Characterizing Mechanical Properties of Silk Films with Atomic Force Microscopy

Abstract

Purpose : The cornea neuro-epithelium is an actively remodeling layer that provides clarity and sensation to the eye. Stem cells derived from the limbus constantly replace epithelial cells in the normal cornea. To appropriately localize, these cells follow physical cues of the microenvironment by converting mechanical stimuli such as matrix stiffness and elasticity to biochemical signals in a process known as mechanotransduction. Here we developed a novel sample preparation technique to study silk film biomechanics, to understand how this biomaterial may influence corneal regeneration and wound repair.

Methods : Silk films were made from processing the cocoons of Bombyx mori silkworms. The films were adhered onto glass slides using a mounting solution made of 10% w/v gelatin, 0.1% w/v chrome alum in dH₂O. Atomic force microscopy (AFM) nanoindentation was used to measure the stiffness (Young’s modulus) of the films once rehydrated in dH₂O. Indentation parameters were fixed and three silk films were each measured in 16 separate areas to account for regional variation. The Young’s modulus was determined by fitting data with the Hertzian model for describing non-adhesive, elastic contact mechanics.

Results : Chrome alum-gelatin provided stable adhesion of silk films under dH₂O immersion as evident from lack of sample drift seen under AFM. The 95% confidence intervals for the three films were 14.36 ± 1.13 MPa, 13.03 ± 0.94 MPa, and 13.25 ± 0.86 MPa. Statistical analysis showed no significant difference between the films. Little hysteresis was seen in force curves which justifies use of the Hertz model and indicates that hydrated silk films have almost purely elastic mechanical behavior.

Conclusions : Here we characterized the Young’s modulus of hydrated silk films using a chrome alum-gelatin mounting solution. Previously, AFM with thin, hydrated samples proved to be particularly challenging as most water resistant adhesives chemically alter the sample and therefore its stiffness. We believe this technique can be extended to biomaterials that have previously proven difficult to measure using AFM. Our next step is to determine how corneal cell behavior changes to silk films of different Young's modulus.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Data of silk films presented as whisker plots subdivided into quartiles.

 

An example force curve. The red and blue lines represent the indentation and retraction phases, respectively. The dotted black line is the fitted Hertz model.