A Prototype Skin Substitute, Made of Recycled Marine Collagen, Improves the Skin Regeneration of Sheep

Abstract

Skin wound healing is a complex and dynamic process that aims to restore lesioned tissues. Collagen-based skin substitutes are a promising treatment to promote wound healing by mimicking the native skin structure. Recently, collagen from marine organisms has gained interest as a source for producing biomaterials for skin regenerative strategies. This preliminary study aimed to describe the application of a collagen-based skin-like scaffold (CBSS), manufactured with collagen extracted from sea urchin food waste, to treat experimental skin wounds in a large animal. The wound-healing process was assessed over different time points by the means of clinical, histopathological, and molecular analysis. The CBSS treatment improved wound re-epithelialization along with cell proliferation, gene expression of growth factors (VEGF-A), and development of skin adnexa throughout the healing process. Furthermore, it regulated the gene expression of collagen type I and III, thus enhancing the maturation of the granulation tissue into a mature dermis without any signs of scarring as observed in untreated wounds. The observed results (reduced inflammation, better re-epithelialization, proper development of mature dermis and skin adnexa) suggest that sea urchin-derived CBSS is a promising biomaterial for skin wound healing in a "blue biotechnologies" perspective for animals of Veterinary interest.

Keywords: 3D scaffolds; biomaterials; circular economy; innovative therapies; marine collagen; regenerative medicine; sea urchin; skin substitute; tissue engineering; wound healing.

Figure 1

Examples of 2D membranes and 3D scaffolds of sea urchin-derived collagen. (a) Top view of a 2D membrane (light microscopy). Asterisks mark macroscopic folds of the thin 2D membrane. (b) Micrograph of a 2D membrane where the random distribution of the single collagen fibrils (arrows) in the two-dimensional network is visible (scanning electron microscopy). (c) Top view of a 3D scaffold (light microscopy). (d) Micrograph of a 3D scaffold where the porous microstructure of the biomaterial is detectable (scanning electron microscopy). Scalebar: (a) = 500 µm; (b) = 2 µm; (c) = 1 cm; (d) = 200 µm.

Figure 2

Representative images of the surgery procedure and biomaterial implantation. (a) Skin full-thickness removal by using surgical scissors for detaching the dermis from the subcutis; (b) macroscopic appearance of the wound after surgery; (c) 3D scaffold after UV sterilization and before implantation; (d) 3D scaffold implantation in the wound, the biomaterial was directly placed onto the wound bed; (e) representative figure of the bandages applied to the back of each sheep after the surgery.

Figure 3

Representative images of the skin ulcers during wound healing.

Figure 4

(a) Percentage of contraction. (b) Percentage of re-epithelialization. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01.

Figure 5

Histopathological microphotographs of skin biopsies at different time points after wounding: control and CBSS-treated wounds comparison. (a,b) Skin wounds at 7 days, in the CBSS-treated wounds (b) is possible to appreciate the presence of the 3D scaffold; (c,d) wounds at 14 days, treated wounds started to show a neoepidermis (NE, characterized by an hyperplastic appearance) and skin adnexa; (e,f) wounds at 21 days after wounding; (g,h) wounds at 42 days. GT = granulation tissue; NE = neoepidermis; NS = neoskin; F = fibrosis; asterisk = 3D sponge-like scaffold. Scalebar = 200 μm.

Figure 6

Epidermal thickness index (ETI) at 21 and 42 days respect to unwounded skin. Data are shown as mean ± SEM. * p < 0.05.

Figure 7

Immunohistochemistry microphotographs for Ki67 immunolabeling. Wounds are showed at (a,b) 7 days, (c,d) 14 days, (e,f) 21 days, and (g,h) 42 days. (i) Quantitative analysis of the percentage of positive area of each sample at 7, 14, 21, and 42 days. Arrowhead = active proliferating keratinocytes in the epidermal basal layer; NE = neoepidermis; NS = neoskin. Scalebar = 200 μm. (i) Data are expressed as mean ± SEM. Statistical differences were measured between the two experimental groups at the same time-point. **** p < 0.0001.

Figure 8

Immunohistochemistry microphotographs for α-SMA immunostaining. Wounds are showed at (a,b) 7 days, (c,d) 14 days, (e,f) 21 days, and (g,h) 42 days. (i) Semi-quantitative analysis based on the score for presence and orientation of myofibroblasts in wounds at 7, 14, 21, and 42 days. Arrowhead = myofibroblasts in the mature dermis; NE = neoepidermis; NS = neoskin. Scalebar = 200 μm; inset = higher magnification of the dermal fibrosis. (i) Data are expressed as mean ± SEM. Statistical differences were measured between the two experimental groups at the same time point * p < 0.05.

Figure 9

Gene expression analysis for collagen genes involved in skin wound healing. (a) Relative expression of the collagen type I (Col1α1) gene and (b) collagen type III (Col3α1) gene at 7, 14, 21, and 42 days after wounding in control and CBSS-treated wounds. Data are shown as mean ± SEM. Unwounded skin was used as the calibrator sample. Statistical differences were measured between the two experimental groups at the same time point. **** p < 0.0001.

Figure 10

Gene expression analysis for VEGF and hKER genes (a) Relative expression of the vascular endothelial growth factor A (VEGF-A) gene and (b) hair-Keratin (hKER) gene at 7, 14, 21, and 42 days after wounding in control and CBSS-treated wounds. Data are shown as mean ± SEM. Unwounded skin was used as the calibrator sample. Statistical differences were measured between the two experimental groups at the same time point. * p < 0.05; **** p < 0.0001.