Zivelonghi G et al. (2024), Sea food by-products valorization for biomedica...

ECB-ART-53449

Front Vet Sci 2024 Nov 18;11:1491385. doi: 10.3389/fvets.2024.1491385.

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Sea food by-products valorization for biomedical applications: evaluation of their wound regeneration capabilities in an Ex vivo skin model.

Zivelonghi G , Melotti L , Carolo A , Venerando A , Roncoroni M , Martinelli G , Maccatrozzo L , Marzorati S , Sugni M , Patruno M .

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INTRODUCTION: The skin is often exposed to harmful stimuli that might compromise its integrity and functionality. After an injury, the skin has a limited capability to restore its complex structure, and in the case of severe skin damage, surgical operations and rapid application of wound dressings are often required to promote optimal wound healing. Nowadays, collagen-based biomaterials are widely used in combination with bioactive molecules able to prevent excessive inflammation and possible infections. In line with a circular economy and blue biotechnology approach, it was recently demonstrated that both collagen and bioactive molecules (i.e., antioxidant compounds) can be sustainably obtained from sea food by-products and effectively used for biomaterial development. Herein, we describe and compare the application of two marine collagen-based wound dressings (CBWDs), produced with materials obtained from sea urchin food waste, for the treatment of skin lesions in a wound healing organ culture (WHOC) model. METHODS: The ex vivo WHOC model was set up starting from rat skin explants and the induced lesions were assigned into three different groups: control (CTRL) group, not treated, marine collagen wound dressing (MCWD) group, and antioxidants-enriched marine collagen wound dressing (A-MCWD) group. After 5 and 10 days, specimens were examined for organ maintenance and assessed for the healing process. RESULTS: Immunohistochemical results showed that both CBWDs were similarly successful in prolonging skin repair, preserving the epidermal barrier up to 5 days under static culture conditions. Histological and gene expression analysis highlighted that the A-MCWD might support and accelerate skin wound healing by exerting antioxidant activity and counteracting inflammation. DISCUSSION: Overall, these findings underline the potential of sea urchin food waste as a novel resource for the development of functional medical devices for the treatment of skin wounds.

