Acceleration of Healing in Full-Thickness Wound by Chitosan-Binding bFGF and Antimicrobial Peptide Modification Chitosan Membrane

Abstract

Skin wound healing is an important clinical challenge, and the main treatment points are accelerating epidermal regeneration and preventing infection. Therefore, it is necessary to develop a wound dressing that can simultaneously cure bacterial infections and accelerate wound healing. Here, we report a multifunctional composite wound dressing loaded with chitosan (CS)-binding bFGF (CSBD-bFGF) and antimicrobial peptides (P5S9K). First, CS was used as the dressing matrix material, and P5S9K was encapsulated in CS. Then, CSBD-bFGF was designed by combining recombinant DNA technology and tyrosinase treatment and modified on the dressing material surface. The results show that the binding ability of CSBD-bFGF and CS was significantly improved compared with that of commercial bFGF, and CSBD-bFGF could be controllably released from the CS dressing. More importantly, the prepared dressing material showed excellent antibacterial activity in vivo and in vitro and could effectively inhibit the growth of E. coli and S. aureus. Using NIH3T3 cells as cellular models, the results showed that the CSBD-bFGF@CS/P5S9K composite dressing was a friendly material for cell growth. After cells were seeded on the composite dressing surface, collagen-1 (COL-1) and vascular endothelial growth factor (VEGF) genes expression in cells were significantly upregulated. Finally, the full-thickness wound of the rat dorsal model was applied to analyse the tissue repair ability of the composite dressing. The results showed that the composite dressing containing CSBD-bFGF and P5S9K had the strongest ability to repair skin wounds. Therefore, the CSBD-bFGF@CS/P5S9K composite dressing has good antibacterial and accelerated wound healing abilities and has good application prospects in the treatment of skin wounds.

Keywords: CSBD-bFGF; P5S9K; antimicrobial peptides; chitosan; skin dressing; skin wound.

FIGURE 1

Preparation of the CSBD-bFGF@CS/P5S9K membrane and its effect on skin wound repair.

FIGURE 2

(A) SDS–PAGE of the bFGF and CSBD-bFGF fusion proteins, (B) immunofluorescence intensity represented the amount of bFGF and CSBD-bFGF, (C) binding efficiency and (D) cumulative release of bFGF and CSBD-bFGF, p < 0.05, n = 3.

FIGURE 3

Photos and numbers of bacterial colonies formed by E. coli (A,B) and S. aureus (C,D) treated with different membranes (P5S9K: 1, 2, 3 mg/ml and CS: Control) (E) H&E staining of different membranes embedded in rat infected tissue. Red asterisks indicate areas of inflammatory cell infiltration, n = 3, p < 0.05.

FIGURE 4

(A) SEM images and (B) FTIR spectra of different membranes. The scale bars represent 2 μm.

FIGURE 5

The water contact angles of the CS, CS/P5S9K, bFGF@CS/P5S9K and CSBD-bFGF@CS/P5S9K membranes, n = 5, p < 0.05.

FIGURE 6

(A) CCK-8 test of NIH3T3 cell proliferation on CS, CS/P5S9K, bFGF@CS/P5S9K and CSBD-bFGF@CS/P5S9K membranes for 1, 3 and 7 days. (B-C) Fluorescent micrographs and cell area analysis of NIH3T3 cells on CS, CS/P5S9K, bFGF@CS/P5S9K and CSBD-bFGF@CS/P5S9K membranes for 2 days. The green fluorescent indicated the cell morphology stained with FITC, the blue fluorescent indicated the cell nucleus stained with DAPI. Small scale bar lengths are 50 μm, large scale bar lengths are 200 μm, n = 3, p < 0.05.

FIGURE 7

Quantitative real-time PCR analysis of tissue repair-related gene expression of (A) COL-I and (B) VEGF after NIH3T3 cells were cultured for 4 days. (C) Immunofluorescent images of COL-I protein for different samples on 4 days. The green fluorescent indicates the COL-I, the blue fluorescent indicates the cell nucleus stained with DAPI and the red fluorescent indicates the cytoskeleton stained with Alexa Fluor 555 labelled phalloidine. Scale bar lengths are 50 μm, n = 3, p < 0.05.

FIGURE 8

(A) Photographic images of the wound healing status and (B) the wound-healing curves.

FIGURE 9

(A) H&E and Masson’s trichrome staining of the skin at day 12 post operation, (B) epithelial gap, (C) wound healing rate at day 12 post operation. Scale bars = 200 μm, n = 3, p < 0.05.

FIGURE 10

At day 12 post operation, H&E was used to stain sections of important organs (liver, spleen, kidney and heart) in experimental animals.