Matrices for wound healing

Abstract

The present invention provides articles for the improved healing of wounds, preferably burn wounds. In some preferred embodiments, the article is a matrix formed from a mixture of two or more polysaccharides, the matrix comprising an active ingredient which is an extract from differentiable cells.

Description

[0001] MATRICES FOR WOUND HEALING

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] The present application claims priority to U.S. Provisional Application No. 63 / 573,553, filed April 3, 2024, which is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The present invention provides articles for the improved healing of wounds, preferably burn wounds.

[0006] BACKGROUND OF THE INVENTION

[0007] Burn injuries are traumatic, can be extremely painful and are associated with slow recovery. Burn injuries may also lead to severe mental and emotional distress caused by massive scarring. Healing of burn wounds follows the same steps as any wound, such as hemostasis, inflammation, proliferation, and remodeling. However, burn wounds differ from other types of wounds by the severity and duration of the inflammatory phased In the inflammatory phase, neutrophils and monocytes are recruited to the wound, where they are responsible for removal of foreign materials and necrotic tissue. Upon activation, monocytes are transformed into macrophages (MO), which will be further transformed into pro- inflammatory (Ml) or anti-inflammatory (M2) state macrophages. The pro-inflammatory Ml macrophages secrete chemokines such as interleukin (IL)- 1 P to sustain the inflammatory response, while anti-inflammatory M2 macrophages suppress inflammation and activates the proliferative phase (1,2). The inflammatory phase is a vital step in healing of all wounds, but in burn wounds, the pro-inflammatory Ml phenotype is predominant, causing an excessive and prolonged inflammatory response (3). This prolonged inflammation may cause secondary necrosis and progression of the burn wounds many days after the actual trauma (4).

[0008] Reactive oxygen species (ROS) are products of natural cellular respiration and act as second messengers in several cellular processes. In wound healing, ROS play a pivotal role in orchestration of several processes such as recruitment of lymphoid cells, effective tissue repair and angiogenesis.5 However, excessive ROS, called oxidative stress, cause damage to DNA, proteins and lipids and cause activation of pro-apoptotic proteins (6,7). Burn wounds are known to generate a massive production of ROS, which is one of the mechanisms responsible for the pathophysiological events observed after burn injury. Together with increased inflammation, increased ROS contributes to the secondary necrosis (6,7). Moreover, ROS may boost the inflammatory response, while the inflammatory response may boost the ROS levels, 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 scarring seen after severe burn wounds (8,9).

[0009] Current standard-of-care dressings for partial thickness burn wounds aim to cover and protect the wound surface from infection, maintain a moist environment, and reduce discomfort for the patient. In Norwegian hospitals, burn wounds are covered by the vaseline compress Jelonet® for the first day (10). Where the treatment does not call for skin transplantation, treatment of partial thickness burn wounds typically entail wound covering with Mepilex® Ag or Aquacel® Ag Burn (10). These dressings will contribute to moist healing, while reducing the risk of bacterial infection (11,12). Silver ions (Ag+) released from these dressing have antibacterial activity. However, studies have shown that high release of Ag+ are correlated with strong cytotoxicity, causing histological damage and delayed healing (12,13). Dressings with incorporated anti-inflammatory and antioxidant activity are being investigated, (14, 15) however, to our knowledge, there are no such dressings in regular, i.e. non-research, clinical use.

[0010] What is needed in the art are new and effective treatments for burn wounds.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides articles for the improved healing of wounds, preferably burn wounds.

[0013] Provided herein are methods of balancing one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof comprising applying an article described herein to a wound of said subject. Further provided is the use of a matrix described herein to balance one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof. In some embodiments, one or more of the level of reactive oxygen species, the level of autophagy, and / or the level of TNF alpha is decreased in cells in said wound. In some embodiments, the level of Matrix metalloprotease 9 is increased in keratinocytes in said wound and reduced in proinflammatory macrophage cells in said wound.

[0014] Accordingly, in some preferred embodiments, the present provides an article comprising a matrix formed from at least a first polysaccharide, said matrix further comprising a differentiable cell extract and wherein the first polysaccharide is from a source different from the differentiable cell extract for use in the above methods. In some preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.5% to 2.5%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.85% to 2.15%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.9% to 2.1%.

[0015] In some preferred embodiments, the article further comprises a second polysaccharide from a source different the differentiable cell extract. In some preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.2% to 2.2%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.5% to 2.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 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 from 1.0% to 10.0%. In other preferred embodiments, the article comprises less than 0.5%, 0.1% or 0.01% w / w nanocellulose or is free from added nanocellulose.

[0016] In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 5.0% to 20.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 7.0% to 17.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 9.0% to 15.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 10.0% to 14.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 11.0% to 13.0%.

[0017] In some preferred embodiments, the differentiable cell extract is a fish egg extract. In some preferred embodiments, the fish egg extract is a salmonid egg extract. In some preferred embodiments, the fish egg extract is a Salmo salar egg extract. In some preferred embodiments, the fish egg extract is an unfertilized egg extract. In some preferred embodiments, the fish egg extract is characterized is characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 600 mOsm, most preferably from 330 to 440 mOsm; and f) a pH of from about 5.0 to 7.7. In some preferred embodiments, the fish egg extract has properties: (a) and (b); (a) and (c); (a) and (d); (a) and

[0018] (e); (a) and (f); (a), (b), and (c); (a), (b), and (d); (a), (b), and (e); (a), (b), and (f); (a), (c), and

[0019] (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

[0020] (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d),

[0021] (e), and (f).

[0022] In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract. In some preferred embodiments, the heat-treated fish egg extract is prepared by heating the fish egg extract to from 90 to 100 degrees Celsius for from 1 to 30 minutes.

[0023] In some preferred embodiments, the first or the first and second polysaccharides are cross-linked.

