Multilayer Electrospun Scaffolds of Opposite-Charged Chitosans

Abstract

Chitosan (CS) is a polysaccharide obtainable by the deacetylation of chitin, which is highly available in nature and is consequently low-cost. Chitosan is already used in the biomedical field (e.g., guides for nerve reconstruction) and has been proposed as a biomaterial for tissue regeneration in different body districts, including bone tissue. The interest in chitosan as a biomaterial stems from its ease of functionalization due to the presence of reactive groups, its antibacterial properties, its ease of processing to obtain porous matrices, and its inherent similarity to polysaccharides that constitute the human extracellular matrix, such as hyaluronic acid (HA). Here, chitosan was made to react with succinic anhydride to develop a negatively charged chitosan (SCS) that better mimics HA. FT-IR and NMR analyses confirmed the presence of the carboxylic groups in the modified polymer. Four different electrospun matrices were prepared: CS, SCS, a layer-by-layer matrix (LBL), and a matrix with both CS and SCS simultaneously electrospun (HYB). All the matrices containing SCS showed increased human osteoblast proliferation, mineralization, and gene expression, with the best results obtained with HYB compared to the control (CS). Moreover, the antibacterial potential of CS was preserved in all the SCS-containing matrices, and the pure SCS matrix demonstrated a significant reduction in bacterial proliferation of both S. aureus and E. coli.

Keywords: E. coli; S. aureus; bone tissue engineering; electrospinning; multilayer matrices; negatively charged chitosan; osteoblasts; succinyl chitosan.

Figure 1

SEM images of electrospun matrices. (a,b) CS; (c,d) SCS; (e,f) LBL; (g,h) HYB. The images on the left column are at 2000× magnification (scale bar = 10 µm), whilst the right column is at 5000× magnification (scale bar = 5 µm).

Figure 2

FT-IR spectra in the 450–4000 cm⁻¹ region of 70% deacetylated chitosan, 70% deacetylated chitosan functionalized with succinic anhydride (C₄H₄O₃), SCS (blue), and CS (orange).

Figure 3

NMR spectrum (400 MHz, T = 343 K) of deacetylated chitosan functionalized with succinic anhydride (C₄H₄O₃), SCS. Glx = Glc or GlNAc.

Figure 4

Proliferation and mineralization assays in primary human osteoblast cells cultured for 7 days. Proliferation was determined by loading cells with CFSE fluorescent probe and flow cytometric analysis (a). Matrix mineralization was determined using Alizarin staining (b). * = p-value < 0.05; ** = p-value < 0.01; *** = p-value < 0.001; **** = p-value < 0.0001.

Figure 5

Gene expression assay (qPCR) determined in RNA isolated from osteoblast cells cultured for 24 h on all types of matrices and assessed for the expression of Vitronectin, VTN (a), Secreted Phosphoprotein 1, SPP1 (b), and Runt-Related Transcription Factor 2, RUNX2 (c). ** = p-value < 0.01; *** = p-value < 0.001; **** = p-value < 0.0001.

Figure 6

Antibacterial assays, on all types of matrices, with S. aureus bacteria (a) and E. coli (b). * = p-value < 0.05; ** = p-value < 0.01; *** = p-value < 0.001; **** = p-value < 0.0001.

Figure 7

Schematic representation of the chemistry used for SCS preparation.

Figure 8

Schematic illustration of the different fiber deposition methods, via electrospinning, for the creation of the LBL matrix (a) and the HYB matrix (b) on top of aluminum foil.