Highly porous gelatin based foam doped with super absorbent polymers and method of preparation thereof

Abstract

A foam comprising starch, gelatin and carboxymethylcellulose having advantageous absorbency for blood, saliva or other fluids and to a method using mechanical mixing to produce the foam and to methods of using the foam for hemostasis and wound repair.

Description

[0001] Highly porous gelatin based foam doped with super absorbent polymers and method of preparation thereof

[0002] This application claims priority to US Provisional Patent Application No.

[0003] 63 / 527,055, filed on July 16, 2023, the entire contents of which are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The invention pertains to the fields of medicine and foam sciences especially to absorbent foams suitable for treating wounds and absorbing blood and other fluids.

[0006] BACKGROUND OF THE INVENTION

[0007] Bleeding is a significant factor in causing death in cases of traumatic injuries. It is crucial to halt bleeding and promote faster blood clotting than what the body can achieve naturally. In the context of emergency care, the term "Golden Hour" highlights the vital first 60 minutes after a serious injury. Such injuries often lead to significant bleeding from the wound site. Acting quickly to control this bleeding can mean the difference between life and death. Bleeding is the biggest threat to survival in the first day after a serious injury, causing 39% of deaths in civilian trauma cases, many occurring before patients reach the hospital. In military situations, severe injuries with bleeding are the leading cause of death among soldiers. Most of these deaths (about 90%) are due to internal bleeding that can't be controlled with standard methods like applying pressure. Currently, first responders have few options beyond giving fluids to manage bleeding in critical areas like the abdomen, chest, or neck. However, giving fluids can sometimes make bleeding worse by diluting the blood's ability to clot. Even for patients who make it to the operating room alive, stopping bleeding from these serious injuries is a major challenge for surgeons, despite having advanced tools and techniques. Thus, there is a pressing need for innovative highly absorbing hemostatic agents to expedite the process of bleeding control and blood clotting.

[0008] These hemostatic agents promote blood clotting, thereby stopping or controlling bleeding. Various hemostatic agents are used, with different effectiveness and levels of evidence supporting their benefits. Some polysaccharides and proteins can also serve as hemostatic agents due to their non-toxic nature and easy absorption by the body. These agents promote stable and rapid clotting due to their long chain length and branching structure.

[0009] In recent years, several polysaccharide and protein-based hemostatic agents have been approved for use. These include CMC -based agents, gelatin-based agents, and starch-based agents. These agents have shown promising results in controlling severe hemorrhage both in pre-hospital settings and animal models.

[0010] Gelatin-based hemostatic agents, such as Gelfoam®, have been widely used in modern operating rooms since the 1940s. Gelfoam®, made from purified porcine skin, utilizes the physical properties of gelatin to promote hemostasis rather than directly affecting the clotting cascade. Gelfoam®, can be used in various forms, either as a dry sponge, moistened with injectable sodium chloride solution, or more commonly, saturated with topical purified thrombin.

[0011] An important challenge with currently available sponges / foams is the requirement of prewetting before application, as well as their limited absorption capacity of only 40 times (40x) their weight.

[0012] The combination of gelatin-based agents with topical thrombin is one of the few advancements in this field. Another significant development in gelatin-based hemostatic agents is the product Floseal®. Approved for commercial use in the US in 1999, Floseal® combines human- derived thrombin with bovine-derived gelatin matrix granules, which are mixed at the time of use.

[0013] Despite the huge effect of thrombin in accelerating the blood clotting, thrombin still stands as a protein that is unstable, highly expensive and difficult to store thus limiting the wide utilization of thrombin-based products.

[0014] Starch-based hemostatic agents, particularly in powder form, have demonstrated effectiveness in managing bleeding conditions.

[0015] Arista™ AH is a surgical hemostatic powder derived from purified plant starch, offering complete absorbability. Its effectiveness lies in the use of microporous polysaccharide hemospheres, a patented technology that promotes blood clotting. Starch acts as a filter, concentrating blood cells and proteins such as thrombin, albumin, and fibrinogen, leading to hemostasis within a few minutes.

[0016] The availability of starch as a powder hemostatic agent was proven efficient in specific surgical operations, especially related to narrow and hard to reach surgical sites. However, the powder form of hemostatic agents can be inconvenient to handle and challenging to apply to various body areas such as the nose, gums during oral surgery, or the back.

[0017] Despite the huge advantages displayed by starch as a hemostatic agent, its inclusion in sponge form is still limited due to the pre-requisite of the gelatinization step that hinders its incorporation with other polymers except in certain concentrations.

[0018] Carboxymethyl cellulose (CMC), is a kind of plant polysaccharide, has good biocompatibility and biodegradability, safety non-toxic, to skin, mucosa nonirritant, without immunoreaction, in the process of tissue injury healing, has agglutinant effect.

[0019] Despite its advantageous effects with regard to hemostasis it is rarely available as foams and sponges due to its high solubility in water. So to attain such foam structure it needs to be highly crosslinked and there are no available functional groups for crosslinking.

[0020] Thus, the combination of starch, gelatin, and CMC with their collective advantageous properties with regard to hemostasis in addition to the advantageous effects of the foam structure as a hemostatic agent is highly needed. The current invention combines starch, CMC and gelatin in a foam structure with optimal pore structure that resulted in maximum absorption capacity and maximum hemostatic ability.