[0024] In some preferred embodiments, the matrix is a gel matrix.

[0025] In some preferred embodiments, the matrix is formed in a grid pattern.

[0026] In some further preferred embodiments, the present invention provides an article comprising a matrix formed from alginate, said matrix further comprising a heat-treated Salmo salar egg extract, wherein the weight / weight percent of the alginate in the article is from 1.0% to 10.0% and the volume / weight percent of the heat-treated Salmo salar egg extract in the article is from 5.0% to 20.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.5% to 2.5%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.85% to 2.15%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.9% to 2.1%.

[0027] In some preferred embodiments, the article further comprises nanocellulose. In some preferred embodiments, the weight / weight percent of nanocellulose in the article is from 1.0% to 10.0%. In some 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 from 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.2% to 2.2%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.5% to 2.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.55% to 1.95%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 7.0% to 17.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 9.0% to 15.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 10.0% to 14.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 11.0% to 13.0%. In other preferred embodiments, the article comprises less than 0.5%, 0.1% or 0.01% w / w nanocellulose or is free from added nanocellulose.

[0028] In some preferred embodiments, the heat-treated Salmo salar egg extract is prepared from unfertilized eggs. In some preferred embodiments, the heat-treated Salmo salar egg extract is characterized is characterized in having one or more of properties (a) to (f): a) from 50 to 500 mg / ml protein and most preferably from 10 to 5000 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w; e) an osmolarity of from 200 to 60 mOsm, most preferably from 330 to 440 mOsm; and f) a pH of from about 5.0 to 7.7. In some preferred embodiments, the fish egg extract has 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). In some preferred embodiments, the heat-treated Salmo salar egg extract is prepared by heating the Salmo salar egg extract to from 90 to 100 degrees Celsius for from 1 to 30 minutes.

[0029] In some preferred embodiments, the matrix is a cross-linked gel matrix. In some preferred embodiments, the matrix is formed in a grid pattern.

[0030] In some preferred embodiments, the present invention provides methods of producing a wound healing article comprising: forming an aqueous mixture of at least a first polysaccharide and a fish egg extract; forming a matrix from the aqueous mixture to provide the wound healing article; and wherein the at least a first polysaccharide is from a source different from the fish egg extract.

[0031] In some preferred embodiments, the methods further comprise the step of cross linking the matrix to provide the wound healing article. In some preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the alginate is included in the mixture at a weight / weight percent of from 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.5% to 2.5%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.85% to 2.15%. In some preferred embodiments, the weight / weight percent of the alginate in the article is from 1.9% to 2.1%.

[0032] In some preferred embodiments, the methods further comprise including a second polysaccharide in the aqueous mixture, wherein the second polysaccharide is from a source different from the fish egg extract. In some preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the nanocellulose is included in the mixture at a weight / weight percent of from 1.0% to 10.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.0% to 3.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.2% to 2.2%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.5% to 2.0%. In some preferred embodiments, the weight / weight percent of the nanocellulose in the article is from 1.55% to 1.95%. In other preferred embodiments, the methods utilize less than 0.5%, 0.1% or 0.01% w / w nanocellulose or do not comprise the addition of nanocellulose.

[0033] In some preferred embodiments, the fish egg extract is included in the mixture at a volume / weight percent of from 5.0% to 20.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 7.0% to 17.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 9.0% to 15.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 10.0% to 14.0%. In some preferred embodiments, the volume / weight percent of the differentiable cell extract in the article is from 11.0% to 13.0%. In some preferred embodiments, the fish egg extract is from unfertilized fish eggs. In some preferred embodiments, the fish egg extract is a Salmo salar egg extract. In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract. In some preferred embodiments, the heat-treated fish egg extract is prepared by heating the fish egg extract to a temperature of from 90 to 100 degrees Celsius for from 1 to 30 minutes. In some preferred embodiments, the heat-treated Salmo salar egg extract is characterized is characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w; e) an osmolarity of from 200 to 60 mOsm, most preferably from 330 to 440 mOsm; and f) a pH of from about 5.0 to 7.7. In some preferred embodiments, the fish egg extract has 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

[0034] (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

[0035] (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d),(e), and (f).

[0036] In some 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 from 1.0% to 10.0%.

[0037] In some preferred embodiments, the aqueous mixture further comprises CaCh- In some preferred embodiments, the CaCh is included in the aqueous mixture at a concentration of from 0.01 to 0.1 M.

[0038] In some preferred embodiments, the matrix is formed by printing the aqueous mixture onto a substrate.

[0039] In some preferred embodiments, the matrix is formed by molding the aqueous mixture.

[0040] In some preferred embodiments, the matrix is cross-linked by treating the matrix with a cross-linking solution comprising CaCh at a concentration of from 0.01 to 0.1 M. In some preferred embodiments, the cross-linking solution further comprises a weight / weight percent of NaCl of from 0.5% to 1.5%. In some preferred embodiments, comprises the fish egg extract at a volume / weight percent of from 5.0% to 20.0%.

[0041] In some preferred embodiments, the matrix is a gel.

[0042] In some preferred embodiments, the present invention provides a matrix made by any of the foregoing methods.

[0043] In some preferred embodiments, the present invention provides an article or matrix as described above for use in treating a wound in a subject. In some preferred embodiments, the wound is a burn wound. In some preferred embodiments, the wound is a chronic wound. In some preferred embodiments, the article or solid matrix is topically applied to the wound.

[0044] In some preferred embodiments, the present invention provides a method of treating a wound in a subject in need thereof comprising applying an article or matrix as described above to the wound. In some preferred embodiments, the wound is a burn wound. In some preferred embodiments, the wound is a chronic wound. In some preferred embodiments, the present invention provides for the use of an article or matrix as described above to reduce reactive oxygen species in a subject in need thereof. In some preferred embodiments, the article or matrix is applied at a site on the subject that exhibits or is 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.