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	Figure 1. (A,B) MCWD biomaterial. (C) SEM image of MCWD (blue arrow: fibril network; red arrow: laminar structure). (D) SEM image of MCWD collagen fibrils. (E,F) A-MCWD biomaterial. (G) SEM image of A-MCWD (blue arrow: fibril network; red arrow: laminar structure). (H) SEM image of A-MCWD collagen fibrils. Other ultrastructural details of the scaffolds are also shown in SEM images in the study by Martinelli et al. (2024, submitted, under review).
	Figure 2. Wound Healing Organ culture (WHOC) Model: experimental set-up. (A) Representative images of excess hair removal and excision of dorsal skin in rats. (B) Scheme representing the 24 – well plate of the WHOC model used in the current study and the culture insert. Illustrations were created with BioRender.com. (C) Macroscopic observation of skin biopsies after 5 and 10 days of culture; scale bar = 1 cm.
	Figure 3. Harris’ hematoxylin and eosin (H&E). Representative skin histological microphotographs H&E-stained: comparison of untreated (CTRL), MCWD- and A-MCWD-treated wounds. (A) Panel of H&E-stained sections at 5 and 10 days post-wounding; scalebar = 1,000 μm, entire sample. In the dotted boxes, microphotographs of A-MCWD-treated wounds showing initial re-epithelization, magnification 50x; scale bar = 200 μm. , marine collagen wound dressing; *, antioxidant-enriched marine collagen wound dressing. (B) Quantification of epidermal thickness (μm). Data are expressed as mean ± SEM. Different letters within time points means statistically significant different values for p < 0.05. (C) Representative microphotographs of rat dorsal skin and its layers (panniculus is missing). Above, skin epithelium, magnification 100x, scalebar = 200 μm. Below, whole skin, magnification 800x, scalebar = 50 μm. E, epithelium; D, dermis; H, hypodermis; Sc, stratum corneum; Sl, stratum lucidum; Sg, stratum granulosum; Sp, stratum spinosum; Sb, stratum basale; l, lamina propria; Pd, papillary dermis; Rd, reticular dermis; SG, sebaceous gland; Hf, hair follicle.
	Figure 4. Masson-Goldner’s Trichrome (MT). Representative skin histological microphotographs MT-stained: comparison of untreated (CTRL), MCWD- and A-MCWD-treated wounds at 5 and 10 days. (A) Dermis regions (ROI) of 8-bit format and RGB images; scalebar = 50 μm. (B) Quantification of collagen fibers relative to total area (%). Data are expressed as mean ± SEM; p < 0.05, *p < 0.01.
	Figure 5. Cytokeratin 10 (K10). Representative skin immunohistochemical microphotographs of K10: comparison of untreated, MCWD- and A-MCWD-treated wounds. (A) Panel of immunohistochemical DAB-stained sections at 5 and 10 days; high magnification images (400x), scalebar = 50 μm. (B) Quantification of DAB-positive area (data are reported as percentages, %). Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. (C) Epidermal barrier at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 6. Cytokeratin 14 (K14). Representative skin immunohistochemical microphotographs of K14: comparison of untreated, MCWD- and A-MCWD-treated wounds. (A) Panel of immunohistochemical DAB-stained sections at 0, 5, and 10 days; high magnification images (400x), scalebar = 50 μm. (B) Quantification of DAB-positive area (data are reported as percentages, %). Data are expressed as mean ± SEM; p < 0.05, p < 0.01, ***p < 0.0001. (C) Epidermal barrier at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 7. Proliferation marker protein Ki-67 (Ki67). Representative skin immunohistochemical microphotographs of Ki67. (A) Panel of Ki67 DAB-stained skin appendages of untreated, MCWD- and A-MCWD-treated wounds at 5 and 10 days, magnification 400x, scalebar = 50 μm. (B) Ki67 score (manual) was evaluated as: negative (0), weak (+), moderate (++), strong (+++). (C) Skin appendages at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 8. Gene expression analysis of NADPH Oxidase 1, Glutathione Peroxidase 1 and Superoxide Dismutase 2. The mRNA levels of (A) NADPH Oxidase 1 (NADPH1), (B) Glutathione Peroxidase 1 (GPx1) and (C) Superoxide Dismutase 2 (SOD2) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, **p < 0.001.
	Figure 9. Gene expression analysis of Tumor Necrosis Factor alpha and Interleukin 6. The mRNA levels of (A) Tumor Necrosis Factor (TNF-α) and (B) Interleukin 6 (IL-6) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.001, **p < 0.0001.
	Figure 10. Gene expression analysis of Matrix Metalloproteinase 9, Tissue Inhibitor of Metalloproteinase 2, Collagen type I and Collagen type III. The mRNA levels of (A) Matrix Metalloproteinase 9 (MMP-9), (B) Tissue Inhibitor of Metalloproteinase 2 (TIMP-2), (C) Collagen type I (COL1A1) and of (D) Collagen type III (COL3A1) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 11. Gene expression analysis of Vascular Endothelial Growth Factor, Platelet Derived Growth Factor Subunit B, Keratinocyte Growth Factor 1 and Keratin 16. The mRNA levels of (A) Vascular Endothelial Growth Factor (VEGF), (B) Platelet Derived Growth Factor Subunit B (PDGFB), (C) Keratinocyte Growth Factor (KGF-1) and (D) Keratin 16 (KRT16) at 0, 5 and 10 days after wounding in untreated, MCWD- and A-MCWD-treated wounds. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, *p < 0.0001.