[0021] The current innovation presents a foam / sponge that possesses a maximum absorption capacity of up to 70 times its weight and eliminates the need for pre-wetting. It combines the hemostatic benefits of starch, gelatin, and CMC (carboxymethylcellulose) within a single foam. The current invention allowed optimal pore structure together with mechanical stability allowing the foam to exhibit maximum absorption while marinating mechanical integrity allowing it to hold the fluid within the foam even while handling the foam after fluid absorption.

[0022] In CN105727345A, a hemostatic membrane is disclosed, which includes starch, gelatin, and CMC. However, it differs from the current invention as it is a membrane rather than a foam. Furthermore, it does not possess the optimized pore structure that allows for the lowest foam density and maximum absorption. Additionally, the preparation process is not a one-pot method.

[0023] CN 105727344 reveals a composite hemostatic gel material that consists of a chitosan / starch / gelatin layer. This differs from the current invention as it describes the preparation of a hemostatic gel not a dried foam with optimized porous structure and absorption capacities.

[0024] US 5,612,052 discloses absorbable polyester copolymers forming hydrogels. The prescribed invention differs from the invention as it highlights an invented copolymer and its utilization in post operation adhesion prevention, while our invention describes a one-pot hemostatic foam with tailored porous structure, substantial absorption capacity while maintaining structural stability.

[0025] W02020021499A1 describes a hemostatic gel composition that comprises polysaccharide, protein, and carbohydrate in a single phase. In the present invention, the hemostatic gel composition includes chitosan as the polysaccharide, gelatin as the protein, and starch as the carbohydrate, all within a single phase. However, the invention is related to development of a gel which lacks the highly porous structure that accompanies foams and their related high absorption capacity. In addition, herein, the inventors highlight a simple one-pot process for the foam preparation.

[0026] EP2233157A1 describes modified starch hemostatic sponge / foam and its use as any of the hemostat, anti-adhesion material, promoting tissue healing material, surgical sealant and tissue adhesive glue. The said modified starch includes any or a composite of more than one of the pregelatinized starch, acid modified starch, oxidized starch, esterified starch, etherized starch, cross-linked starch, and grafted starch, composite modified starch.

[0027] The present invention differs mainly in that it is not starch-based foam but rather a gelatinbased foam that combines the advantages of having starch, gelatin, and CMC in the same foam matrix. Additionally, the current invention describes an advantageous porous structure with high absorption capacity while maintaining mechanical stability even after blood / fluid absorption.

[0028] None of the prior art references discloses a haemostatic foam comprising CMC, starch and gelatin prepared via one pot preparation with an optimized process that allowed a low density of 15, 20, or 25-30mg / cm3, high water adsorption and mechanical stability, homogenous and narrow porous size distribution (e.g. 180-240 microns) and over 70% of the total foam volume is air.

[0029] Embodiments of the present invention also provide an efficient process for preparation of the disclosed hemostatic foam, wherein the foam was carried out in a one-pot formation protocol for an easier manufacturing process that incudes high optimized propellers and mechanical stirring with control over the viscosity. And over 70% of the composite content is gelatin that allows the homogenous composite hydrogel to increase in viscosity with lowering the temperature and so form a stable foam. BREIF DESCRIPTION OF THE INVENTION

[0030] The present invention relates to low density of 30 mg / cm or below superabsorbent, adhesive, biocompatible, biodegradable, mechanically stable foam based on gelatin, starch and carboxy methyl cellulose (CMC) that can be produced in a one-pot reaction. One objective of the invention is to provide a haemostatic / superabsorbent foam composition comprising gelatin, starch and CMC in a single phase. This can provide a haemostatic / superabsorbent foam composition comprising the optimal pore structure that allows the lowest density allowing maximum absorption and mechanical stability.

[0031] Embodiments of the invention disclose herein allow the development of a stable foam with maximum air entrapment that reached up to 50, 60, 70, or >70% of the total foam volume. The hemostatic foam / sponge disclosed herein, displays an advantageous or optimal pore structure with regard to pore number, size and homogenous distribution aided with high sheer mechanical mixing that allowed reduction of the air bubble size to only 180 microns that was a significant factor in enhancing its absorption capacity and mechanical stability. Notably, the yielded pores were homogenously distributed and of narrow size distribution between 180-240 microns in diameter. The disclosed hemostatic foams / sponges have superior absorption capacity that reaches to 70X its weight, strong adhesion to the bleeding site allowing minimal blood loss and maximum absorption capacity of blood while maintaining its mechanical integrity.

[0032] Another objective of the invention is to provide a process for preparing haemostatic foam, via one pot synthesis. This method of manufacturing provides an advantageous pore structure that results in the lowest density and super absorption capacity and stable mechanical properties. The method includes a specific design of the propeller and specific control over viscosity, temp and stirring. Over 70% of the composite content is gelatin that allowed the homogenous composite hydrogel to increase in viscosity with lowering the temperature and so form a stable foam. The specific design of the propeller allows control over the specific pore diameter and distribution.

[0033] Aspects of this invention include but are not limited to the following embodiments.

[0034] One aspect of this invention is a foam comprising gelatin, starch, and carboxymethylcellulose (CMC) that has a density of no more than < 5, 5, 10, 15, 20, 25, 30, or > 30 mg / cm and that comprises pores ranging in average diameter from 160, 170, 180, 190, 200, 210, 220, 230, 240, or > 240 microns. These ingredients may be crosslinked with glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker.

[0035] The foam may comprise 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 to 1 wt.% starch, 1, 2, 3, 4, 5, 6, 7, 8, 9 to 10 wt.% gelatin, and 0.1, 0.2, 0.3, 0.4 to 0.5 wt.% CMC.