[0045] In some preferred embodiments, the present invention provides for the use of a fish egg extract or formulation thereof to reduce reactive oxygen species in a subject in need thereof, wherein the fish egg extract is characterized in having one or more of properties (a) to (f): a) from 50 to 500 mg / ml protein and most preferably from 10 to 5000 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 600 mOsm, most preferably from 330 to 440 mOsm; and f) a pH of from about 5.0 to 7.7. In some preferred embodiments, the extract is applied at a site on the subject that exhibits or is 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. In some preferred embodiments, the article or matrix is applied at a site on the subject that exhibits or is at risk of inflammation. In some preferred embodiments, the fish egg extract has 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

[0046] (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

[0047] (f); (a), (c), (d), (e) and (f); or (a), (b), (c), (d),(e), and (f). In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract. In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract.

[0048] Additional embodiments are described herein.

[0049] BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1. Collex containing HTX has buffer capacity and the ability to stabilize the pH in a solution.

[0051] FIG. 2A-C. HS707 was seeded at a density of 1.2- 105 viable cells / ml in optimal (10% FBS) and adherent (1% FBS) adherent medium. Cells were administered HTX to a final concentration of 0 or 5%. ROS was assessed through CellROX staining 2 hours (A), 24 hours (B), and 3 days (C) after HTX administration. The values are given as the relative change (fold-change) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t- tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0052] FIG. 3A-C. HaCaT was seeded at a density of 0.6405 viable cells / ml in optimal (10% FBS) and adherent (1% FBS) adherent medium. Cells were administered HTX to a final concentration of 0 or 5%. ROS was assessed through CellROX staining 2 hours (A), 24 hours (B), and 3 days (C) after HTX administration. The values are given as the relative change (fold-change) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t- tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0053] FIG. 4A-C. THP-1 was seeded at a density of 2405 viable cells / ml in optimal (10% FBS) and starvation (1% FBS) adherent medium. Cells were administered HTX to a final concentration of 0 or 5%. ROS was assessed through CellROX staining 2 hours (A), 24 hours (B), and 3 days (C) after HTX administration. The values are given as the relative change (fold-change) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t- tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0054] FIG. 5A-C. HS707 was seeded at a density of 1.2405 viable cells / ml in optimal (10% FBS) and adherent (1% FBS) adherent medium. Cells were administered HTX to a final concentration of 0 or 5%. Autophagy was assessed through CytoID staining 2 hours (A), 24 hours (B), and 3 days (C) after HTX administration. The values are given as the relative change (fold-change) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t-tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. FIG. 6A-C. HaCaT was seeded at a density of 0.6-105 viable cells / ml in optimal (10% FBS) and adherent (1% FBS) adherent medium. Cells were administered HTX to a final concentration of 0 or 5%. Autophagy was assessed through CytoID staining 2 hours (A), 24 hours (B), and 3 days (C) after HTX administration. The values are given as the relative change (fold-change) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t-tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0055] FIG. 7A-B. THP-1 was seeded at a density of 2-105 viable cells / ml in optimal (10% FBS) and starvation (1% FBS) suspension medium. Cells were administered HTX to a final concentration of 0 or 5%. Autophagy was assessed through CytoID staining 2 hours (A) and 3 days (B) after HTX administration. The values are given as the relative change (foldchange) to the optimal control of the corresponding day, and each bar represents the mean of three biological replicates. The statistical significance was determined using paired t-tests, where each treatment was compared to the control within the same day and medium conditions (solid line). Dotted lines imply significance between the controls of optimal and starvation medium. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0056] FIG. 8A-B. (A) HaCat cells were seeded at a density of 0.6-105 viable cells / ml in optimal (10% FBS) and starvation (1% FBS) adherent medium. (B) THP-1 cells were seeded at a density 2-105 viable cells / ml in optimal (10% FBS) and starvation (1% FBS) suspension medium. All three cell lines were administered HTX to final concentrations of 0 and 5%. MMP9 levels were measured through ELISA. The graph presents the mean MMP9 level for two technical replicates within three biological replicates. The statistical significance was determined using paired t-tests, where each treatment was compared to the control within the same medium condition. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0057] FIG. 9. THP-1 cells were seeded at a density 2-105 viable cells / ml in optimal (10% FBS) and starvation (1% FBS) suspension medium. Cells were administered HTX to final concentrations of 0 and 5%. TNF-alpha levels were measured through ELISA. The graph presents the mean TNF-alpha level for two technical replicates within three biological replicates. The statistical significance was determined using paired t-tests, where each treatment was compared to the control within the same medium condition. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. DEFINITIONS

[0058] As used herein, the term “matrix” when used in reference to an article of the present invention refers to a material in which an agent (e.g., HTX; heat-treated egg extract) is incorporated into. The matrix will be understood to have a length, breadth, and depth and may assume a variety of shapes and patterns, including, but not limited to, circular, rectangular, triangular or square sheets, as well as grid and other patterns (see, e.g., FIG. 9).

[0059] As used herein, the term “HTX” refers to a heated treated extract of salmon eggs (See, e.g., PCT / IB2013 / 003177 and as described elsewhere herein).

[0060] As used herein, “cell” means the smallest structural unit of living matter capable of functioning autonomously, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable membrane. Cells include all somatic cells obtained or derived from a living or deceased animal body at any stage of development as well as germ cells, including sperm and eggs (animal reproductive body consisting of an ovum or embryo together with nutritive and protective envelopes). Included are both general categories of cells: prokaryotes and eukaryotes. The cells contemplated for use in this invention include all types of cells from all organisms in all kingdoms: plants, animals, protists, fungi, archaebacteria and eubacteria. Stem cells are cells capable, by successive divisions, of producing specialized cells on many different levels. For example, hematopoietic stem cells produce both red blood cells and white blood cells. From conception until death, humans contain stem cells, but in adults their power to differentiate is reduced.

