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.

Highlights

  • The use of Bombyx mori silk fibroin (SF) as a biomaterial of choice for the development of medical applications is a growing field of study (Holland et al, 2019)

  • Dissolution with 9.3 M LiBr, followed by dialysis and casting, gave a reconstituted film with the formic acid (FA) carbonyl and skeletal bands still present with the same intensity of the original SF mat (Figure 1C), confirming the stability of the FA-SF bond

  • In the presence of FA, a fraction of the hydroxyl groups of Ser and Thr residues underwent esterification resulting in the formation of formyl esters

Read more

Summary

Introduction

The use of Bombyx mori silk fibroin (SF) as a biomaterial of choice for the development of medical applications is a growing field of study (Holland et al, 2019). A preliminary dissolution of native SF fibers is usually required to obtain regenerated scaffolding materials. In the 50s of the last century Earland and Raven (1954) proposed the use of formic acid (FA), mixed with small quantities of water and inorganic salts (e.g. CaCl2), for the dissolution of SF fibers until a concentration of 20% w/v. The resulting solution was used to produce high strength and high-quality films and electrospun mats (Liu et al, 2015; Zhang et al, 2015a). Changing the salt from CaCl2 to LiBr allowed producing electrospun SF mats with satisfactory mechanical stability, good biocompatibility, slow degradability, and promising new bone regeneration ability (Lu et al, 2015). Refinement of the FA-LiBr solvent system allowed controlling the degree of hydrogen bonding among the silk fibroin molecules composing the nanofibers so that they kept an amorphous state and remained stable in aqueous solution even after removal of FA and LiBr by dialysis (Dong et al, 2016)

Objectives
Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call