WO2018060994A1

WO2018060994A1 - Extracellular matrix comprising type ii collagen and uses thereof

Info

Publication number: WO2018060994A1
Application number: PCT/IL2017/051084
Authority: WO
Inventors: Max Herzberg; Sharon VIGODMAN
Original assignee: A.G.M. Biological Products Development Ltd.
Priority date: 2016-09-28
Filing date: 2017-09-27
Publication date: 2018-04-05

Abstract

This disclosure relates to a method for producing a native, non-soluble, non-crossed- linked extracellular matrix, containing collagens, mainly Type II collagen, derived from jellyfish sources. A short extraction procedure with acid and in the absence of cross-linking agents during the procedure results in unique features of the matrix. Although cross-linking materials are not added, the innovative matrix is stable and has shown increased resistance to degradation induced by in vivo enzymes (proteases) present in the wound. In addition, the innovative non-cross-linked matrix demonstrates the ability to not be degraded or absorbed rapidly, but rather to adhere and persist in the wound. This innovative non-cross-linked matrix can support cellular infiltration and proliferation including fibroblasts, which are essential for wound healing as the cells produce new collagen and proteins to heal the wound. Moreover, this innovative non-cross-linked matrix enables rapid transition of the wound from the inflammatory stage to the healing stage.

Description

EXTRACELLULAR MATRIX COMPRISING TYPE II COLLAGEN AND

USES THEREOF

FIELD OF THE INVENTION

[001] This disclosure relates to an extracellular matrix comprising jellyfish proteins, mainly Type II collagen, methods of making the compositions, methods of using the compositions for therapeutic purposes, and kits related to such compositions.

BACKGROUND OF THE INVENTION

[002] Most of the collagen matrices that are currently used on the market utilize bovine, porcine, or equine collagen, which pose the risk of zoonotic infections such as BSE or Mad Cow Disease, Hoof and Mouth Disease, Hog Cholera, Avian Flu and others (see References 1-7). Treating these infections is costly. There have been no reports related to transferring diseases to humans by using biomaterials derived from marine sources (1 ,4) mainly since most marine animals are invertebrates (9) and due to a large "species barrier" between marine creatures and humans (1). Jellyfish belong to the invertebrate phylum and are evolutionary and genetically distant from humans and other mammals (9). Jellyfish are relatively easy to clean, and do not require de-hairing, blood removal, or fat removal, as in animal skins, nor do they depend on animal or fish skin removal and de-lipidation.

[003] Most collagen matrices require the addition of cross-linking agents, which may induce cytotoxic effects as well as inflammatory and immune responses when applied on or within the human body (1). While non-cross-linked products can be associated with rapid revascularization without scar tissue formation and a low inflammatory or immunological response, they rapidly disintegrate in an active-wound environment, need to be replaced often, and slow down the wound-healing process (8). In addition, the development of collagen as a topically applied biomaterial requires enhanced stability. The native collagen triple-helix structure is likely an ideal substrate, as it is not rapidly degraded under physiological conditions. The degradation rate and mechanical properties of collagen can be manipulated via cross-linking and sterilization methods. Chemical cross-linking may increase stability; however, the literature has shown that chemically cross-linked collagen matrices result in inflammation, and the covalent bonds between the polymeric chains of these matrices may be cytotoxic when the material is degraded. Moreover, although cross-linking delays degradation, it has been shown to decrease the bioavailability of the matrix, to reduce cellular engraftment, and to prolong inflammation of the wound (10).

[004] Most available collagen matrices are made of a soluble form of collagen. Moreover, collagen matrices from jellyfish tissue have only been produced using solubilized collagen gels, which are generally not stable. They are also not thermally stable without the introduction of cross-linking agents, which can potentially have adverse effects for wound- dressing applications. Thus, the common treatment for wounds involves repeated replacement or addition of the wound dressing since most dressings are absorbed due to their solubility; this procedure causes pain, increases the possibility of morbidity and infection of the wound, demands time and human resources, and increases treatment costs.

[005] Collagen is a structural protein that is naturally present in almost every part of the human body and especially in skin and connective tissue. Collagen can be very effective as an occlusive agent in wound healing, as collagen has useful properties for wound treatment: it is homeostatic and enhances skin fibroblast proliferation (11). In wound healing and fibrosis, a variety of processes are crucial, such as inflammation, cell proliferation, cell migration, and extracellular matrix (ECM) remodeling. Two major cellular players in these processes are macrophages and fibroblasts (12). During the proliferation phase of wound healing, fibroblasts proliferate and migrate into the wound site to form granulation tissue (12). Fibroblasts are responsible for the creation of collagen in the skin, which is necessary to support cellular ingrowth and plays a critical role in wound healing (1 1-12). Thus, treatment with collagen can help the development of a supporting network in the wound for remodeling of new epidermal structures. A jellyfish matrix has been shown to increase the proliferation of dermal fibroblasts, which produce collagen preferentially than collagen derived from bovine (1 1). Therefore, the jellyfish collagen matrix would play an active role in remodeling the skin architecture during dermal- wound healing.

[006] Macrophages are another major player in wound healing and fibrosis (12).They exist in two opposite activation states— classically activated (Ml) and alternatively activated (M2) macrophages. The Ml macrophage is pro-inflammatory and is often associated with tissue injury and inflammation, whereas the M2 macrophage is associated with tissue repair and fibrosis (12). It has been suggested that a transition from a pro-inflammatory (Ml) phenotype to a more regulatory or anti-inflammatory M2 phenotype is a key aspect of tissue remodeling, which promotes functional outcomes as opposed to scar-tissue formation (13). The pro-inflammatory activity of Ml macrophages during the inflammatory phase is required for efficient pathogen control (13) and is associated with chronic inflammatory and foreign- body reactions (14). Conversely, the M2 macrophage phenotype is considered to be an immunomodulatory and tissue-remodeling phenotype (14). M2 macrophage activity during the late inflammatory phase is required for the resolution of inflammation and the recruitment of cells, which facilitate granulation-tissue formation and wound re-epithelization (13). In general, macrophages that invade the tissue in the inflammatory phase of wound healing adopt an Ml phenotype and give rise to fibroblasts with a pro-inflammatory and ECM-degrading profile. Under the direction of paracrine signals of Ml macrophages, fibroblasts contribute to a pro-inflammatory environment by secreting cytokines and chemokines (such as CCL2, CCL7, and IL6) in the inflammatory phase of wound healing. M2 macrophages induce fibroblast proliferation. Fibroblasts with an inflammatory phenotype (initiated by stimulation with secreted factors of Ml macrophages) can be reversed to an anti- inflammatory phenotype with secreted factors of M2 macrophages. In these fibroblasts, the previously upregulated proinflammatory cytokines, chemokines, and MMPs are completely downregulated after stimulation with paracrine signals from M2 macrophages. Therefore, in wound repair, it is thought that M2 macrophages are responsible for reversing the inflammatory response, thereby initiating the healing process (12).These M2 macrophage-depleted cutaneous wounds resemble chronic wounds typically associated with the pathogenesis of chronic venous ulcers (CVU) and diabetes. Failure of cutaneous wound macrophages to undergo the Ml-to-M2 phenotypic transition represents a hallmark of those chronic inflammatory diseases. Multiple studies have shown that ECM-based scaffold materials that are properly prepared facilitate a transition from an Ml to M2 phenotype around 7-14 days post-implantation. Moreover, if the material has been chemically cross-linked to prevent degradation, an extendedMl type immune response with no transition to an M2 response is observed, and the lack of this transition is associated with poor remodeling outcomes or encapsulation (13).