[0036] In one embodiment, the foam comprises 0.1 wt.% starch, 5 wt.% gelatin, and 0.2% CMC, wherein each of these values may vary by ± 1, 2, 5, 10 or 20 wt.%.

[0037] In some embodiments, up to 30, 40, 50, 60, 70, >70% of the foam volume is trapped air or gas.

[0038] In some embodiments, the foam is highly stable. Foam stability pertains to the ability of the viscous solution to uniformly retain trapped air within the mixture, comprising up to 70% of the total final volume.

[0039] In some embodiments, the foam absorbs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, or >70 -times its original dry weight in water or other liquid, such as blood, plasma, tissue fluid, CSF, saliva, mucous, urine, or other biological fluids.

[0040] In some embodiments, the foam advantageously has structural stability even after water or other liquid, such as blood, plasma, saliva, mucous or other biological fluids. In addition, the foam shows mechanical strength exceeding that of starch-based foams and other commercial foams.

[0041] In some embodiments, the foam of has a break stress value ranging from 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, or 0.19 to 0.2 N / mm2. In some embodiments, the foam is stable at 25°C and maintains at least 70, 80, 90, < 100, or 100% % of its break stress value and / or adsorption capacity for 90 days.

[0042] The foam as disclosed above that, in its final form after freeze-drying, is stable at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or > 60°C and maintains at least 50, 60, 70, 80, 90, 95, or 100% , of its mechanical strength and / or adsorption capacity and / or other functional properties for 10, 20, 30, 40, 50, 60 70, 80, 90, or > 90 days.

[0043] In some embodiments, the foam is produced by a process comprising dissolving the starch in water at an alkaline pH ranging from pH 8 to 14, separately dissolving gelatin in water, mixing the dissolved starch and dissolved gelatin, adding the CMC to the mixture of dissolved starch and dissolved gelatin until it is dissolved, and then adding glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker and freezing and lyophilizing the resulting mixture, thereby obtaining a foam. Alternatively, in some embodiments, two or three of these ingredients may be simultaneous dissolved in water or an aqueous solvent.

[0044] Another aspect of the invention is a medical or pharmaceutical product comprising the foam as disclosed herein, preferably in the form of a sheet, pad, sponge, powder, granule, or customized form; optionally containing at least one other active agent including but not limited to a hemostatic drug, antibiotic, a vitamin such as vitamin A, a B complex vitamin, C, D, E or K, or an analgesic. The medical product may be directly applied to a wound without prewetting.

[0045] Another aspect of the invention is directed to a method for treating a wound comprising contacting the wound with the foam, foam-containing or foam-derived product disclosed herein that comprises starch, gelatin and CMC.

[0046] In some embodiments of this method the wound is a bleeding wound that is severe, spontaneous, or chronic; the wound may comprise a puncture wound, laceration, abrasion, or avulsion.

[0047] In some embodiments the wound may be a sport injury wound, surgical wound, internal bleeding such as in the gastrointestinal tract or reproductive organs, a bum, a bite or claw wound, a shaving wound, a wound from an vehicular accident, wound from a tool, a gunshot wound, a battlefield wound, wound from a fall, explosion, or other impact, or other traumatic wound, a wound resulting from a medical condition such as from hemophilia, leukemia, liver disease, lupus, Marfan syndrome, meningitis, platelet disorder, vitamin K deficiency, use of a blood thinner, vasculitis or autoimmune condition, prolonged staining such as weightlifting.

[0048] In some embodiments the foam or foam-derived product disclosed herein may be used to absorb perspiration or other non-hematological fluids, for example, clothing, diapers, and other wearable products, wipes, anti-perspirants, or lotions and other cosmetics.

[0049] Another aspect of the invention is directed to a process for making the foams disclosed herein comprising: dissolving the starch in water or another aqueous medium at an alkaline pH ranging from pH 8 to 14, separately dissolving gelatin in water or another aqueous medium mixing the dissolved starch and dissolved gelatin, adding the CMC to the mixture of dissolved starch and dissolved gelatin until it is dissolved, and then adding glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker and freezing and lyophilizing the resulting mixture, thereby obtaining a foam. In some embodiments of this process the starch is dissolved in water or another aqueous medium at a pH ranging from 9.5 to 11.5, preferably at about pH 10.5 and / or is dissolved at a temperature ranging from > 25 to 100°C; and / or the gelatin is dissolved in in water or another aqueous medium at a temperature ranging from > 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, or > 100°C, preferably at about 50, 60, to 70°C; and / or the pH of the mixture of the starch solution and gelatin solution is adjusted to pH 6 to pH 8, preferably to about pH 7 prior to the addition of the CMC.

[0050] In some embodiments the glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is added as a solution containing 20, 50, 100, 150, 200, or > 200 pl / lOOml glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker, preferably about 100 pl / 100ml.

[0051] In some embodiments of the process of making, the mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is stirred at a speed ranging from 500, 1,000, 1,500, 2,000, 2,500, 3,000, or > 3,000 rpm.

[0052] In other embodiments of this process the stirred mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is frozen at its freezing point or at a temperature ranging from -5°C, -25°C, <-25°C, preferably at about -14°C.

[0053] In some embodiments of this process, the stirred mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is frozen in a preselected form such as being frozen in a mold, sheet, or other substrate suitable for lyophilization; or alternatively after being lyophilized is shaped or cut into a desired form.

[0054] Other aspects of the invention include more rapid or higher blood or fluid absorption than a same or similar quantity or configuration of a commercially available hemostatic products such as CUTANPLAST®, rapid swelling or expansion of the foam capable of filling voids in a wound and production of a faster clotting response.

