Fibronectin Functionalized Electrospun Fibers by Using Benign Solvents: Best Way to Achieve Effective Functionalization

Abstract

The aim of this study is to demonstrate the feasibility of different functionalization methods for electrospun fibers developed using benign solvents. In particular three different approaches were investigated to achieve the functionalization of poly(epsilon caprolactone) (PCL) electrospun fibers with fibronectin. Protein surface entrapment, chemical functionalization and coaxial electrospinning were performed and compared. Moreover, bilayered scaffolds, with a top patterned and functionalized layer with fibronectin and a randomly oriented not functionalized layer were fabricated, demonstrating the versatility of the use of benign solvents for electrospinning also for the fabrication of complex graded structures. Besides the characterization of the morphology of the obtained scaffolds, ATR-FTIR and ToF-SIMS were used for the surface characterization of the functionalized fibers. Cell adhesion and proliferation were also investigated by using ST-2 cells. Positive results were obtained from all functionalized scaffolds and the most promising results were obtained with bilayered scaffolds, in terms of cells infiltration inside the fibrous structure.

Keywords: benign solvents; electrospinning; fibronectin; functionalization; nanofibers; scaffolds; tissue engineering.

Figure 1

SEM micrographs at different magnifications of PCL electrospun fibers obtained by using acetic acid (PCL AA) and dichloromethane/methanol (PCL DCM/MeOH) as solvents. EDX analysis is reported at the bottom for both samples.

Figure 2

SEM micrographs of PCL fibers before (A,B), after hydrolysis and protonation (C,D), and after EDC-NHS treatment (E,F).

Figure 3

SEM micrographs of PCL fibers before (A,B), after hydrolysis and protonation (C,D), and after EDC-NHS treatment (E,F).

Figure 4

SEM micrographs with different magnification of bilayered scaffolds with the top layer functionalized with fibronectin by using the hydrolysis and protonation (Bilayer Hy_FN) (A–C) and EDC-NHS treatment (Bilayer EN_FN) (D–F).

Figure 5

ATR-FTIR spectra of the scaffolds after hydrolysis and EDC_NHS treatment (A); spectra of samples after the functionalization with fibronectin with three methods (B) with inset with a focus in the wavenumber range 1,800–1,400 cm⁻¹. ATR-FTIR spectrum of neat PCL was used as control for all samples. Main bands discussed in the text are reported on the spectra.

Figure 6

PCA comparing the fibronectin coated samples to linker coated PCL: scores (with 95% confidence limits; dotted lines) of (A) PCL Hy and (B) PCL EN mediated protein adsorption; (C,D) corresponding loadings.

Figure 7

PCA comparing the coaxial samples to bare PCL: (A) scores (with 95% confidence limits; dotted lines) and (B) corresponding loadings.

Figure 8

ToF-SIMS negative polarity images of the fragments characteristic for fibronectin (left) and PCL (right) for bilayer samples Hy_FN (A) and EN_FN (B).

Figure 9

Fibronectin release profile and SEM micrographs of coaxial, PCL EN_FN and PCL Hy_FN samples after immersion in PBS.

Figure 10

WST-8 analysis: histograms of OD at 450 nm for all samples 1 day and 7 days after the seeding. A table reporting the increase of the OD values expressed as ratio between the measured OD 7 days after the seeding respect to the OD measured after 1 day. Asterisk denotes significant difference, p < 0.05.

Figure 11

Fluorescence images of actin filaments (red) and cell nuclei (blue) for the samples PCL, PCL Hy_FN, PCL EN_FN, and coaxial 7 days after the seeding.

Figure 12

Fluorescence images of actin filaments (red) and cell nuclei (blue) for the bilayered samples Bilayer Hy_FN (A,B) and Bilayer EN_FN (C,D) with two magnifications.