[007] In the 1960s and 1970s, several US and Canadian patents were issued to Battista and others, in which they disclosed that by using a particular concentration of acid with cattle skin collagen, and with strong agitation, it possible to obtain viscous collagen salts. The viscosity of these collagen salts remained stable over a period of several weeks. These patents include Canadian Patent Number 806621, entitled "Coatings of microcrystalline collagen," Canadian Patent Number 814301, entitled "Colloidal compositions and methods," Canadian Patent Number 856216, entitled "Foods, pharmaceuticals and cosmetics containing a salt of collagen," US Patent Number 3,628,974 entitled, "Microcrystalline collagen, an ionizable partial salt of collagen and foods, pharmaceuticals and cosmetics containing same," US Patent Number 3,742,955, entitled "Fibrous collagen derived product having hemostatic and wound binding properties," Canadian Patent Number 953054, entitled "Method of forming structures from microcrystalline collagen, " and Canadian Patent Number 964131 to Zeleznick, entitled "Microcrystalline collagen structures and method of preparing same. "

[008] All of the methods disclosed in the above-referenced patents disclose preparation of collagen from animal collagen sources. To date, although it is clear that matrices produced from jellyfish collagen would provide enhanced fibroblast proliferation and thus enhanced wound healing, there are no known wound dressings produced from jellyfish collagen. This is at least partially due to the lack of thermal stability and/or lack of resistance to protease activity in the absence of cross-linking agents. It would therefore be advantageous to have a method of preparation of a collagen-salt matrix from jellyfish, thus maximizing the benefits of jellyfish, while retaining a thermally stable collagen matrix without the need for adding cross-linking agents to the process. The method of preparation should be performed within a short period of time.

Table of Abbreviations

DM Diabetes Mellitus

DW Distilled water

JF Jellyfish

JFE Jellyfish extracts

JFPE Jellyfish protein extract

HP Hydroxyproline

OUS study Clinical study conducted outside the US

PBS Phosphate buffered saline

SUMMARY OF THE INVENTION

[009] This disclosure provides, in accordance with one embodiment of the invention, a method of producing a jellyfish collagen matrix that can be used for the treatment of wounds. The method includes providing a jellyfish tissue, cutting the jellyfish tissue to small pieces, desalinating the jellyfish tissue, centrifugation, and lyophilizing ground raw material. The method further comprises adding an acid to the jellyfish tissue to extract the collagen, dispersing the collagen solution to form a viscous gel, and forming a wound-dressing pad from the viscous gel.

[010] A short extraction procedure with acid and in the absence of cross-linking agents during the procedure results in unique features of the matrix. Although cross-linking materials are not added, the innovative matrix is stable and has shown increased resistance to degradation induced by in vivo enzymes (proteases) present in the wound. In addition, the innovative non-cross-linked matrix demonstrates the ability to not be degraded or absorbed rapidly, but rather to adhere and persist in the wound. This innovative non-cross-linked matrix can support cellular infiltration and proliferation including fibroblasts, which are essential for wound healing as the cells produce new collagen and proteins to heal the wound. Moreover, this innovative non-cross-linked matrix enables rapid transition of the wound from the inflammatory stage to the healing stage.

BRIEF DESCRIPTION OF THE DRAWINGS

[011] Figure 1 is a flowchart showing steps in a production process, consistent with disclosed embodiments.

[012] Figure 2 shows the morphology of JF collagen scaffolds at different protein concentrations without cells and seeded with cells, consistent with disclosed embodiments.

[013] Figure 3 shows the morphology of different collagen scaffolds 3 days after cell seeding, consistent with disclosed embodiments: (A) Jellyfish collagen of Coll- Med. (B) Fish skin collagen of Kolspan. (C) Bovine collagen of J&J. (D) Horse tendon collagen of Bio-Pad.

[014] Figure 4 shows the appearance of wounds treated with JF collagen pads (Coll- Med) or bovine collagen dressings (PROMOGRAN®) from different experiments, consistent with disclosed embodiments: (A) 7 days post-application. (B) 8 days post-application.

[015] Figure 5 shows adhesion and proliferation of fibroblasts on JF collagen scaffolds at different collagen concentrations, consistent with disclosed embodiments.

[016] Figure 6 shows comparisons of fibroblast proliferation on collagen scaffolds from different sources, consistent with disclosed embodiments. [017] Figure 7 shows the polarization indexes of the Coll-Med JF scaffold and three commercially available ECM scaffolds, which represent the tendency towards expression of Ml or M2 macrophages.

[018] Figure 8 shows a comparison of resistance to collagenase degradation after four hours of incubation for the Coll-Med JF scaffold ("#102") versus a commercially available scaffold made from type I equine collagen.

DETAILED DESCRIPTION OF THE INVENTION

[019] Consistent with disclosed embodiments, it has now been found that extraction of high-quality ECM derived from jellyfish collagen is possible by rapidly adding a volume of weak acid to lyophilized tissue taken from the three layers of the jellyfish umbrella. This acid addition step is completed on the order of minutes, rather than hours as previous groups have demonstrated. Patents related to producing collagen from marine sources, including jellyfish (Allard and Wolfinbarger), are based on extraction of collagen with acid for a long time, such as 3 days (Wolfinbarger) or 5-7 days (Allard), followed by salt precipitation (Wolfinbarger) or homogenization (Allard). The present disclosure teaches how to perform collagen extraction very quickly (approximately 5 min, or less).

