Matrices for wound healing

Abstract

The present invention provides an article for improving wound healing, preferably burn healing, in some preferred embodiments, the article is a matrix formed from a mixture of two or more polysaccharides, the matrix containing an active ingredient that is an extract from differentiable cells.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 429,705 (filed December 2, 2022), the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention provides an article for improving wound healing, preferably burns.

[0003] BACKGROUND OF THE INVENTION Thermal injuries are traumatic, can be extremely painful, and require a long recovery time. Thermal injuries can also result in severe psychological and emotional distress due to extensive scarring. Burn healing follows the same steps as other wounds: hemostasis, inflammation, proliferation, and remodeling. However, burns differ from other types of wounds by the severity and duration of the inflammatory phase. During the inflammatory phase, neutrophils and monocytes are recruited to the wound, where they are responsible for removing foreign material and necrotic tissue. Upon activation, monocytes transform into macrophages (M0), which then transform into proinflammatory (M1) or anti-inflammatory (M2) macrophages. The proinflammatory M1 macrophages secrete chemokines, such as interleukin (IL)-1β, to sustain the inflammatory response, whereas the anti-inflammatory M2 macrophages reduce inflammation and activate the proliferative phase (1, 2). The inflammatory phase is an essential step in all wound healing, but in burns, the proinflammatory M1 phenotype predominates, leading to an exaggerated and prolonged inflammatory response (3), which can lead to secondary necrosis and progression of the burn injury many days after the actual injury (4).

[0004] Reactive oxygen species (ROS) are natural products of cellular respiration and act as second messengers in multiple cellular processes. In wound healing, ROS play a crucial role in orchestrating multiple processes, such as lymphocyte recruitment, effective tissue repair, and angiogenesis. 5 However, excessive ROS, referred to as oxidative stress, can cause damage to DNA, proteins, and lipids, as well as the activation of proapoptotic proteins (6, 7). Burns are known to produce large amounts of ROS, which is one of the mechanisms underlying the pathophysiological events observed after thermal injury. Increased ROS, along with increased inflammation, contribute to the secondary necrosis (6, 7). Furthermore, ROS enhance the inflammatory response, and the inflammatory response can increase the amount of ROS, thus reinforcing each other. Increased inflammation and increased ROS not only delay wound healing but also play a major role in the formation of hypertrophic scars seen after severe burns (8, 9).

[0005] Current standard-of-care dressings for partial-thickness burns aim to cover and protect the wound surface from infection, maintain a moist environment, and reduce patient discomfort. In Norwegian hospitals, burns are covered with Jelonet® petrolatum compresses on the first day (10). If treatment does not require skin grafting, partial-thickness burn treatment typically involves covering the wound with Mepilex® Ag or Aquacel® Ag Burn (10). These dressings may contribute to moist healing while reducing the risk of bacterial infection (11, 12). Silver ions (Ag+) released from these dressings have antibacterial activity. However, studies have shown that high Ag+ release correlates with strong cytotoxicity, causing histological damage and delayed healing (12, 13). Dressings incorporating anti-inflammatory and antioxidant properties have been investigated (14, 15), but to our knowledge, such dressings are not in routine, i.e., non-investigational, clinical use.

[0006] What is needed in the art are new and effective treatments for burns.

[0007] Summary of the Invention The present invention provides an article for improving wound healing, preferably burns.

[0008] Thus, in some preferred embodiments, the present invention provides articles comprising a matrix formed from at least a first polysaccharide, said matrix further comprising an extract of differentiable cells, and wherein said first polysaccharide is from a different source than said extract of differentiable cells.

[0009] In some preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the weight / weight percent of the alginate in the article is 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is 1.5% to 2.5%. In some preferred embodiments, the weight / weight percent of the alginate in the article is 1.85% to 2.15%. In some preferred embodiments, the weight / weight percent of the alginate in the article is 1.9% to 2.1%.

[0010] In some preferred embodiments, the article further comprises a second polysaccharide from a source different from the extract of the differentiable cells. In some preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is 1.2% to 2.2%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is 1.5% to 2.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is 1.55% to 1.95%. In some further preferred embodiments, the nanocellulose is supplemented with mannitol. In some preferred embodiments, the weight / weight percent of the mannitol used to supplement the nanocellulose is 1.0% to 10.0%. In other preferred embodiments, the article contains less than 0.5%, less than 0.1%, or less than 0.01% nanocellulose, or no added nanocellulose, weight / weight.

[0011] In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 5.0% to 20.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 7.0% to 17.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 9.0% to 15.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 10.0% to 14.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 11.0% to 13.0%.

[0012] In some preferred embodiments, the extract of differentiable cells is a fish roe extract. In some preferred embodiments, the fish roe extract is a salmonid roe extract. In some preferred embodiments, the fish roe extract is a Salmo salar roe extract. In some preferred embodiments, the fish roe extract is an unfertilized roe extract. In some preferred embodiments, the fish roe extract is characterized by having one or more of the following properties (a) to (f): a) in aqueous solution, 10 to 500 mg / ml protein, and most preferably 50 to 200 mg / ml protein; b) 0.1 to 10 mg / ml RNA; c) 0.1 to 10 mg / ml DNA; d) 0.1-10% lipid by weight; e) an osmolality of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.7. In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and and (f);(a),(b),(c) and (d);(a),(b),(c) and (e);(a),(b),(c) and (f);(a),(c),(d) and (e);(a),(c),(d) and (f);(a),(c),(e) and (f);(a),(b),(c),(d) and (e);(a),(b),(c),(d) and (f);(a),(c),(d),(e) and (f);or (a),(b),(c),(d),(e) and (f).

[0013] In some preferred embodiments, the fish roe extract is a heat-treated fish roe extract, which is prepared by heating the fish roe extract at 90 to 100°C for 1 to 30 minutes.

[0014] In some preferred embodiments, the first or the first and second polysaccharides are cross-linked.

[0015] In some preferred embodiments, the matrix is ​​a gel matrix.

[0016] In some preferred embodiments, the matrix is ​​formed in a lattice pattern.

[0017] In some further preferred embodiments, the present invention provides an article comprising a matrix formed from alginate, the matrix further comprising heat-treated Salmo salar egg extract, wherein the weight / weight percentage of the alginate in the article is 1.0% to 10.0% and the volume / weight percentage of the heat-treated Salmo salar egg extract in the article is 5.0% to 20.0%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.5% to 2.5%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.85% to 2.15%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.9% to 2.1%.

[0018] In some preferred embodiments, the article further comprises nanocellulose. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is 1.0% to 10.0%. In some preferred embodiments, the nanocellulose is supplemented with mannitol. In some preferred embodiments, the weight / weight percent of mannitol used to supplement the nanocellulose is 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is 1.2% to 2.2%. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is 1.5% to 2.0%. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is 1.55% to 1.95%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is 7.0% to 17.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is 9.0% to 15.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is 10.0% to 14.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is 11.0% to 13.0%. In other preferred embodiments, the article contains less than 0.5%, less than 0.1%, or less than 0.01% nanocellulose by weight, or no added nanocellulose.

[0019] In some preferred embodiments, the heat-treated Salmo salar egg extract is prepared from unfertilized eggs and is characterized by having one or more of the following properties (a) through (f): a) in aqueous solution, 50 to 500 mg / ml protein, and most preferably 10 to 5000 mg / ml protein, and most preferably 50 to 200 mg / ml protein; b) 0.1 to 10 mg / ml RNA; c) 0.1 to 10 mg / ml DNA; d) 0.1-10% lipid by weight; e) osmolality of 200 to 60 mOsm, most preferably 330 to 440 mOsm; and f) pH of about 5.0 to 7.7. In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f). In some preferred embodiments, the heat-treated Salmo salar egg extract is prepared by heating the Salmo salar egg extract to 90 to 100°C for 1 to 30 minutes.

[0020] In some preferred embodiments, the matrix is ​​a cross-linked gel matrix, hi some preferred embodiments, the matrix is ​​formed into a lattice.

[0021] In some preferred embodiments, the present invention provides a method of manufacturing an article for wound healing, the method comprising: forming an aqueous mixture of at least a first polysaccharide and a fish roe extract; and forming a matrix from the aqueous mixture to provide the article for wound healing; wherein the at least first polysaccharide is from a source different from the fish roe extract.

[0022] In some preferred embodiments, the method further comprises the step of crosslinking the matrix to prepare the article for wound healing.

[0023] In some preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the alginate is present in the mixture at a weight / weight percentage of 1.0% to 10.0%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.5% to 2.5%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.85% to 2.15%. In some preferred embodiments, the weight / weight percentage of the alginate in the article is 1.9% to 2.1%.

[0024] In some preferred embodiments, the method further comprises including a second polysaccharide in the aqueous mixture, wherein the second polysaccharide is from a different source than the fish roe extract. In some preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the nanocellulose is present in the mixture at a weight / weight % of 1.0% to 10.0%. In some preferred embodiments, the weight / weight % of the nanocellulose in the article is 1.0% to 3.0%. In some preferred embodiments, the weight / weight % of the nanocellulose in the article is 1.2% to 2.2%. In some preferred embodiments, the weight / weight % of the nanocellulose in the article is 1.5% to 2.0%. In some preferred embodiments, the weight / weight % of the nanocellulose in the article is 1.55% to 1.95%. In other preferred embodiments, the method utilizes less than 0.5%, less than 0.1%, or less than 0.01% weight / weight of nanocellulose, or does not include the addition of nanocellulose.

[0025] In some preferred embodiments, the fish roe extract is present in the mixture at a volume / weight percentage of 5.0% to 20.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 7.0% to 17.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 9.0% to 15.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 10.0% to 14.0%. In some preferred embodiments, the volume / weight percentage of the differentiable cell extract in the article is 11.0% to 13.0%. In some preferred embodiments, the fish roe extract is derived from unfertilized fish roe. In some preferred embodiments, the fish roe extract is Salmo salar roe extract. In some preferred embodiments, the fish roe extract is heat-treated fish roe extract. In some preferred embodiments, the heat-treated fish roe extract is prepared by heating the fish roe extract at 90 to 100° C. for 1 to 30 minutes. In some preferred embodiments, the heat-treated Salmo salar roe extract is characterized by having one or more of the following properties (a) to (f): a) in aqueous solution, 10 to 500 mg / ml protein, and most preferably 50 to 200 mg / ml protein; b) 0.1 to 10 mg / ml RNA; c) 0.1 to 10 mg / ml DNA; d) 0.1-10% lipid by weight; e) osmolality of 200 to 60 mOsm, most preferably 330 to 440 mOsm; and f) pH of about 5.0 to 7.7.In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f);(a),(b),(c) and (d);(a),(b),(c) and (e);(a),(b),(c) and (f);(a),(c),(d) and (e);(a),(c),(d) and (f);(a),(c),(e) and (f);(a),(b),(c),(d) and (e);(a),(b),(c),(d) and (f);(a),(c),(d),(e) and (f);or (a),(b),(c),(d),(e) and (f).

[0026] In some preferred embodiments, the nanocellulose is supplemented with mannitol. In some preferred embodiments, the wt / wt% of mannitol used to supplement the nanocellulose is between 1.0% and 10.0%.

[0027] In some preferred embodiments, the aqueous mixture further comprises CaCl. In some preferred embodiments, the CaCl is present in the aqueous mixture at a concentration of 0.01 to 0.1M.

[0028] In some preferred embodiments, the matrix is ​​formed by printing the aqueous mixture onto a substrate.

[0029] In some preferred embodiments, the matrix is ​​formed by molding the aqueous mixture.

[0030] In some preferred embodiments, the matrix is ​​crosslinked by treating the matrix with a crosslinking solution comprising CaCl2 at a concentration of 0.01 to 0.1 M. In some preferred embodiments, the crosslinking solution further comprises NaCl at 0.5% to 1.5% w / w. In some preferred embodiments, the crosslinking solution comprises the fish roe extract at 5.0% to 20.0% v / w.

[0031] In some preferred embodiments, the matrix is ​​a gel.

[0032] In some preferred embodiments, the present invention provides a matrix made by any of the aforementioned methods.

[0033] In some preferred embodiments, the present invention provides the above-described article or matrix for use in treating a wound in a subject. In some preferred embodiments, the wound is a burn. In some preferred embodiments, the wound is a chronic wound. In some preferred embodiments, the article or solid matrix is ​​applied topically to the wound.

[0034] In some preferred embodiments, the present invention provides a method of treating a wound in a subject in need thereof, comprising applying the above-described article or matrix to the wound. In some preferred embodiments, the wound is a burn. In some preferred embodiments, the wound is a chronic wound.

[0035] In some preferred embodiments, the present invention provides use of the above-described article or matrix to reduce reactive oxygen species in a subject in need thereof. In some preferred embodiments, the article or matrix is ​​applied to a site on the subject exhibiting inflammation or at risk of inflammation. In some preferred embodiments, the subject has a wound and the matrix is ​​applied to the wound. In some preferred embodiments, the subject has skin inflammation and the matrix is ​​applied to the site of skin inflammation.

[0036] In some preferred embodiments, the present invention provides use of a fish roe extract or a formulation thereof for reducing reactive oxygen species in a subject in need thereof, wherein the fish roe extract is characterized by having one or more of the following properties (a) to (f): a) in aqueous solution, 50 to 500 mg / ml of protein, and most preferably 10 to 5000 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1-10% lipid by weight; e) an osmolality of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.7. In some preferred embodiments, the extract is applied to a site on the subject exhibiting or at risk of inflammation. In some preferred embodiments, the subject has a wound, and the extract is applied to the wound. In some preferred embodiments, the subject has skin inflammation and the extract is applied to the site of skin inflammation, hi some preferred embodiments, the article or matrix is ​​applied to a site on the subject exhibiting or at risk of inflammation. In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f). In some preferred embodiments, the fish roe extract is a heat-treated fish roe extract. In some preferred embodiments, the fish roe extract is a heat-treated fish roe extract.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Burn treatments were used on Goettingen minipigs. (A) Diagram of burn distribution on the pig's back. There were overlapping burns on both sides of the spine, with eight burns on each side. Representative treatments were arranged so that all animals received all treatments, and distribution near the head and tail was even for all treatments. (B) Photograph of the custom-made burn apparatus used to create partial-thickness burns on the pig. The aluminum rod (creating the overlapping burns) was connected directly to the aluminum block to ensure a stable temperature. The block was attached to a heat-resistant Plexiglas plate for easy handling. (C) Diagram of the dressings used on the minipigs. Orange circles indicate the burns. Collex Matrix, Collex Matrix without HTX, and Jelonet® are non-adhesive dressings, so they were secured in place with Mefix. Tegaderm was included to ensure the dressings remained in place throughout the experiment. (D) Schematic diagram of the timeline for the minipig burn study. Numbers above the arrows indicate days.

[0038] Figure 2. The burns inflicted were partial-thickness burns, and the Collex Matrix shows signs of biocompatibility. (A) and (B) Hematoxylin and eosin-stained biopsy slides of wounds on days 4 (A) and 23 (B). (A) Burn depth revealed by denaturation of dermal collagen (deep eosinophilic staining). (B) Two representative wounds from each treatment at the end of the experiment, based on evaluation by a pathologist: 1 and 2: Collex Matrix, 3 and 4: Collex Matrix without HTX, and 5 and 6: Jelonet®. (C) Reactivity scores for Collex Matrix and Collex Matrix without HTX compared to Jelonet®, based on evaluation by a pathologist at Scantox A / S. Scores of 0.0-2.9 indicate no reactivity, 3.0-8.9 indicate a mild reaction, 9-15.0 indicate a moderate reaction, and >15.1 indicates a severe reaction. Data presented are mean ± SEM, N = 3 (days 4 and 10), and N = 9 (day 23). Blue bars represent the reactivity of wounds treated with Collex matrix, and green bars represent the reactivity of wounds treated with Collex matrix without HTX.