	Figure 1. (A,B) MCWD biomaterial. (C) SEM image of MCWD (blue arrow: fibril network; red arrow: laminar structure). (D) SEM image of MCWD collagen fibrils. (E,F) A-MCWD biomaterial. (G) SEM image of A-MCWD (blue arrow: fibril network; red arrow: laminar structure). (H) SEM image of A-MCWD collagen fibrils. Other ultrastructural details of the scaffolds are also shown in SEM images in the study by Martinelli et al. (2024, submitted, under review).
	Figure 2. Wound Healing Organ culture (WHOC) Model: experimental set-up. (A) Representative images of excess hair removal and excision of dorsal skin in rats. (B) Scheme representing the 24 – well plate of the WHOC model used in the current study and the culture insert. Illustrations were created with BioRender.com. (C) Macroscopic observation of skin biopsies after 5 and 10 days of culture; scale bar = 1 cm.
	Figure 3. Harris’ hematoxylin and eosin (H&E). Representative skin histological microphotographs H&E-stained: comparison of untreated (CTRL), MCWD- and A-MCWD-treated wounds. (A) Panel of H&E-stained sections at 5 and 10 days post-wounding; scalebar = 1,000 μm, entire sample. In the dotted boxes, microphotographs of A-MCWD-treated wounds showing initial re-epithelization, magnification 50x; scale bar = 200 μm. , marine collagen wound dressing; *, antioxidant-enriched marine collagen wound dressing. (B) Quantification of epidermal thickness (μm). Data are expressed as mean ± SEM. Different letters within time points means statistically significant different values for p < 0.05. (C) Representative microphotographs of rat dorsal skin and its layers (panniculus is missing). Above, skin epithelium, magnification 100x, scalebar = 200 μm. Below, whole skin, magnification 800x, scalebar = 50 μm. E, epithelium; D, dermis; H, hypodermis; Sc, stratum corneum; Sl, stratum lucidum; Sg, stratum granulosum; Sp, stratum spinosum; Sb, stratum basale; l, lamina propria; Pd, papillary dermis; Rd, reticular dermis; SG, sebaceous gland; Hf, hair follicle.
	Figure 4. Masson-Goldner’s Trichrome (MT). Representative skin histological microphotographs MT-stained: comparison of untreated (CTRL), MCWD- and A-MCWD-treated wounds at 5 and 10 days. (A) Dermis regions (ROI) of 8-bit format and RGB images; scalebar = 50 μm. (B) Quantification of collagen fibers relative to total area (%). Data are expressed as mean ± SEM; p < 0.05, *p < 0.01.
	Figure 5. Cytokeratin 10 (K10). Representative skin immunohistochemical microphotographs of K10: comparison of untreated, MCWD- and A-MCWD-treated wounds. (A) Panel of immunohistochemical DAB-stained sections at 5 and 10 days; high magnification images (400x), scalebar = 50 μm. (B) Quantification of DAB-positive area (data are reported as percentages, %). Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. (C) Epidermal barrier at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 6. Cytokeratin 14 (K14). Representative skin immunohistochemical microphotographs of K14: comparison of untreated, MCWD- and A-MCWD-treated wounds. (A) Panel of immunohistochemical DAB-stained sections at 0, 5, and 10 days; high magnification images (400x), scalebar = 50 μm. (B) Quantification of DAB-positive area (data are reported as percentages, %). Data are expressed as mean ± SEM; p < 0.05, p < 0.01, ***p < 0.0001. (C) Epidermal barrier at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 7. Proliferation marker protein Ki-67 (Ki67). Representative skin immunohistochemical microphotographs of Ki67. (A) Panel of Ki67 DAB-stained skin appendages of untreated, MCWD- and A-MCWD-treated wounds at 5 and 10 days, magnification 400x, scalebar = 50 μm. (B) Ki67 score (manual) was evaluated as: negative (0), weak (+), moderate (++), strong (+++). (C) Skin appendages at day 0 (T0), magnification 400x, scalebar = 50 μm.
	Figure 8. Gene expression analysis of NADPH Oxidase 1, Glutathione Peroxidase 1 and Superoxide Dismutase 2. The mRNA levels of (A) NADPH Oxidase 1 (NADPH1), (B) Glutathione Peroxidase 1 (GPx1) and (C) Superoxide Dismutase 2 (SOD2) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, **p < 0.001.
	Figure 9. Gene expression analysis of Tumor Necrosis Factor alpha and Interleukin 6. The mRNA levels of (A) Tumor Necrosis Factor (TNF-α) and (B) Interleukin 6 (IL-6) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.001, **p < 0.0001.
	Figure 10. Gene expression analysis of Matrix Metalloproteinase 9, Tissue Inhibitor of Metalloproteinase 2, Collagen type I and Collagen type III. The mRNA levels of (A) Matrix Metalloproteinase 9 (MMP-9), (B) Tissue Inhibitor of Metalloproteinase 2 (TIMP-2), (C) Collagen type I (COL1A1) and of (D) Collagen type III (COL3A1) at 0, 5 and 10 days after wounding. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 11. Gene expression analysis of Vascular Endothelial Growth Factor, Platelet Derived Growth Factor Subunit B, Keratinocyte Growth Factor 1 and Keratin 16. The mRNA levels of (A) Vascular Endothelial Growth Factor (VEGF), (B) Platelet Derived Growth Factor Subunit B (PDGFB), (C) Keratinocyte Growth Factor (KGF-1) and (D) Keratin 16 (KRT16) at 0, 5 and 10 days after wounding in untreated, MCWD- and A-MCWD-treated wounds. Relative gene expression levels were normalized using two reference genes (TBP and RPLP0) and uninjured skin was used as the calibrator sample. Data are expressed as mean ± SEM; p < 0.05, p < 0.01, p < 0.001, *p < 0.0001.