[020] This disclosure teaches how to produce a collagen matrix, which would not require additional cross-linking agents or biochemical and genetic modifications to increase the stability. This innovative collagen matrix will keep its structure and will not shrink. Moreover, it will enable fibroblast proliferation as well as Ml and M2 macrophage response and therefore, will increase wound healing.

[021] By minimizing exposure time to the acid, and potentially, by using a weak rather than a strong acid, it has been found that less structural damage is suffered by the matrix. Matrices produced with this rapid acid addition protocol have been found to be more resistant to protease degradation, and to persist for longer time periods in wound healing applications than matrices and resultant wound dressings generated by other methods.

[022] Wound dressings may be made from the viscous ECM gel formed by the methods described herein. In clinical application, these wound dressings may be indicated for the treatment and management of a variety of wound types, including but not limited to partial and full-thickness wounds, pressure ulcers, venous ulcers, chronic vascular ulcers, diabetic ulcers, trauma wounds (including but not limited to abrasions, lacerations, second-degree burns, and skin tears), surgical wounds (including but not limited to donor sites/grafts, post- Mohs surgery, post-laser surgery, podiatric wounds, and wound dehiscence), and draining wounds.

EXAMPLES

Example 1: Method of making or formulating matrix— full procedure

[023] The following steps were used in formulating the matrix (Fig. 1):

[024] Storage of raw material: Jellyfish (JF) Umbrellas (U's) containing all three layers of tissue (the endoderm, ectoderm and the mesoglea)were cut into pieces and frozen at - 32°C in a freezer.

[025] Defrosting procedure: Pieces of JF U's were defrosted under running faucet water while maintaining a temperature of less than 10±2°C. Semi-defrosted U's pieces were transferred into large basins containing cold distilled water (DW) at approximately 4°C. The DW was exchanged three times until the U's were completely defrosted. The defrosted U's were further cut into 3 cm² pieces and transferred to a strainer to remove excess water. The weight of the initial defrosted U's was measured.

[026] The defrosted U's pieces were then divided between basins containing a large egg-shaped magnetic stirring bar. The basin was placed on a stirrer in a 4°C refrigerator.

[027] Desalination procedure: This step is performed for the purpose of removing salt from defrosted JF U's pieces. Desalination was performed by washing the U's 8 times in cooled DW, while stirring on a stirrer using a magnetic bar in a 4°C refrigerator. The distilled water volume required for each wash was determined according to the defrosted U's weight as measured in each basin (ratio of -200 ml DW for each lOOg of the initial defrosted U's). Salt level was estimated at the end of each wash (after 15 mins, 30 mins, 45 mins, 60 mins, 90 mins, and 105 mins) using C strips. If, after the last 105-min step, the salt level reached -500 (mg CIVIL), then the final DW volume was added and left to stir at 4 °C for approximately 16- 18 hours (overnight). If the salt level was still above 500 (mg CIVIL), then the water was exchanged and left for an additional 105 mins to stir at 4°C. The salt level was tested again, and this process was repeated until a level of ~500(mg CIVIL) was reached. Then, a final DW volume was added and left to stir at 4 °C for approximately 16-18 hours (overnight). At the end of the desalination procedure, the salt level should be no more than 500mg C17L.

[028] Draining procedure: This optional step is performed for the purpose of removing excess water and reducing the volume of the raw material. The desalinated JF U's pieces were combined, and their weight was measured. Then, desalinated JF U's pieces were transferred to a strainer, which was placed on a basin at 4°C, and left for draining. Every 30-40 minutes, the U's were hand-stirred to ease the draining process, and their weight was measured. The percent of the remaining dried weight was calculated from the original desalinated JF U's weight. Generally, the recommended percentage of the drained U's weight from initial defrosted U's weight should be between 35-50%. The percentage of the drained U's weight from the initial defrosted U's weight should be no less than 35% in order to avoid difficulties in the grinding procedure. If the drained U's percentage is lower than 35%, DW should be added to make JF U's weight equal to 35% of the initial defrosted U's weight.

[029] Grinding procedure: The drained JF U's pieces were transferred to a grinder and ground five times for 5 minutes for a total grinding time of 25 minutes. The material's temperature was measured after each grinding. Generally, the material's temperature should be no more than 15°C. Otherwise, the grinder was placed in a -32 °C freezer in order to decrease the material's temperature. Exposure of the U's in the freezer is minimized to avoid re- freezing. While in the freezer, the grinder vessel was shaken every 10 minutes to avoid peripheral material frosting.

[030] Centrifugation procedure— first round: The ground U's were transferred into pre-weighed centrifuge bottles (e.g., 250-ml bottles) and centrifuged at 13,000g (9,300 rpm) for 30 minutes at 4 °C. After centrifugation, the DW supernatant was collected. The salt level was estimated in the collected DW supernatant. Generally, the salt level in the supernatant should be less than 500 mg Cl/L. Otherwise, the pellets should be re-suspended in DW (e.g., 250 ml DW in each 250-ml centrifuge bottle) and centrifuged at 9,300 rpm (-13,000 g) for an additional 30 minutes at 4 °C. The weight of the centrifuge bottles containing the pellets was measured. The total weight of the pellets was calculated by subtracting the centrifuge bottle's weight from the final weight of the bottles plus the pellets.

[031] Feed-stock preparation: The U's pellets were resuspended in DW at a ratio of 2.5ml DW for each lg of pellet by vortexing. The suspension was transferred to a grinder and re-ground once for 5 minutes. The ground U's, now named "feed stock," were recast into dishes (e.g., 40ml feed stock for each 100-mm petri dish), and the dishes containing the feed stock were placed on lyophilizer racks and frozen at -32°C for no less than about 6 hours.

[032] Freeze drying (lyophilization) procedure for feed stock— first round: After casting and freezing, the feed stock was freeze-dried for a total of 30 hours. The dishes containing the lyophilized material, named "Freeze Dried Feed Stock" (FDFS), were stored in a parafilm-wrapped box overnight at room temperature. The FDFS was stored at room temperature for no more than 2 weeks before preparing insoluble gels.

[033] Re-suspension of FDFS in DW: The FDFS was weighed. Depending on the required concentration, the FDFS was suspended in a known volume of DW. Generally, in order to obtain a low protein concentration gel (0.7%), 100 ml of pre-cooled DW was added to every 0.7g of FDFS. If a total volume of about 1,000 ml or less is desired, no more than about 5.6 g of FDFS should be used per one grinder. The weighed FDFS was cut and placed in each grinder. Depending on the required concentration, DW was added in accordance to the weight of the FDFS, and the material was blended for 30 sees to achieve a homogenous mixture. The mixture was incubated from about 4 hours up to overnight at 4°C for re-hydration of insoluble particles. Alternatively, the mixture was not incubated at all.