[0039] Figure 3: Collex matrix accelerates healing of partial-thickness burns. (A) Development of wound area for all wounds calculated from plan views. Data presented are mean ± SEM. Because wounds were terminated at biopsy on days 4 and 10, the number of wounds is not the same across days: N = 15 (days 1 and 4), N = 12 (days 8 and 10), and N = 9 (days 12 through 23). Blue circles represent the area of ​​wounds treated with Collex matrix, green triangles represent the area of ​​wounds treated with Collex matrix without HTX, and gray squares represent the area of ​​wounds treated with Jelonet®. Gray stars indicate P < 0.05 between Collex and Jelonet®. Green stars indicate P < 0.05 between Collex matrix and Collex matrix without HTX. Significance was determined by unpaired t-test. (B) Photographs of two representative wounds from each treatment on day 19. The wound areas shown are closest to the mean area. The wound areas of the representative wounds were: Collex matrix: 65 mm 2 and 52mm 2 (Average: 58mm 2 ), Collex matrix without HTX: 63mm 2 and 62mm 2 (Average: 62mm 2 ), and Jelonet®: 72mm 2 and 81mm 2 (Average: 79mm 2 ).

[0040] Figure 4. Burn wounds treated with Collex matrix showed reduced inflammation. Inflammation scoring of (A) the wound edge and (B) the surrounding skin based on macroscopic assessment by Scantox A / S. (A and B) Scoring system: 0: absent, 1: minimal, 2: mild, 3: moderate, 4: marked. Because wounds were terminated with biopsy on days 4 and 10, wound numbers were not equal across days: N = 15 (day 4) and N = 12 (days 6 and 8). Data shown are means ± SEM. P values ​​were determined by unpaired t-test. (C) Photographs of two representative wounds from each treatment on day 4. The wounds are the same as those shown in Figure 3B.

[0041] Figure 5. In vitro studies demonstrate the anti-inflammatory and antioxidant effects of Collex matrix and HTX on M1-polarized macrophages. (A) Schematic diagram of the method used to differentiate THP-1 monocytes into macrophages, M0, and M1. (B) IL-1β secreted from macrophages activated according to (A) was detected by ELISA assay. Data presented were normalized to the levels secreted from cells treated with PMA, IFNγ, and LPS (M1+Medium). (C) Cell death determined by PI-positive cells. Cells are from the same experiment as in (B). (D) ROS levels based on CellROX measured by flow cytometry. Data were normalized to the ROS levels from cells treated with PMA, IFNγ, and LPS (M1+Medium). Cells are from the same experiment as in (B and C). (B, C, and D) Data presented are means ± SD, N = 2 (PMA treatment only, M0) and N = 3 (M1). P values ​​were determined by unpaired T-test.

[0042] [Figure 6] A model explaining the suggested mode of action of Collex Matrix. Burn wounds are highly inflamed and have abundant secretion of proinflammatory IL-1β and excessive production of ROS. Our data suggest that Collex Matrix (containing HTX) attenuates the inflammatory response in vivo, likely by reducing levels of the proinflammatory cytokine IL-1β and ROS. During both the inflammatory and subsequent proliferative phases of wound healing, Collex Matrix appears to accelerate wound closure by ensuring a moist and protective environment.

[0043] Figure 7. Protein release from Collex matrix in 1 ml DMEM supplemented with 10% FBS and 1% Pen / Strep was determined by absorbance at 280 nm. During the analysis, patches were incubated at 37°C in a humidified atmosphere with 5% CO2.

[0044] Figure 8. Procollagen-1 α 1 ELISA assay results normalized to medium control. Data are presented as mean and SD. ** indicates p<0.01. Collex 4 weeks 20°C (N=4) and Collex 4 weeks 4°C (N=2) were stored at room temperature and in a refrigerator for 4 weeks, respectively.

[0045] FIG. 9 is a schematic diagram illustrating a printed grid design in an article of the present invention.

[0046] Figure 10. (a) Viscosity measurements performed on the bioink, where viscosity (Pa s) versus shear rate (s -1 ), (b) Shear stress (Pa) versus shear rate (s -1 ) are shown. Mean values ​​are presented together with SD (n=3).

[0047] [Figure 11] Storage modulus G' (Pa) vs. angular frequency ω (rad s -1 Frequency sweep showing the viscoelastic properties of the ink in relation to the IR. Mean values ​​with SD are presented (n=3).

[0048] Figure 12: CAD schematic of the serial interlocking grid design (a) and in-row printing of the second layer with R4 ink (b). The CAD grid dimensions are 16mm x 16mm x 1.37mm, with hole sizes of 1.4mm x 1.4mm.

[0049] Figure 13. Release from Collex matrix: Release in DMEM supplemented with 10% FBS and 1% Pen / Strep incubated at room temperature: a) Collex matrix cast without nanocellulose (unpublished), b) Collex matrix 3D printed with nanocellulose.

[0050] Figure 14. Procollagen-1 ELISA results: a) Collex matrix cast without nanocellulose, b) Collex matrix 3D printed with nanocellulose.

[0051] Figure 15. ROS detection by CellROX Deep Red performed on the fibroblast cell line HS707 at 2, 24, and 72 hours after HTX treatment. The cells were starved (1% serum instead of 10% serum) for 24 hours before HTX treatment.

[0052] Figure 16. ROS detection by CellROX Deep Red performed on the keratinocyte cell line HaCaT at 2, 24, and 72 hours after HTX treatment. The cells were starved (1% serum instead of 10% serum) for 24 hours before HTX treatment.

[0053] [Definition] As used herein, the term "matrix," when used in reference to an article of the present invention, refers to a material into which an ingredient (e.g., HTX; heat-treated egg extract) is incorporated. The matrix is ​​understood to have a length, width, and depth, and may be envisioned in a variety of shapes and patterns, including, but not limited to, circular, rectangular, triangular, or square sheets, as well as grids and other patterns (see, e.g., FIG. 9).

[0054] As used herein, the term "HTX" refers to a heat-treated extract of salmonid roe (see, e.g., PCT / IB2013 / 003177 and elsewhere herein).

[0055] As used herein, "cell" refers to the smallest structural unit of an organism capable of autonomous function, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable membrane. Cells include not only germ cells, including sperm and eggs (animal gametes consisting of an egg or embryo, along with nutritive and protective envelopes), but also all somatic cells obtained or derived from living or dead animals at any stage of development. Included are the general categories of cells: prokaryotes and eukaryotes. The cells contemplated for use in the present invention include all types of cells from all living organisms, belonging to all kingdoms: plants, animals, protists, fungi, archaea, and eubacteria. Stem cells are cells capable of producing cells with many different levels of specialization through continuous division. For example, hematopoietic stem cells produce both red and white blood cells. From conception to death, humans possess stem cells, but their capacity for differentiation is reduced in adults.

[0056] As used herein, the term "differentiation" in relation to cells refers to the process by which cells become structurally and functionally specialized, which is the gradual restriction of developmental potential that occurs during embryonic development and results in increasing functional specialization that leads to the formation of specialized cells, tissues, and organs.

[0057] The term "dedifferentiation" in relation to cells refers to the reverse process of differentiation, in which a cell becomes less structurally and functionally specialized, increasing the cell's developmental potential.

[0058] "Differentiable" refers to the ability of a cell to differentiate into a desired cell type. As used herein, the term "differentiate" refers to specialization (differentiation) or reversion to a more primitive cell type (dedifferentiation).

[0059] In the context of "cell extract" or "egg extract" in the present invention, "extract" refers to a preparation of any type of cell as defined above, obtained by chemical or mechanical action, such as by pressing, distillation, evaporation, etc. The extract may contain all of the components of the cell, including a concentrated preparation of the active ingredient, or any single component or combination. Such components of the extract include, but are not limited to, RNA, DNA, microRNA, lipids, free amino acids, all amino acid-based structures, including peptides and proteins, carbohydrates, minerals, or combinations thereof. Extracts contemplated by the present invention include, but are not limited to, extracts of fish eggs, sea urchin eggs, frog eggs, adult stem cells, plant seeds, and plant stem cells.

[0060] The term "manage," when used in connection with a disease or condition, means providing a beneficial effect to a subject receiving a prophylactic or therapeutic agent, but does not result in a cure of the disease. In certain embodiments, a subject is administered one or more prophylactic or therapeutic agents to manage the disease and prevent the progression or worsening of the disease.

[0061] As used herein, the terms "prevent" and "preventing" include prevention of recurrence, spread, or onset. It is not intended that the present invention be limited to complete prevention. Onset may be delayed or the severity of the disease may be reduced.

[0062] As used herein, the terms "treat" and "treating" are not limited to cases where a subject (e.g., a patient) is cured and the disease is eradicated. Rather, the present invention also contemplates treatments that merely reduce symptoms and / or slow the progression of the disease.

[0063] Detailed Description of the Invention The present invention provides an article for improving wound healing, preferably burn healing, in some preferred embodiments, the article is a matrix formed from a mixture of two or more polysaccharides, the matrix containing an active ingredient that is an extract from differentiable cells.

[0064] In some particularly preferred embodiments, the articles of the present invention are utilized in the treatment of burns. Partial-thickness burns are characterized by prolonged inflammatory responses, oxidative stress, tissue damage, and secondary necrosis. Therefore, an optimal dressing for burns can provide moisturizing and absorbent coverage, protecting the wound while reducing inflammation and oxidative stress. In some preferred embodiments, the articles of the present invention contain an extract from unfertilized salmon roe, called HTX, which contains a component with potential anti-inflammatory and antioxidant properties. See, for example, PCT applications PCT / IB2011 / 001488 and PCT / IB2013 / 003177 (both of which are incorporated herein by reference in their entirety). In the articles of the present invention, HTX is combined with alginate derived from brown algae and nanocellulose derived from sea squirts, and three-dimensionally printed into an all-marine hydrogel wound dressing, referred to herein as Collex Matrix. Thus, the present invention provides a technical solution for effectively delivering HTX as an active ingredient to wounds, such as partial-thickness burns.

[0065] The data presented herein describe the testing of Collex Matrix in partial-thickness burns in Göttingen minipigs. Collex Matrix was compared with the petrolatum compress Jelonet® and an HTX-free variant of Collex Matrix. Results showed that treatment of burned skin with Collex Matrix accelerated healing compared with wounds treated with Jelonet®. Compared with HTX-free Collex Matrix, Collex Matrix improved healing during the first week after injury, when secondary necrosis was prominent. Notably, Collex Matrix attenuated the inflammatory response early after injury. The anti-inflammatory response of Collex Matrix was examined in more detail against activated M1 macrophages. Results further showed that, similar to HTX alone, Collex Matrix significantly reduced intracellular levels of oxidative stress as well as the secretion of proinflammatory interleukin-1β. The results of this study suggest that Collex matrix is ​​a potential dressing material for the treatment of burn wounds, with the anti-inflammatory effect of HTX being beneficial in the early stages of wound healing and the moisturizing properties of the hydrogel being favorable in both the early and advanced proliferative phases of wound healing.

[0066] Thus, in some embodiments, the present invention provides matrices, most preferably hydrogels, for delivery of differentiable cell (e.g., HTX) extracts to wounds. In some preferred embodiments, the differentiable cell extract components provided in the matrix are characterized by having one or more of the following properties (a) through (f): a) 10 to 500 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 60 mOsm, most preferably 330 to 440 mOsm; f) pH of about 5.0 to 7.7.

[0067] In some preferred embodiments, the egg cell extract is characterized by having two or more of properties (a) through (f). In some preferred embodiments, the differentiable cell extract is characterized by having three or more of properties (a) through (f). In some preferred embodiments, the differentiable cell extract is characterized by having four or more of properties (a) through (f). In some preferred embodiments, the differentiable cell extract is characterized by having five or more of properties (a) through (f). In some preferred embodiments, the differentiable cell extract is characterized by having all six of properties (a) through (f). In some preferred embodiments, the differentiable cell extract is characterized by having properties (a) and (b). In some preferred embodiments, the differentiable cell extract is characterized by having properties (a) and (c). In some preferred embodiments, the differentiable cell extract is characterized by having properties (a) and (d). In some preferred embodiments, the differentiable cell extract is characterized by having properties (a) and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a) and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), and (c). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), and (d). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), and (d). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), and (e).In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (d), and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (d), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), and (d). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), (d), and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), (d), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), (e), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), (d), and (e). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), (d), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (c), (d), (e), and (f). In some preferred embodiments, the extract of the differentiable cells is characterized by having properties (a), (b), (c), (d), (e), and (f).

[0068] In some embodiments, the extract of differentiable cells is selected from the group consisting of activated fish roe cell extract and unactivated fish roe cell extract. In some embodiments, the fish roe cell extract is derived from fertilized eggs. In some embodiments, the fish roe cell extract is derived from unfertilized eggs. In some embodiments, the cell extract is heat-treated by heating the extract to greater than 80°C, 90°C, 95°C, or 100°C. In some embodiments, the heat treatment is for about 1 minute to about 30 minutes.

[0069] As mentioned above, the compositions of the present invention utilize extracts of cells, eggs, and embryos from vertebrates, and include those from the Superclass Gnathostomata (jawed vertebrates), Euteleostomi (bony vertebrates), Class Actinopterygii (ray-finned vertebrates), Class Sarcopterygii (lobe-finned vertebrates and terrestrial vertebrates), Tetrapoda (tetrapods), Amniota (amniotes), Synapsida (synapsids), Class Mammalia (mammals), Early Therapsida (early therapsids), Class Reptilia (reptiles), Anapsida (land and sea turtles), Order Testudines (land and sea turtles), Diapsida (birds, crocodiles, lizards, snakes, and related animals), Archosauria (birds and crocodiles), Order Acropia ... Examples of suitable eggs include, but are not limited to, Crocodilia (caimans, crocodiles, and related animals), Lepidosauria (water lizards, lizards, snakes, and tuatara), Order Rhynchocephalia (tuatara), Order Squamata (water lizards, lizards, and snakes), Class Amphibia (amphibians), Subclass Dipnoi (lungfish), Actinistia, Order Coelacanthiformes (coelacanths), Class Chondrichthyes (rays, sharks, and related animals), Placodermi (armored fish and placoderms), and Class Cephalaspidomorphi, and more preferably, eggs or embryos of fish, shrimp, sea urchin, or amphibians. In some embodiments, unfertilized but activated eggs of fish, shrimp, sea urchin, or amphibians are used. The present invention is not limited to the use of any particular type of egg. Indeed, a variety of eggs are contemplated for use, including, but not limited to, eggs from Xenopus, shrimp, sea urchin, salmon, trout, or zebrafish. In some embodiments, the eggs are collected from mature females and spontaneously activate upon contact with water. In some embodiments, the eggs are washed in Ringer's saline. In some embodiments, the eggs are not from avian species.In some particularly preferred embodiments, the eggs are from a salmonid species. In some particularly preferred embodiments, the salmonid fish is Salmo salar.

[0070] The extracts of the present invention are prepared from any of the sources described herein. In some embodiments, the extract is a cellular extract. The cellular extracts of the present invention are preferably compositions of cells, such as disrupted eggs. The cells can be disrupted by a variety of methods, including, but not limited to, mechanical shearing or blending, sonication, or osmotic lysis. In some embodiments, the extract contains less than about 1% and preferably less than about 0.1% cholesterol or ovalbumin. Thus, in some embodiments, the cellular extract contains carbohydrates, proteins, glycosylated or otherwise modified proteins, peptides, amino acids, RNA (mRNA, sRNA, miRNA, rRNA), DNA, water, etc., and combinations thereof. In some embodiments, the cellular extract may contain small amounts of lipids naturally associated with the cells, as well as nuclear components such as chromosomes, nucleic acids, and nuclear proteins. In some embodiments, the cellular extract is preferably a cytoplasmic extract or fraction prepared by removing nuclei, cell membranes, and other water-insoluble materials naturally associated with the cells. In some embodiments, these components are removed by centrifugation or fractionation of the disrupted cells. In some embodiments, the cellular extract is preferably an aqueous extract or fraction containing water-soluble cellular components (such as proteins, mRNA, and carbohydrates).

