(C-117) Silk Fibroin and Hyaluronic Acid Electrospun Material Encourages Regeneration of Peripheral Nerves

Axonal gaps in peripheral nerves from disease or injury cause pain as well as loss of movement, sensory input, and autonomic nerve function. Neuropathy is common affecting over twenty-million Americans ^[1]. Injuries with gaps less than 1 cm in length can be healed with Wallerian degeneration ^[2]. This process, while effective, is only able to repair a small percentage of injuries. Long gap injuries (over 1 cm) are equally as common and cannot heal without intervention. Currently, autografts are the gold standard treatment for long gap injuries ^[1]. This entails a section of healthy nerve tissue from the patient’s body being implanted into the injury site. However, this requires multiple surgeries, potential for donor site morbidity, and may not lead to full functional recovery. An alternative treatment is a nerve growth conduit for healing long gap injuries. In this case, a hollow cylinder of biomaterial is implanted into the injury site encouraging the severed ends to grow onto the material effectively reconnecting the nerve overtime. Research has shown that silk fibroin (SF) from the Bombyx mori silkworm possess qualities, such as tensile strength, biocompatibility, and controlled degradation that make it a suitable replacement to autografts ^[3]. SF, however, can be improved in combination with a material like hyaluronic acid (HA) to maintain structural integrity and favorable water properties. SF-HA nanofibers were collected through electrospinning producing a material conducive to peripheral nerve regeneration. The goal of the study was to develop the SF-HA material and study its mechanical and cellular behavior.

A 5% silk fibroin solution (Biomatrix) was first methacrylated with 50 µL of glycidol methacrylate heated to 70 °C while stirring at 300 rpm ^[5]. This allowed for control over the material’s mechanical properties and solubility. Powdered hyaluronic acid (Sigma) was also methacrylated with methacrylic anhydride at a pH of 8 over ice for 24 hours ^[4]. The resulting solutions were dialyzed for at least 48 hours and lyophilized before further use. To make a composite spin solution, SF was dissolved using a solvent of CaCl₂/EtOH/H₂O (1:2:8 M ratio) ^[5] and added to HA, 6 % polyethylene oxide (PEO), and I2959 photocrosslinking agent. The spin solution was then vortexed, sonicated, and stored at 4 °C for 24 hours. Finally, 1.0 mL of diH₂0 was added before electrospinning. The resulting spin solution was approximately 1.0% w/v SF and 0.62% w/v HA, 3.9% w/v PEO and 0.03% w/v Irgacure 2959.  Electrospinning of SF-HA was conducted with a mandrel speed of 1931 rpm, 24 kV of applied voltage, and a flow rate of 1.2 mL/hr. The resulting fibers were UV crosslinked for 30 minutes after collection. As an experimental control, an HA only material that has proven successfully in peripheral nerve regeneration was also spun at 1931 rpm, 28 kV, and at a rate of 1.8 mL/hr. Material was spun into mats and onto methacrylated glass coverslips for testing. Scanning electron microscopy (SEM) images (Figure 1) display fiber alignment and diameter. 

Mechanical testing was conducted with 30x10x0.05 mm electrospun mats of both SF-HA and HA pulled in tension at a rate of 1 mm/min until failure in both the dry and hydrated state. The SF-HA material was significantly more (p< 0.001) elastic than the HA material in both conditions (Figure 2). The elongation of the samples was also significantly higher for SF-HA (p=0.006 dry and p=0.04 hydrated). Additionally, SF-HA was significantly stronger when hydrated (p< 0.03) but not when dry (p=0.1) (Figure 2). Material strength and elasticity are crucial to conduit implantation and recovery, and therefore, this added strength is a benefit of the SF-HA material.

 
Fluorescence microscopy was used to test the biocompatibility of the SF-HA material. Human Schwann cells (ipn 02.8) were seeded onto SF-HA and HA fibers and stained with a S100 primary and DAPI counterstain to measure the cell aspect ratio (AR) (Figure 3). Greater AR of cells indicates a stronger interaction with the material increasing its viability. Cells on the SF-HA fibers were significantly (p< 0.001) more elongated than those on the HA fibers meaning SF improves cellular interactions with the biomaterial.

Schwann cell (SC) proliferation was measured with an Alamar blue assay. SCs were plated onto SF-HA and HA and cell density was measured calorimetrically at 24 and 48 hours of incubation. It was found that SC proliferation was greater on the SF-HA fibers than the HA fibers (Figure 4) and is approaching statistical significance (p=0.14). Proliferation of SCs is quintessential to nerve regeneration making the SF-HA material preferable to the HA control.

Cell migration of rodent fibroblasts (L929s) was captured using a microscope time lapse. Images were captured every ten minutes over 8 hours and analyzed to fit a random walk model calculating the motility coefficient and persistence time of each cell. For regeneration, cells should move efficiently and uniformly. There was no significant difference (Figure 5) in the cell migration behavior between the two materials meaning they are both conducive to regeneration.

From these results it can be concluded that SF-HA encourages peripheral nerve regeneration and is a viable conduit material.

[1] U.S. Department of Health and Human Services. (n.d.). Peripheral neuropathy fact sheet. National

Institute of Neurological Disorders and Stroke. Retrieved November 28, 2022, from https://www.ninds.nih.gov/peripheral-neuropathy-fact- sheet#:~:text=More%20than%2020%20million%20people,for%20all%20forms%20of%20 neuropathy.

[2] Wallerian degeneration. Physiopedia. (n.d.). Retrieved November 28, 2022, from

https://www.physio-pedia.com/Wallerian_Degeneration

[3] Millesi, F., Weiss, T., Mann, A., Haertinger, M., Semmler, L., Supper, P., ... & Radtke, C.

(2021). Defining the regenerative effects of native spider silk fibers on primary Schwann

cells, sensory neurons, and nerve‐associated fibroblasts. The FASEB Journal, 35(2), e21196

[4] Bae, S. B., Kim, M. H., & Park, W. H. (2020). Electrospinning and dual crosslinking of

water-soluble silk fibroin modified with glycidyl methacrylate. Polymer Degradation and

[5] Burdick, J. A., Chung, C., Jia, X., Randolph, M. A., & Langer, R. (2005). Controlled degradation

and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules, 6(1), 386-391.