[034] Extraction of JF collagen procedure: Protein extraction from the re-hydrated FDFS/DW mixture was achieved by gradually adding a weak acid (for example, glacial acetic acid (GHAc)) to the FDFS/DW mixture while maintaining a constant increase rate in the acid concentration within 5 minutes of addition, starting from the final concentration divided by 5 until obtaining the final and desired concentration of acetic acid. Therefore, the total volume of acid to be added to the total volume of FDFS/DW ground solution should be divided by 5. The result of the division should be added within 1 minute allowing the maintenance of acid concentration increase rate during the 5 minutes of acid addition. For example, if the desired acid concentration is 0.6M then a total volume of 3.5ml of a weak acid should be added to every 100ml of the FDFS/DW mixture while grinding. In order to gradually increase the concentration until reaching the desired 0.6M of weak acid, a volume of 0.7ml weak acid (3.5ml divided by 5) was gradually added every 1 minute resulting in a total addition time of 5 minutes. The FDFS/DW mixture was ground for at least about 30 seconds, using a grinder. A calculated volume of the acid was gradually added to the FDFS/DW mixture within 5 minutes, while the grinder was working, until obtaining the desired concentration of acetic acid in the FDFS/DW mixture. The tables below show examples of how the acid should be added to FDFS/DW mixtures that are 100 ml and 500 ml in volume throughout a 5-minute duration in order to obtain a final 0.6M concentration. At the end of the acid addition procedure, a white solution gel was formed. Table 1. Acid addition protocol for 100 ml volume, final acid concentration 0.6M

Note - The desired acid concentration at each time point is the final concentration divided by 5. As a result the volume of acid to be added every minute is the final volume divided by 5. Therefore, in case the desired concentration of the weak acid is 0.1M the acid will be added gradually as presented in the table below. Table 3. Acid addition protocol for 100 ml volume, final acid concentration 0.1M

[035] Dispersing procedure: A disperser was prepared by placing an empty beaker in a dish containing distilled water and ice cubes, at a distance (e.g., 2cm) from the bottom of the disperser blade. The milky gel was transferred (e.g., about 500 ml at a time) to a separate glass beaker. The beaker was placed in a cooling basin (containing water and ice cubes) under the disperser shaft to which a blade (e.g., 4-cm-diameter, saw-toothed blade) was attached at a distance (e.g., 2 cm) from the beaker's bottom. Each milky gel volume (e.g., about 500 ml)was dispersed for 15 minutes at from about 4,500 to about 4,700 rpm to obtain a viscous, milky gel. All of the received gel was combined together.

[036] Centrifugation procedure— second round: The milky gel was poured into centrifuge bottles and centrifuged at 13,000g (9,300 rpm) for 45 minutes at 4°C. A clear, homogeneous, viscous hydrogel was obtained. After centrifugation, all hydrogel was transferred into a glass bottle and stored at 4°C. All the remaining small un-extracted material pellets from the centrifuge bottles were discarded. Prior to insoluble collagen scaffold preparation, the hydrogel' s total protein concentration and viscosity were tested.

[037] Preparation of JF collagen scaffolds: The hydrogel was cast into dishes (e.g., 10 ml hydrogel per 60-mm petri dish), and placed on a lyophilizer rack for freezing at -32°C for no less than 6 hours. [038] Freeze drying (lyophilization) procedure for hydrogel— second round: The hydrogel was freeze-dried for a total of 30 hours. Finally, the petri dishes containing the lyophilized hydrogel (named scaffolds in general) were taken out and separately weighed. Then, each scaffold was placed into a sterilization envelope and stored at room temperature until sterilized by ethylene oxide (EtO).

Example 2: Method of making or formulating matrix without dispersing procedure

[039] Storage of the raw material is carried out as in Example 1.

[040] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[041] The extraction of JF collagen procedure is performed as in Example 1. Then, the centrifugation procedure— second round is performed as in Example 1 without performing the dispersing procedure (Fig 1 , arrow with circles).

[042] Preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1.

Example 3: Method of making or formulating matrix without centrifugation procedure— second round

[043] Storage of the raw material is carried out as in Example 1.

[044] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[045] The extraction of JF collagen and dispersing procedures are performed as in Example 1. The centrifugation procedure— second round is not performed (Fig 1 , arrow with triangles).

[046] Preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1. Example 4: Method of making or formulating matrix without the first lyophilization procedure

[047] Storage of the raw material is carried out as in Example 1.

[048] The defrosting, desalination, draining, grinding, and centrifugation are performed as in Example 1.

[049] The pellet of the desalted ground JF is resuspended in DW depending on the required concentration and transferred to a grinder and re-ground once for 5 minutes.

[050] The pellet suspension is used immediately for extraction with acid (Fig 1 , arrow with diamonds).

[051] The extraction of JF collagen from the pellet/DW suspension is performed as in Example 1 with the same ratio of acid and DW.

[052] The dispersing and centrifugation procedures are performed as in Example 1.

[053] The preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1.

Example 5: Method of making or formulating matrix with rapid acid addition or addition of dilute acid

[054] In one embodiment, storage of the raw material is carried out as in Example 1.

[055] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[056] The extraction of JF collagen from the pellet/DW suspension is performed as in Example 1 with the same ratio of acid and DW, but the acid (at concentration 0.6M), is added all at once rather than gradually. After the rapid acid addition, the extracted collagen was ground for 5 min.

[057] The dispersing and centrifugation procedures are performed as in Example 1.

[058] The preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1. [059] In another embodiment, storage of the raw material is carried out as in Example

1.

[060] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[061] The extraction of JF collagen from the pellet/DW suspension is performed as in Example 1 , but the acid is at a concentration of 0.01M rather than 0.6M. As in Example 1 , the 0.01M acid is added gradually over 5 min, and may be added during grinding or without grinding.

[062] The dispersing and centrifugation procedures are performed as in Example 1.

[063] The preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1.

[064] In yet another embodiment, storage of the raw material is carried out as in Example 1.

[065] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[066] The extraction of JF collagen from the pellet/DW suspension is performed as in Example 1, but the acid is at a concentration of 0.1M rather than 0.6M. As in Example 1 , the 0.1M acid is added gradually over 5 min, and may be added during grinding or without grinding.