[0071] A variety of methods can be used to prepare extracts, including those described in the Examples below. For example, in some embodiments, eggs are "dried" in a 15 mL glass centrifuge tube and disrupted by sedimentation at 15,000 g for 15 minutes. This produces three layers: an upper lipid fraction (collected, aliquoted, and frozen); a middle cellular or cytoplasmic fraction (also collected, aliquoted, and frozen); and a pellet fraction (discarded). In some embodiments, the cellular fraction or extract contains primarily cytoplasmic contents. The cellular fraction is used as the extract. In some embodiments, the cellular fraction may be used in combination with the lipid fraction. The cytoplasmic fraction can be further clarified by sedimentation at 50,000, 100,000, or 200,000 g to produce additional cellular extracts, which are primarily aqueous extract fractions. Regardless of the fraction used, the extract may be diluted to about 300 mOsm with cell lysis buffer (see above), if necessary. Thus, in some preferred embodiments, a water-soluble extract prepared from eggs or embryos is utilized.

[0072] In another embodiment, eggs are suspended in 0.5 volumes of cell lysis buffer and sonicated on ice until all eggs are lysed. Particulate matter is sedimented at 15,000 g for 15 minutes at 4°C. The supernatant is the extract. As above, the osmolality can be adjusted to 300 mOsm, if necessary. The extract can be clarified as above.

[0073] In yet another embodiment, eggs are suspended in cell lysis buffer as in Method 2. Eggs are lysed by Dounce homogenization using a glass mortar and pestle (Kontes, Type A or Type B). The lysate is sedimented and processed as above.

[0074] In some embodiments, the homogenate and extract may be stabilized by adding one or more stabilizers, such as lipid stabilizers, or by packaging in a package designed to prevent oxidation. In some embodiments, an antioxidant, such as vitamin E, is added to the extract to reduce the rate of lipid oxidation. In some embodiments, the extract is packaged in a container under an inert atmosphere. In some embodiments, the extract is packaged in an air-free container, such as an aluminum-coated bag (less than 10 kg per bag for efficient oxygen removal), or in a container filled with nitrogen to remove oxygen, to reduce the rate of lipid oxidation. In other embodiments, the extract is packaged in a vacuum-packed container using a pump delivery system.

[0075] In some embodiments, the present invention provides powders prepared from extracts of the cells described above. In some embodiments, the extracts of the cells used to produce the powders are prepared from salmonid eggs. In some embodiments, the extracts of the cells used to produce the powders are prepared from salmon or trout eggs. In some embodiments, the powders are biologically active. In some preferred embodiments, the powders are lyophilized. In some embodiments, the powder contains less than about 10% moisture, and most preferably less than about 5% moisture; protein at a concentration of about 500 to about 800 mg / g powder, preferably about 600 to about 700 mg / g powder, and most preferably about 640 mg / g powder; DNA at a concentration of about 1 to about 50 μl / mg powder, preferably about 5 to about 25 μl / mg powder, and most preferably about 16 μl / mg powder; total RNA (e.g., including mRNA, rRNA, and microRNA) at a concentration of about 1 to about 50 μl / mg powder, preferably about 5 to about 20 μl / mg powder, and most preferably about 12 μl / mg powder; and lipids at a concentration of about 100 to about 200 mg / g powder, most preferably about 150 mg / g powder. Powders can preferably be used as a substitute for non-powdered cellular extracts to produce the formulations described herein.

[0076] In some particularly preferred embodiments, the cell extract is incorporated into a matrix formed from one or more polysaccharides or a mixture of polysaccharides. Thus, in some preferred embodiments, the present invention provides a method of producing an article for wound healing, the method comprising: forming an aqueous mixture of at least a first polysaccharide and an extract of differentiable cells; forming a matrix from the aqueous mixture; and optionally crosslinking the matrix to provide an article for wound healing. In some preferred embodiments, a second polysaccharide is included in the aqueous mixture.

[0077] In some particularly preferred embodiments, the extract of differentiable cells is a fish roe extract. In some preferred embodiments, the fish roe extract is a salmonid roe extract. In some more preferred embodiments, the salmonid roe extract is a Salmo salar roe extract. In some preferred embodiments, the fish roe extract, such as a Salmo salar roe extract, is prepared from unfertilized eggs. In some preferred embodiments, the cell extract is present in the mixture at 5.0% to 20.0% volume / weight, where volume is the volume of the cell extract in milliliters and weight is the weight of the remaining components of the mixture in grams. In some preferred embodiments, the cell extract is present in the mixture at 8.0% to 16.0% volume / weight. In some preferred embodiments, the cell extract is present in the mixture at 10.0% to 14.0% volume / weight. In some preferred embodiments, the cell extract may be reconstituted from the powder prepared above. In some preferred embodiments, the fish roe extract is a heat-treated fish roe extract. In some preferred embodiments, the heat-treated fish roe extract is prepared by heating the fish roe extract at 90 to 100° C. for 1 to 30 minutes. In some embodiments, the cell extract, which may preferably be heat-treated Salmo salar roe extract, is characterized by having one or more of the following properties (a) to (f): a) 10 to 500 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 60 mOsm, most preferably 330 to 440 mOsm; f) pH of about 5.0 to 7.7.

[0078] In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f);(a),(b),(c) and (d);(a),(b),(c) and (e);(a),(b),(c) and (f);(a),(c),(d) and (e);(a),(c),(d) and (f);(a),(c),(e) and (f);(a),(b),(c),(d) and (e);(a),(b),(c),(d) and (f);(a),(c),(d),(e) and (f);or (a),(b),(c),(d),(e) and (f).

[0079] In some particularly preferred embodiments, at least two polysaccharides are from a source different from the source of the extract of the differentiable cells, e.g., a source different from Salmo salar, hi some preferred embodiments, the polysaccharides are from a marine source different from the source of the cell extract.

[0080] In some particularly preferred embodiments, the first polysaccharide is alginate, hi some preferred embodiments, the alginate is present in the aqueous mixture at a weight / weight percentage of 1.0% to 10.0%, and most preferably 1.0% to 3.0%, where weight / weight is the weight of alginate per total weight of the aqueous mixture.

[0081] In some particularly preferred embodiments, the second polysaccharide is nanocellulose, which is present in the aqueous mixture at a weight / weight percentage of 1.0% to 10.0%, and most preferably 1.0% to 3.0%, where weight / weight is the weight of nanocellulose per total weight of the aqueous mixture.

[0082] In some preferred embodiments, the nanocellulose used to form the matrix further comprises mannitol, hi some preferred embodiments, the nanocellulose is optionally supplemented with mannitol at a weight percent of 1.0% to 10.0%, and most preferably 2.0% to 7.0%, where weight / weight is the weight of mannitol per total weight of nanocellulose.

[0083] In some preferred embodiments, the aqueous mixture used to form the matrix further comprises CaCl. In some preferred embodiments, the CaCl is present in the aqueous mixture at a concentration of 0.01 to 0.1 M.

[0084] The present invention is not limited to any particular method for forming a matrix from the aqueous mixture. In some preferred embodiments, the matrix is ​​formed by printing the aqueous mixture onto a substrate. In other preferred embodiments, the matrix is ​​formed by molding the aqueous mixture. Suitable molds include, for example, release molds formed from polydimethylsiloxane (PDMS) or release-coated polymers. Suitable release coatings are known in the art and include silicone release coatings.

[0085] The present invention is not limited to any particular method for crosslinking the polymer used to form the matrix. In some preferred embodiments, the matrix is ​​crosslinked by treating the matrix with a crosslinking solution containing CaCl2 at a concentration of 0.01 to 0.1 M. In some preferred embodiments, the crosslinking solution further contains NaCl at 0.5% to 1.5% w / w. In some preferred embodiments, the crosslinking solution further contains fish roe extract at 5.0% to 20.0%, and most preferably 5.0% to 17.0% v / w. In some preferred embodiments, the fish roe extract is present in the crosslinking solution at 8.0% to 16.0% v / w. In some preferred embodiments, the cell extract is present in the crosslinking solution at 10.0% to 14.0% v / w.

[0086] In some preferred embodiments, the matrix is ​​a gel.

[0087] In some preferred embodiments, the matrix is ​​formed in a lattice pattern.

[0088] In some preferred embodiments, the present invention provides matrices made by the above methods. Accordingly, in some embodiments, the present invention provides articles comprising a matrix (e.g., a gel matrix, and most preferably a hydrogel matrix) formed from at least a first polysaccharide, the matrix further comprising an extract of differentiable cells, and wherein the first polysaccharide is from a source different from the extract of differentiable cells. In some preferred embodiments, a second polysaccharide is included in the aqueous mixture, and wherein the first polysaccharide is from a source different from the extract of differentiable cells.

[0089] In some particularly preferred embodiments, the extract of differentiable cells is a fish roe extract. In some preferred embodiments, the fish roe extract is a salmonid roe extract. In some more preferred embodiments, the salmonid roe extract is a Salmo salar roe extract. In some preferred embodiments, the fish roe extract, such as Salmo salar roe extract, is prepared from unfertilized eggs. In some preferred embodiments, the cell extract is present in the mixture at 5.0% to 20.0% volume / weight. In some preferred embodiments, the cell extract is present in the mixture at 8.0% to 16.0% volume / weight. In some preferred embodiments, the cell extract is present in the mixture at 10.0% to 14.0% volume / weight. In some preferred embodiments, the fish roe extract is a heat-treated fish roe extract. In some preferred embodiments, the heat-treated fish roe extract is prepared by heating the fish roe extract at 90 to 100°C for 1 to 30 minutes. In some embodiments, the cell extract, which may preferably be heat-treated Salmo salar egg extract, is characterized by having one or more of the following properties (a) through (f): a) 10 to 500 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 60 mOsm, most preferably 330 to 440 mOsm; f) pH of about 5.0 to 7.7.

[0090] In some preferred embodiments, the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f);(a),(b),(c) and (d);(a),(b),(c) and (e);(a),(b),(c) and (f);(a),(c),(d) and (e);(a),(c),(d) and (f);(a),(c),(e) and (f);(a),(b),(c),(d) and (e);(a),(b),(c),(d) and (f);(a),(c),(d),(e) and (f);or (a),(b),(c),(d),(e) and (f).

[0091] In some particularly preferred embodiments, the polysaccharides (e.g., the first polysaccharide and / or the second polysaccharide) are from a source different from the source of the extract of the differentiable cells, e.g., a source different from Salmo salar. In some preferred embodiments, the polysaccharides are from a marine source different from the source of the cell extract.

[0092] In some particularly preferred embodiments, the first polysaccharide is alginate, hi some preferred embodiments, the alginate is present in the aqueous mixture at a weight / weight percentage of 1.0% to 10.0%, and most preferably 1.0% to 3.0%.

[0093] In some particularly preferred embodiments, the second polysaccharide is nanocellulose, hi some preferred embodiments, the nanocellulose is present in the aqueous mixture at a weight / weight percentage of 1.0% to 10.0%, and most preferably 1.0% to 3.0%.

[0094] In some preferred embodiments, the nanocellulose used to form the matrix further comprises mannitol, hi some preferred embodiments, the nanocellulose is optionally supplemented with mannitol at a weight percent of 1.0% to 10.0%, and most preferably 2.0% to 7.0%.

[0095] In some particularly preferred embodiments, the present invention provides articles comprising a solid matrix formed from a mixture of alginate and nanocellulose, said matrix further comprising heat-treated salmonid egg extract, wherein the wt / wt% of alginate in the article is from 1.0% to 10.0%, most preferably from 1.0% to 3.0%, the wt / wt% of nanocellulose in the article is from 1.0% to 10.0%, most preferably from 1.0% to 3.0%, and the vol / wt% of heat-treated salmonid egg extract in the article is from 5.0% to 20.0%, most preferably from 8.0% to 16.0%.

[0096] In some preferred embodiments, one or more additional active agents may be included in the matrix. In some preferred embodiments, one or more additional active agents are incorporated into the aqueous mixture prior to matrix formation.

[0097] Suitable additional active agents include nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g., the NSAIDS are selected from the following categories: (e.g., propionic acid derivatives, acetic acid derivatives, fenamic acid derivatives, diphenylcarboxylic acid derivatives, and oxicams)); steroidal anti-inflammatory drugs, including hydrocortisone and the like; antihistamines (e.g., chlorpheniramine, triprolidine); antitussives (e.g., dextromethorphan, codeine, carmiphen, and carbetapentane); antipruritics (e.g., methidyl anticholinergics (e.g., scopolamine, atropine, homatropine, levodopa); antiemetics and antinausea drugs (e.g., cyclozine, meclizine, chlorpromazine, buclizine); appetite suppressants (e.g., benzphetamine, phentermine, chlorphentermine, fenfluramine); central nervous system stimulants (e.g., amphetamine, methamphetamine, dextroamphetamine, and methylphenidate); minoxidil; antiarrhythmics (e.g., propranolol, procainamide, disopyramide, quinidine) P-adrenergic blocking agents (e.g., metoprolol, acebutolol, betaxolol, labetalol, and timolol); cardiac inotropes (e.g., milrinone, amrinone, and dobutamine); antihypertensives (e.g., enalapril, clonidine, hydralazine, minoxidil, guanadrel, guanethidine); diuretics (e.g., amiloride and hydrochlorothiazide); vasodilators (e.g., diltiazem, amiodarone, isosprin, nylidrin, tolazoline, and verapamil); vasoconstrictors (e.g., dihydrazine, antiulcer drugs (e.g., ranitidine and cimetidine); anesthetics (e.g., lidocaine, bupivacaine, chloroprocaine, dibucaine); antidepressants (e.g., imipramine, desipramine, amitriptyline, nortriptyline); PDE5 inhibitors such as Viagra® or Cialis®; tranquilizers and sedatives (e.g., chlordiazepoxide, benacitidine, benzquinamide, flurazepam, hydroxyzine, loxapine, and promazine);Antipsychotics (e.g., chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine, and trifluoperazine); antibacterials (antibacterial, antifungal, antiprotozoal, and antiviral);

[0098] Preferred antibacterial agents for incorporation into the present compositions include, for example, pharmaceutically acceptable salts of beta-lactam drugs, quinolones, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole, pentamidine, gentamicin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, miconazole, and amanfadine.

[0099] Other drugs that can be used in the practice of the present invention include antineoplastic agents (e.g., antiandrogens (e.g., leuprorelin or flutamide), cytotoxic agents (adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, alpha-2 interferon), antiestrogens (tamoxifen), antimetabolites (fluorouracil, methotrexate, mercaptopurine, thioguanine).

[0100] The compositions may also contain other compounds such as hormones (e.g., medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide, or somatostatin); muscle relaxants (e.g., cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, diphenoxylate, dantrolene, and azumolene); antispasmodics; bone active agents (e.g., diphosphonate and phosphonoalkylphosphinate drug compounds); endocrine modulating agents (e.g., For example, these may include contraceptives (e.g., ethynodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone), diabetes control drugs (e.g., glyburide or chlorpropamide), anabolics such as testolactone or stanozolol, androgens (e.g., methyltestosterone, testosterone, or fluoxymesterone), antidiuretics (e.g., desmopressin), and calcitonin.