[0055] Another aspect of this technology is a one-pot manufacturing method as disclosed herein for hemostatic foams comprising gelatin, starch and CMC preferably without introduction of extraneous chemicals such as surfactants, wetting agents, hardening agents, or blowing agents and to the products so produced.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The swelling kinetics of the Gelatin / Starch / CMC sponge as compared to the Cutanplast® hemostatic foam / sponge. Unless otherwise noted, Cutanplast® Standard 70 x 50 x 10 mm, Catalog No. 0517050) was used herein.

[0057] Figures 2A and 2B. Scanning electron microscope (SEM) micrographs showing the internal structure of Gelatin / Starch / CMC (A), and Cutanplast® (Standard Catalog No. 0517050 70 x 50 x 10 mm) (B).

[0058] Figure 3. Stress-strain curves of ColiSorB® and Cutanplast® after repeated mechanical compression.

[0059] Figure 4. Water contact angle of Gelatin / Starch / CMC and Cutanplast®.

[0060] Figure 5. In vitro degradation rate of Gelatin / Starch / CMC sponge as compared to Cutanplast via immersion in phosphate buffer saline and shaking at 37°C.

[0061] Figurea 6A and 6B. In vitro Assessment of Blood uptake and blood coagulation.

[0062] Fig. 6A: A photograph was taken after the blood uptake ratio test demonstrating the structural stability of the Gelatin / Starch / CMC sponge as compared to the commercial Cutanplast® Sponges (1cm x 1cm x 1cm) were immersed in 1.5 ml of blood for 3 min and evaluated with slight pressure application.

[0063] Fig. 6B: Blood uptake ratio of Gelatin / Starch / CMC and Cutanplast® (a ****P<0.0001).

[0064] Fig. 6C: The clotting time (c) of Gelatin / Starch / CMC and Cutanplast® was significant compared to the untreated blood ( ***P<0.001, ****P<0.0001).

[0065] Fig. 6D: (d) The blood clotting index (BCI) for both Gelatin / Starch / CMC and Cutanplast® was significant compared to the control, there was no difference between Gelatin / Starch / CMC and Cutanplast®. Coagulation (prothrombin time, PT) was not significantly different (e) between all groups.

[0066] Fig. 6E: a PTT time (e) was significantly higher in Gelatin / Starch / CMC and Cutanplast® samples compared to the control (untreated blood).

[0067] Figure 7. In vitro Hemolysis Assay. Blood hemolysis was minimal after using Gelatin / Starch / CMC and Cutanplast®. Compared to the positive control (10 pl triton-treated blood). Normal Saline was used as a negative control.

[0068] Figure 8. Cytocompatibility of the Gelatin / Starch / CMC. Cell viability of L929 cells assessment with Gelatin / Starch / CMC, and Cutanplast®, no significant difference was seen between the tested samples and the Control (untreated cells) Figure 9A and 9B. The amount of blood loss from the liver In vivo (Fig. 9 A) was significantly lower compared to the untreated animals. Fig. 9B: Blood Clotting Time (BCT) was also significantly lower after using Gelatin / Starch / CMC and Cutanplast® compared to untreated control. Blood loss and clotting time were significantly lower after using Gelatin / Starch / CMC compared to Cutanplast®.

[0069] Figure 10A-10J. Histological examination of the liver following laceration and hemostasis. Normal liver (a,f) showed normal histology of the hepatic lobules and hepatic cords. Non-treated control (b,g) liver samples showed dense fibrous connective tissue proliferation at the line of the incision (arrow). Gelatin / Starch-CMC foam treated (d, i) samples showing fibrous tissue with vacuolar degeneration of hepatocytes and few numbers of apoptotic bodies (arrow). Cutanplast® treated samples (e, j) show a thin layer of fibrous tissue mixed with inflammatory cells and embedded sponge material (arrow).

[0070] Figure 11. Time of Hemostasis for the negative control, commercial sponge group and ColiSorB® group.

[0071] Figure 12. VAS scores for the negative control, commercial sponge group and ColiSorB® group.

[0072] Figure 13. Sealing of the Wound for the negative control group, commercial sponge group and ColiSorB® group.

[0073] DETAILED DESCRIPTION OF THE INVENTION.

[0074] More specific description of the foam, its components and properties is provided below. Although specific terms are used in the following description for the sake of clarity, these terms are not intended to define or limit the scope of the disclosure.

[0075] Starch. A starch is a complex carbohydrate comprising glucose molecules linked together. It exists in at least two forms: amylose and amylopectin. Amylose is linear, while amylopectin has a branched structure. Herein, we describe the utilization of soluble starch.

[0076] Gelatin. The term “gelatin” is well defined in the art and encompasses its usual meaning. It typically refers to a translucent, colorless, flavorless food ingredient, commonly derived from collagen taken from animal body parts. It is sometimes known as hydrolyzed collagen.

[0077] Gelatin can be classified into type A and type B, based on acid and alkaline pretreatment, respectively. Type A gelatin is created by treating collagen with acid. It is derived from pig skin, fish skin / scales, beef hides or beef bones and is typically used in food-grade products. It has a lower viscosity but higher bloom than Type B. Type B gelatin is manufactured using a high-pH treatment and is most often derived from pig skin, fish skin / scales, beef hides or beef bones. Type B alkaline gelatin is generally for industrial uses. It has a higher viscosity and lower bloom strength. A suitable form of gelatin may be selected for production of the foams disclosed herein.

