Micropatterned composite membrane guides oriented cell growth and vascularization for accelerating wound healing

Abstract

Skin defect is common in daily life, but repairing large skin defects remains a challenge. Using biomaterials to deliver biochemical or physical factors to promote skin tissue regeneration is of great significance for accelerating wound healing. Specific surface micropatterns on biomaterials could affect cell behavior and tissue regeneration. However, few studies have focused on the construction of wound healing biomaterials with surface micropatterns and their role in skin tissue regeneration. In the present study, gelatin-polycaprolactone/silk fibroin composite membranes with different micropatterns were fabricated by photolithography, including line, grid and plane micropatterns. In vitro cell experiments demonstrated that the line micropattern on the composite membrane could guide cell-oriented growth, and more importantly, promote the expression of angiogenesis-related markers and α-smooth muscle actin (α-SMA) at both gene level and protein level. In the rat full-thickness skin defect model, the composite membrane with line micropatterns increased α-SMA production and neovascularization in wounds, leading to accelerated wound contraction and healing. The current study not only suggests that composite membranes with specific micropatterns can be promising wound repair materials but also provides new insights into the importance of biomaterial surface topology for tissue regeneration.

Keywords: cell behavior; gelatin; polymer biomaterials; topology; wound healing.

Graphical abstract

Scheme 1.

Procedure of patterned gelatin–PCL/SF composite membrane for repairing full-thickness skin defects in rats.

Figure 1.

(a) Preparing process of patterned gelatin–PCL/SF composite membranes; (b and c) microscope images of gelatin membranes with different patterns; (d) depth of the patterns on gelatin membranes; (e and f) SEM images of PCL/SF electrospun fibers; (g and h) pseudo color SEM images of cross sections in composite membrane. Gelatin was marked as purple. (i–k) Tensile strength, tensile strain at break and tensile modulus of patterned gelatin and gelatin–PCL/SF composite membranes. **P < 0.01; (l) Tensile strength–strain curve of patterned gelatin–PCL/SF composite membranes and gelatin membranes.

Figure 2.

(a and b) Confocal images of F-actin from L-929 cells and MSCs on patterned gelatin membranes (scale bars: 120 μm); (c) proliferation of L-929 cells and MSCs grown on patterned gelatin membranes.

Figure 3.

Western blot and rt-PCR analysis results of MSCs and L-929 cells. (a and b) rt-PCR analysis results of MSCs cultured for 1 and 3 days; (c) rt-PCR analysis results of L-929 cells cultured for 3 days; (d and e) immunoblotting results of MSCs cultured for 1 and 3 days; (f and g) immunoblotting results of L-929 cells cultured for 1 and 3 days; (h) schematic representation of protein expression changes in L-929 cells and MSCs cultured on line patterned membranes. *P < 0.05, **P < 0.01.

Figure 4.

(a) Wound images and (b) relative wound area on the 3rd, 7th and 14th day after operation. *P < 0.05, **P < 0.01.

Figure 5.

Images of H&E and Masson’s trichrome staining (scale bars: images in Days 3, 7 and 14 columns, 250 μm; images in Day 7-magnified column, 50 μm).

Figure 6.

(a) Images of nucleus, α-SMA and CD31 in new tissue on the seventh postoperative day. (b) Cell amount in visual field; (c) ratio of α-SMA fluorescence intensities; (d) amount of blood vessels in visual field; (e) cross-sectional area of blood vessel in visual field (%) (* indicates that there was a significant difference between this group and the gauze group, P < 0.05; **indicates that there was a very significant difference between this group and the gauze group, P < 0.01).