[0101] Also usable in the present invention are estrogens (e.g., diethylstilbestrol), glucocorticoids (e.g., triamcinolone, betamethasone, etc.), and progestins such as norethindrone, ethynodiol, norethindrone, and levonorgestrel; thyroid drugs (e.g., liothyronine or levothyroxine) or antithyroid drugs (e.g., methimazole); antihyperprolactinergic drugs (cabergoline); hormone suppressants (e.g., danazol or goserelin), tocolytics (e.g., methylergonovine or oxytocin), and prostaglandins such as misoprostol, alprostadil, or dinoprostone.

[0102] Other useful compounds include immunomodulators (e.g., antihistamines, mast cell stabilizers such as lodoxamide and / or cromoglycate, steroids (e.g., triamcinolone, beclomethasone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone, or clobetasol), histamine H2 receptor antagonists (e.g., famotidine, cimetidine, ranitidine), immunosuppressants (e.g., azathioprine, cyclosporine), and the like. Anti-inflammatory agents such as sulindac, etodolac, ketoprofen, and ketorolac may also be used. Other agents that can be used in combination with the present invention will be apparent to those skilled in the art.

[0103] In some preferred embodiments, the articles of the present invention find use in treating wounds. Thus, in some preferred embodiments, the articles of the present invention may preferably be applied topically to a wound on a subject. The present invention is not limited to the treatment of a particular type of wound. In some preferred embodiments, the wound is a burn. Types of burns that can be treated include partial-thickness burns (second-degree burns), superficial burns (first-degree burns), and full-thickness burns (third-degree burns). In some preferred embodiments, the burn is a partial-thickness burn. In other embodiments, the wound is a chronic wound. In particular, the chronic wound may be associated with diabetes (i.e., diabetic ulcers, such as diabetic foot ulcers), obesity, spinal cord injury (pressure ulcers), venous ulcers, and the like. Thus, in some preferred embodiments, the chronic wound is a diabetic ulcer, a venous ulcer, a pressure ulcer, or an ischemic ulcer. In yet other embodiments, the wound may be an abrasion, a skin laceration, a puncture wound, a surgical wound or incision, or a laceration. In other preferred embodiments, the wound may be an insect bite or sting.

[0104] [Example] Example 1: Preparation of fish roe extract Fresh, unfertilized salmon (Salmo salar) eggs collected from breeding females (late autumn) were kept on ice and preferably immediately used for extract preparation. The eggs were crushed and the eggshells were removed by sieving. 50% by volume of 0.9% NaCl was added to the filtrate. The diluted filtrate was heated to 90°C for 20 minutes with stirring every 5 minutes. The mixture was then centrifuged at 4500 rpm for 10 minutes. The supernatant was then transferred to a Stericup Quick Release, vacuum-driven, disposable filtration system (0.22 mm) and vacuum was applied. The resulting filtrate (HTX) was aliquoted and frozen for use. The HTX contained 100 to 110 mg / ml of protein.

[0105] Example 2: Use of HTX-containing 3-d matrix (Collex matrix) in burn treatment [method] (Collex and HTX-free Collex manufacturing) The ink used to fabricate the Collex matrix contained alginate (2% wt / wt, MVG, NovaMatrix AS), 4.6% mannitol-supplemented nanocellulose (1.7% wt / wt, OceanTunicell AS), HTX (12% vol / wt, Regenics AS), NaCl (0.9% wt / vol, Fresenius Kabi), and CaCl2 (0.02 M, Sigma-Aldrich). Approximately 200 μl (approximately 200 mg) of this ink was three-dimensionally printed in four layers at a size of 16 x 16 mm using a 4th generation 3D Bioplotter from EnvisionTEC (Gladbeck, Germany). The patch was crosslinked with a 0.02 M CaCl2 solution supplemented with saline (0.9% NaCl) and HTX (12%). The Collex matrix was stored in a polyethylene / aluminum sachet under argon and refrigerated until analysis. The preparation of the HTX-free Collex matrix was identical to the Collex matrix, except that HTX was replaced with saline (0.9% NaCl, Fresenius Kabi).

[0106] (Endotoxin assay) Five Collex matrix dressings, ranging in size from 190 to 197 mg, were tested for endotoxin levels. The Collex matrix was extracted with 0.9% NaCl, a total volume of 2 ml (1:10 dilution), and incubated at 37°C and 50 rpm for 1 hour. The tubes (containing the dressing and extract) were then centrifuged at 4000 rpm for 30 minutes. 1 ml of the supernatant / extract, at a 1:10 ratio, was transferred to a new tube. The supernatant / extract was then further diluted 1:100 (0.1 ml extract + 0.9 ml water (Biowest)) and tested for endotoxin levels using a PyroGene® kit (Lonza, #50-658U) and WinKQCL® Endotoxin Detection & Analysis Software. Analysis was completed according to European Pharmacopoeia 2.6.32: Bacterial Endotoxin Test Using Recombinant Factor C.

[0107] Animals: Housing, Anesthesia, and Pain Management The study was conducted on three female Göttingen minipigs from Ellegaard Göttingen Minipigs A / S. The animals were cared for in accordance with EU Directive 2010 / 63 / EU of September 22, 2010, on the protection of animals used for scientific purposes. On the day of wounding, anesthesia was administered by intramuscular injection into the neck (1.0 mL / 10 kg) of a mixture of Zoletil 50® Vet., Virbac, France (125 mg tyletamine and 125 mg zolazepam), 20 mg xylazine / mL (6.25 mL), 100 mg ketamine / mL (1.25 mL), and 10 mg butorphanol / mL (2.5 mL). After induction of anesthesia and preparation of the animals for surgery, the animals were intubated, and anesthesia was maintained with isoflurane.

[0108] For postoperative pain management, the animals received a transdermal dressing containing fentanyl (75 μg / hour) from the day before surgery until 72 hours later. In connection with wound preparation, the animals received an intramuscular injection of methadone (10 mg / mL, 0.02 mL / kg). Additionally, on the day of wounding (day 1), the animals received an intramuscular injection of 20 mg meloxicam / mL (0.02 mL / kg). Treatment with 15 mg meloxicam / mL oral suspension was continued orally once daily for the next two days. In connection with biopsy collection during the first week after wounding and on day 10, the animals received an intramuscular injection of buprenorphine (0.3 mg / mL, 0.04 mL / kg) prior to biopsy collection, medication, and dressing changes.

[0109] Medication and dressing changes were performed under general anesthesia. Before biopsy collection, medication, and dressing changes, the animals were anesthetized with propofol administered intravenously (using a continuous infusion line) through a catheter inserted in an ear vein until the desired effect was achieved (loss of corneal reflex and satisfactory muscle relaxation, usually 2–2.5 mg / kg, 10 mg / mL, i.e., 0.2–0.25 mL / kg). Before the use of propofol, the TauroLock® Hep500 was removed. On the day of necropsy (day 23), the animals were weighed, examined externally, and anesthetized with a mixture of Zoletil 50 Vet., Virbac, France (125 mg tyletamine and 125 mg zolazepam), 20 mg xylazine / mL (6.25 mL), 100 mg ketamine / mL (1.25 mL), and 10 mg butorphanol / mL (2.5 mL) intramuscularly (0.3 mL / kg body weight) into the neck or left hind leg. The animals were exsanguinated and not fasted prior to necropsy.

[0110] (burn treatment) Wound treatment was performed while the animals were under anesthesia. The dorsal lateral areas of both sides of the animal's back were clipped using an electric shaver / trimmer and then shaved. The area was washed with soap and water and rinsed with gauze soaked in sterile water. The area was disinfected with 70% ethanol and 5% iodine ethanol. On day 1, 16 circular, partial-thickness burns (8 mm in diameter) were instituted on the back of each animal, eight on each side of the spine (Figure 1A). The burns were created using a custom-made aluminum block consisting of two cylinders (8 mm in diameter, Figure 1B). For easy handling, the aluminum block was attached to an acrylic plate with screws, and the assembly weighed approximately 111 g. The aluminum block was first preheated in boiling water. The heated aluminum block was then wiped dry. Immediately afterwards, the heated aluminum block was placed on the animal, and the two cylinders were placed in direct contact with the skin surface for 25 ± 5 seconds.

[0111] (Dressing materials and dressing changes) Collex matrix and HTX-free Collex matrix were prepared as described above. Jelonet® petrolatum compress dressings were cut into squares sized to fit the Collex matrix and HTX-free Collex matrix (1.6 x 1.6 cm). All three dressings were non-adhesive and secured with Moelnlycke's Mefix (5 x 3.5 cm) and 3M's Tegaderm (10 x 10 cm), as outlined in Figure 1C. BSN's Fixumull was used over these supportive dressings, and a BSN net body stocking was attached to the neck collar.

[0112] Dressings were changed on days 4, 6, 8, 10, 12, 14, 16, 19, and 23. At each dressing change, wounds were evaluated macroscopically by plan view and photography. On days 4 and 10, three wounds from each treatment were biopsied and terminated. At the end of the experiment, all wounds were biopsied and sent for histopathological evaluation. A timeline is shown in Figure 1D.

[0113] (Processing and Microscopy) On days 4 and 10, biopsies were taken while the animals were under anesthesia for dressing changes (Figure 1D). These biopsies were taken with 8-mm punch biopsies and included tissue from the center and edge of the wound, as well as tissue from a portion of unaffected skin surrounding the wound. At necropsy, all wounds were sectioned as separate blocks (approximately 1 cm around the wound and, if possible, a 0.5 cm margin to the bottom) from skeletal muscle tissue. All biopsies were fixed in phosphate-buffered neutral 4% formaldehyde. After fixation, representative specimens were cut and processed from designated tissues for microscopic examination. The specimens were embedded in paraffin and cut at a nominal thickness of approximately 5 μm. Slides were stained with hematoxylin and eosin and examined under a light microscope by scientists at Scantox A / S. Slides were evaluated according to ISO 10993-6:2016. The scoring system was semiquantitative, and microscopic evaluation was performed using a light microscope equipped with a grid attached to the eyepiece to aid in counting features in tissue sections. Burn depth was assessed based on damage to the epidermis and dermis, and damage to the pilosebaceous appendages. Reactivity scores were based on the extent of several factors: polymorphonuclear leukocytes, lymphocytes, macrophages, giant cells, necrosis, neovascularization, fatty infiltration, edema, hemorrhage, calcification, dilated blood vessels, crust, and hyperkeratosis.

[0114] (Floor evaluation and photos) The outline of the wound margins and the areas covered by slough / eschar, tough eschar, granulation tissue, or epithelium were drawn on a transparent sheet on the designated date (Figure 1D). The sheet was scanned, and an algorithm was developed to automatically measure the wound area. Photographs were taken with a digital camera using flash at a fixed distance in a standardized manner. A rectangular metal box was used to calibrate the distance.

[0115] (Differentiation and culture of monocytes into macrophages) The THP-1 monocytic cell line (ATCC, TIB-202) was cultured in RPMI (Lonza, 12-702F) supplemented with 10% fetal bovine serum (ATCC, 30-2025) and 1% penicillin-streptomycin (Sigma Aldrich, P4333), hereafter referred to as "medium." Cells were kept at 37°C, 5% CO2, in a humidified atmosphere. For differentiation of THP-1 to M0 and M1, the protocol used was essentially as described in 22: cells were plated in 24-well plates at 2x10 5 Cells were seeded at a concentration of 1000 cells / ml and exposed to phorbol 12-myristate 13-acetate (PMA, 200 nM, Sigma-Aldrich, P8139) for 24 hours. This treatment transformed monocytes in suspension into adherent macrophages (M0). The medium was removed from M0 cells and replaced with medium containing interferon (IF)-g (20 ng / ml, Sigma-Aldrich, GF305) and lipopolysaccharide (LPS, 250 ng / ml, Sigma-Aldrich, P4391). After 24 hours of incubation, M0 differentiated into M1 cells. Collex matrix (approximately 200 mg) and HTX-free Collex matrix (approximately 200 mg) were immersed in medium (1 ml) and incubated at 37°C for 4 hours. This resulted in the release of HTX from the Collex matrix into the medium at a protein concentration similar to that of 2% HTX. In parallel, HTX (2%) and IL-4 (40 ng / ml) were prepared. The IFNγ and LPS-containing medium was replaced with medium containing Collex matrix release, Collex matrix without HTX release, HTX, IL-4, or medium alone and incubated for an additional 48 hours. The supernatants from these cells were tested for levels of interleukin (IL)-1β, and the cells were tested for cell death and oxidative stress.

[0116] (Interleukin-1 beta ELISA assay) IL-1β levels were measured from cell culture medium using the Human IL-1 beta ELISA Kit (Abcam, ab214025) according to the manufacturer's recommendations.

[0117] (Propidium iodide (PI) staining) The fluorescent DNA-binding dye PI (Sigma Aldrich, P4170) was used to determine cell death. PI penetrates the plasma membrane of dying and dying cells. Forty-eight hours after treatment with HTX, Collex matrix, or Collex matrix without HTX, cells were detached by scraping and pipetting to obtain single cells, which were then transferred to Eppendorf tubes on ice. PI was added to the tubes (final concentration 1 mg / ml), and the signal was immediately analyzed by flow cytometry (MACSQuant Analyzer 10 Flow Cytometer; Miltenyi Biotec) with excitation at λ = 488 nm and detection at λ = 585 / 40 nm.

[0118] (Reactive oxygen species (ROS) staining) CellROX green (Thermo Fisher, C10492) is a dye used for detecting ROS, which is converted to a fluorescent molecule upon intracellular oxidation. Forty-eight hours after treatment with HTX, Collex matrix, or Collex matrix without HTX, cells were detached by scraping and pipetting to obtain single cells, which were then transferred to Eppendorf tubes. CellROX green was added to the tubes (final concentration 5 μM) and incubated at 37°C for 30 minutes. Signal intensity was analyzed by flow cytometry (MACSQuant Analyzer 10 Flow Cytometer; Miltenyi Biotec) with excitation at λ = 488 nm and detection at λ = 525 / 50 nm.

[0119] (statistical analysis) All statistical analyses were performed using GraphPad Prism software, version 9.4 (GraphPad Software, La Jolla, CA). Significance between treatment groups was determined by unpaired t-test, and statistical significance was defined as a P value of <0.05. In the animal study, planimetric assessments from day 6 were excluded from analysis because the plans were drawn by a different veterinarian and all wounds had significantly reduced wound area compared to other days. In addition, two outlier wounds (treated with Collex matrix and Jelonet®) were identified and excluded from analysis in agreement with Scantox A / S. One of the outliers appeared significantly more burned than the other 47 wounds, and the other had an incorrectly drawn wound border during planimetric assessment.

[0120] [result] Collex is sterile and demonstrated biocompatibility when applied to burns in minipigs. The Collex matrix tested in this study is a marine-derived, three-dimensionally printed dressing made from alginate and nanocellulose bound with HTX, a salmon roe extract. When the Collex matrix is ​​added to any solution, HTX is released from the dressing into the surrounding solution (data not shown). We previously demonstrated that HTX accelerates partial-thickness burn healing in a human excised skin model,23 and here we combine the wound-healing properties of HTX with those of alginate and nanocellulose.

[0121] HTX contains high concentrations of proteins and lipids and is sensitive to oxidation and excessive heat. Therefore, traditional medical device sterilization methods, such as exposure to ethylene oxide and autoclaving, could not be used with Collex matrix (24). Instead, sterile components were used, and the manufacturing process was completed under aseptic conditions as possible. Prior to animal testing of Collex matrix, the sterility of the final product was tested by detection of endotoxin. Endotoxin is a lipopolysaccharide (LPS) derived from the outer membrane of bacteria and can estimate not only the sterility of the product at the time of testing but also the presence of bacterial contamination during manufacturing (25). As shown in Table 1, the analysis showed endotoxin concentrations of less than 0.005 EU / ml in all five Collex matrix samples analyzed, significantly below the medical device limit (0.5 EU / ml according to the U.S. Food and Drug Administration's Center for Devices and Radiological Health and European Pharmacopeia), strongly suggesting that Collex is sterile.