[0061] As used herein, the term “differentiation” related to cells means the process by which cells becomes structurally and functionally specialized, which is a progressive restriction of the developmental potential and increasing specialization of function which takes place during the development of the embryo and leads to the formation of specialized cells, tissues, and organs.

[0062] The term “dedifferentiation” related to cells means the reverse process of differentiation, where cells become less structurally and functionally specialized, which increases the developmental potential of the cell.

[0063] “Differentiable” means the ability of a cell to differentiate into a desired cell type. As used herein, the term “differentiates” means specialization (differentiation) or return to a more primitive cell type; dedifferentiation).

[0064] An “extract” as used in the context of “cell extract” and “egg extract” in this invention means a preparation of any type of cell as defined above obtained by chemical or mechanical action, as by pressure, distillation, evaporation etc. Extracts can include all or any single component or combination of components of the cells, including concentrated preparations of the active components. Such components of the extracts include but are not limited to RNA, DNA, micro RNA, lipids, free amino acids, all amino acid base structures including peptides and proteins, carbohydrates, minerals or combinations thereof. Extracts contemplated by this invention include but are not limited to extracts of fish eggs, urchin eggs, frog eggs, adult stem cells, plant seeds and plant stem cells.

[0065] The term "manage" when used in connection with a disease or condition means to provide beneficial effects to a subject being administered with a prophylactic or therapeutic agent, which does not result in a cure of the disease. In certain embodiments, a subject is administered with one or more prophylactic or therapeutic agents to manage a disease so as to prevent the progression or worsening of the disease.

[0066] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present invention be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0067] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, the present invention also contemplates treatment that merely reduces symptoms, and / or delays disease progression.

[0068] DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention provides articles for the improved healing of wounds, preferably burn wounds. In some preferred embodiments, the article is a matrix formed from a mixture of two or more polysaccharides, the matrix comprising an active ingredient which is an extract from differentiable cells.

[0070] In some particularly preferred embodiments, the articles of the present invention are utilized to treat burn wounds. Partial thickness thermal burn wounds are characterized by prolonged inflammatory response, oxidative stress, tissue damage, and secondary necrosis. An optimal dressing for burn wounds can therefore reduce inflammation and oxidative stress while it provides a moist and absorbent cover, protecting the wound. In some preferred embodiments, the articles of the present invention comprise an extract from unfertilized salmon roe containing components with potential anti-inflammatory and antioxidative properties, called HTX. See, e.g., PCT Applications PCT / IB2011 / 001488 and PCT / IB 2013 / 003177 which are both incorporated by reference herein their entirety. In the articles of the present invention, HTX has been combined with alginate from brown algae and nanocellulose from tunicates, and 3D printed into an all-marine hydrogel wound dressing, which is referred to herein as the Collex matrix. Thus, the present invention provides a technical solution for effective delivery of HTX as an active ingredient to a wound, for example a partial thickness burn wound.

[0071] Data provided herein describes testing of the Collex matrix on partial thickness burn wounds in Gottingen minipigs. The Collex matrix was compared to the vaseline compress Jelonet®, and a variant of the Collex matrix without HTX. It was found that dermal treatment of burn wounds with the Collex matrix resulted in accelerated healing compared to wounds treated with Jelonet®. Compared to the Collex matrix without HTX, the Collex matrix improved healing in the first week after trauma where secondary necrosis was pronounced. Notably, the Collex matrix reduced the inflammatory response in the early postinjury phase. The anti-inflammatory response of the Collex matrix was investigated in more detail on activated Ml macrophages. It was further found that the Collex matrix, as well as HTX alone, significantly reduced secretion of pro-inflammatory interleukin- ip as well as the intracellular level of oxidative stress. The results from this study suggest the Collex matrix is a potent dressing for treatment of burn wounds, with the anti-inflammatory effect of HTX beneficial in the initial phase, and the moist qualities of the hydrogel being favorable both in the initial and the proceeding proliferative phase of the wound healing.

[0072] Accordingly, in some embodiments, the present invention provides the present invention provides matrices, most preferably hydrogels, for delivery of an extract of differentiable cells (e.g., HTX) to a wound. In some preferred embodiments, the differentiable cell extract component provided in the matrix is characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 60 mOsm, most preferably from 330 to 440 mOsm; f) a pH of from about 5.0 to 7.7.

[0073] In some preferred embodiments, the egg cellular extract is characterized in having two or more of properties (a) to (f). In some preferred embodiments, the differentiable cell extract is characterized in having three or more of properties (a) to (f). In some preferred embodiments, the differentiable cell extract is characterized in having four or more of properties (a) to (f). In some preferred embodiments, the differentiable cell extract is characterized in having five or more of properties (a) to (f). In some preferred embodiments, the differentiable cell extract is characterized in having all six of properties (a) to (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a) and (b). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a) and (c). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a) and (d). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a) and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a) and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), and (c). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), and (d). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), and (f). In some preferred embodiments, the differentiable cell is characterized in having properties (a), (c), and (d). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (d), and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (d), and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), and (d). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), (d), and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), (d), and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), (e), and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), (d) and (e). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), (d) and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (c), (d), (e) and (f). In some preferred embodiments, the differentiable cell extract is characterized in having properties (a), (b), (c), (d),(e), and (f).

[0074] In some embodiments, the differentiable cell extract is selected from the group consisting of an extract of an activated fish egg cellular extract and an unactivated fish egg cellular extract. In some embodiments, the fish egg cellular extract is from a fertilized egg. In some embodiments, the fish egg cellular extract is from an unfertilized egg. In some embodiments, the cellular extract is heat treated by heating the extract to greater than 80C, 90C, 95C or 100C. In some embodiments, the heat treatment is from about 1 minute to about 30 minutes.