[067] The dispersing and centrifugation procedures are performed as in Example 1.

[068] The preparation of JF collagen scaffolds and freeze-drying (lyophilization) for the hydrogel— second round procedures are performed as in Example 1.

[069] JF collagen scaffolds obtained by these three variant methods had similar appearance and properties to scaffolds produced by the methods of Example 1, as shown below.

Table 4. Comparison of properties of JF scaffolds produced by various methods

Acetic acid 0.6M 0.6M 0.1M 0.01M concentration

Acid addition Gradually at once Gradually Gradually

Appearance proper proper proper Proper with some uniform tendency to sink by hand pressure

Pad's weight 57.3mg/pad 61.3mg/pad 65.6mg/pad 62.2mg/pad

Gel's pH 2 2 3 4

Gel's viscosity

1130 1260 1580 1760

Protein 4.67 mg/ml 6.24 mg/ml 5.54 mg/ml 4.91 mg/ml concentration

Example 6: Method of making or formulating a non-lyophilized material (hydrogel)

[070] Storage of the raw material is carried out as in Example 1.

[071] The defrosting, desalination, draining, grinding, centrifugation, feed stock preparation, first round of freeze drying, and re-suspension of the FDFS in DW procedures are performed as in Example 1.

[072] The extraction of JF collagen procedure is performed as in Example 1.

[073] Dispersing and centrifugation procedure- second round are performed as in Example 1 , resulting in a hydrogel.

[074] In this embodiment, "freeze-drying (lyophilization) of the hydrogel-second round" procedures are not performed (see Fig. 1). Thus, preparation of JF collagen scaffolds proceeds as in Example 1 , but with a hydrogel as the final product instead of a lyophilized substance cast into pads. The hydrogel may be useful for topical application, and may be used in medical treatment, for skin care, or as a cosmetic product. Example 7: Stability of the structure of JF collagen scaffold

[075] JF collagen scaffolds with different protein concentrations were seeded with fibroblasts or were left unseeded. Morphology of the scaffold was monitored by evaluating the shrinkage of the scaffold. In medium concentrations of 4.2 mg/ml and 6.2 mg/ml, the hydrated collagen showed only moderate shrinkage after incubation with the medium; in a medium concentration of 5.2 mg/ml, the hydrated collagens lightly shrank when blotted dry following one day of cell loading and MTS assay or in unloaded scaffolds (Fig. 2). Shrinkage of scaffolds was not dependent on the collagen concentration.

Example 8: Stability of the structure of JF collagen scaffolds compared to collagen scaffolds from different sources

[076] JF collagen scaffolds were compared with collagen scaffolds from other sources— fish skin collagen (Kolspon™ sponge, Eucare Pharmaceuticals (P) Ltd.), bovine collagen (PROMOGRAN®, Systagenix), and horse tendon collagen (BIOPAD® wound dressing, Euroresearch S.R.L.)— for the ability to maintain their structure after they were seeded with cells and exposed to liquid. The scaffolds' ability to maintain their structures was measured up to seven days post-cell seeding. Figure 3 summarizes the morphology of scaffolds three days post-cell seeding. Kolspon (Fig. 3B) and PROMOGRAN (Fig. 3C) dressing scaffolds shrank significantly; the Bio-Pad dressing scaffold (Fig. 3D) also shrank, but to a lesser extent than others. In contrast, the JF collagen scaffold (Fig. 3A) appeared not to have shrunk at all. At seven days post-cell seeding, the JF collagen scaffold still maintained its structure.

Example 9: Stability of JF collagen pad during second-degree burn treatment

[077] The stability of JF collagen pads was compared to that of a bovine collagen dressing (PROMOGRAN®, Systagenix) for second-degree burn treatment. A pig model was chosen for this study because of the similarities between pig skin and human skin (15-19). After proper sedation, the animal's back was shaved, and its skin was disinfected. Five symmetrical partial thickness wounds were made on each side of the pig's back (10 wounds total) running from shoulders to rump. The wounds were made using a 2-cm-diameter metal bar heated in boiling water (100°C), dried and applied on the pig's back for 7 or 10 seconds. Following debridement, each wound was either treated with JF collagen pads, dipped in phosphate buffered saline prior to application, or with bovine-collagen dressings (PROMOGRAN®, Systagenix), dipped in saline. The PROMOGRAN® dressings were absorbed/dissolved by the wounds so that additional dressings were added on days 3 and 7 after the initial application. However, the JF collagen pads adhered to the wound area but not to the healthy surrounding tissue, and they were not dissolved by the wounds. Therefore, there was no need to add to or change the dressing until it fell off as a scab when the wound healed (Fig. 4).

Example 10: Adhesion and proliferation of fibroblasts on JF collagen scaffolds at different collagen concentrations

[078] Human foreskin fibroblast cells were seeded on JF collagen scaffolds (50,000 or 100,000 cells were loaded onto each scaffold). Cell proliferation on JF collagen scaffolds of different concentrations was evaluated by an MTS assay on days 1 , 4, 7 and 11 (Fig. 5). The results indicate that the JF collagen scaffold can support fibroblast proliferation. Thus, JF collagen scaffolds could be useful in wound healing, especially in cases such as third-degree burns and chronic wounds.

Example 11 : Proliferation of human fibroblasts on collagen from different sources

[079] Human fibroblast cells were seeded on different collagen scaffolds for 3 days and assayed for cell proliferation using Ki-67 staining. The highest proliferation levels were observed on JF collagen scaffolds (Fig. 6).

Example 12: Expression of Ml and M2 macrophages on JF collagen scaffolds compared to commercial ECM scaffolds

[080] CD14⁺ cells were isolated and characterized from human peripheral blood (PB) obtained from the New York Blood Center (leukopack). PB-derived CD14⁺cells were differentiated towards macrophage linage using established methods. Ml , M2a, and M2c macrophages were polarized using established methods as controls for gene-expression analysis. JF collagen scaffolds (Coll-Med) and commercially available scaffolds were glued using fibrin clots to the bottom of 48 -well plates and seeded at 0.5 x 10⁶ macrophages per scaffold (n = 3 per scaffold, 2 trials). Seeded macrophages were cultured for 2 and 7 days under standard culture conditions. Cells were harvested at 2 and 7 days of culture, and their RNA was isolated. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on the isolated RNA for the following genes, which were selected based on historical data from the lab: CD206 (MRC1), CD163, CCL22, CCL17, TNF-a, CCR7, HLA- DR, MMP9, and GAPDH. The qRT-PCR results were analyzed using the comparative cycle threshold (CT) method. ACT was obtained by normalizing CT values to the housekeeping gene, GAPDH, and AACT was obtained by normalizing to the internal control (MO for the polarization results, and tissue culture plastic (TCP) for the scaffolds). Ml and M2 indexes were calculated. Puracol and PROMOGRAN demonstrated significantly stronger in vitro Ml responses rather than an M2 response (Fig. 7). Coll-Med demonstrated the same response for Ml and M2.