[0078] Bloom value. The gelatin bloom value or number indicates the firmness or softness of a specific type of gelatin and is mainly related to its viscosity. Gelatin viscosity determines the texture and creaminess of the final product. The bloom strength of gelatin can range from 30 to 325 with 30 being the softest and 325 being the stiffest. Bloom strength ranges tend to fall into three categories. Low bloom gelatin typically has a bloom value of 50-15, medium bloom gelatin from 175-225 and high bloom gelatin from 225-335. Preferably, a high bloom gelatin (>240) with viscosity range between 40-50 cP is used to produce the foams disclosed herein.

[0079] Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative that typically has a molecular weight of about 15,000 to 300,000 Daltons and a degree of polymerization of 80 to 1,500. Preferably, a CMC with an average molecular weight of 70,000, 80,000, 90,000, 100,000, or 110,000 Da is used to produce the foams disclosed herein.

[0080] Absorbable hemostatic gelatin product refers to an absorbable or re-absorbable, hemostatic material Cutanplast® is one example of such a material. This gelatin product may be in a sponge-like form or in other physical forms such as in a form of a cube, sheet, powder, foam, paste or other specialized shape or form. Typically it is manufactured from animal gelatin and is sterile. Gelatin sponges are sterile, water-insoluble, and porcine gelatin absorbable foams / sponges, widely used as a hemostatic material.

[0081] Cutanplast® is a sterile re-absorbable gelatin sponge haemostats manufactured by Mascia Brunelli in Italy. Cutanplast comes in 10 easy to modify formats specifically designed for a range of disciplines . Cutanplast® is commercially available gelatin haemostatic sponge from pigs in cubes with a rapid haemostatic effect, disposable and fully reabsorbable. Unless otherwise specified Cutanplast® Standard is used herein. In case of ambiguity, the trademarked products identified herein correspond to their formulations as of the filing date of this application.

[0082] ColiSorB® hemostatic gelatin sponges and powder flakes developed by InCurA are made from highly purified bovine gelatin and offer best performance in blood absorption and bleeding control and highest biocompatibility. Some embodiments of the gelatin / starch / CMC product disclosed herein have been identified under, but not limited by, the trademarked term

[0083] ColiSorB®

[0084] Foam. This term refers to a material composed of gas bubbles dispersed in a liquid or solid medium. The foams of the invention are typically produced by mechanical agitation of their components, but in some alternative embodiments may be produced by injection of a gas into a composition comprising starch, gelatin and CMC or also comprising glutaraldehyde, genepin, polyethylene glycol digly cidyl ether or other crosslinker or crosslinks provided by a crosslinker. It may also be produced by addition of a foaming agent or surfactant to a mixture as disclosed herein comprising starch, gelatin, and CMC or said mixture including glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker. The foams disclosed herein are dry, lyophilized foams. However, this term includes wet versions of these foams for example as prepared as intermediate products to dry foams or dry foams which have been exposed to liquid. The foams described herein when visualized by SEM exhibit a swellable pore like structure.

[0085] Cross-linking. Crosslinking is induced by the addition of, or exposure to, a crosslinking agent, such as an aldehyde like formaldehyde or glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker, in amounts sufficient to induce crosslinking. For example, glutaraldehyde (50%) or other aldehydes, genepin, polyethylene glycol diglycidyl ether or other crosslinker may be used in concentrations ranging from about 0.01%, 0.02%, 0.03%, 0.04%, 0 05%, 0.06%, 0.07%, 0 08%, 0.09%, to 0.5% (v / v). In addition to aldehyde crosslinkers, epoxy crosslinkers such as polyethylene glycol diglycidyl ether may be used in concentrations spanning approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, to 0.5% (v / v), as well as genipin, can be employed at concentrations covering approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, to 0.5% (wt. / v) to achieve the desired crosslinking effect.

[0086] Foam stability may be measured by a variety of parameters or metrics. These include foam half-life or the time it takes for a foam volume to reduce to half of its initial volume, drainage halflife of the time it takes for half of the liquid in the foam to drain out; foam height including the initial height of the foam and subsequent heights overtime; and bubble size distribution, including the degree of uniformity or non-uniformity of bubble size; and / or foam volume including initial foam volume and foam volume over time. Preferably, the foams disclosed herein have a half-life of at least 5, 10, 15, 30, 45, or 60 minutes, or 1, 2, 3, 4, 5, or >5 hours.

[0087] The term “q stable refers to a foam that is stable over time. A q stable foam is one in which gas diffusion between bubbles in foam remains steady which allows the overall foam structure to persist without collapse or significant coarsening. A q stable foam can be described as rich, thick and stable or as dense frothy and able to provide stable foam / froth textures for 10, 20, 30, 40, 50, 60, 90, or 120 minutes or more A q stable foam may be stable when heated to 25, 30, 35, 40, 45, 50-, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or >120 for an hour.

[0088] A q stable foam may also be stable at a pH ranging from 2, 3, 4, 5, 6, 7 or 8, preferably at an oral pH such as between pH 5.5, 6.0, 6.5, 7.0, 7.5, and pH 8.

[0089] In one embodiment the q stabile foam is stable in the presence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or >10% (vol / vol) ethanol or stable in up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or >10 % (vol / vol) citric acid.

[0090] The term foam density' refers to the density of the foam, for example in grams / cm3. Preferably, the foams described herein have a density ranging from 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09-0.10 g / cm3which allows them to be lightweight and easily manipulated.

