Neurons and neural stem cells are sensitive to their mechanical and topographical environment, and cell-substrate binding contributes to this sensitivity to activate signaling pathways for basic cell functions. Many transmembrane proteins transmit signals into and out of the cell, including integrins, growth factor receptors, G-protein-coupled receptors, cadherins, cell adhesion molecules, and ion channels. Specifically, integrins are one of the main transmembrane proteins that transmit force across the cell membrane between a cell and its extracellular matrix, making them critical in the study of cell-material interactions. This review focuses on mechanotransduction, defined as the conversion of force a cell generates through cell-substrate bonds to a chemical signal, of neural cells. The chemical signals relay information via pathways through the cellular cytoplasm to the nucleus, where signaling events can affect gene expression. Pathways and the cellular response initiated by substrate binding are explored to better understand their effect on neural cells mechanotransduction. As the results of mechanotransduction affect cell adhesion, cell shape, and differentiation, knowledge regarding neural mechanotransduction is critical for most regenerative strategies in tissue engineering, where novel environments are developed to improve conduit design for central and peripheral nervous system repair in vivo.

2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

Cells are sensitive to physical cues in their environment, such as the stiffness of the substrate, peptide density, and peptide affinity. Understanding how neural stem cells (NSCs) sense and respond to these matrix cues has the potential to improve disease outcome, particularly if a regenerative response can be exploited. While the material properties are known to influence other stem cells, little is known about how NSC differentiation is altered by this interplay of mechanical, or bulk properties, with peptide concentration and affinity, or microscale properties. We are interested in the combined effect of bulk and microscale features in an in vitro hydrogel model and therefore we investigated NSC differentiation by focusing on integrin interactions via RGD peptide affinity and concentration. Our studies demonstrated that the peptide concentration affected adhesion as there were more cells on scaffolds with 1 mM RGD than 2.5 mM RGD. The hydrogel stiffness affected neurite length in differentiating NSCs, as 0.1-0.8 kPa substrates promoted greater neurite extension than 4.2-7.9 kPa substrates. The NSCs differentiated towards β-ΙΙΙ tubulin positive cells on scaffolds with RGD after 7 days and those scaffolds containing 1 mM linear or cyclic RGD had longer neurite extensions than scaffolds containing 0.1 or 2.5 mM RGD. While peptide affinity had a lesser effect on the NSC response in our hydrogel system, blocking actin, myosin II, or integrin interactions resulted in changes to the cell morphology and focal adhesion assembly. Overall, these results demonstrated NSCs are more responsive to a change in tissue stiffness than peptide affinity in the range of gels tested, which may influence design of materials for neural tissue engineering.

Delivery of bioactive molecules is a critical step in fabricating materials for regenerative medicine, yet, this step is particularly challenging in hydrated scaffolds such as hydrogels. Although bulk photocrosslinked poly(ethylene glycol) (PEG) hydrogels have been used for a variety of tissue engineering applications, their capability as drug delivery scaffolds has been limited due to undesirable release profiles and reduction in bioactivity of molecules. To solve these problems, this article presents the fabrication of degradable PEG microgels, which are micron-sized spherical hydrogels, to deliver bioactive nerve growth factor (NGF). NGF release and activity was measured after encapsulation in microgels formed from either 3 kDa or 6 kDa PEG to determine the role of hydrogel mesh size on release. Microgels formed from 6 kDa PEG were statistically larger and had a higher swelling ratio than 3 kDa PEG. The 6 kDa PEG microgels provided a Fickian release with a reduced burst effect and 3 kDa microgels provided anomalous release over ≥20 days. Regardless of molecular weight of PEG, NGF bioactivity was not significantly reduced compared to unprocessed NGF. These results demonstrate that microgels provide easy mechanisms to control the release while retaining the activity of growth factors. As this microgel-based delivery system can be injected at the site of nerve injury to promote nerve repair, the potential to deliver active growth factors in a controlled manner may reduce healing time for neural tissue engineering applications.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e. low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds. Video LinkThe video component of this article can be found at