Dual growth factor releasing multi-functional nanofibers for wound healing

Abstract

The objective of this research is to develop a dual growth factor-releasing nanoparticle-in-nanofiber system for wound healing applications. In order to mimic and promote the natural healing procedure, chitosan and poly(ethylene oxide) were electrospun into nanofibrous meshes as mimics of extracellular matrix. Vascular endothelial growth factor (VEGF) was loaded within nanofibers to promote angiogenesis in the short term. In addition, platelet-derived growth factor-BB (PDGF-BB) encapsulated poly(lactic-co-glycolic acid) nanoparticles were embedded inside nanofibers to generate a sustained release of PDGF-BB for accelerated tissue regeneration and remodeling. In vitro studies revealed that our nanofibrous composites delivered VEGF quickly and PDGF-BB in a relayed manner, supported fibroblast growth and exhibited anti-bacterial activities. A preliminary in vivo study performed on normal full thickness rat skin wound models demonstrated that nanofiber/nanoparticle scaffolds significantly accelerated the wound healing process by promoting angiogenesis, increasing re-epithelialization and controlling granulation tissue formation. For later stages of healing, evidence also showed quicker collagen deposition and earlier remodeling of the injured site to achieve a faster full regeneration of skin compared to the commercial Hydrofera Blue® wound dressing. These results suggest that our nanoparticle-in-nanofiber system could provide a promising treatment for normal and chronic wound healing.

Keywords: Dual-release; Growth factors; Nanofibers; Nanoparticles; Wound healing.

Fig. 1

Schematic illustration of the nanoparticle embedded electrospun nanofibers loaded with two growth factors for the wound healing. By applying these nanofiber/nanoparticle complexes on skin wound site, the chitosan/PEO nanofibers will act as a scaffold to support tissue regeneration, while fast releasing vascular endothelial growth factor (VEGF) and slow releasing platelet-derived growth factor-BB (PDGF-BB) will be the therapeutic agents that come into play at different healing stages.

Fig. 2

Characterization of the nanoparticle embedded electrospun nanofibers. SEM images of nanofiber scaffolds: 2:1 CS/PEO-NPs (A) and 1:1 CS/PEO-NPs (B). Fluorescent image merge monochrome image of ICG loaded NPs in CS/PEO fibers as indicated by arrows (C). Diameter distribution of the nanoparticle embedded electrospun nanofibers (D).

Fig. 3

Growth factor release kinetics from nanofibers and nanoparticles within fibers as determined by ELISA. VEGF was released from 1:1 CS/PEO-NPs nanofibers in PBS at 37 °C. PDGF-BB was release from PLGA nanoparticles encapsulated in 1:1 CS/PEO-NPs nanofibers at the same conditions.

Fig. 4

Cell proliferation on nanofiber scaffolds: 1:1 CS/PEO, 2:1 CS/PEO without growth factors nanoparticles, 2:1 CS/PEO with PLGA nanoparticles loaded with PDGF-BB (2:1 CS/PEO-NPs). Tissue culture plate and free PDGF-BB were served as negative and positive controls respectively. Nanofiber meshes and controls were seeded with adult human dermal fibroblast (HDFs) and MTS assay was used to quantify the cell viability (* p<0.01).

Fig. 5

Antibacterial assessment of chitosan/PEO-NP scaffolds comparing to negative controls (cell suspension and PEO scaffold) and a positive control (Penstrep solution). 1:1 CS/PEO-NPs and 2:1 CS/PEO-NPs scaffolds showed antibacterial activities against both E.Coli and S. aureus compared to negative controls(* p<0.05).

Fig. 6

Wound healing evaluation using a full skin rat wound model. A) Representative macroscopic appearance of wound closure at 0, 1, 2, and 4 weeks after treatment of skin wound of control, 2:1 CS/PEO, 2:1 CS/PEO-NPs, and Hydrofera Blue after 0, 1, 2, and 4 weeks; B) quantitative measurement of wound size reduction (* p<0.01).

Fig. 7

Histological evaluation of wounds treated by CS/PEO-NP meshes. A) H&E staining for skin wound samples of control (open wound), 2:1 CS/PEO, 2:1 CS/PEO-NPs, Hydrofera Blue after 1 and 2 weeks of treatment; inflammatory cells were indicated by arrows; B) epithelial length after 1 week of treatment; C) capillary density at wound site after 1 and 2 weeks of treatment; and D) granulation tissue thickness after 1 and 2 weeks of treatment. (* p<0.05, ** p<0.01)

Fig. 8

Collagen staining images and quantification of wounds treated by CS/PEO-NP meshes. A) Masson’s Trichrome staining of each wound samples: control, 2:1 CS/PEO, 2:1 CS/PEO-NPs, and Hydrofera Blue at 2 and 4 weeks of treatment; inflammatory cells were indicated by arrows; collagen quantification of each wound area at 2 (B) and 4 (C) weeks of treatment (* p<0.05).