[0091] The term “porosity” or “void fraction” is the ratio of the volume of voids in a foam to the total volume of the foam. Preferably, the foams described herein have a porosity ranging from 75, 80, 85, 90-95% (ASTM C272) which contributes to their ability to absorb blood, saliva, and other biological fluids.

[0092] The term “foam absorbency” refers to the ability of the foams disclosed herein to absorb blood, saliva, and other biological fluids, for example, as compared to cotton, dental papers, or other absorbent materials. Absorbency may be measured in units of grams of water absorbed by grams of an absorbent material. Absorbency of foams described herein may range from 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or > 120 gr / gr, preferably at least 90 gr / gr.

[0093] A “foam softness” or “foam firmness” may be measured by Indentation Force Deflection (ASTM D3574) and may range from about 8 to about 150 ILD, preferably the foams disclosed herein having an ILD of about 66, 70, 75, 80, 85 to 90 lbs. Preferably, the foams disclosed herein have a degree of softness that characterizes a foam that is non-abrasive and does not irritate sensitive teeth or gum tissue. “Durability” of a foam may be measured by a compression test or by a tensile strength and elongation test, a tear resistance test, dynamic fatigue test or other test described by ASTM D3574.

[0094] “Biocompatibility” . Preferably, the foams disclosed herein are biocompatible and induce little or no cytotoxicity in in vitro or in vivo tests. In vitro testing includes implantation of a unit amount of a foam and comparison to a control, such as 100% cotton or Cutanplast® and evaluation of inflammation. Preferably, the foams as described herein induce fewer or equivalent responses to other material such as cotton. Preferably, the foams disclosed herein are biocompatible and induce little or no cytotoxicity in in vitro or in vivo tests. In vitro testing includes exposure of a cell culture to a unit amount of foam and to a unit amount of a control, such as another type of foam or 100% cotton and measurement of cytotoxicity or mutagenesis of the cells. In vivo testing includes implantation or exposure of a unit amount of a foam and comparison to unit amount of a control, such as 100% cotton or an alternative type of foam, and evaluation of inflammation. Preferably, the foams as described herein induce fewer or equivalent responses to other material such as cotton especially as measured in oral tissues. Preferably, a foam of as described herein will induce no more than or less cytotoxicity (e.g., 0, < 1, 1, 2, 5, or 10%) or inflammatory responses (e.g., 0, <1, 1, 2, 5, or 10%) than a control material such as a conventional foam or absorbent material such as 100% cotton, or Cutanplast®.

[0095] “ Release properties” . In some embodiments, the foams disclosed herein may contain other active agents such as drugs, vitamins, bleaches (e.g. hydrogen or other peroxides), fluorides, antimicrobials or other functional ingredients. Such foams may release these ingredients as a burst or continuously over a period of 5, 10, 20, 30, or 60 minutes, or 1, 2, 3, 4, 5, or > 5 hours.

[0096] Blood. In the context of this disclosure, the term “blood” is used generally to refer to whole blood, which is a water-based fluid containing diverse solutes, suspended polypeptides, growth factors, and blood cells. Examples of blood cells are white blood cells, red blood cells and thrombocytes. White blood cells (leukocytes) are immunocompetent cells in the circulation of blood characterized by their central role of maintaining the humoral and innate immune systems. Red blood cells (erythrocytes) are the major oxygen carrying cells in the blood. The thrombocytes (platelets) are smaller than both white and red blood cells and mediate certain types of coagulation of blood. In some examples, the blood is mammalian blood, such as for example human blood.

[0097] Plasma. The term “plasma” refers to the yellow liquid component of whole blood, in which the blood cells in whole blood would normally be suspended. Put another way, plasma is whole blood minus the blood cells. Plasma is mostly water and comprises dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide. Plasma may be prepared by spinning a tube of fresh blood containing an anti-coagulant in a centrifuge until the blood cells fall to the bottom of the tube. The plasma is then poured or drawn off from the blood cells

[0098] Saliva is a viscid, watery fluid, secreted into the mouth by the salivary glands, that functions in the tasting, chewing, and swallowing of food, moistens the mouth, and starts the digestion of starches. Typically, saliva has a pH range of 6.2 to 7.6 which can be impacted by consumption of sugars, acidic foods or liquids, oral microorganisms, or smoking. Saliva may contain enzymes such as amylases and antibodies such as IgA and IgG. Bodily fluids such as blood, plasma, tissue fluid, pus, fluids produced by bleeding or inflammation, may also be controlled or absorbed using a foam as described herein.

[0099] “Molar extraction refers to the removal of one of the molar teeth, which are the large, flat teeth located at the back of the mouth. These teeth are primarily used for grinding food. The procedure can be performed for various reasons, including severe decay, infection, periodontal disease, or impaction. Molar extractions may be performed for patients with severe tooth decay or where molar repair is not feasible, for patients with oral infections or periodontal diseases, for impaction, such as impaction of wisdom teeth, or for orthodontic reasons. The products disclosed herein may, of course, be used during simple or surgical extraction of molar or non-molar teeth.

[0100] Treatment and Prevention. The materials disclosed herein may be used in conjunction with such molar extractions or other dental or periodontal diseases. Dental diseases in which these products may be used include, but are not limited to tooth decay, dental caries, oral cancer, halitosis or conditions associated with halitosis. Subjects with poor oral hygiene, who smoke or use alcohol, or who have medical conditions like diabetes may benefit from treatments using the products disclosed herein. The products disclosed herein may be used in conjunction with other treatments such as professional dental cleanings, including deep cleanings, placement of fillings, crowns or root canal treatments, or treatment for oral cancer.