Example 13: CoUagenase resistance of JF collagen scaffolds compared to commercial ECM scaffolds

[081 ] The objective of this assay was to compare the stability of JF collagen scaffolds (Coll-Med) to that of scaffolds derived from horse tendon type I collagen (BIOPAD® wound dressing, Euroresearch S.R.L.) via a collagenase resistance assay. 1.5mg punches of JF collagen scaffold and BIOPAD® scaffold were weighed and transferred into 1.7ml Eppendorf tubes. A volume of 1.425 ml of reaction buffer (lOmM Tris + 25 mM CaC^) was added to each tube, and the tubes were incubated in a water bath at 37 °C for 30 min. After incubation, 75 μΐ of reaction buffer containing 1 12.5 units of collagenase type VII (Sigma) were added to reach a ratio of 75 units collagenase per lmg of scaffold. Tubes were then incubated again under the same conditions as previously described. After four hours of incubation, samples were mixed and centrifuged (30 seconds, 10,000 rpm). The supernatants (comprising the scaffold proteins digested by the collagenase) were collected, and the pellets (comprising the undigested scaffolds) were washed with DW. The pellets were dissolved in 1.5 ml of 0.5M NaOH for 15 min at 70°C. Both the supernatants and the dissolved pellets were diluted 1 : 10 in DW and tested for protein concentration using the Lowry method. A collagenase resistance

Pvoteitx in. pellet

rate (CRR) was calculated according to the formula X 100. After four

Protein in (sup +pellet)

hours of incubation with the collagenase, 61% of the JF collagen scaffold (from batch #102) was not degraded, while only 22% of the BIOPAD® scaffold remained intact (Fig. 8). These results indicate ECM scaffolds made from jellyfish collagen are highly stable and resistant to protease degradation. References

US Patents

Foreign Patents

CA806621 Orlando A. Battista "Coatings of microcrystalline collagen"

CA814301A Orlando A. Battista "Colloidal compositions and methods"

CA856216A Orlando A. Battista "Foods, pharmaceuticals and cosmetics containing a salt of collagen"

CA953054A1 Orlando A. Battista "Method of forming structures from microcrystalline collagen"

CA964131 A Lowell D. Zeleznick "Microcrystalline collagen structures and method of preparing same"

Other References

1. Matsumoto Y, Ikeda K, Yamaya Y, Yamashita K, Saito T, Hoshino Y, Koga T, Enari H, Suto S, Yotsuyanagi T. The usefulness of the collagen and elastin sponge derived from salmon as an artificial dermis and scaffold for tissue engineering. Biomed Res 2011, 32(l):29-36.

2. Pang S, Chang YP, Woo KK. The evaluation of the suitability of fish wastes as a source of collagen. International conference on nutrition and food sciences. 2013, 53(15):77-81.

3. Lee KJ, Park HY, Kim YK, Park JI, Yoon HD. Biochemical characterization of collagen from the starfish Asterias amurensis. J Korean Soc. Chem. 2009, 52(3):221-226

4. Nagai T, Suzuki N, Tanoue Y, Kai N, Nagashima T. Characterization of Acid-Soluble Collagen from Skins of Surf Smelt {Hypomesuspretiosus japonicus Brevoort). Food and Nutrition Sciences. 2010, 1 : 59-66

5. Ogawa M, Portier RJ, Moody MW, Bell J, Schexnayder MA, Losso JN. Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonias cromis) and sheepshead sea bream {Archosargus probatocephalus). Food

Chem2004, 88:495-501.

6. Krishnan S, Perumal P. Preparation and biomedical characterization of jellyfish (Chrysaora quinquecirrha) collagen from southeast coast of India. Int J Pharm PharmSci 2013, 5(3):698-701.

7. Sudharsan S, Seedevi P, Saravanan R, Ramasamy P, Kumar SV, Vairamani S, Srinivasan A Shanmugam A. Isolation, characterization and molecular weight determination of collagen from marine sponge Spirastrella inconstans (Dendy). African Journal of Biotechnology 2013, 12(5):504-511.

8. Acellular matrices for the treatment of wounds. Wounds International (2011). http://www.woundsinternational.com/pdf/content_9732.pdf

9. Addad S, Exposito J-Y, Faye C, Ricard-Blum S, Lethias C. Isolation, Characterization and Biological Evaluation of jellyfish collagen for Use in Biomedical Applications. Mar Drugs 2011, (9):967-83.

10. Gould LJ. Topical collagen-based biomaterials for chronic wounds: rationale and clinical application. Advances in wound care 2014, 5 (1): 19-31. 11. Song E, Kim SY, Chun T, Byun H-J, Lee YM. Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials 2006, (27): 2951-61.

12. Ploeger D-TA, Hosper N-A, Schipper M, Koerts J-A, Rond S and Bank R-A. Cell plasticity in wound healing: paracrine factors of Ml/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts. Cell Communication and Signaling 2013,

(11,29): 1 -17.

13. Brown BN., SicariBM. and Badylak SF.Rethinking regenerative medicine: a macrophage - centered approach. Frontiers in immunology 2014, (5), article 510: 1-11.

14. Valentin JE., B-S.. Stewart-Akers A-M., Gilbert T-W., and Badylak S-F., D.V.M.

Macrophage participation in the degradation and remodeling of ECM scaffolds. Tissue

Engineering 2009, part A, 15 (00): 1 -8.

15. Middelkoop E, Bogaerdt AJ„ Lamme EN, Hoekstra MJ, Brandsma K, Ulrich MMW.

Porcine wound models for skin substitution and burn treatment. Biomaterials 2004:25: 1559-1567

16. Singer AJ, McClain SA, Taira BR, Romanov A, Rooney J, Zimmerman T. Validation of a porcine comb burn model. Am J Emerg Med 2009;27:285-8.

17. Singer AJ, McClain SA. A porcine burn model. Methods Mol Med 2003;78:107-19.

18. Singer AJ, Taira BR, Anderson R, McClain SA, Rosenberg L. Does pressure matter in creating burns in a porcine model. J Burn Care Res 2010;31 (4):646-51.