[0075] As just described, the compositions of the present invention utilize cell, egg and embryo extracts from vertebrates, including but not limited to Superclass Gnathostomata (jawed vertebrates), Euteleostomi (bony vertebrates), Class Actinopterygii (ray-finned fishes), Class Sarcopterygii (lobe-finned fishes and terrestrial vertebrates), Tetrapoda (tetrapods), Amniota (amniotes), Synapsida (synapsids), Class Mammalia (mammals), Early Therapsida (early therapsids), Class Reptilia (reptiles), Anapsida (tortoises and turtles), Order Testudines (tortoises and turtles), Diapsida (birds, crocodiles, lizards, snakes, and relatives), Archosauria (birds and crocodiles), Order Crocodilia (caimans, crocodiles, and relatives), Lepidosauria (amphisbaenians, lizards, snakes, and tuataras), Order Rhynchocephalia (tuataras), Order Squamata (amphisbaenians, lizards, and snakes), Class Amphibia (amphibians), Subclass Dipnoi (lungfishes), Actinistia, Order Coelacanthiformes (coelacanths), Class Chondrichthyes (rays, sharks, and relatives), Placodermi (armored fishes and placoderms), Class Cephalaspidomorphi, more preferably fish, shrimp, sea urchin or amphibian eggs or embryos. In some embodiments, unfertilized but activated fish, shrimp, sea urchin or amphibian eggs are used. The present invention is not limited to the use of any particular types of eggs. Indeed, the use of a variety of eggs is contemplated, including, but not limited to eggs from Xenopus, shrimp, sea urchin, salmon, trout or zebrafish. In some embodiments, eggs are collected from mature females and spontaneously activate upon contact with water. In further embodiments, the eggs are washed in Ringer’s saline. In some embodiments, the eggs are not from an avian species. In some particularly preferred embodiments, the eggs are from a salmonid species. In some especially preferred embodiments, the salmonid is Salmo salar.

[0076] Extracts of the present invention are prepared from any of the sources described herein. In some embodiments, the extracts are cellular extracts. Cellular extracts of the present invention are preferably compositions of disrupted cells such as eggs. The cells may be disrupted by a variety of methods, including, but not limited to, mechanical shearing or blending, sonication, or osmotic lysis. In some embodiments, the extracts comprise less than about 1% and preferably less than 0.1% cholesterol or ovalbumin. Accordingly, in some embodiments, the cellular extract comprises 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 extracts can comprise 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 nuclear, cell membrane 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 comprising water soluble cellular components such as proteins, mRNA, and carbohydrates.

[0077] A variety of methods may be used to prepare extracts, including those described in the examples below. In some embodiments, eggs are placed “dry” in a glass 15 ml centrifuge tube, and crushed by sedimentation at 15,000 g for 15 min. This produces three layers: a lipid top fraction, which is collected, aliquoted and frozen; a middle cellular or cytoplasmic fraction, which is also collected, aliquoted and frozen; and a pellet fraction, which is discarded. In some embodiments, the cellular fraction or extract primarily comprises contents of the cytoplasm. The cellular fraction is used as extract. In some embodiments, the cellular fraction may be used in combination with a lipid fraction. The cytoplasmic fraction may be cleared further by sedimentation at 50,000, 100,000 or 200,000 g to yield a further cellular extract which is primarily a water soluble extract fraction. Regardless of the fraction used, the extract can be diluted to about 300 mOsm with cell lysis buffer (see above), if necessary. Accordingly, in some preferred embodiment’s, a water soluble extract prepared from eggs or embryos is utilized.

[0078] In other embodiments, the eggs are suspended in 0.5 volume of cell lysis buffer and sonicated on ice until all eggs are lysed. The particulate material is sedimented at 15,000 g for 15 min at 4°C. The supernatant constitutes the extract. As above, osmolarity can be adjusted to 300 mOsm if needed. The extract can also be cleared as above.

[0079] In still other embodiments, the 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 B). The lysate is sedimented and treated as described above. In some embodiments, the homogenates and extracts may be stabilized by the addition of one or more stabilizing agents, such as a lipid stabilizing agent, or by packaging in a package designed to prevent oxidation. In some embodiments, antioxidants such as vitamin E are added to the extract to reduce rate of lipid oxidation. In some embodiments, the extracts are packaged in a container under an inert atmosphere. In some embodiments, the extract is packaged to reduce rate of lipid oxidation in air-free containers such as aluminum coated bags (less than 10 kg per bag for efficient removal of oxygen), or containers filled with nitrogen to remove oxygen. In other embodiments, the extracts are packaged in vacuum packed containers with a pump delivery system.

[0080] In some embodiments, the present invention provides powders prepared from the cellular extracts described above. In some embodiments, the cellular extracts used in the production of the powders are prepared from salmonid eggs. In some embodiments, the cellular extracts used in the production of the powders are prepared from salmon or trout eggs. In some embodiments, the powders are biologically active. In some preferred embodiments, the powders are freeze-dried. In some embodiments, the powders have less than about 10% moisture and most preferably less than about 5% moisture; protein in a concentration of from about 500 to about 800 mg / g powder, preferably from about 600 to about 700 mg / g powder, most preferably about 640 mg / g powder; DNA in a concentration of from about 1 to about 50 pl / mg powder, preferably from about 5 to about 25 pl / mg powder, and most preferably about 16 pl / mg powder; total RNA (e.g., including mRNA, rRNA, and microRNA) in a concentration of from about 1 to about 50 pl / mg powder, preferably from about 5 to about 20 pl / mg powder, and most preferably about 12 pl / mg powder; and lipids in a concentration of from about 100 to about 200 mg / g powder, most preferably about 150 mg / g powder. The powders may preferably be used to make the formulations described herein as an alternative to the non-powdered cellular extracts.

