(J-365) One-pot assembly of NGF-loaded silk coatings for nerve tissue regeneration

Introduction:: Peripheral nervous system injury resulting from disease or trauma impacts over 20 million individuals in the United States alone and can significantly reduce the patient’s quality of life¹. Aligned electrospun fibers can provide topographical cues and local therapeutic delivery to facilitate robust peripheral nerve regeneration²; however, current functionalization using multi-step biorthogonal chemistries or physical adsorption can lead to loss of biomolecule bioactivity^3,4. Here we demonstrate a novel approach for enhancing the bioactive properties of synthetic electrospun scaffolds though the interfacial co-assembly of protein payloads into conformal silk fibroin (SF) nano-thin coatings. In our approach, protein payloads are added into the coating solution, which allows for their non-covalent entrapment onto the scaffold surface during coating growth. We generated co-assembled SF coatings containing nerve growth factor (NGF) on aligned electrospun fibers and assessed their effect on neurite outgrowth from rat dorsal root ganglia explants. Ultimately, this work demonstrates a new modular approach for controlling the surface bioactivity of synthetic electrospun scaffolds that provides more tuneability and better outcomes than conventional approaches.


Materials and Methods:: Aligned fibers were electrospun onto glass coverslips using a 12% (w/w) solution of poly-L-lactic acid (PLLA)/chloroform pumped through a 22G needle at 2 mL/h with an applied voltage of 15kV for 30 min. PLLA electrospun scaffolds were immersed for 24h in solutions containing 0.5 mg/mL silk fibroin (MW~100 kDa), 500 ng/mL NGF, and 200 mM KH2PO4/K2HPO4 to generate the self-assembled coatings. Cellular response of the co-assembled coatings was compared to scaffolds loaded with NGF via physical adsorption: bare PLLA or 24h SF-coated scaffolds (no NGF) soaked with 500 ng/mL NGF diluted in PBS for 24h. Primary rat dorsal root ganglia (DRG) explants were cultured onto the fiber scaffolds for 4 days, then fixed and stained against neurofilament. Confocal images of the DRG explants were analyzed for neurite length and area coverage.


Results, Conclusions, and Discussions:: Results and Discussion: The total area of neurite outgrowth from DRG cultured on scaffolds with NGF co-assembled SF coatings was significantly enhanced compared to bare PLLA scaffolds with adsorbed NGF and SF-coated scaffolds with adsorbed NGF (Figure 1). In addition to total neurite area, the ten longest neurites from the DRG on both scaffolds with NGF co-assembled SF coatings and scaffolds with NGF adsorbed to SF coatings had significantly longer neurite extension than the bare PLLA scaffold with adsorbed NGF.
Conclusions: Overall, these results suggest that delivery of NGF at the scaffold surface was more effective for enhancing both the length and density of extending neurites from primary DRG explants when a co-assembly process was used for loading rather than physical adsorption of NGF onto either bare PLLA or SF-coated PLLA surfaces.


Acknowledgements (Optional): : NSF Award [245510] to R.H.Z., Veteran’s Affairs [I01RX003502-01A1] to R.J.G., and NSF GRFP [DGE-1744655] to J.L.F.

References (Optional): : [1] Peripheral Neuropathy Fact Sheet | National Institute of Neurological Disorders and Stroke (2021). [2] S. Stojanov, et al. Front Bioeng Biotechnol. 8, 130 (2020).
[3] Shen, H. & Hu, X. Rsc Adv 11, 6735–6747 (2021).
[4] Wong, L. S., et al. Chem Rev 109, 4025–4053 (2009).