19. Singer AJ, Berruti L, Thode HC, McClain SA. Standardized Burn Model Using a Multiparametric Histologic Analysis of Burn Depth. Acad. Emer. Med. 2000:7:1 -6.

Claims

1. A method of producing a biological matrix, comprising:

cutting jellyfish umbrella tissue into pieces;

freezing the jellyfish umbrella tissue;

thawing the jellyfish umbrella tissue;

desalting the jellyfish umbrella tissue;

grinding the jellyfish umbrella tissue;

centrifuging the jellyfish umbrella tissue to obtain a pellet;

resuspending the pellet to obtain a suspension;

casting the suspension to obtain a first cast material;

lyophilizing the first cast material to obtain a lyophilized material;

weighing the lyophilized material;

resuspending the lyophilized material to obtain a suspension;

adding a weak acid to form a suspension gel;

dispersing the suspension gel to obtain a viscous gel;

centrifuging the viscous gel to yield a hydrogel;

casting the hydrogel to obtain a second cast material; and

lyophilizing the second cast material to form the biological matrix.

2. The method of claim 1 , wherein the addition of the weak acid is performed gradually over a period of no more than five minutes.

3. The method of claim 1 , wherein the addition of the weak acid is performed all at once.

4. The method of any of claims 1 -3, wherein the addition of the weak acid is performed while grinding.

5. The method of any of claims 1-3, wherein the step of desalting the jellyfish umbrella tissue further comprises:

washing the jellyfish umbrella tissue while stirring the jellyfish umbrella tissue at 4°C; testing the salt level of the jellyfish umbrella tissue; and

repeating the washing and testing steps until the salt level is no more than about 500 mg C17L.

6. The method of any of claims 1-3, further comprising:

transferring the jellyfish umbrella tissue to a strainer at 4°C;

stirring the jellyfish umbrella tissue;

weighing the jellyfish umbrella tissue; and

repeating the stirring and weighing steps until the weight of the jellyfish umbrella tissue is between about 35% and about 50% of the weight of the jellyfish umbrella tissue immediately after the defrosting step.

7. The method of any of claims 1-3, wherein the step of resuspending the lyophilized material comprises adding about 100 ml of distilled water for every 0.7 g of the lyophilized material to obtain a solution with a desired concentration.

8. The method of any of claims 1-3, wherein the weak acid is glacial acetic acid.

9. The method of any of claims 1-3, further comprising sterilizing the biological matrix with ethylene oxide.

10. The method of any of claims 1-3, wherein the jellyfish umbrella tissue is cut into pieces approximately 3 cm² in size.

11. A wound dressing comprising the biological matrix formed by any of the methods of claims 1 -10.

12. A method of treating a wound, comprising administering to a subject in need thereof the wound dressing of claim 11.

13. The method of claim 12, wherein the wound is selected from the group consisting of burns, partial wounds, full-thickness wounds, pressure ulcers, venous ulcers, chronic vascular ulcers, diabetic ulcers, trauma wounds, abrasions, lacerations, skin tears, surgical wounds, donor site/graft wounds, post-Mohs surgery wounds, post-laser surgery wounds, podiatric wounds, wound dehiscence, and draining wounds.

14. The method of any of claims 1-3, wherein the biological matrix is resistant to protease degradation for one day.

15. The method of any of claims 1-3, wherein the biological matrix is resistant to protease degradation for three days.

16. The method of any of claims 1-3, wherein the biological matrix is resistant to protease degradation for five days.

17. The method of any of claims 1-3, wherein the biological matrix is resistant to protease degradation for seven days.

18. A method of producing a biological matrix, comprising:

cutting jellyfish umbrella tissue into pieces;

freezing the jellyfish umbrella tissue;

thawing the jellyfish umbrella tissue;

desalting the jellyfish umbrella tissue;

grinding the jellyfish umbrella tissue;

centrifuging the jellyfish umbrella tissue to obtain a pellet;

resuspending the pellet to obtain a suspension;

casting the suspension to obtain a first cast material;

lyophilizing the first cast material to obtain a lyophilized material;

weighing the lyophilized material;

resuspending the lyophilized material to obtain a suspension;

adding a weak acid to form a suspension gel;

centrifuging the viscous gel to yield a hydrogel;

casting the hydrogel to obtain a second cast material; and

lyophilizing the second cast material to form the biological matrix.

19. The method of claim 18, wherein the addition of the weak acid is performed gradually over a period of no more than five minutes.

20. The method of claim 18, wherein the addition of the weak acid is performed all at once.

21. The method of any of claims 18-20, wherein the addition of the weak acid is performed while grinding.

22. The method of any of claims 18-20, wherein the step of desalting the jellyfish umbrella tissue further comprises:

23. The method of any of claims 18-20, further comprising:

transferring the jellyfish umbrella tissue to a strainer at 4°C;

stirring the jellyfish umbrella tissue;

weighing the jellyfish umbrella tissue; and

24. The method of any of claims 18-20, wherein the step of resuspending the lyophilized material comprises adding about 100 ml of distilled water for every 0.7 g of the lyophilized material to obtain a solution with a desired concentration.

25. The method of any of claims 18-20, wherein the weak acid is glacial acetic acid.

26. The method of any of claims 18-20, further comprising sterilizing the biological matrix with ethylene oxide.

27. The method of any of claims 18-20, wherein the jellyfish umbrella tissue is cut into pieces approximately 3 cm² in size.

28. A wound dressing comprising the biological matrix formed by any of the methods of claims 18-27.

29. A method of treating a wound, comprising administering to a subject in need thereof the wound dressing of claim 28.

30. The method of claim 29, wherein the wound is selected from the group consisting of burns, partial wounds, full-thickness wounds, pressure ulcers, venous ulcers, chronic vascular ulcers, diabetic ulcers, trauma wounds, abrasions, lacerations, skin tears, surgical wounds, donor site/graft wounds, post-Mohs surgery wounds, post-laser surgery wounds, podiatric wounds, wound dehiscence, and draining wounds.

31. The method of any of claims 18-20, wherein the biological matrix is resistant to protease degradation for one day.

32. The method of any of claims 18-20, wherein the biological matrix is resistant to protease degradation for three days.

33. The method of any of claims 18-20, wherein the biological matrix is resistant to protease degradation for five days.

34. The method of any of claims 18-20, wherein the biological matrix is resistant to protease degradation for seven days.

