Effect of Electrical and Electromechanical Stimulation on PC12 Cell Proliferation and Axon Outgrowth.

Peripheral nerve injuries have become a common clinical disease with poor prognosis and complicated treatments. The development of tissue engineering pointed a promising direction to produce nerve conduits for nerve regeneration. Electrical and mechanical stimulations have been incorporated with tissue engineering, since such external stimulations could promote nerve cell proliferation, migration and differentiation. However, the combination of electrical and mechanical stimulations (electromechanical stimulation) and its effects on neuron proliferation and axon outgrowth have been rarely investigated. Herein, silver nanowires (AgNWs) embedded polydimethylsiloxane (PDMS) electrodes were developed to study the effects of electromechanical stimulation on rat pheochromocytoma cells (PC12 cells) behaviors. AgNWs/PDMS electrodes demonstrated good biocompatibility and established a stable electric field during mechanical stretching. PC12 cells showed enhanced proliferation rate and axon outgrowth under electrical stimulation alone, and the cell number significantly increased with higher electrical stimulation intensity. The involvement of mechanical stretching in electrical stimulation reduced the cell proliferation rate and axon outgrowth, compared with the case of electrical stimulation alone. Interestingly, the cellular axons outgrowth was found to depend on the stretching direction, where the axons prefer to align perpendicularly to the stretch direction. These results suggested that AgNWs/PDMS electrodes provide an in vitro platform to investigate the effects of electromechanical stimulation on nerve cell behaviors and can be potentially used for nerve regeneration in the future.

Effect of Cyclic Stretch on Neuron Reorientation and Axon Outgrowth.

The directional alignment and outgrowth of neurons is a critical step of nerve regeneration and functional recovery of nerve systems, where neurons are exposed to a complex mechanical environment with subcellular structures such as stress fibers and focal adhesions acting as the key mechanical transducer. In this paper, we investigate the effects of cyclic stretch on neuron reorientation and axon outgrowth with a feasible stretching device that controls stretching amplitude and frequency. Statistical results indicate an evident frequency and amplitude dependence of neuron reorientation, that is, neurons tend to align away from stretch direction when stretching amplitude and frequency are large enough. On the other hand, axon elongation under cyclic stretch is very close to the reference case where neurons are not stretched. A mechanochemical framework is proposed by connecting the evolution of cellular configuration to the microscopic dynamics of subcellular structures, including stress fiber, focal adhesion, and microtubule, yielding theoretical predictions that are consistent with the experimental observations. The theoretical work provides an explanation of the neuron's mechanical response to cyclic stretch, suggesting that the contraction force generated by stress fiber plays an essential role in both neuron reorientation and axon elongation. This combined experimental and theoretical study on stretch-induced neuron reorientation may have potential applications in neurodevelopment and neuron regeneration.

Direct 3D printing of a tough hydrogel incorporated with carbon nanotubes for bone regeneration.

The emerging three-dimensional (3D) printing technique has shown prominent advantages to fabricate hydrogel-based tissue scaffolds for the regeneration of bone defects. Here, a tough polyion complex (PIC) hydrogel was synthesized, and multiwalled carbon nanotubes (MWCNTs) were incorporated into the PIC matrix to form the PIC/MWCNT biohybrid hydrogel, which was manufactured into 3D scaffolds by extrusion-based 3D printing for bone defect repair. To the best of our knowledge, this is the first study to combine CNTs with PIC hydrogels as biohybrid scaffolds for bone repair. The results from the in vitro cell culture demonstrated that the PIC/MWCNT scaffolds exhibited good biocompatibility with rat bone marrow-derived mesenchymal stem cells (rBMSCs) and facilitated the osteogenic differentiation of rBMSCs. Moreover, rBMSCs cultured on the PIC/MWCNT scaffolds exhibited a higher degree of osteogenic differentiation than those cultured on PIC scaffolds in terms of mineralized matrix formation and osteogenesis-related gene upregulation. The in vivo experiments in a calvarial defect model of Sprague-Dawley (SD) rats revealed that the PIC/MWCNT scaffolds significantly promoted the regeneration of calvarial defect healing. These findings suggest that the PIC hydrogel is a potential scaffold material for bone regeneration, and the addition of MWCNTs provides further enhancement in bone repair efficiency by the PIC/MWCNT scaffolds.

The influence of the stiffness of GelMA substrate on the outgrowth of PC12 cells.

Recent studies have shown the importance of cell-substrate interaction on neurone outgrowth, where the Young's modulus of the matrix plays a crucial role on the neurite length, migration, proliferation, and morphology of neurones. In the present study, PC12 cells were selected as the representative neurone to be cultured on hydrogel substrates with different stiffness to explore the effect of substrate stiffness on the neurone outgrowth. By adjusting the concentration of gelatin methacryloyl (GelMA), the hydrogel substrates with the variation of stiffnesses (indicated by Young's modulus) from approximately 3-180 KPa were prepared. It is found that the stiffness of GelMA substrates influences neuronal outgrowth, including cell viability, adhesion, spreading, and average neurite length. Our results show a critical range of substrate's Young's modulus that support PC12 outgrowth, and modulate the cell characteristics and morphology. The present study provides an insight into the relationship between the stiffness of GelMA hydrogel substrates and PC12 cell outgrowth, and helps the design and optimization of tissue engineering scaffolds for nerve regeneration.