[0122] To evaluate the safety and efficacy of Collex Matrix preclinically, partial-thickness burns were produced on the backs of Göttingen minipigs. Burn depth was determined by microscopic evaluation of hematoxylin-eosin-stained slides of biopsies taken from the wounds on day 4 and is illustrated in Figure 2A for two independent wounds. Burn depth, visualized by deep eosinophilic staining of denatured dermal collagen, extended down to the hair follicles, confirming the burn was a partial-thickness burn.

[0123] The Collex matrix was compared with Jelonet®, a traditional nonadhesive dressing, and an alginate and nanocellulose dressing similar to Collex but using 0.9% NaCl instead of HTX. The HTX-free Collex matrix possesses the beneficial properties of alginate and nanocellulose hydrogels without the effects of HTX, isolating the potential impact of HTX on the healing process. Collex matrix was developed in accordance with medical device guidelines, and in this regard, hematoxylin-eosin-stained biopsy slides were analyzed for local effects on skin reactivity (in accordance with ISO 10993-6:2016). Figure 2B shows two representative wounds from each treatment at day 23. According to pathologist examination at Scantox A / S, all wounds except one treated with Jelonet® were completely reepithelialized by day 23. Photographs show intact epidermis over granulation tissue and low reactivity. Jelonet® is currently used as an initial treatment for burns in Norwegian hospitals (10) and was used as a benchmark for reactivity. Based on a semiquantitative scoring system, Collex matrix showed minimal to no reactivity compared with Jelonet® (Figure 2C), suggesting a better fit to the lacerated skin. Although the HTX-free Collex matrix showed a slight reaction at 23 days, this dressing was not developed for use in clinical or clinical trials. Gross evaluation by veterinarians at Scantox A / S revealed no obvious infection in any of the wounds, supporting the sterility demonstrated by the endotoxin assay and demonstrating Collex's ability to protect the wound from environmental microorganisms.

[0124] Collex matrix accelerates healing of partial-thickness burns in minipigs. During the healing process, each wound was evaluated macroscopically, using plan views and photographs. Macroscopic evaluation by Scantox A / S showed that on days 10, 12, and 14, firm scabs formed in wounds treated with Jelonet®, but not in wounds treated with Collex or Collex without HTX (data not shown). This suggests that the alginate and nanocellulose dressings successfully kept the wounds moist, thereby preventing scab formation.

[0125] Wound area at each time point was calculated from plan views. Figure 3A shows the average wound area for all wounds from day 1 through day 23 (end of experiment). As expected for partial-thickness burns, the average wound size initially increased over the first week and then gradually decreased. At all time points tested, wounds treated with Collex matrix had a smaller average area compared to wounds treated with Jelonet® (particularly) and Collex matrix without HTX. The difference in wound area between Collex matrix and Jelonet® was significant at days 4, 10, 12, and 19 (gray asterisks in Figure 3A, p<0.05) and approached significance at days 8 and 14 (p=0.058 and 0.055, respectively). The area of ​​wounds treated with Collex matrix was smaller than that of wounds treated with Collex matrix without HTX at all time points except day 23, when histopathological evaluation showed that the wounds had completely healed. However, the difference was statistically significant only on day 8 (green asterisk in Figure 3A, p<0.05), and near significance on day 4 (p=0.078). The reduction in wound area in the Collex matrix-treated group suggests accelerated wound healing.

[0126] Digital photographs were taken using standard methods to visually assess the wounds. Figure 3B shows two representative wounds from each treatment on day 19, when the wounds were nearing complete healing. The wounds were selected based on planar assessment and had an area closest to the mean area in each treatment category. The photographs visually demonstrate a reduction in wound area and wound redness in the Collex matrix-treated wounds compared to both the wounds treated with Collex matrix without HTX and the wounds treated with Jelonet®.

[0127] Collex matrix reduces inflammation in partial-thickness burns in minipigs. In partial-thickness and full-thickness burns, wound progression can occur after heat exposure due to necrosis secondary to excessive inflammation. (4) From days 1 to 4, wounds treated with Collex matrix increased by only 8%, while wounds treated with Collex matrix without HTX and Jelonet® increased by 14% and 40%, respectively (Figure 3A). Furthermore, from days 1 to 8, wounds treated with Collex matrix increased by 42%, while wounds treated with Collex matrix without HTX and Jelonet® increased by 59% and 66%, respectively. Furthermore, the difference in size between wounds treated with Collex matrix and wounds treated with Collex matrix without HTX and Jelonet® was most pronounced during the first week. On day 4, treatment with Collex matrix reduced wound size by 23% compared to wounds treated with Jelonet® and by 11% compared to wounds treated with Collex matrix without HTX. On day 8, treatment with Collex matrix reduced wound size by 15% and 16% compared to wounds treated with Jelonet® and Collex matrix without HTX, respectively.

[0128] Between each dressing change, wounds were macroscopically evaluated by a veterinarian from Scantox A / S. During this evaluation, inflammation was assessed for each wound and given a score of 0 (absent) to 4 (marked). Figure 4A shows the mean wound edge inflammation score. The inflammatory response was assessed as being reduced by 17% on day 4 in Collex matrix-treated wounds compared to both HTX-free Collex matrix-treated wounds and Jelonet®-treated wounds. On day 6, overall inflammation was reduced but more significantly in Collex matrix-treated wounds, with reductions of 51% and 62% compared to HTX-free Collex matrix-treated wounds and Jelonet®-treated wounds, respectively. On days 8 and 10, inflammation levels were similar to those on day 6, with Collex Matrix-treated wounds showing the lowest levels. Although inflammation was low in the surrounding skin (Figure 4B), there was a significant reduction in inflammation around Collex Matrix-treated wounds compared to Jelonet®-treated wounds on days 4 and 6. On days 8 and 10, there was low level of inflammation in the skin surrounding all wounds, with no differences observed between treatments.

[0129] In addition to macroscopic evaluation, histopathological evaluation was performed on hematoxylin-eosin-stained biopsy slides of wounds on days 4 and 10 (a time series is shown in Figure 1D). This analysis demonstrated a significantly reduced inflammatory response in wounds treated with Collex matrix compared with wounds treated with Collex matrix without HTX and with Jelonet® (data not shown). Due to the small sample size (N = 3) and high variability, statistical significance was not achieved. However, the subtle signs of reduced inflammation are consistent with the reduced inflammation observed in the macroscopic evaluation. Excessive inflammation on day 4 can also be visualized by the red wound borders in photographs. Figure 4C shows two representative wounds from each treatment category on day 4 (same wounds as those shown in Figure 3B). The photographs showed a reduced inflammatory response in Collex matrix-treated wounds compared to HTX-free Collex matrix-treated wounds and Jelonet®-treated wounds, confirming the observations suggested by the macroscopic and histopathological evaluations.

[0130] Overall, the results of this minipig burn study suggest that Collex Matrix accelerates the healing of partial-thickness burns, likely by attenuating the inflammatory response during the early stages of wound healing.

[0131] Collex matrix and HTX reduce the inflammatory response and oxidative stress levels in activated M1 macrophages. Because the results of our burn injury study using minipigs strongly suggest that Collex matrix accelerates wound healing by reducing inflammation, we aimed to study the anti-inflammatory response in more detail. To this end, we employed the immortalized human monocyte cell line, THP-1. THP-1 cells have been widely used in the literature and can be easily differentiated into M0 macrophages with phorbol 12-myristate 13-acetate (PMA) and into pro-inflammatory M1 macrophages with interferon-γ (IFNγ) and LPS. 22 M1 macrophages secrete pro-inflammatory cytokines such as IL-1β. 26 To determine the effect of Collex matrix alone on inflammation, we stimulated THP-1 cells into a pro-inflammatory M1 state and treated these cells with the following conditions: medium exposed to Collex matrix; medium exposed to Collex matrix without HTX; HTX; or interleukin-4 (IL-4) for 48 hours (Figure 5A). The levels of secreted IL-1β in the culture medium were then analyzed by ELISA. HTX diluted to 2% in the medium yielded a concentration comparable to that of 0.2 g of Collex matrix soaked in 1 ml of medium. IL-4 has been reported to promote the transition of proinflammatory M1 macrophages to an anti-inflammatory M2 phenotype, resulting in reduced IL-1β levels. 27, 28 Therefore, it was used as a reference for this assay. As shown in Figure 5B, differentiation of cells from M0 (PMA only) to M1 (PMA + IFNγ + LPS) significantly increased IL-1β levels, as expected. Importantly, treatment with both Collex matrix and HTX significantly reduced IL-1β secretion by 45% and 43%, respectively. However, HTX-free Collex matrix did not reduce secretion, but rather increased IL-1β secretion, strongly suggesting that HTX mediates the anti-inflammatory response of the Collex matrix. Treatment of M1 cells with IL-4 slightly reduced IL-1β secretion (14%). The reduction was below the expected level, since the dose of IL-4 was at the upper end of that used in references (22, 27) for the same cell line.However, optimizing the IL-4 dosage is beyond the scope of this paper. To confirm that the observed decrease in IL-1β secretion was not simply due to a decrease in cell viability, cells from the same experiment were collected, stained with propidium iodide (PI), a cell death stain, and analyzed by flow cytometry. As shown in Figure 5C, neither HTX nor Collex matrix caused an increase in cell death. In fact, the extent of cell death was slightly reduced in cells exposed to medium derived from Collex.

[0132] It is widely known that burns cause the excessive release of reactive oxygen species (ROS), which subsequently damage DNA, proteins, lipids, and surrounding tissues (7). Furthermore, ROS are known to have pro-inflammatory effects (29). Because burns treated with Collex matrix appeared to have reduced inflammation, and because HTX (12% of Collex) contains chemicals with antioxidant properties, we hypothesized that Collex matrix might also reduce ROS in activated macrophages. To test this, we stained the same M1-polarized macrophages used in the IL-1β and cell death assays with CellROX, a ROS staining agent. As shown in Figure 5D, both Collex and HTX significantly reduced ROS levels by 48% and 41%, respectively. Treatment with IL-4 did not reduce ROS levels, suggesting that the reduction in IL-1β by IL-4 treatment is independent of ROS levels. Furthermore, treatment with HTX-free Collex matrix did not result in a reduction in ROS levels, strongly suggesting that the antioxidant effect of Collex is driven by HTX. Collectively, the anti-inflammatory effects of Collex matrix suggested by animal studies are supported by in vitro findings showing a reduction in both IL-1β and ROS levels.

[0133] [discussion] We have shown that treatment of partial-thickness porcine burn wounds with Collex matrix reduces inflammation, inhibits wound progression, provides a moist wound environment, prevents scab formation, and accelerates wound closure. Additionally, Collex matrix is ​​biocompatible and appears to protect wounds from infection. In vitro data on proinflammatory macrophages support the anti-inflammatory effects suggested by the minipig study. Figure 6 shows a model suggesting the mechanism of action of Collex matrix. Burn injury induces an exaggerated inflammatory response accompanied by high levels of IL-1β and ROS. Both inflammation and ROS are closely associated with increased apoptosis and necrosis, thus contributing to burn wound progression (4, 5, 30, 31). HTX released from Collex matrix appears to inhibit inflammation by reducing IL-1β and ROS levels. A 2% (vol / vol) concentration of stock HTX results in a similar HTX concentration to Collex in culture medium. Because stored HTX and Collex matrix reduced IL-1β and ROS production to similar degrees, the anti-inflammatory and antioxidant effects of Collex matrix are likely due to HTX. During the advanced proliferative phase of burn wound healing, Collex matrix provides a moist environment favorable for keratinocyte migration (32). These two mechanisms of Collex matrix may combine to accelerate wound healing in partial-thickness burns.

[0134] Among current dressings indicated for partial-thickness burns, several common properties are generally considered important: the dressing must cover and protect the wound, be absorbent to manage exudate, and be moist to promote re-epithelialization. These are all important mechanical properties for promoting an optimal environment for wound closure and reducing the risk of infection during healing (1, 2). Collex matrix possesses all of these properties, which are crucial to the wound-healing properties of the dressing; the authors hypothesize that collectively they constitute the primary mechanism of action. Evaluation of the graph in Figure 3A reveals that both Collex matrix and Collex matrix without HTX demonstrated a reduction in wound area compared to Jelonet®, although the differences were not significant. The ability of Collex matrix to maintain a moist wound bed was supported by macroscopic assessment, which revealed firm eschars in wounds treated with Jelonet® but none in wounds treated with Collex matrix or Collex matrix without HTX. The association with moist wound healing and prevention of crust formation, as well as increased rates of re-epithelialization, is well established ( 32 , 33 ).

[0135] Therefore, the anti-inflammatory and antioxidant effects demonstrated in this study appear to be secondary mechanisms of action for the Collex matrix. However, these properties remain important because they fulfill an unmet medical need in wound management and are not found in dressings commonly used in burn management today (10, 34). Excessive ROS are known to activate the inflammatory response and vice versa; therefore, reducing ROS may itself reduce inflammation. Both inflammation and ROS not only contribute to burn progression and impaired wound healing, but are also associated with the formation of hypertrophic scars seen after burns (35, 36). Therefore, it is not surprising that the development of novel burn treatments focuses on both reducing the inflammatory response (3, 4, 37) and reducing ROS levels (5, 9, 14, 38, 39). Due to the anti-inflammatory effects of Collex matrix, which reduces secondary necrosis, wounds treated with Collex matrix appear to be approximately one week ahead in terms of wound closure compared to wounds treated with Jelonet®. This accelerated healing of severe burns may result in shorter treatment times and more rapid transition of patients from intensive care to lower levels of care, such as inpatient and outpatient care. These benefits could improve patient burn care and result in significant savings in medical costs.

[0136] Jelonet® is the standard of care in Norwegian hospitals for the initial management of burns. 10 Given the strong anti-inflammatory properties of Collex matrix, replacing Jelonet® with Collex matrix early after injury may reduce secondary necrosis and accelerate wound closure. Furthermore, Collex matrix may be an efficient dressing for the treatment of chronic wounds. Chronic wounds suffer from chronic inflammation and excessive ROS (15, 46), which persist not only for the first week, as in partial-thickness burns, but also for weeks and months (47). Although Collex matrix has not yet been tested in chronic wounds, based on the results of minipig studies, in vitro data, and the nature of chronic wounds, there is reason to believe that topical treatment of chronic wounds with Collex matrix may reduce inflammation and accelerate healing.

[0137] Table 1: Endotoxin analysis. Endotoxin levels were within the medical device limit (0.5 EU / ml according to the Center for Devices and Radiological Health and European Pharmacopeia of the U.S. Food and Drug Administration) for all five dressings tested. PCC recoveries between 50 and 200% indicate reliable testing.

[0138] [Table 1]

[0139] Example 3: Release of HTX from Collex matrices Total protein release from Collex matrix into DMEM supplemented with 10% FBS and 1% Pen / Strep is shown in Figure 7. Unmodified HTX was encapsulated in Collex patches. Total protein content was measured using absorbance at 280 nm, and the absorbance from the medium (DMEM-F with Pen / Strep) was subtracted for each time point.

[0140] Example 4: Measurement of Collex Matrix Bioactivity by Procollagen-1 Assay The bioactivity of released HTX was tested in a procollagen Iα1 ELISA assay by extracting the contents of the Collex matrix into cell culture medium (DMEM-F supplemented with Pen / Strep) and exposing fibroblasts to this cell culture medium for 7 days. As expected, Collex matrix without HTX (Collex placebo) did not affect procollagen-1α1 production. Collex matrix with HTX significantly increased procollagen-1α1 production compared to the negative control (p<0.01). The Collex matrix maintained its bioactivity after 4 weeks of storage at refrigerated and room temperature (see Figure 8).

