Ferrario C et al. (2020), From Food Waste to Innovative Biomaterial: Sea ...

ECB-ART-49682

Mar Drugs 2020 Aug 06;188:. doi: 10.3390/md18080414.

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From Food Waste to Innovative Biomaterial: Sea Urchin-Derived Collagen for Applications in Skin Regenerative Medicine.

Ferrario C , Rusconi F , Pulaj A , Macchi R , Landini P , Paroni M , Colombo G , Martinello T , Melotti L , Gomiero C , Candia Carnevali MD , Bonasoro F , Patruno M , Sugni M .

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Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

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	Figure 1. Percentage of bovine serum albumin (BSA) passing through the 2D collagen membranes. Time is expressed in hours. Mean ± standard error (SE).
	Figure 2. SEM micrographs of the bacteria infiltration test showing the upper and the lower surfaces of the 2D collagen membranes at 48 h. A: E. coli, upper surface. B: P. aeruginosa, upper surface. C: S. aureus, upper surface. D: E. coli, lower surface. E: P. aeruginosa, lower surface. F: S. aureus, lower surface. Bacteria are present on the upper surfaces of the 2D collagen membranes, but they are never detectable on the lower surfaces. Inserts in A, B and C, showing bacteria shapes, are pseudo-colored with the Software Adobe Photoshop CS3 Extended. Scale bars: A–D,F = 10 µm; E = 2 µm.
	Figure 3. SEM micrographs of 3D scaffolds ([collagen] = 6 mg/mL) with different ethanol concentrations (0%, 2.8% and 6%) and freezing temperatures (−196 °C and −80 °C). First column: 3D scaffold thickness. Second column: 3D scaffold upper surface. Third column: 3D scaffold lower surface. Arrows: vertical channels; arrowheads: horizontal laminae; asterisks: scaffold macroscopic ruptures. Scale bars: A,D,F,I = 100 µm; B,C,E,G,H,K,L = 200 µm; J = 300 µm.
	Figure 4. XTT assay. Cells cultured with and without sea urchin-derived collagen progressively proliferate from 24 to 72 h. Differences are not statistically significant (p > 0.05). Values: mean ± standard deviation (SD).
	Figure 5. Cells within the 3D scaffolds at short-, medium-, and long-term period (A,D,G: 3 days; B,E,H: 7 days; C,F,I: 14 days). Thick paraffin sections and haematoxylin/eosin staining (A–C 4x; D–F 10x), and transmission electron microscopy (TEM) micrographs (G–I). Fibroblasts easily infiltrate and migrate within the porous 3D scaffolds and directly contact collagen fibrils via cell processes. Arrows: cells; arrowheads: collagen fibrils. Abbreviations: c: cytoplasm; m: mitochondrion; n: nucleus; us: 3D scaffold upper surface. Top-right inserts in G, H and I show details of collagen fibril ultrastructure, namely the D-period.
	Figure 6. Viability and proliferation of fibroblasts within the 3D scaffolds at short-, medium-, and long-term period using the KI67 marker on thick paraffin sections. A: 3 days; B: 7 days; C: 14 days. At all time-points, proliferating cells are widespread in the 3D scaffolds. Abbreviations: d: days. Arrows: KI67-positive cells.
	Figure 7. Experimental setup of the 2D collagen membrane permeability tests. A: Permeability to distilled water in “dry-wet” conditions (mimicking a “dry” skin wound). B: Permeability to distilled water in “wet-wet” conditions (mimicking a “moist” skin wound). C: Permeability to proteins (BSA) in “wet-wet” conditions (mimicking a “moist” skin wound). D: Bacteria infiltration test. Red lines: 2D collagen membranes. I: insert of the modified Boyden chamber. W: well below the insert of the modified Boyden chamber.

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