35. A method of producing a biological matrix, comprising:

cutting jellyfish umbrella tissue into pieces;

freezing the jellyfish umbrella tissue;

thawing the jellyfish umbrella tissue;

desalting the jellyfish umbrella tissue;

grinding the jellyfish umbrella tissue;

centrifuging the jellyfish umbrella tissue to obtain a pellet;

resuspending the pellet to obtain a suspension;

casting the suspension to obtain a first cast material;

lyophilizing the first cast material to obtain a lyophilized material;

weighing the lyophilized material;

resuspending the lyophilized material to obtain a suspension;

adding a weak acid to form a suspension gel;

dispersing the suspension gel to obtain a viscous gel;

casting the viscous gel to obtain a second cast material; and

lyophilizing the second cast material to form the biological matrix.

36. The method of claim 35, wherein the addition of the weak acid is performed gradually over a period of no more than five minutes.

37. The method of claim 35, wherein the addition of the weak acid is performed all at once.

38. The method of any of claims 35-37, wherein the addition of the weak acid is performed while grinding.

39. The method of any of claims 35-37, wherein the step of desalting the jellyfish umbrella tissue further comprises:

40. The method of any of claims 35-37, further comprising:

transferring the jellyfish umbrella tissue to a strainer at 4°C;

stirring the jellyfish umbrella tissue;

weighing the jellyfish umbrella tissue; and

41. The method of any of claims 35-37, wherein the step of resuspending the lyophilized material comprises adding about 100 ml of distilled water for every 0.7 g of the lyophilized material to obtain a solution with a desired concentration.

42. The method of any of claims 35-37, wherein the weak acid is glacial acetic acid.

43. The method of any of claims 35-37, further comprising sterilizing the biological matrix with ethylene oxide.

44. The method of any of claims 35-37, wherein the jellyfish umbrella tissue is cut into pieces approximately 3 cm in size.

45. A wound dressing comprising the biological matrix formed by any of the methods of claims 35-44.

46. A method of treating a wound, comprising administering to a subject in need thereof the wound dressing of claim 45.

47. The method of claim 46, wherein the wound is selected from the group consisting of burns, partial wounds, full-thickness wounds, pressure ulcers, venous ulcers, chronic vascular ulcers, diabetic ulcers, trauma wounds, abrasions, lacerations, skin tears, surgical wounds, donor site/graft wounds, post-Mohs surgery wounds, post-laser surgery wounds, podiatric wounds, wound dehiscence, and draining wounds.

48. The method of any of claims 35-37, wherein the biological matrix is resistant to protease degradation for one day.

49. The method of any of claims 35-37, wherein the biological matrix is resistant to protease degradation for three days.

50. The method of any of claims 35-37, wherein the biological matrix is resistant to protease degradation for five days.

51. The method of any of claims 35-37, wherein the biological matrix is resistant to protease degradation for seven days.

52. A method of producing a biological matrix, comprising:

cutting jellyfish umbrella tissue into pieces;

freezing the jellyfish umbrella tissue,

thawing the jellyfish umbrella tissue;

desalting the jellyfish umbrella tissue;

grinding the jellyfish umbrella tissue;

centrifuging the jellyfish umbrella tissue to obtain a pellet;

resuspending the pellet to obtain a suspension;

adding a weak acid to form a suspension gel;

dispersing the suspension gel to obtain a viscous gel;

centrifuging the viscous gel to yield a hydrogel;

casting the hydrogel to obtain a cast material; and

lyophilizing the cast material to form a biological matrix.

53. The method of claim 52, wherein the addition of the weak acid is performed gradually over a period of no more than five minutes.

54. The method of claim 52, wherein the addition of the weak acid is performed all at once.

55. The method of any of claims 52-54, wherein the addition of the weak acid is performed while grinding.

56. The method of any of claims 52-54, wherein the step of desalting the jellyfish umbrella tissue further comprises:

57. The method of any of claims 52-54, further comprising:

transferring the jellyfish umbrella tissue to a strainer at 4°C;

stirring the jellyfish umbrella tissue;

weighing the jellyfish umbrella tissue; and

58. The method of any of claims 52-54, wherein the step of resuspending the lyophilized material comprises adding about 100 ml of distilled water for every 0.7 g of the lyophilized material to obtain a solution with a desired concentration.

59. The method of any of claims 52-54, wherein the weak acid is glacial acetic acid.

60. The method of any of claims 52-54, further comprising sterilizing the biological matrix with ethylene oxide.

61. The method of any of claims 52-54, wherein the jellyfish umbrella tissue is cut into pieces approximately 3 cm in size.

62. A wound dressing comprising the biological matrix formed by any of the methods of claims 52-61.

63. A method of treating a wound, comprising administering to a subject in need thereof the wound dressing of claim 62.

64. The method of claim 63, wherein the wound is selected from the group consisting of burns, partial wounds, full-thickness wounds, pressure ulcers, venous ulcers, chronic vascular ulcers, diabetic ulcers, trauma wounds, abrasions, lacerations, skin tears, surgical wounds, donor site/graft wounds, post-Mohs surgery wounds, post-laser surgery wounds, podiatric wounds, wound dehiscence, and draining wounds.

65. The method of any of claims 52-54, wherein the biological matrix is resistant to protease degradation for one day.

66. The method of any of claims 52-54, wherein the biological matrix is resistant to protease degradation for three days.

67. The method of any of claims 52-54, wherein the biological matrix is resistant to protease degradation for five days.

68. The method of any of claims 52-54, wherein the biological matrix is resistant to protease degradation for seven days.

69. A biological matrix, comprising a cast, lyophilized collagen gel derived from tissue from the endoderm, ectoderm and the mesoglea layers of jellyfish umbrellas,

wherein the lyophilized collagen gel is formed by the addition of a weak acid to the jellyfish tissue.

70. The biological matrix of claim 69, wherein the weak acid is gradually added to the jellyfish tissue over a period of no more than five minutes.

71. The biological matrix of claim 69, wherein the weak acid is added to the jellyfish tissue all at once.

72. The biological matrix of any of claims 69-71 , wherein the weak acid is added to the jellyfish tissue while grinding the jellyfish tissue.

73. The biological matrix of any of claims 69-71, wherein the jellyfish tissue is desalted prior to addition of the weak acid.

74. The biological matrix of any of claims 69-71, wherein the jellyfish tissue is lyophilized prior to addition of the weak acid.

75. The biological matrix of any of claims 69-71, wherein the weak acid is glacial The method of any of claims 3, 20, 37, 54 and 71 , further comprising grinding