Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction

Abstract

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm−1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Silk Vascular Grafts with Optimized Mechanical Properties for the Repair and Regeneration of Small Caliber Blood Vessels

“…The crystallinity index of the ES nanofibers, expressed as an intensity ratio between the two amide III components (CI = I 1227 /I 1262 ), are reported in Table 3 . The values fall in the range characteristics of native SF microfibers (CI ≅ 0.60) [ 21 ] and regenerated SF nanofibers (CI ≅ 0.59) [ 22 ]. Interestingly, a slight increase in the crystallinity index can be observed for the SILKGraft designs of group 2.…”

Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering

“…Finally, thanks to availability of various procedures to depose silk fibroin fibers (e.g., 3D printing or electrospinning), it is relatively easy to obtain three-dimensional structures such as tubular scaffolds able to reproduce the geometrical characteristics of blood vessels (41,42). The chosen electrospinning process, in particular, exploited the use of formic acid as solvent during scaffold fabrication to ensure maximal biocompatibility and retention of bioactivity and morphology of the scaffold's structure (43). The compliance of these tubular scaffolds have also a mechanical advantage compared to equivalent tubular structures with similar dimension, with a 2.4%/100 mmHg of diameter compliance for 10.3389/fcvm.2022.1013183 silk electrospun graft vs. 2%/100 mmHg of diameter compliance for P(LLA-CL) tubular structures with similar geometry (40).…”

Cross‐Linking Strategies for Silk‐Protein‐Based Inks for 3D Printing

“…This suggests that SF is able to accelerate the regeneration of β-sheets and increase intermolecular interactions by mechanical stirring, ultrasonic oscillation or shear stress, thereby accelerating ink sol-gel changes. [108][109][110] In addition, the ways that induce physical cross-linking include factors such as temperature, pH, thickening agents, surfactants and metal ion reagents. [111][112][113][114][115][116][117][118] A simple method of physical cross-linking is the addition of thickening agents.…”