A Novel Toolkit for Characterizing the Mechanical and Electrical Properties of Engineered Neural Tissues

Abstract

We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature, however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method, spherical indenters are positioned on top of the fibrin samples, generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods.

Physical and Mechanical Characterization of Fibrin-Based Bioprinted Constructs Containing Drug-Releasing Microspheres for Neural Tissue Engineering Applications

“…The indention method consistently produced robust measurements, which indicated that the white matter tissue was approximately one third stiffer than gray matter tissue. These studies validated the use of indentation methods to interpret the mechanical properties of neural tissues [19,21,23,26,[29][30][31][32][33].…”

“…The mechanical integrity and stability of engineered tissues depend upon the hydrogel-based bioinks used during fabrication. The adjustable concentration of biomaterials can appropriately tune the mechanical and rheological properties of hydrogels [19]. It is important to understand the effect of mechanical environment on neurite outgrowth for understanding neural tissue regeneration strategies.…”

“…Given that neural tissues have low mechanical stiffness-with Young s elastic moduli (E) in the order of 0.1 kilopascal (kPa)-compared to other tissues present in the body [21,22], it is challenging to measure the mechanical properties using standard techniques, such as atomic force microscopy (AFM), impact indentation, and rheometry because these methods are indirect and require several assumptions that lead to variances in data across groups [19,21,[23][24][25][26][27][28]. Such methods use mathematical modeling to correlate experimental measurements, such as displacement and load to stress and strain, to evaluate elastic modulus [19]. Alternatively, some researchers have directly measured the rheological properties of white and gray matter using indentation experiments on various regions of the brain, such as the midbrain, cerebrum, and thalamus [29,30].…”

“…However, bioinks that only contain fibrinogen pass through the microfluidic print head too fast to be polymerized, even at the lowest pressure, due to low viscosity. [19,62,63]. To overcome this issue, the naturally derived materials alginate and chitosan were added to the bioink to increase its viscosity and gelation speed and, thus, the printability of the ink.…”

Application of fibrin-based hydrogels for nerve protection and regeneration after spinal cord injury

“…Apart from commercial fibrin hydrogels products [49,69], the isolation and purification of autologous plasma can also prepare fibrin hydrogels commonly used in neural tissue engineering. However, the cost is only materials, without traffic expense and patent fee [70].…”