[0141] Example 5: Comparison of Collex Matrix Formulations This example describes the testing of various formulations of Collex containing different levels of alginate and nanocellulose.

[0142] [material and method] (Ink formulation and optimization) The different inks were formulated by mixing medical-grade TUNICELL ETC + M, enzyme-pretreated cellulose nanofibrils (E-CNF) supplemented with 4.6% mannitol (OceanTunicell AS, Bergen, Norway), H O, and 0.9% NaCl (Fresenius Kabi AG, Bad Homburg, Germany). Sodium alginate (10% wt / vol in saline, PRONOVA UP MVG (NovaMatrix DuPont Nutrition Norge AS, Sandvika, Norway), molecular weight (Mw) >200 kDa, mannuronic acid / guluronic acid (M / G) ratio ≥1.5) was then added and mixed for 2 h to equilibrate. After that, purified salmon roe extract (Regenics AS, Oslo, Norway) was added and mixed again before adding 12% (vol / vol) HTX. All mixing steps were performed manually for 5 min, after which the ink was centrifuged to release air bubbles. The ink composition was systematically varied as shown in Table 2. Ink properties were evaluated based on rheology, printability, shape fidelity, handling, and protein release profile. To better understand and model the response, experiments were planned according to a full factorial design (R1-R4) with R5 as the center point. Data were presented in Statistica ver. The evaluation was carried out using the design of experiments (DOE) module of StatSoft Europe GmbH, Hamburg, Germany.

[0143] Table 2. Compositions of the inks evaluated, expressed as percentages (volume / volume).

[0144] [Table 2]

[0145] (Increologia) Characterization of the flow and viscoelastic properties of different inks was performed using a DHR-30 rheometer (TA instruments, New Castle, DE, USA) with a 20 mm upper plate and a lower Peltier plate. All rheological characterizations were performed at 20 °C, and a 500 μm gap was used for all experiments. TRIOS software version #5.5.0.323 (TA instruments, New Castle, DE, USA) was used to process the data and acquire 10 points per decade for each test. A 30-second soak time was used to allow the sample to relax on the plate before testing. Three replicate measurements were performed for each ink composition, and the reported data in the graphs are the mean ± standard deviation.

[0146] Flow behavior in response to shear was measured using steady-state sensing with a maximum equilibration time of 10 seconds and a sample period of 10 seconds. -1 to 0.1 seconds -1 The stresses were determined by flow sweeps in the range of 0.1 to 30 Hz consecutively within three measurements with a 5% tolerance. The stresses from the flow sweeps were fitted to the Herschel-Bulkley model using the software TRIOS version #5.5.0.323 (TA instruments, New Castle, DE, USA). The parameter fits of the Herschel-Bulkley model are shown as mean ± standard error (SE). The viscoelastic properties with respect to frequency were characterized using an oscillatory frequency sweep between 0.1 and 30 Hz within the linear viscoelastic region.

[0147] (3D printing of hydrogels) Alginate, nanocellulose, saline, and HTX were mixed in the ratios shown in Table 1 to prepare the ink. Using a fourth-generation 3D Bioplotter (EnvisionTEC, Gladbeck, Germany), a target ink patch of 200 mg was three-dimensionally printed in an interlaced 90-degree continuous lattice pattern consisting of four layers measuring 16 mm x 16 mm. The inner lattice was created using a 22-gauge nozzle with a lateral spacing of 1.8 mm between the filaments and a distance of 0.7 mm from the contour. The printing pressure and speed ranged from 0.1 to 0.4 bar and 4 to 40 mm / s, respectively. After the four layers were printed, the patch was imaged with the 3D Bioplotter, and printability was evaluated using the results obtained by stitching together nine images. The ink patch was then crosslinked for at least 30 minutes in a 0.02 M CaCl2 solution (Sigma-Aldrich, Darmstadt, Germany) supplemented with HTX (12% v / v). Meanwhile, the ink patches were cooled and kept covered with aluminum foil to protect them from light. They were then transferred to ethanol-cleaned polyethylene / aluminum sachets under argon and stored in a refrigerator before analysis. For experiments involving hydrogel control patches, HTX in both the ink and crosslinking solution was replaced with 0.9% NaCl. To estimate patch height, the final construct was transferred and imaged again using the same protocol as before, after wiping off excess crosslinking solution. The printability of each material was evaluated by flow behavior, filament size, pore size, lattice uniformity, and patch height. This was performed by comparing lattice structures imaged immediately after printing and after crosslinking. Three patches of each ink type were evaluated, and size estimation was performed at three random sites each using ImageJ 1.53t (NIH, Bethesda, MD, USA). The average size (width) of the major filaments and pores was estimated, excluding small pores along the contour.

[0148] (Release Profile) HTX is a complex liquid rich in proteins. Because proteins are easily detectable in small amounts, protein release was used as an indicator of total HTX release. Protein (HTX) release from crosslinked hydrogel structures was systematically studied using solid discs as a starting point during ink formulation optimization, where protein release was analyzed using a BCA protein assay kit. Release from solid discs crosslinked with selected R4 inks was then evaluated in different media of increasing complexity: 0.9% NaCl, phosphate-buffered saline (PBS), chemically mimicked wound fluid (CSWF), cell culture medium with 10% fetal bovine serum (DMEM-F), and FBS plus peptone medium (FBSpept). Finally, protein release was assessed for the selected R4 ink 3D printing hydrogel, Collex Matrix, whose fabrication method is described in the 3D printing section. Collex Matrix (approximately 200 mg), stored in sachets for 1 to 4 weeks, was individually transferred to a 24-well plate and 1 mL of cell culture medium (DMEM supplemented with 10% FBS and 1% Pen / Strep) was added. Antibiotics were added according to the release medium used in the bioactivity assay. Plates were incubated at 37°C in a humidified atmosphere with 5% CO2. At the indicated time points, 2 μL of cell culture medium was removed, and protein concentration was measured at 280 nm using a NanoDrop™ (One / OneC, Thermo Scientific, Madison, WI, USA).

[0149] (Chemical Characterization) Extraction of Collex matrix was performed according to ISO 10993-12:2021 using Milli-Q water and isopropanol containing 0.9% NaCl as the extraction medium, at an extraction ratio of 0.2 g / mL, with 5–10 patches per extraction in duplicate. Excess crosslinking solution was washed from the patches by immersion in saline for less than 1 second. Extraction was performed at 50°C for 72 ± 2 hours.

[0150] Chemical analyses by gas chromatography-mass spectrometry (GC-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) were performed within 24 hours after the end of the extraction. Following the extraction procedure, the extracted organic and inorganic components were quantified by GC-MS and ICP-MS, respectively, according to ISO 10993-18:2020.

[0151] (bioburden) The microbial cleanliness (bioburden) of Collex matrix was assessed according to European Pharmacopoeia 2.6.12: Microbial Enumeration Test. The 12% (vol / vol) HTX solution and Collex matrix were tested for their inhibitory (antimicrobial) effect on microbial growth (see Table 3), including corrections where necessary. All samples were handled in a laminar airflow cabinet (LAF) equipped with a high-efficiency particulate absorption (HEPA) air filtration system.

[0152] Table 3. Microorganisms evaluated

[0153] [Table 3]

[0154] Bioburden for HTX and Collex was assessed using total aerobic microbial counts (TAMC) and total yeast and mold counts (TYMC). Both products were tested in triplicate. Environmental sterility was assessed in parallel with tryptone soy broth (TSB), TSB-100, and negative controls of 0.9% saline spread on TSB, tryptic soy agar (TSA), and Sabouraud sugar agar (SDA).

[0155] (Bioactivity and biocompatibility) Cytotoxicity. The cytotoxic potential of the Collex matrix was analyzed by extracting test articles according to ISO 10993-12:2021 and by cytotoxicity testing according to ISO 10993-5:2009 Annex C (MTT cytotoxicity test) in a GLP-accredited testing facility.

[0156] Cell culture. Human fibroblast cell line Hs 707 (ATCC, CRL-7449) was cultured at 37°C in a humidified atmosphere supplemented with 5% CO2. Cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, D0822) supplemented with 10% FBS (ATCC, 30-2025) and 1% penicillin-streptomycin (Sigma-Aldrich, P4333). Cells from passages 2 to 5 were used for the pro-collagen Iα1 assay.

[0157] Pro-collagen Iα1 assay. 12 × 10 Hs 707 cells 4 The cells were diluted to a concentration of 1000 cells / mL, and 1 mL was seeded into a 24-well plate and cultured in a cell incubator for 24 hours. The next day (Day 1), Collex (approximately 200 mg) was transferred to cell culture medium (1.2 mL) and cultured in a cell incubator for 4 hours to release HTX. The absorbance at 280 nm was measured to confirm that the protein concentration was equal to or higher than that of a 1% stock solution of HTX. On the cell plate, the cell culture medium (1 mL) was replaced with the following: fresh cell culture medium, cell culture medium supplemented with extract from the Collex matrix, cell culture medium supplemented with extract from a print control (no HTX), or cell culture medium supplemented with 1% HTX (stock solution). The cells were further cultured in the cell incubator, and on Days 4 and 6, fresh cell culture medium was prepared as on Day 1, except that only 300 μL of cell culture medium was replaced on these days. On day 8, cell culture medium was collected from each treatment and pro-collagen I levels were measured using a human pro-collagen Iα1 ELISA kit (ab210966, Abcam, Cambridge, UK) according to the manufacturer's recommendations.

[0158] [result] (Increologia) Extrusion 3D printing relies on the rheological behavior of the ink to enable printability through a syringe. To achieve adequate printability, the ink must be shear thinning and flow through the nozzle when low force is applied. Five ink formulations were characterized by viscosity measurements to assess the printability and flowability of the ink formulations. In Figure 10a, the flow sweep shows that R1 had the lowest viscosity of the inks tested, and at a shear rate of 0.1 s -1 The initial viscosity at low shear was approximately 20 Pa·s. Additionally, R1 did not exhibit clear shear thinning, and viscosity did not decrease with increasing shear rate, likely due to the low amounts of both nanocellulose and alginate. This could make it difficult to extrude the ink in a controlled manner on the build plate. The viscosities of R2, R3, and R5 at low shear were similar and nearly indistinguishable from one another (see Figure 10a). Although the ratios of alginate and nanocellulose differed between R3 and R5, they appeared balanced in terms of viscosity. Meanwhile, R2, which contains a lower nanocellulose content, showed a slight decrease in initial viscosity despite the increased alginate content (see Table 2). The R4 ink formulation contained the highest amounts of both alginate and nanocellulose, resulting in increased shear thinning compared to the others, as well as increased viscosity at low shear (see Figure 10a). The ink viscosity increased with increasing polymer concentration, and shear thinning is explained by the unwinding of polymer chains when subjected to high shear rates. This combination of properties is believed to be beneficial for printing in terms of extrudability.

[0159] Additionally, the ink must have sufficient yield stress to retain the shape of the printed structure on the build plate. High shear thinning ability allows for increased printing speeds, while yield stress is related to the ink's ability to retain its shape on the build plate. In general, increased yield stress supports the printing of overhanging structures with minimal risk of collapse. To gain further insight into the ink's non-Newtonian behavior, the shear stress was fitted to the Herschel-Bulkley model (see Figure 10b and Table 4). The results revealed that R4 had a significantly higher yield stress than the other inks. R1 exhibited the lowest yield stress value, while R2, R3, and R5 had similar values. R4's yield stress increased more than threefold compared to the other inks, suggesting better shape retention after printing. The factor model indicated a significant positive interaction effect between alginate and nanocellulose.

[0160] Table 4. Calculated yield stresses along with SD for different ink compositions.

[0161] [Table 4]

[0162] While flow behavior describes the rheological behavior during printing and the ink's ability to retain its shape on the build plate, the viscoelastic properties of the ink describe additional properties of the formulation. In this study, the storage modulus (G') evaluated by frequency sweep was plotted against angular frequency (see Figure 11). It was found that the ink formulations exhibited similar behavior across angular frequency, typically related to the level of entanglement in the physical gel. Tests were performed before alginate crosslinking, and no crossover point was observed, meaning the ink exhibited a gel-like appearance within the tested frequency range (data not shown). The primary contribution to G' was the nanocellulose concentration, and no additional modeling of the data was performed. R1 and R2 exhibited similar absolute G' values, which were lower compared to R3–R5. Samples R3–R5 exhibited a similar range in terms of G', likely due to the high nanocellulose content (see Table 2). Nanocellulose is known to have a reinforcing ability that affects the viscoelastic properties of bioinks, indicating favorable printability. Cellulose nanofibers can support ink stability through entanglement at low concentrations.

[0163] (3D printing and structural characterization) The inks listed in Table 2 were evaluated for printability and shape fidelity based on the resulting overall lattice uniformity, filament width, hole size, and patch height. The printing parameters for each ink were individually optimized prior to evaluation, and four-layer patches were printed with a given design and compared to a template CAD drawing (see Figure 12a, b).

[0164] The observations made during printing of inks R1–R5 were consistent with the rheological results. Compared to R1 and R3, the higher alginate content in R2 and R4 improved the ink's printability and shape fidelity. This is likely due to increased viscosity, shear-thinning behavior, and yield stress. R2 and R3 exhibited similar shear-thinning capabilities and yield stress levels, but the R3 ink exhibited improved printability and shape retention, likely due to increased G'. R1 and R2 tended to drip during printing and exhibited poor to no shape retention. This was associated with their lower yield stress and G', consistent with the rheological observations that R3–R5 exhibited improved viscoelastic properties. To achieve continuous flow through the needle, the required printing pressure increased as the ink number progressed from R1 to R4 due to increased viscosity and viscoelastic behavior. R5 required similar pressure to R3 but required a much higher printing speed due to the increased flow rate through the syringe. We also observed that HTX content slightly improved printability compared with inks in which HTX was replaced with saline, as well as with control patches used in other experiments, such as bioactivity testing. The HTX-containing ink R4 exhibited better shape retention than its non-HTX counterpart, but exhibited a higher tendency for air entrapment and increased hydrophobicity. This is likely explained by the surface activity of the proteins and polar lipids contained in HTX, which tend to concentrate at the water-air interface, resulting in the stabilization of air bubbles and increased hydrophobicity. This became apparent when the patches were exposed to a crosslinking solution. Patches based on R1, R3, and R5, which contained lower amounts of alginate, were somewhat soft and sensitive, risking deformation when handled.

[0165] Patch properties for R1–R4, shown in Table 4, demonstrated increased lattice uniformity, filament consistency, and conformance as the ink count increased compared to the CAD design. The structural properties were consistent with rheological observations, with the combination of high shear thinning, yield stress, and G' enhancing three-dimensional printing properties, as in R4, and subsequently decreasing as the ink count decreased. All inks exhibited dye swelling, as determined by increased filament width compared to a nozzle size with an inner diameter of 0.41 mm (Table 4). The printing protocol included a 20% overlap of filaments between layers and targeted a total height of 1.37 mm. The resulting patch heights estimated after crosslinking the constructs ranged between 0.5 and 1.0 mm (see Table 5), suggesting some structural collapse and flow. The patches showed slight shrinkage upon crosslinking (not quantified). The inks likely achieved denser lattice structures, and further compositional tuning and optimization, for example, by increasing biopolymer content or additional additives, could have been achieved. However, considering the overall printability and properties in combination with the structural stability maintained during storage (patches stored in solution for 4 weeks were used in bioactivity assays), the lattice structure achieved by the R4 ink was deemed suitable for the given application.

[0166] Table 5. Characterization of ink printability and shape fidelity. Shown as mean SD, n = 3 for patch height (crosslinked), n = 9 for filaments and holes (non-crosslinked).