[0081] In some particularly preferred embodiments, the cell extracts described above are incorporated into a matrix formed from one or more polysaccharides or a mixture of polysaccharides. Accordingly, in some preferred embodiments, the present invention provides methods of producing a wound healing article comprising: forming an aqueous mixture of at least a first polysaccharide and a differentiable cell extract; forming a matrix from the aqueous mixture; and optionally cross-linking matrix to provide a wound healing article. In some preferred embodiments, a second polysaccharide is included in the aqueous mixture. In some particularly preferred embodiments, the differentiable cell extract is a fish egg extract. In some preferred embodiments, the fish egg extract is a salmonid egg extract. In some more preferred embodiments, the salmonid egg extract is a Salmo salar egg extract. In some preferred embodiments, the fish egg extracts, such as Salmo salar egg extracts, are prepared from unfertilized eggs. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 5.0% to 20.0%, where volume is the volume of the cellular extract in milliliters and weight is the weight of the remainder of the components of the mixture in grams. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 8.0% to 16.0%. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 10.0% to 14.0%. In some embodiments, the cellular extract may be reconstituted from a powder prepared as described above. In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract. In some preferred embodiments, the heat-treated fish egg extract is prepared by heating the fish egg extract to a temperature of from 90 to 100 degrees Celsius for from 1 to 30 minutes. In some embodiments, the cell extract, which may preferably be a heat-treated Salmo salar egg extract, is characterized is characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 60 mOsm, most preferably from 330 to 440 mOsm; f) a pH of from about 5.0 to 7.7.

[0082] In some preferred embodiments, the fish egg extract has properties: (a) and (b); (a) and (c);

[0083] (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),

[0084] (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).

[0085] In some particularly preferred embodiments, the at least two polysaccharides are from a source different from the source of the differentiable cell extract, for example, from a different source than Salmo salar. In some preferred embodiments, the polysaccharides are from a marine source that is different than the source of the cell extract. In some particularly preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the alginate is included in the aqueous mixture at a weight / weight percent of from 1.0% to 10.0%, and most preferably from 1.0% to 3.0%, where weight / weight is the weight of the alginate per the total weight of the aqueous mixture.

[0086] In some particularly preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the nanocellulose is included in the aqueous mixture at a weight / weight percent of from 1.0% to 10.0%, and most preferably from 1.0% to 3.0%, where weight / weight is the weight of the nanocellulose per the total weight of the aqueous mixture.

[0087] In some preferred embodiments, the nanocellulose used to form the matrix further comprises mannitol. In some preferred embodiments, the nanocellulose is optionally supplemented with mannitol at weight percent of from 1.0% to 10.0% and most preferably from 2.0% to 7.0% where weight / weight is the weight of the mannitol per the total weight of the nanocellulose.

[0088] In some preferred embodiments, the aqueous mixture used to form the matrix further comprises CaCh- In some preferred embodiments, the CaCh is included in the aqueous mixture at a concentration of from 0.01 to 0.1 M.

[0089] The present invention is not limited to any particular method of forming the 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 release molds formed from, for example, polydimethylsiloxane (PDMS) or a release-coated polymer. Suitable release coatings are known in the art in the art and include silicone release coatings.

[0090] The present invention is not limited to any particular method of cross-linking the polymers used to form the matrix. In some embodiments, the matrix is cross-linked by treating the matrix with a cross-linking solution comprising CaCh at a concentration of from 0.01 to 0.1 M. In some preferred embodiments, the cross-linking solution further comprises a weight / weight percent of NaCl of from 0.5% to 1.5%. In some preferred embodiments, the cross-linking solution further comprises the fish egg extract at a volume / weight percent of from 5.0% to 20.0%, and most preferably from 5.0% to 17.0%. In some preferred embodiments, the fish egg extract is included in the cross-linking solution at a volume / weight percent of from 8.0% to 16.0%. In some preferred embodiments, the cellular extract is included in the cross-linking solution at a volume / weight percent of from 10.0% to 14.0%.

[0091] In some preferred embodiments, the matrix is a gel. In some preferred embodiments, the matrix is formed into a grid pattern.

[0092] In some preferred embodiments, the present invention provides a matrix made by the methods described above. According, in some embodiments, the present invention provides an article comprising a matrix (e.g., a gel matrix and most preferably a hydrogel matrix) formed from at least a first polysaccharide, said matrix further comprising a differentiable cell extract and wherein the first polysaccharide is from a source different from the differentiable cell extract. In some preferred embodiments, a second polysaccharide is included in the aqueous mixture, wherein the first polysaccharide is from a source different from the differentiable cell extract.

[0093] In some particularly preferred embodiments, the differentiable cell extract is a fish egg extract. In some preferred embodiments, the fish egg extract is a salmonid egg extract. In some more preferred embodiments, the salmonid egg extract is a Salmo salar egg extract. In some preferred embodiments, the fish egg extracts, such as Salmo salar egg extracts, are prepared from unfertilized eggs. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 5.0% to 20.0%. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 8.0% to 16.0%. In some preferred embodiments, the cellular extract is included in the mixture at a volume / weight percent of from 10.0% to 14.0%. In some preferred embodiments, the fish egg extract is a heat-treated fish egg extract. In some preferred embodiments, the heat-treated fish egg extract is prepared by heating the fish egg extract to a temperature of from 90 to 100 degrees Celsius for from 1 to 30 minutes. In some embodiments, the cell extract, which may preferably be a heat-treated Salmo salar egg extract, is characterized is characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 60 mOsm, most preferably from 330 to 440 mOsm; f) a pH of from about 5.0 to 7.7.

[0094] In some preferred embodiments, the fish egg extract has properties: (a) and (b); (a) and (c);

[0095] (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),

[0096] (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).