[0101] The materials disclosed herein may be used in various medical and surgical procedures where hemostasis or fluid control is required including, but not limited to general surgery, for example, for control bleeding from parenchymal organs; thoracic surgery, neurosurgery, orthopedic surgery, gynocological procedures, ENT surgery, vascular surgery, plastic surgery, spleen surgery, in conjunction with dialysis, endoscopy, biopsy, wound care, and dental procedures. The form, shape and size of the materials disclosed herein, such as sponge-like form, sheet form, powder form, paste form may be selected for use with a particular procedure. Particular shapes and sizes and uses of the foams described herein, which may be similar to those of Cutanplast®, are incorporated by reference to those described at worldwide web.uniplexuk.com / cutanplast (last accessed July 15, 2024).

[0102] The products disclosed herein may be used for treatment of periodontal diseases such as gingivitis, periodontitis, aggressive periodontitis, chronic or systemic periodontitis or necrotizing periodontal disease. The products disclosed herein may be used during or post-operatively a dental implant, bone graft, periodontal surgery, root canal therapy, orthognathic (Jaw) surgery, surgery for sleep apnea, cleft lip or palate repair surgery, facial trauma surgery, cosmetic surgery including dental bonding, teeth whitening, or facial implant surgery, apicoectomy (removal of a tip of a tooth’s root to treat infection), frenectomy including removal or repositioning of the frenum, the tissue connecting the lips and gums or the tongue and gums; gingivectomy including removal of gum tissue to treat gum disease or improve aesthetics; odontectomy including removal of impacted teeth trapped under bone and soft tissue; crown lengthening including reshaping of gum and bone tissue to expose more of the tooth's crown; surgical removal of pathology including the removal of tumors, cysts, or other pathological growths in the oral cavity; orthodontic surgeries such as procedures to correct teeth and jaw alignment, often in conjunction with braces; full-arch restoration including implant-supported dentures for patients missing all teeth in an arch; maxillomandibular advancement surgery such as surgery to move the upper and lower jaws forward to treat obstructive sleep apnea and other conditions; and distraction osteogenesis including gradual bone lengthening procedure for correcting jaw deformities.

[0103] The products disclosed herein may be used for prevention or during treatment of dry socket including loss of a blood clot in a socket which may expose bone or nerves. It may be used in conjunction with treatment of infection at an extraction site of a molar or non-molar tooth. These products may be used to prevent or treat damage to nerves, including damage to nerves in the lower molars which can affect sensations to the lips, tongue or chin. These products may be used to prevent bleeding or swelling during a post-operative period after tooth extradition or oral surgery. In some embodiments, the products disclosed herein may be used during an initial post-operative period of 1-2 weeks when a socket is healing. In other embodiments, these products may be used until partial, substantial, or full recovery is attained while bone and oral tissue is healing. EXAMPLE 1

[0104] Production of the fabricated foam

[0105] Fabrication of the Gelatin / Starch / CMC foam. A starch solution containing 0.2 wt.% starch in water is produced by admixing starch with water in a beaker. The pH of the mixture is adjusted to pH 10.5, and the mixture is left under continuous stirring at 300 rpm for 45 minutes at 90°C, see Table 1 below.

[0106] Subsequently, gelatin is added to the starch solution to reach a final concentration of 5 wt.%. The mixture is left under continuous stirring at 300 rpm for 45 minutes at 60°C.

[0107] Then, the pH of this solution was adjusted to pH 7.0.

[0108] An amount of CMC is added to the mixture of the starch and gelatin solutions to bring the CMC concentration to 0.2 wt.%, and the starch, gelatin, and CMC mixture is stirred at 300 rpm until the CMC is fully dissolved, about 45 minutes.

[0109] Then, glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker (50%) is added to the starch, gelatin, and CMC solution to reach a concentration of 100 pl / lOOmL solution and stirred at 300 rpm for 45 minutes at 50°C.

[0110] In order to generate foam, the resulting solution is then mechanically stirred for 30 minutes at a vigorous speed of 2,000 rpm. The solution's temperature was gradually lowered at a rate of 1°C every 3 minutes until it reached 18°C, forming a stable foam and samples are taken in 15cm petri-dishes.

[0111] Samples are frozen overnight at -14°C and then lyophilized in a freeze dryer to obtain the final fabricated foam product. Typically, the amount of glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker added is completely reacted leaving no traces in the resulting foam. As disclosed herein, the inventors utilized the DLAB OS20-S Overhead Stirrer with a propeller from DLAB catalogue number 18900071. Other structurally similar stirrers and / or reactors or commercially available propeller stirrers and / or reactors which produce similar results may also be employed.

[0112] Characterization of fabricated foam

[0113] The fabricated sponge was cut into 1 cm3cubes. The cubes were weighed, and distilled water was added. The sponge was allowed to swell in the water for 30 seconds. The sponge was removed from the water, and any excess water on the surface was gently removed. The swollen sponge was weighted, and the percentage swelling of the sponge was calculated using the following formula: Percentage swelling = (Wt - Wd) / Wd x 100), Where Wt is the weight of the swollen sponge, and Wd is the initial weight of the dry sponge cube22.

[0114] SEM morphology and measurement of pore size.

[0115] The surface morphology of the sponge was examined using a scanning electron microscope. The sample was cut into small pieces and coated with a thin layer of gold under vacuum at 15kV. The surface of the sample was examined.