[0167] [Table 5]

[0168] (Release Profile) The release of HTX from crosslinked hydrogels was systematically studied, starting with a solid-disk design to support the optimization of the ink formulation. Protein release appeared similar regardless of the hydrogel composition within the range evaluated. Release was then evaluated in various complex release media. HTX loading into the hydrogel was nonspecific (noncovalent), and various types of physical intermolecular interactions are expected to affect the binding strength of HTX components to the gel structure. Therefore, pH, ionic strength, and protein content may affect the release rate of HTX from the hydrogel. Therefore, the release profile may vary depending on the composition of the surrounding medium. For wound healing applications, it is important that the bioactive component is released into wound exudate, which consists of a crude mixture of proteins, salts, nutrients, inflammatory cells, and their components. To establish a controlled release system, saline, specifically 0.9% NaCl (aqueous solution), was used as the release medium. The purpose was not to mimic the actual wound environment but to use it as a control system for initial understanding and characterization without interactions with other medium components. Additionally, phosphate-buffered saline (PBS) was used as a more physiologically relevant buffer. Methods for preparing chemically simulated wound fluid (CSWF) were found in the literature. CSWF is composed of ions associated with the wound environment and the protein bovine serum albumin (BSA) in amounts equivalent to the total protein content of the wound. This chemically defined medium provides a high level of control for in vitro assays. For more complex protein mixtures, fetal bovine serum (FBS) was added in small amounts (10%) to a nutrient-complete cell culture medium (DMEM-F) or in large amounts (50%) directly to peptone water (FBSpept); the latter is referred to in the literature as simulated wound fluid. Substantial release of HTX also occurs in protein-free PBS and protein-rich media such as CSWF, DMEM-F, and FBSpept. This is significant.Because the hydrogel was designed as a dressing to be applied directly to the wound, the surrounding environment is rich in both proteins and other biological components, and this environment has a high similarity to serum. The release results confirmed that the construct made from R4 ink released HTX into the surrounding fluid, which resembles the actual wound environment.

[0169] Finally, the release of HTX into complex media was studied in selected hydrogels, Collex, three-dimensionally printed from R4 ink. Unlabeled HTX was used to construct the three-dimensionally printed Collex patches. To avoid HTX release loss during the crosslinking process, crosslinking was performed in a CaCl2 solution supplemented with 12% (vol / vol) HTX, the same concentration as in R4 ink. A time-dependent release of Collex into DMEM-F was also observed. Figure 7 shows the total protein release from Collex patches into DMEM supplemented with 10% FBS and 1% Pen / Strep, consistent with the medium used in the bioactivity assay. Total protein content was measured using absorbance at 280 nm, and the absorbance from the medium (DMEM-F supplemented with Pen / Strep) was subtracted for each time point. Compared to solid disks, protein release from the three-dimensionally printed Collex patches was faster and the total amount released was significantly higher. Approximately 90% of the protein was released from 3D-printed patches crosslinked in a CaCl2 solution containing HTX. Higher levels may be released in wounds where protein exchange occurs and the equilibrium is altered. There were several differences between the solid disk and printed patch experiments that may explain the differences in release profiles. Printing allowed for more precise control of patch size, and weighing the patches after preparation ensured consistency between samples. Additionally, the patches were stored in sachets with storage solution for 1 to 4 weeks before use. The crosslinking and storage solutions contained added HTX to prevent release from the patches. However, we cannot rule out the possibility that the patches absorbed HTX from the surrounding environment during preparation and storage, resulting in higher than expected total HTX levels in the samples. If this were the case, the percent release of total protein calculated based on the amount of HTX in the ink before printing would overestimate the actual percentage of protein released. While it would be difficult to accurately account for potential absorption during storage, this is an interesting area to investigate further. However, such evaluation does not detract from the usefulness of this patch in wound care, as long as storage conditions are clearly defined and the patch has the desired effect when used.Important factors affecting the release rate and total amount released are the structure and surface area of ​​the patch; lattice structures have a larger surface area than solid disks, reducing the distance proteins must travel within the gel structure before reaching the surrounding medium. Measuring protein release solely by absorbance at 280 nm makes it difficult to gain detailed insight into the release mechanism. However, as well as using a complex medium representative of the wound environment, it is also important to study the behavior of unlabeled HTX to allow for its natural interaction with the gel structure, a combination that limits the choice of detection method. The results, which demonstrate effective release, are promising.

[0170] (Chemical Characterization) The chemical composition of Collex was characterized, focusing exclusively on volatile and semivolatile organic compounds and inorganic elements, to assess its safety profile. Both saline and isopropanol extracts from Collex were clear and colorless, without visible particles. The hydrogel remained visibly unchanged after extraction. The extracts were analyzed in triplicate. The eluted organic compounds were screened using GC-MS, and primarily fatty acids, cholesterol, and sugars (mostly mannitol) were identified in the isopropanol extract. The concentrations measured are considered nontoxic. The eluted inorganic elements identified by ICP-MS in the saline extract of Collex patches are listed in Table 6. The reason for using two different extracts, saline and isopropanol, is that inorganic elements are much more soluble in saline than in isopropanol, while the opposite is true for organic compounds. For this reason, organic compounds and inorganic elements were not identified in the saline and isopropanol extracts, respectively. A toxicological risk assessment following chemical characterization revealed a margin of safety that indicated no risk of acute systemic toxicity from the use of Collex matrix. Furthermore, the assessment found no leaching of compounds known to cause material-mediated pyrogenesis and concluded that leaching of compounds under clinical conditions is unlikely to cause skin irritation or sensitization.

[0171] Table 6. Major elements and their amounts identified by ICP-MS in saline extracts of R4 ink hydrogel. Extractions were performed in duplicate (a, b), and extracts were analyzed in triplicate. Values ​​are shown as mean SD, n = 3.

[0172] [Table 6]

[0173] Wound healing and nutrition are closely related, and nutrient deficiency disrupts normal healing by prolonging the inflammatory phase and reducing fibroblast proliferation and collagen synthesis. Iron homeostasis in the skin is important in the wound healing process, and both iron deficiency and iron overload have been shown to have negative effects. Strontium salts have been shown to suppress TNF-α levels, thereby reducing inflammation. Meanwhile, both zinc and manganese have been shown to regulate integrin expression, which influences the proliferative phase of wound healing. Zinc supplementation has been shown to promote wound healing, particularly when administered topically, where zinc ions stimulate epithelialization and reduce the amount of excessive infection and necrotic material. Additionally, zinc has been shown to be particularly effective in healing diabetic foot ulcers.

[0174] (Bioburden analysis) Bioburden analysis was performed to determine the sterility of the final product. The suitability of the test method and growth promotion tests were successful, as low amounts (<100 CFU) of microorganisms inoculated onto the agar medium were recovered. No inhibitory (antibacterial) effect was demonstrated when inoculated with either the HTX solution or the Collex patch. Both the HTX solution and Collex patches were found to be sterile with no growth in any samples, indicating that proper procedures were followed in their sterile formulation, manufacturing and packaging.

[0175] (Bioactivity and biocompatibility) Collagen is converted from procollagen and plays an important role in wound healing. HTX stimulates the production of procollagen Iα1. Therefore, the bioactivity of released HTX was determined based on its ability to stimulate collagen production, as measured by a procollagen Iα1 ELISA assay. HTX-containing Collex significantly increased procollagen Iα1 production compared to the negative control (p<0.01). The R4 ink-printed control without HTX had no effect on procollagen Iα1 production. Collex maintained its bioactivity after 4 weeks of storage in the refrigerator and at room temperature (see Figure 8). The release assay measured only protein; release of fatty acids and other compounds that may contribute to the HTX effect was not detected. However, the procollagen Iα1 assay showed that the increase in procollagen was similar in the Collex matrix.

[0176] Example 6: Preparation of a molded Collex matrix This example describes a method for fabricating a Collex wound-healing matrix by molding. A mixture was created containing, by volume / weight (vw), 12% HTX (heat-treated salmon egg extract containing: protein: 90–130 mg / ml; DNA: 1.4 mg / ml; pH: 6.5–7.5); 0.8% NaCl; and 2.0% alginate (Pronova UP MVG; viscosity [mPa*s]: >200; approximate Mw [kDa]: >200; G / M ratio: ≥1.5). The mixture was then transferred to a shallow rectangular mold. The mixture was then crosslinked by the addition of 0.1% CaCl2, and the resulting Collex sheet was removed from the mold. Protein release from the molded matrix was compared to that from the three-dimensionally printed matrix described above. The data are shown in Figures 13 and 14. Release from the molded Collex matrix in water was similar to that from the three-dimensionally printed matrix containing nanocellulose. Figure 13. Furthermore, the increase in procollagen-1 by the molded Collex matrix was similar to that of the three-dimensionally printed Collex matrix containing nanocellulose, whereas the molded matrix was 4o C for at least 10 weeks (Figure 14).

[0177] Example 7: Reduction of reactive oxygen species (ROS) This example provides data demonstrating that HTX reduces reactive oxygen species in fibroblast (Hs707) and keratinocyte (HaCaT) cells and thus exhibits antioxidant activity. Briefly, HTX (5% v / v) was applied to fibroblast (Hs707) and keratinocyte (HaCaT) cells in vitro under conditions that induce cellular stress. The data are shown in Figures 15 and 16. The antioxidant effect of HTX application was rapid, with ROS reduction already observed 2 hours after HTX treatment. The antioxidant effect of HTX was transient and less pronounced after 3 days. Finally, ROS reduction was more pronounced in stressed cells (stressed by serum starvation).

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Tuning the Cytokine Responses: An Update on Interleukin (IL)-4 and IL-13 Receptor Complexes. Front. Immunol. 2018, 9, 888. https: / / doi.org / 10.3389 / fimmu.2018.00888. (29) Rendra, E.; Riabov, V.; Mossel, D. M.; Sevastyanova, T.; Harmsen, M. C.; Kzhyshkowska, J. Reactive Oxygen Species (ROS) in Macrophage Activation and Function in Diabetes. Immunobiology 2019, 224 (2), 242-253. https: / / doi.org / 10.1016 / j.imbio.2018.11.010. (30) Matylevitch, N. P.; Schuschereba, S. T.; Mata, J. R.; Gilligan, G. R.; Lawlor, D. F.; Goodwin, C. W.; Bowman, P. D. Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury. Am. J. Pathol. 1998, 153 (2), 567-577. https: / / doi.org / 10.1016 / S0002-9440(10)65599-X. (31) Iwasaki, K.; Izawa, M.; Mihara, M. Thermal Injury Induces Both Necrosis and Apoptosis in Rat Skin. Br. J. Dermatol. 1997, 137 (4), 647-648. https: / / doi.org / 10.1111 / j.1365-2133.1997.tb03805.x. (32) Nuutila, K.; Eriksson, E. Moist Wound Healing with Commonly Available Dressings. Adv. Wound Care 2021, 10 (12), 685-698. https: / / doi.org / 10.1089 / wound.2020.1232. (33) Fonder, M. A.; Mamelak, A. J.; Lazarus, G. S.; Chanmugam, A. Occlusive Wound Dressings in Emergency Medicine and Acute Care. Emerg. Med. Clin. North Am. 2007, 25 (1), 235-242. https: / / doi.org / 10.1016 / j.emc.2007.01.012. (34) El Ayadi, A.; Jay, J. W.; Prasai, A. Current Approaches Targeting the Wound Healing Phases to Attenuate Fibrosis and Scarring. Int. J. Mol. Sci. 2020, 21 (3), E1105. https: / / doi.org / 10.3390 / ijms21031105. (35) Carney, B. C.; Chen, J. H.; Kent, R. A.; Rummani, M.; Alkhalil, A.; Moffatt, L. T.; Rosenthal, D. S.; Shupp, J. W. Reactive Oxygen Species Scavenging Potential Contributes to Hypertrophic Scar Formation. J. Surg. Res. 2019, 244, 312-323. https: / / doi.org / 10.1016 / j.jss.2019.06.006. (36) Wang, Z.-C.; Zhao, W.-Y.; Cao, Y.; Liu, Y.-Q.; Sun, Q.; Shi, P.; Cai, J.-Q.; Shen, X. Z.; Tan, W.-Q. The Roles of Inflammation in Keloid and Hypertrophic Scars. Front. Immunol. 2020, 11, 603187. https: / / doi.org / 10.3389 / fimmu.2020.603187. (37) Dolgachev, V. A.; Ciotti, S.; Liechty, E.; Levi, B.; Wang, S. C.; Baker, J. R.; Hemmila, M. R. Dermal Nanoemulsion Treatment Reduces Burn Wound Conversion and Improves Skin Healing in a Porcine Model of Thermal Burn Injury. J. Burn Care Res. Off. Publ. Am. Burn Assoc. 2021, 42 (6), 1232-1242. https: / / doi.org / 10.1093 / jbcr / irab118. (38) Kim, Y. E.; Kim, J. ROS-Scavenging Therapeutic Hydrogels for Modulation of the Inflammatory Response. ACS Appl. Mater. Interfaces 2021. https: / / doi.org / 10.1021 / acsami.1c18261. (39) Thi, P. L.; Lee, Y.; Tran, D. L.; Thi, T. T. H.; Kang, J. I.; Park, K. M.; Park, K. D. In Situ Forming and Reactive Oxygen Species-Scavenging Gelatin Hydrogels for Enhancing Wound Healing Efficacy. Acta Biomater. 2020, 103, 142-152. https: / / doi.org / 10.1016 / j.actbio.2019.12.009. (40) Calder, P. C. Marine Omega-3 Fatty Acids and Inflammatory Processes: Effects, Mechanisms and Clinical Relevance. Biochim. Biophys. Acta 2015, 1851 (4), 469-484. https: / / doi.org / 10.1016 / j.bbalip.2014.08.010. (41) Djuricic, I.; Calder, P. C. Beneficial Outcomes of Omega-6 and Omega-3 Polyunsaturated Fatty Acids on Human Health: An Update for 2021. Nutrients 2021, 13 (7), 2421. https: / / doi.org / 10.3390 / nu13072421. (42) Jarosz, M.; Olbert, M.; Wyszogrodzka, G.; Mlyniec, K.; Librowski, T. Antioxidant and Anti-Inflammatory Effects of Zinc. Zinc-Dependent NF-ΚB Signaling. Inflammopharmacology 2017, 25 (1), 11-24. https: / / doi.org / 10.1007 / s10787-017-0309-4. (43) Lansdown, A. B. G.; Mirastschijski, U.; Stubbs, N.; Scanlon, E.; Agren, M. S. Zinc in Wound Healing: Theoretical, Experimental, and Clinical Aspects. Wound Repair Regen. Off. Publ. Wound Heal. Soc. Eur. Tissue Repair Soc. 2007, 15 (1), 2-16. https: / / doi.org / 10.1111 / j.1524-475X.2006.00179.x. 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[0179] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations in the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in medicine, medicinal chemistry, organic chemistry, virology, biology, genetics, or related fields are intended to be within the scope of the following claims. [Brief explanation of the drawings]