[0097] 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 differentiable cell extract, for example, from a different source than Salmo salar. In some preferred embodiments, the polysaccharides are from a marine source that is different than the source of the cell extract.

[0098] In some particularly preferred embodiments, the first polysaccharide is alginate. In some preferred embodiments, the alginate is included in the aqueous mixture at a weight / weight percent of from 1.0% to 10.0% and most preferably from 1.0% to 3.0%.

[0099] In some particularly preferred embodiments, the second polysaccharide is nanocellulose. In some preferred embodiments, the nanocellulose is included in the aqueous mixture at a weight / weight percent of from 1.0% to 10.0% and most preferably from 1.0% to 3.0%.

[0100] In some preferred embodiments, the nanocellulose used to form the matrix further comprises mannitol. In some preferred embodiments, the nanocellulose is optionally supplemented with mannitol at a weight percent of from 1.0% to 10.0% and most preferably from 2.0% to 7.0%.

[0101] In some particularly preferred embodiment, the present invention provides an article comprising a solid matrix formed from a mixture of alginate and nanocellulose, said matrix further comprising a heat-treated salmonid egg extract, wherein the weight / weight percent of the alginate in the article is from 1.0% to 10.0%, most preferably from 1.0% to 3.0%, the weight / weight percent of the nanocellulose in the article is from 1.0% to 10.0%, most preferably from 1.0% to 3.0%, and the volume / weight percent of the heat-treated Salmo salar egg extract in the article is from 5.0% to 20.0%, most preferably from 8.0% to 16.0%.

[0102] In some preferred embodiments, one or more additional active agents may be included in the matrix. In some preferred embodiments, the one or more additional active agents are incorporated into the aqueous mixture prior to formation of the matrix.

[0103] Suitable additional active agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDS)(the NAIDS can, for example, be selected from the following categories: (e.g., propionic acid derivatives, acetic acid derivatives, fenamic acid derivatives, biphenylcarboxylic acid derivatives and oxicams)); steroidal anti-inflammatory drugs including hydrocortisone and the like; antihistaminic drugs (e.g., chlorpheniramine, triprolidine); antitussive drugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane); antipruritic drugs (e.g., methidilizine and trimeprizine); anticholinergic drugs (e.g., scopolamine, atropine, homatropine, levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs (e.g., benzphetamine, phentermine, chlorphentermine, fenfluramine); central stimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamine and methylphenidate); minoxidil; antiarrhythmic drugs (e.g., propanolol, procainamide, disopyraminde, quinidine, encainide); P-adrenergic blocker drugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol); cardiotonic drugs (e.g., milrinone, amrinone and dobutamine); antihypertensive drugs (e.g., enalapril, clonidine, hydralazine, minoxidil, guanadrel, guanethidine);diuretic drugs (e.g., amiloride and hydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone, isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs (e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs (e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine, bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g., imipramine, desipramine, amitryptiline, nortryptiline); PDE5 inhibitors such as Viagra® or Cialis®; tranquilizer and sedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide, flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs (e.g., chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine); antimicrobial drugs (antibacterial, antifungal, antiprotozoal and antiviral drugs).

[0104] Antimicrobial drugs which are preferred for incorporation into the present composition include, for example, pharmaceutically acceptable salts of P-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole; pentamidine, gentamycin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmycin, paromomycin, streptomycin, tobramycin, miconazole, and amanfadine.

[0105] Other drug moieties of use in practicing the present invention include antineoplastic drugs (e.g., antiandrogens (e.g., leuprolide or flutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate, mercaptopurine, thioguanine).

[0106] The compositions can also comprise hormones (e.g., medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide or somatostatin); muscle relaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, dephenoxylate, dantrolene and azumolen); antispasmodic drugs; bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinate drug compounds); endocrine modulating drugs (e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone), modulators of diabetes (e.g., glyburide or chlorpropamide), anabolics, such as testolactone or stanozolol, androgens (e.g., methyltestosterone, testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) and calcitonins).

[0107] Also of use in the present invention are estrogens (e.g., diethylstilbesterol), glucocorticoids (e.g., triamcinolone, betamethasone, etc.) and progenstogens, such as norethindrone, ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g., liothyronine or levothyroxine) or anti-thyroid agents (e.g., methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormone suppressors (e.g., danazol or goserelin), oxytocics (e.g., methylergonovine or oxytocin) and prostaglandins, such as mioprostol, alprostadil or dinoprostone, can also be employed.

[0108] Other useful active compounds include immunomodulating drugs (e.g., antihistamines, mast cell stabilizers, such as lodoxamide and / or cromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone, or clobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine, ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc. Groups with anti-inflammatory activity, such as sulindac, etodolac, ketoprofen and ketorolac, are also of use. Other drugs of use in conjunction with the present invention will be apparent to those of skill in the art.

[0109] In some preferred embodiments, the articles of the instant invention find use in the treatment of wounds. Accordingly, in some preferred embodiments, the articles of the instant invention may preferably be topically applied to a wound of a subject. The present invention is not limited to the treatment of any particular type of wound. In some preferred embodiments, the wound is a burn wound. Types of burn wounds that may be treated include partial thickness burn wounds (second degree burns), superficial burn wounds (first degree burns) and full thickness burn wounds (third degree burns). In some particularly preferred embodiments, the burn wound is a partial thickness burn wound. In other embodiments, the wound is a chronic wound. In particular, the chronic wounds 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. As such, in some preferred embodiments, the chronic wound is a diabetic ulcer, venous ulcer, pressure ulcer, or ischemic ulcer. In still other preferred embodiments, the wound may be an abrasion, a skin tear, a puncture wound, a surgical wound or incision, or a laceration. In other preferred embodiments, the wound may be an insect bite or sting.

[0110] The effect of moist and absorbent healing on re-epithelialization and closure of burn wounds is well established. Furthermore, burn wounds with poor progression are associated with increased pH. Chronic wounds often exhibit elevated pH levels, tending towards alkalinity, impairing the healing process.

