brain. To improve the chronic performance of neural probes, efforts need to be made on two fronts: enhancing the physiochemical properties of the electrode materials and mitigating the undesired host tissue response.In this Account, we discuss our efforts in developing silica-nanoparticle-based (SiNP) coatings aimed at enhancing neural probe electrochemical properties and promoting device-tissue integration. Our work focuses on three approaches:(1) SiNPs' surface texturization to enhance biomimetic protein coatings for promoting neural integration. Through covalent immobilization, SiNP introduces biologically relevant nanotopography to neural probe surfaces, enhancing neuronal cell attachments and inhibiting microglia. The SiNP base coating further increases the binding density and stability of bioactive molecules such as L1CAM and facilitates the widespread dissemination of biomimetic coatings. (2) Doping SiNPs into conductive polymer electrode coatings improves the electrochemical properties and stability. As neural interface devices are moving to subcellular sizes to escape the immune response and high electrode site density to increase spatial resolution, the electrode sites need to be very small. The smaller electrode size comes at the cost of a high electrode impedance, elevated thermal noise, and insufficient charge injection capacity. Electrochemically deposited conductive polymer films reduce electrode impedance but do not endure prolonged electrical cycling. When incorporated into conductive polymer coatings as a dopant, the SiNP provides structural support for the polymer thin films, significantly increasing their stability and durability. Low interfacial impedance maintained by the conducting polymer/SiNP composite is critical for extended electrode longevity and effective charge injection in chronic neural stimulation applications. (3) Porous nanoparticles are used as drug carriers in conductive polymer coatings for local drug/neurochemical delivery. When triggered by external electrical stimuli, drug molecules and neurochemicals can be released in a controlled manner. Such precise focal manipulation of cellular and vascular behavior enables us to probe brain circuitry and develop therapeutic applications.We foresee tremendous opportunities for further advancing the functionality of SiNP coatings by incorporating new nanoscale components and integrating the coating with other design strategies. With an enriched nanoscale toolbox and optimized design strategies, we can create customizable multifunctional and multimodal neural interfaces that can operate at multiple spatial levels and seamlessly integrate with the host tissue for extended applications.
Asunto(s)
Materiales Biocompatibles , Nanopartículas , Dióxido de Silicio , Dióxido de Silicio/química , Nanopartículas/química , Materiales Biocompatibles/química , Humanos , Animales , Propiedades de Superficie , Neuronas/metabolismo , Neuronas/citologíaRESUMEN
We developed a nanoparticle base layer technology capable of maintaining the bioactivity of protein-based neural probe coating intended to improve neural recording quality. When covalently bound on thiolated nanoparticle (TNP) modified surfaces, neural adhesion molecule L1 maintained bioactivity throughout 8 weeks of dry storage at room temperature, while those bound to unmodified surfaces lost 66% bioactivity within 3 days. We tested the TNP + L1 coating in mouse brains on two different neural electrode arrays after two different dry storage durations (3 and 28 days). The results show that dry-stored coating is as good as the freshly prepared, and even after 28 days of storage, the number of single units per channel and signal-to-noise ratio of the TNP + L1 coated arrays were significantly higher by 32% and 40% respectively than uncoated controls over 16 weeks. This nanoparticle base layer approach enables the dissemination of biomolecule-functionalized neural probes to users worldwide and may also benefit a broad range of applications that rely on surface-bound biomolecules.
Asunto(s)
Biomimética , Sistema Nervioso , Ratones , Animales , Proteínas , Materiales Biocompatibles RevestidosRESUMEN
Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. STATEMENT OF SIGNIFICANCE: Surface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.
Asunto(s)
Nanopartículas , Molécula L1 de Adhesión de Célula Nerviosa , Antiinflamatorios/farmacología , Células Cultivadas , Citocinas/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Microglía/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/química , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/farmacología , Neuronas , SuperóxidosRESUMEN
Neural implants enable bidirectional communications with nervous tissue and have demonstrated tremendous potential in research and clinical applications. To obtain high fidelity and stable information exchange, we need to minimize the undesired host responses and achieve intimate neuron-device interaction. This paper highlights the key bio-integrative strategies aimed at seamless integration through intelligent device designs to minimize the immune responses, as well as incorporate bioactive elements to actively modulate cellular reactions. These approaches span from surface modification and bioactive agent delivery, to biomorphic and biohybrid designs. Many of these strategies have shown effectiveness in functional outcome measures, others are exploratory but with fascinating potentials. The combination of bio-integrative strategies may synergistically promote the next generation of neural interfaces.
Asunto(s)
Tejido Nervioso , Neuronas , Prótesis e ImplantesRESUMEN
An effective strategy for sustained neurotrophic factor (NTF) delivery to sites of peripheral nerve injury (PNI) would accelerate healing and enhance functional recovery, addressing the major clinical challenges associated with the current standard of care. In this study, scaffold-free cell sheets were generated using human dental pulp stem/progenitor cells, that endogenously express high levels of NTFs, for use as bioactive NTF delivery systems. Additionally, the effect of fibroblast growth factor 2 (FGF2) on NTF expression by dental pulp cell (DPC) sheets was evaluated. In vitro analysis confirmed that DPC sheets express high levels of NTF messenger RNA (mRNA) and proteins, and the addition of FGF2 to DPC sheet culture increased total NTF production by significantly increasing the cellularity of sheets. Furthermore, the DPC sheet secretome stimulated neurite formation and extension in cultured neuronal cells, and these functional effects were further enhanced when DPC sheets were cultured with FGF2. These neuritogenic results were reversed by NTF inhibition substantiating that DPC sheets have a positive effect on neuronal cell activity through the production of NTFs. Further evaluation of DPC sheets in a rat facial nerve crush injury model in vivo established that in comparison with untreated controls, nerves treated with DPC sheets had greater axon regeneration through the injury site and superior functional recovery as quantitatively assessed by compound muscle action potential measurements. This study demonstrates the use of DPC sheets as vehicles for NTF delivery that could augment the current methods for treating PNIs to accelerate regeneration and enhance the functional outcome. Impact statement The major challenges associated with current treatments of peripheral nerve injuries (PNIs) are prolonged repair times and insufficient functional recovery. Dental pulp stem/progenitor cells (DPCs) are known to endogenously express high levels of neurotrophic factors (NTFs), growth factors that enhance axon regeneration. In this study, we demonstrate that scaffold-free DPC sheets can act as effective carrier systems to facilitate the delivery and retention of NTF-producing DPCs to sites of PNIs and improve functional nerve regeneration. DPC sheets have high translational feasibility and could augment the current standard of care to enhance the quality of life for patients dealing with PNIs.