[0180] [Figure 1]Burn treatments were used on Goettingen minipigs. (A) Diagram of burn distribution on the pig's back. There were overlapping burns on both sides of the spine, with eight burns on each side. Representative treatments were arranged so that all animals received all treatments, and distribution near the head and tail was even for all treatments. (B) Photograph of the custom-made burn apparatus used to create partial-thickness burns on the pigs. The aluminum rod (creating the overlapping burns) was connected directly to the aluminum block to ensure a stable temperature. The block was attached to a heat-resistant Plexiglas plate for easy handling. (C) Diagram of the dressings used on the minipigs. Orange circles indicate the burns. Collex Matrix, Collex Matrix without HTX, and Jelonet® are non-adhesive dressings, so they were secured in place with Mefix. Tegaderm was included to ensure the dressings remained in place throughout the experimental period. (D) Schematic of the timeline for the minipig burn study. The numbers on the arrows indicate the number of days. [Figure 2]The burns inflicted were partial-thickness burns, and the Collex matrix shows signs of biocompatibility. (A) and (B) Hematoxylin and eosin-stained biopsy slides of wounds on days 4 (A) and 23 (B). (A) Burn depth revealed by denaturation of dermal collagen (deep eosinophilic staining). (B) Two representative wounds from each treatment at the end of the experiment, based on evaluation by a pathologist: 1 and 2: Collex matrix, 3 and 4: Collex matrix without HTX, and 5 and 6: Jelonet®. (C) Reactivity scores for Collex matrix and Collex matrix without HTX compared to Jelonet®, based on evaluation by a pathologist at Scantox A / S. Scores of 0.0-2.9 indicate no reactivity, 3.0-8.9 indicate a mild reaction, 9-15.0 indicate a moderate reaction, and >15.1 indicates a severe reaction. Data presented are mean ± SEM, N = 3 (days 4 and 10), and N = 9 (day 23). Blue bars represent the reactivity of wounds treated with Collex matrix, and green bars represent the reactivity of wounds treated with Collex matrix without HTX. [Figure 3]Collex matrix accelerates the healing of partial-thickness burns. (A) Development of wound area for all wounds calculated from plan views. Data presented are mean ± SEM. Because wounds were terminated at biopsy on days 4 and 10, the number of wounds is not the same across days: N = 15 (days 1 and 4), N = 12 (days 8 and 10), and N = 9 (days 12–23). Blue circles represent the area of ​​wounds treated with Collex matrix, green triangles represent the area of ​​wounds treated with Collex matrix without HTX, and gray squares represent the area of ​​wounds treated with Jelonet®. Gray stars indicate P<0.05 between Collex and Jelonet®. Green stars indicate P<0.05 between Collex matrix and Collex matrix without HTX. Significance was determined by unpaired t-test. (B) Photographs of two representative wounds from each treatment on day 19. The wound areas shown were closest to the mean areas. Wound areas for representative wounds were: Collex matrix: 65 mm and 52 mm (mean: 58 mm), Collex matrix without HTX: 63 mm and 62 mm (mean: 62 mm), and Jelonet®: 72 mm and 81 mm (mean: 79 mm). [Figure 4] Burn wounds treated with Collex matrix showed reduced inflammation. Inflammation scoring of (A) the wound edge and (B) the surrounding skin based on macroscopic assessment by Scantox A / S. (A and B) Grading system: 0: absent, 1: minimal, 2: mild, 3: moderate, 4: marked. Because wounds were terminated with biopsy on days 4 and 10, the number of wounds was not the same across days: N = 15 (day 4) and N = 12 (days 6 and 8). Data shown are mean ± SEM. P values ​​were determined by unpaired t-test. (C) Photographs of two representative wounds from each treatment on day 4. The wounds are the same as those shown in Figure 3B. [Figure 5]In vitro studies demonstrate the anti-inflammatory and antioxidant effects of Collex matrix and HTX on M1-polarized macrophages. (A) Schematic diagram of the method used to differentiate THP-1 monocytes into macrophages, M0, and M1. (B) IL-1β secreted from macrophages activated according to (A) was detected by ELISA assay. Data presented were normalized to the levels secreted from cells treated with PMA, IFNγ, and LPS (M1+Medium). (C) Cell death determined by PI-positive cells. Cells are from the same experiment as in (B). (D) ROS levels based on CellROX measured by flow cytometry. Data were normalized to the ROS levels from cells treated with PMA, IFNγ, and LPS (M1+Medium). Cells are from the same experiment as in (B and C). (B, C, and D) Data presented are means ± SD, N = 2 (PMA treatment only, M0) and N = 3 (M1). P values ​​were determined by unpaired T-test. [Figure 6] A model explaining the suggested mode of action of Collex Matrix. Burn wounds are highly inflamed and have abundant secretion of proinflammatory IL-1β and excessive production of ROS. Our data suggest that Collex Matrix (containing HTX) attenuates the inflammatory response in vivo, likely by reducing levels of the proinflammatory cytokine IL-1β and ROS. During both the inflammatory and subsequent proliferative phases of wound healing, Collex Matrix appears to accelerate wound closure by ensuring a moist and protective environment. [Figure 7] Protein release from Collex matrix in 1 ml DMEM supplemented with 10% FBS and 1% Pen / Strep was determined by absorbance at 280 nm. During the analysis, patches were incubated at 37°C in a humidified atmosphere with 5% CO2. [Figure 8]Procollagen-1 alpha 1 ELISA assay results normalized to medium control. Data are presented as mean and SD. ** indicates p<0.01. Collex 4 weeks 20°C (N=4) and Collex 4 weeks 4°C (N=2) were stored at room temperature and in the refrigerator, respectively, for 4 weeks. [Figure 9] Schematic diagram showing a printed grid design in an article of the present invention. [Figure 10] (a) Viscosity measurements performed on the bioink, shown here in terms of viscosity (Pa s) versus shear rate (s-1), and (b) shear stress (Pa) versus shear rate (s-1). Mean values ​​with SD are presented (n=3). [Figure 11] Frequency sweep showing the viscoelastic properties of the ink, relating the storage modulus G' (Pa) to the angular frequency ω (rad s-1). Mean values ​​with SD are presented (n=3). [Figure 12] CAD schematic of the sequential interdigitated grid design (a) and ongoing printing of the second layer with R4 ink (b). The CAD grid dimensions are 16mm x 16mm x 1.37mm, with hole sizes of 1.4mm x 1.4mm. [Figure 13] Release from Collex matrix: Release in DMEM supplemented with 10% FBS and 1% Pen / Strep incubated at room temperature: a) Collex matrix molded without nanocellulose (unpublished), b) Collex matrix 3D printed with nanocellulose. [Figure 14] Procollagen-1 ELISA results: a) Collex matrix cast without nanocellulose, b) Collex matrix 3D printed with nanocellulose. [Figure 15] ROS detection by CellROX Deep Red was performed on the fibroblast cell line HS707 at 2, 24, and 72 hours after HTX treatment. The cells were starved (1% serum instead of 10% serum) for 24 hours before HTX treatment. [Figure 16]ROS detection by CellROX Deep Red performed on the keratinocyte cell line HaCaT at 2, 24, and 72 hours after HTX treatment. The cells were starved (1% serum instead of 10% serum) for 24 hours before HTX treatment.

Claims

1. An article comprising a matrix formed from at least a first polysaccharide, said matrix further comprising an extract of differentiable cells, and wherein said first polysaccharide is from a source different from the extract of differentiable cells.

2. 10. The article of claim 1, wherein the first polysaccharide is an alginate. Goods.

3. 3. The article of claim 2, wherein the weight / weight percent of the alginate in the article is from 1.0% to 10.0%.

4. 4. The article of any one of claims 1 to 3, further comprising a second polysaccharide from a source different from the extract of the differentiable cells.

5. 5. The article of claim 4, wherein the second polysaccharide is nanocellulose.

6. 6. The article of claim 5, wherein the wt / wt% of the nanocellulose in the article is from 1.0% to 10.0%.

7. 7. The article of any one of claims 1 to 6, wherein the nanocellulose is supplemented with mannitol.

8. 7. The article of claim 6, wherein the wt / wt% of mannitol used to supplement the nanocellulose is between 1.0% and 10.0%.

9. 9. The article of any one of claims 1 to 8, wherein the volume / weight percentage of the extract of differentiable cells in the article is between 5.0% and 20.0%.

10. 10. The article of any one of claims 1 to 9, wherein the extract of differentiable cells is a fish egg extract.

11. 11. The article of claim 10, wherein the fish roe extract is a salmonid roe extract.

12. 12. The article of any one of claims 10 to 11, wherein the fish roe extract is an unfertilized roe extract.

13. 13. The article according to any one of claims 10 to 12, wherein the fish roe extract is characterized by having one or more of the following properties (a) to (f): a) 10 to 500 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein, in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids; e) an osmolarity of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.

7.

14. 14. The article of claim 13, wherein the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f).

15. 15. The article of any one of claims 10 to 14, wherein the fish roe extract is a heat-treated fish roe extract.

16. 16. The article of claim 15, wherein the heat-treated fish roe extract is prepared by heating the fish roe extract at 90 to 100°C for 1 to 30 minutes.

17. 17. The article of any one of claims 1 to 16, wherein the first or the first and second polysaccharides are crosslinked.

18. 18. The article of any one of claims 1 to 17, wherein the matrix is ​​a gel matrix.

19. 19. The article of any one of claims 1 to 18, wherein the matrix is ​​formed in a grid pattern.

20. 1. An article comprising a matrix formed from an alginate, the matrix further comprising a heat-treated Salmo salar egg extract, wherein the weight / weight percentage of the alginate in the article is from 1.0% to 10.0% and the volume / weight percentage of the heat-treated Salmo salar egg extract in the article is from 5.0% to 20.0%.

21. 21. The article of claim 20, further comprising nanocellulose.

22. 22. The article of claim 21, wherein the weight / weight percent of nanocellulose in the article is from 1.0% to 10.0%.

23. 7. The article of any one of claims 1 to 6, wherein the nanocellulose is supplemented with mannitol.

24. 7. The article of claim 6, wherein the wt / wt% of mannitol used to supplement the nanocellulose is between 1.0% and 10.0%.

25. 25. The article of any one of claims 20 to 24, wherein the heat-treated Salmo salar egg extract is prepared from unfertilized eggs.

26. 26. The article of any one of claims 20 to 25, wherein the heat-treated Salmo salar egg extract is characterized by having one or more of the following properties (a) through (f): a) 50 to 500 mg / ml of protein, and most preferably 10 to 5000 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.

7.

27. 27. The article of claim 26, wherein the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f).

28. 28. The article of any one of claims 20 to 27, wherein the heat-treated Salmo salar egg extract is prepared by heating the Salmo salar egg extract at 90 to 100°C for 1 to 30 minutes.

29. 29. The article of any one of claims 20 to 28, wherein the matrix is ​​a cross-linked gel matrix.

30. 30. The article of any one of claims 20 to 29, wherein the matrix is ​​formed in a grid pattern.

31. 1. A method of manufacturing an article for wound healing, comprising: forming an aqueous mixture of at least a first polysaccharide and a fish roe extract; forming a matrix from the aqueous mixture to provide the wound healing article; and wherein said at least a first polysaccharide is from a source different from said fish roe extract.

32. 32. The method of claim 31, further comprising the step of crosslinking the matrix to prepare the article for wound healing.

33. 33. The method of any one of claims 31 to 32, wherein the first polysaccharide is alginate.

34. 34. The method of claim 33, wherein the alginate is present in the mixture at a weight / weight percentage of 1.0% to 10.0%.

35. 35. The method of any one of claims 31 to 34, further comprising including a second polysaccharide in the aqueous mixture, wherein the second polysaccharide is from a different source than the fish roe extract.

36. 36. The method of claim 35, wherein the second polysaccharide is nanocellulose.

37. 37. The method of claim 36, wherein the nanocellulose is present in the mixture at a weight / weight percentage of 1.0% to 10.0%.

38. 38. The method of any one of claims 31 to 37, wherein the fish roe extract is contained in the mixture at a volume / weight percentage of 5.0% to 20.0%.

39. 39. The method of any one of claims 31 to 38, wherein the fish roe extract is derived from unfertilized fish roe.

40. 40. The method of any one of claims 31 to 39, wherein the fish roe extract is a Salmo salar roe extract.

41. 41. The method of any one of claims 31 to 40, wherein the fish roe extract is a heat-treated fish roe extract.

42. 42. The method of claim 41 , wherein the heat-treated fish roe extract is prepared by heating the fish roe extract at a temperature of 90 to 100° C. for 1 to 30 minutes.

43. 43. The method of any one of claims 40 to 42, wherein the heat-treated Salmo salar egg extract is characterized by having one or more of the following properties (a) to (f): a) 10 to 500 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein, in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.

7.

44. 44. The method of claim 43, wherein the fish roe extract has the following properties: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f).

45. 45. The method of any one of claims 31 to 44, wherein the nanocellulose is supplemented with mannitol.

46. 46. ​​The method of claim 45, wherein the wt / wt% of mannitol used to supplement the nanocellulose is 1.0% to 10.0%.

47. 47. The method of any one of claims 31 to 46, wherein the aqueous mixture contains CaCl ２ The method further comprises:

48. 48. The method of claim 47, wherein the aqueous mixture contains the CaCl ２ at a concentration of 0.01 to 0.1 M.

49. 49. The method of any one of claims 31 to 48, wherein the matrix is ​​formed by printing the aqueous mixture onto a substrate.

50. 49. The method of any one of claims 31 to 48, wherein the matrix is ​​formed by shaping the aqueous mixture.

51. 51. The method of any one of claims 31 to 50, wherein the matrix is ​​CaCl at a concentration of 0.01 to 0.1 M. ２ wherein the matrix is ​​crosslinked by treating the matrix with a crosslinking solution comprising:

52. 52. The method of claim 51 , wherein the cross-linking solution further comprises 0.5% to 1.5% w / w NaCl.

53. 53. The method of any one of claims 51 to 52, wherein the cross-linking solution further comprises the fish roe extract at a volume / weight percentage of 5.0% to 20.0%.

54. 54. The method of any one of claims 31 to 53, wherein the matrix is ​​a gel.

55. 55. A matrix made by the method of any one of claims 31 to 54.

56. 56. An article according to any one of claims 1 to 30 or a matrix according to claim 55 for use in treating a wound in a subject.

57. 57. The use of claim 56, wherein the wound is a burn.

58. 57. The use of claim 56, wherein the wound is a chronic wound.

59. 59. The use of any one of claims 56 to 58, wherein the article or solid matrix is ​​applied topically to the wound.

60. 56. A method of treating a wound in a subject in need thereof, comprising applying to said wound an article of any one of claims 1 to 30, or a solid matrix of claim 55.

61. 61. The method of claim 60, wherein the wound is a burn.

62. 61. The method of claim 60, wherein the wound is a chronic wound.

63. 56. Use of an article according to any one of claims 1 to 30, or a matrix according to claim 55, for reducing reactive oxygen species in a subject in need thereof.

64. 64. The use of claim 63, wherein the subject has a wound and the matrix is ​​applied to the wound.

65. 65. The use of claim 64, wherein the subject has skin inflammation and the matrix is ​​applied to the site of skin inflammation.

66. 1. Use of a fish roe extract or a preparation thereof for reducing reactive oxygen species in a subject in need thereof, wherein said fish roe extract is characterized by having one or more of the following properties (a) to (f): a) 50 to 500 mg / ml of protein, and most preferably 10 to 5000 mg / ml of protein, and most preferably 50 to 200 mg / ml of protein in aqueous solution; b) 0.1 to 10 mg / ml of RNA; c) 0.1 to 10 mg / ml of DNA; d) 0.1 to 10% by weight of lipids e) an osmolarity of 200 to 600 mOsm, most preferably 330 to 440 mOsm; and f) a pH of about 5.0 to 7.

7.

67. 67. The use of claim 66, wherein the subject has a wound and the fish roe extract is applied to the wound.

68. 67. The use according to claim 66, wherein the subject has skin inflammation and the fish roe extract is applied to the site of skin inflammation.

69. 69. The use according to any one of claims 66 to 68, wherein the fish roe extract has the following properties: : (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (b), (c) and and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f).

70. 70. The use according to any one of claims 66 to 69, wherein the fish roe extract is a heat-treated fish roe extract.

71. 71. The use according to any one of claims 66 to 70, wherein the fish roe extract is a heat-treated fish roe extract.

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