[0111] Provided herein is a dressing with outstanding moisturizing and absorbing properties, while balancing wound pH and providing a nourishing wound healing environment. To minimize environmental impact, the dressing is made from naturally sourced, sustainable raw materials.

[0112] An all-marine dressing, containing alginate, nanocellulose and purified, unfertilized salmon roe extract was developed (See above for details). This bio-ink was 3D printed and crosslinked with calcium, creating a porous, moist, solid hydrogel dressing. The buffer capacity and the impact of this dressing on porcine burn wounds was determined to evaluate its safety, functionality, and efficacy.

[0113] It was found that the marine dressing had the ability to stabilize pH and to facilitate the healing of porcine burn wounds. The wounds treated with the marine dressing had less rubor and showed accelerated wound closure compared to a standard-of-care dressing. Importantly, the marine dressing showed low reactivity, was sterile, and biocompatible.

[0114] Accordingly, also provided herein is a method of balancing one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof comprising applying an article described herein to a wound of said subject. Further provided is the use of a matrix described herein to balance one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof. In some embodiments, one or more of the level of reactive oxygen species, the level of autophagy, and / or the level of TNF alpha is decreased in cells in said wound. In some embodiments, the level of Matrix metalloprotease 9 is increased in keratinocytes in said wound and reduced in proinflammatory macrophage cells in said wound.

[0115] EXAMPLES

[0116] Example 1 Experiments were performed to demonstrate that Collex containing HTX has buffer capacity and the ability to stabilize the pH in a solution. Experiment: 3 ml Saline (0.9% B. Braun) added to 1 g Collex or 1 g Collex NaCl (without HTX) Incubated at 37°C (shaker incubator) for 2 hours. pH measured (pH meter). NaOH or Acetic acid added, incubated for 10 min. pH measured (pH meter)

[0117] Results are shown in FIG. 1 and show the pH buffering capability of Collex.

[0118] Example 2

[0119] Experiments were performed on the on the activity of HTX. Experimental methods are shown in legends of FIGs. 2-9.

[0120] Results demonstrated that ROS is rapidly decreased with HTX treatment in three different cell lines tested. The effect is more pronounced in starved cells. Results for fibroblast cells (FIG. 2), keratinocyte cells (FIG. 3), and proinflammatory macrophage cells (FIG. 4) are shown.

[0121] Further experiments demonstrated that the level of Autophagy is reduced with HTX treatment in three different cell lines tested. The effect is more pronounced in starved cells. Results for fibroblast cells (FIG. 5), keratinocyte cells (FIG. 6), and proinflammatory macrophage cells (FIG. 7) are shown.

[0122] Additional experiments (FIG. 8) demonstrated that the level of Matrix metalloprotease 9 is influenced by HTX treatment in that it is increased in keratinocytes and reduced in proinflammatory macrophage cells.

[0123] In was also demonstrated (FIG. 9) that the level of Matrix metalloprotease 9 is influenced by HTX treatment in that it is increased in keratinocytes and reduced in proinflammatory macrophage cells

[0124] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method 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 which 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.

Claims

CLAIMSWhat is claimed is1. A method of balancing one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof comprising: applying to a wound of said subject an article comprising a matrix formed from at least a first polysaccharide, said matrix further comprising a differentiable cell extract, and wherein the first polysaccharide is from a source different from the differentiable cell extract.

2. Matrix formed from at least a first polysaccharide, said matrix further comprising a differentiable cell extract and wherein the first polysaccharide is from a source different from the differentiable cell extract for use to balance one or more of pH, moisture and / or absorbance in a wound in a subject in need thereof.

3. Method of claim 1 or use of claim 2, wherein the wound is a burn wound.

4. Method of claim 1 or use of claim 2, wherein the wound is a chronic wound.

5. Method of claim 1 or use of claim 2, wherein one or more of the level of reactive oxygen species, the level of autophagy, and / or the level of TNF alpha is decreased in cells in said wound.

6. Method of claim 1 or use of claim 2, wherein the level of Matrix metalloprotease 9 is increased in keratinocytes in said wound and reduced in proinflammatory macrophage cells in said wound.

7. Method of claim 1 or use of claim 2, wherein the differentiable cell extract is a salmon egg extract.

8. Method of claim 1 or use of claim 2, wherein the differentiable cell extract is a heat- treated salmon egg extract.

9. Method of claim 1 or use of claim 2, wherein the differentiable cell extract is a salmon egg extract characterized in having one or more of properties (a) to (f): a) from 10 to 500 mg / ml protein and most preferably from 50 to 200 mg / ml protein, in an aqueous solution; b) from 0.1 to 10 mg / ml RNA; c) from 0.1 to 10 mg / ml DNA; d) from 0.1 -10% lipids w / w e) an osmolarity of from 200 to 600 mOsm, most preferably from 330 to 440 mOsm; and f) a pH of from about 5.0 to 7.7.

10. Method of claim 1 or use of claim 2, wherein the first polysaccharide is alginate.

11. Method of use of claim 10, wherein the weight / weight percent of the alginate in the matrix is from 1.0% to 10.0%.

12. Method of use of any one of claims 1 to 11, wherein the matrix further comprises nanocellulose.

13. Method or use of claim 12, wherein the weight / weight percent of the nanocellulose in the matrix is from 1.0% to 10.0%.

14. Method or use of any one of claims 12 to 13, wherein the nanocellulose is supplemented with mannitol.

15. Method or use of claim 14, wherein the weight / weight percent of the mannitol used to supplement the nanocellulose is from 1.0% to 10.0%.

16. Method or use of any one of claims 1 to 15, wherein the volume / weight percent of the differentiable cell extract in the article is from 5.0% to 20.0%.

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