Prevention of excessive scar formation using nanofibrous meshes made of biodegradable elastomer poly(3-hydroxybutyrate- co-3-hydroxyvalerate)

Abstract

To reduce excessive scarring in wound healing, electrospun nanofibrous meshes, composed of haloarchaea-produced biodegradable elastomer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), are fabricated for use as a wound dressing. Three PHBV polymers with different 3HV content are used to prepare either solution-cast films or electrospun nanofibrous meshes. As 3HV content increases, the crystallinity decreases and the scaffolds become more elastic. The nanofibrous meshes exhibit greater elasticity and elongation at break than films. When used to culture human dermal fibroblasts in vitro, PHBV meshes give better cell attachment and proliferation, less differentiation to myofibroblasts, and less substrate contraction. In a full-thickness mouse wound model, treatment with films or meshes enables regeneration of pale thin tissues without scabs, dehydration, or tubercular scar formation. The epidermis of wounds treated with meshes develop small invaginations in the dermis within 2 weeks, indicating hair follicle and sweat gland regeneration. Consistent with the in vitro results, meshes reduce myofibroblast differentiation in vivo through downregulation of α-SMA and TGF-β1, and upregulation of TGF-β3. The regenerated wounds treated with meshes are softer and more elastic than those treated with films. These results demonstrate that electrospun nanofibrous PHBV meshes mitigate excessive scar formation by regulating myofibroblast formation, showing their promise for use as wound dressings.

Keywords: Elastomer; PHBV; electrospun nanofiber; mechanical properties; scar formation.

Figure 1.

Schematic illustration of elastomeric nanofibrous PHBV meshes for preventing excessive scar formation. The biodegradable PHBV are produced by haloarchaeon Haloferax mediterranei. Electrospun nanofibrous meshes are fabricated using haloarchaea-produced PHBVs with distinct 3HV contents and mechanical properties. The effects of these scaffolds on reducing excessive scar formation are evaluated by culturing HDFs in vitro and by using a full-thickness wound mouse model in vivo, respectively.

Figure 2.

Morphological and thermal properties of PHBV solution-cast films and electrospun nanofibrous meshes. (a) Chemical structure of the random polymer PHBV. Three PHBV polymers with 10, 30, and 60 mol% 3HV were used. (b) SEM images (scale bar, 10 µm). (c) DSC curves and thermal properties.

Figure 3.

Surface and mechanical properties of PHBV solution-case films and electrospun nanofibrous meshes. (a) Water contact angles. ^*p < 0.05 by one-way ANOVA. (b–e) Mechanical analysis of scaffolds: stress-strain curves (b), yield strength (c), elastic modulus (d), and elongation at break (e).

Figure 4.

In vitro HDF culture on PHBV films or nanofibrous meshes (NF). (a) Cell adhesion on PHBV scaffolds after 12 h. (b) Cell fold-expansion after 7 days of culture. (c) Cell morphology on PHBV scaffolds 12 h after seeding. Overlay of DAPI (blue) and F-actin (green) images (scale bar, 100 µm). (d-e) Analysis of contraction of collagen gels and PHBV meshes. Surface area changes (d) were evaluated by comparing sizes at days 0 and 10 in images (e) (scale bar, 1 cm). (f) α-SMA expression in cells cultured on PHBV scaffolds for 7 days. (g) Fluorescence images of cells on scaffolds stained with anti-α-SMA antibody (green) and DAPI (blue) (scale bar, 200 µm). ^*p < 0.05 by one-way ANOVA.

Figure 5.

Wound regeneration in a full-thickness in vivo mouse wound model. Wounds were treated with PHBV 30 and PHBV 60 scaffolds (film or mesh), or with Tegaderm^TM (positive control). (a) Gross appearance of wounds, indicating open wound areas (%) at days 7 and 14 after wound dressing (scale bar, 1 cm). (b) Histological analysis of regenerated wounds at day 28, staining with H&E, anti-cytokeratin antibody (CK), and Masson’s trichrome (MT) (scale bar, 100 µm). ^*p < 0.05 by one-way ANOVA.

Figure 6.

Effect of PHBV scaffolds on wound healing and scarring at 28 days after treatment: (a–c) Mechanical properties of regenerated wounds: stress-strain curves (a), strain at break (b), and elastic modulus (c). For each group, three independent samples were used for the stress-strain measurements and one representative result was presented. (d) Expression of scarring-associated markers (α-SMA, TGF-β1, and TGF-β3) in regenerated wounds. ^*p < 0.05 by one-way ANOVA.