Peptide-modified chitosan hydrogels promote skin wound healing by enhancing wound angiogenesis and inhibiting inflammation

Abstract

Cutaneous wound healing following trauma is a complex and dynamic process involving multiple overlapping events following trauma. Two critical elements affecting skin wound healing are neovascularization and inflammation. A nascent vessel can provide nutrition and oxygen to a healing wound. Therefore, treatments strategies that enhance angiogenesis and inhibit inflammation can promote skin wound healing. Previous studies have shown that the SIKVAV peptide (Ser-Ile-Lys-Val-Ala-Val) from laminin can promote angiogenesis in vitro. This study evaluated the effects of peptide SIKVAV-modified chitosan hydrogels on skin wound healing. We established skin wounds established in mice and treated them with SIKVAV-modified chitosan hydrogels. H&E staining showed that peptide-modified chitosan hydrogels accelerated the reepithelialization of wounds compared with the negative and positive controls. Immunohistochemistry analysis demonstrated that more myofibroblasts were deposited at wounds treated with peptide-modified chitosan hydrogels that at those treated with negative and positive controls. In addition, peptide-modified chitosan hydrogels promoted angiogenesis as well as keratinocyte proliferation and differentiation, but inhibited inflammation in skin wounds. Taken together, these results suggest that SIKVAV-modified chitosan hydrogels are a promising treatment component for healing-impaired wounds.

Keywords: SIKVAV; angiogenesis; chitosan hydrogel; inflammation; re-epithelialization; wound healing.

Figure 1

Peptide-modified chitosan hydrogels significantly promote skin wound healing in mice. A: Representative photographs of wounds in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups taken 3, 5, and 7 days after the initial treatment in mice. B: The residual wound percentages in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups (n = 3). C: α-SMA expression in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups detected on days 3, 5, and 7 after the initial treatments as detected by immunohistochemical staining (scale bar: 50 μm). D: Mean α-SMA expression in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups measured 3, 5, and 7 days after the initial treatments (n = 3).

Figure 2

Peptide-modified chitosan hydrogels promote skin re-epithelialization in mice. H&E staining showed the re-epithelialization process in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (scale bar: 500 μm).

Figure 3

Peptide-modified chitosan hydrogels significantly promote angiogenesis in mouse skin wounds. A: Immunohistochemical staining for CD31 expression in vascular endothelial cells of wounded mouse skin 5 and 7 days after the initial treatments (scale bar: 50 μm). B: Mean numbers of new blood capillaries per field of view in mouse skin wounds in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups (n = 3).

Figure 4

Peptide-modified chitosan hydrogels promote the proliferation and differentiation of keratinocytes in skin wounds. A: Immunohistochemical staining to show K6 expression in keratinocytes in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups 3, 5, and 7 days after the initial treatments (scale bar: 50 μm). B: K6 expression in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (n = 3). C: Immunohistochemical staining of K1 expression in keratinocytes in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on day 14 after the initial treatments (scale bar: 50 μm). D: K1 expression in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on day 14 after the initial treatments (n = 3).

Figure 5

Peptide-modified chitosan hydrogels inhibit inflammation in skin wounds. A: Immunohistochemical staining of CD86-positive cells in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (scale bar: 50 μm). B: Mean numbers of CD86-positive cells per field of view in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (n = 3). C: Immunohistochemical staining of CD163-positive cells in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (scale bar: 50 μm). D: Mean numbers of CD163-positive cells in the control, peptide, chitosan hydrogel, and peptide + chitosan hydrogel groups on days 3, 5, and 7 after the initial treatments (n = 3).