[0116] Porosity Analysis. The dried samples underwent a 1-hour immersion in alcohol, and the weight was recorded. The porosity was determined using the following equation:

[0117] (Eq): □ □ □ □ □ □ □ □ (%) = (12 - > 1 / □ * □) x 100%

[0118] In this equation, W 1 represents the weight of the sample before immersion in alcohol, while W2 represents the weight of the sample after immersion. V denotes the volume of the samples, and p represents the density of alcohol (0.79 g / cm3at room temperature).

[0119] Mechanical Analysis. The mechanical characteristics of sponges were examined utilizing a universal testing machine (UTM, Shimadzu-AGX-10 kN, Kyoto, Japan). Rectangular sponge samples measuring around 10 mm x 40 mm x 5 mm were tested with a crosshead speed of 50 mm / minute. During the testing process, the tensile strength of the sponge was recorded.

[0120] Contact angle measurement. The contact angle of sponges was measured using a contact angle goniometer (Drop shape Analyzer DSA25, Kruss, Germany) to evaluate the wettability of the sponge. For this test, water was carefully dropped on the top surface of the sponge, and the change of contact angle with time was measured.

[0121] Degradation assay To investigate the process of biodegradation, we began by measuring the weight of a cubic sponge. The sponge was then placed in phosphate-buffered saline (PBS) and kept inside an incubator set at a temperature of 37 °C for 8 days. The weight of the sponge was recorded after drying every day. The weight of the sponges before butting in PBS was also noted. To calculate the remaining weight as a percentage, the following equation was used: = (Wd -Wt) / Wd x 100.

[0122] Where Wt represents the weight of the dried sponge, and Wd represents the initial weight of the dry sponge cube23

[0123] Blood Uptake Ratio. The sponge (1cm x 1cm x 1cm) was incubated at 37°C for 5 minutes with 1 mb blood (VO) (Citrated rabbit whole blood from rabbits) in 24-well plates. The sponges were removed, and the volume of blood remaining in the wells (VI) was measured.24The absorbed blood volume (VB) was calculated using the following formula: ( VB = VO - VI). Additionally, the sponges were visually evaluated for their mechanical stability following blood intake after 5 min.

[0124] Blood Clotting Time [BCTJ. One ml of citrated whole blood was added to the plate wells, followed by 100 pl of 0.2 CaCl2, and mixed and incubated at 37 °C for 5 minutes. Sponges (1cm x 1cm x 1cm) were added to the wells, and the time to initiate blood clotting was measured. The wells were gently tilted every 10 seconds to examine the blood f

[0125] Blood Clotting Index [BCI]. Sponges were cut into 1 cm3squares and placed in a prewarmed 24-well plate. 200 pl of 3.2% citrated whole blood were added to sponges synchronously with 20 pl of 0.2 CaCl2and incubated for 5 minutes at 37°C. Sponge samples were then immersed in 25 ml distilled water and allowed to incubate for 10 minutes at 37°C with 30 rpm shaking. The commercial sponge, Cutanplast® (Mascia Brunelli S.p.a., Milan, Italy), was used as a positive control, and citrated whole blood with deionized water was used as blank. Finally, 500 pl of each sample mixture was measured for absorbance at 545 nm.l’25BCI was calculated using the following formula: BCI index = 100 x (Abs. of the sample) / (Abs. of blank).

[0126] In Vitro Hemolysis Test. A preweighed sponge sample (20 mg) was immersed in 2 ml saline and incubated for 30 minutes at 37°C. RBCs were prepared by adding 4 ml whole citrated blood to 5 ml saline followed by mixing and centrifuging at 2000 rpm for 10 minutes. 1 ml of each saline-sponge mixture was added to 20 pl RBCs. 10 pl triton + 20 pl RBCs + 1 ml saline were used as a positive control. Saline was used as a negative control (0.9 NaCl %). All sample mixtures were centrifuged at 2000 rpm for 5 minutes. The absorbance of the supernatant was then measured at 545 nm24The hemolysis rate (HR %) was calculated using the following formula: HR% = (Dsample - Ddist) / (Ddist - Dsaline) x 100.

[0127] Prothrombin Time (PT) Analysis. Platelet-poor plasma was obtained by centrifugation of citrated whole blood at 2000g for 15 minutes at 4 °C. 100 pl of platelet-poor plasma, and 100 pl of PT reagent was incubated at 37 °C for 3 minutes. After incubation, 2 mg of sponge samples were added to 200 pl plasma and incubated for 5 minutes at 37°C. Finally, the PT reagent was added to the plasma-sponge mixture. For aPTT measurement, 100 pL of aPTT reagents and 100 pL of PPP were incubated at 37 °C for 3 min. Then, 100 pL of 0.025 mol / L CaCh and samples were added. Then, PT and aPTT were measured using a semi-automatic coagulation analyzer (TS6000, MD Pacific Biotechnology Co., Ltd, China).

[0128] Cell viability (MTT) Assay. The cell viability was measured using the 3-(4,5- dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) reduction assay. Mouse fibroblast L929 cells were seeded at a density of 50,000 cells / well in 500 pl complete DMEM media for 24 hours before adding 10 mg of each sponge (ColiSorB® and Cutanplast®).

[0129] Sponges were left in direct contact with the cells for 24 hours. Then, the media was discarded, and 100 pl MTT and 500 pl media were added to each well. The plate was incubated for 2 hours at 37°C. After incubation, the media was discarded, and 600 pl DMSO was added, and incubated at room temperature for 10 minutes before measuring the absorbance at 570 nm using a SPECTROstar® Nano plate reader.

[0130] In vivo Biological Assessment

[0131] In vivo bleeding assessment. Female Sprague Dawley rats (125-150 gm) were obtained from Theodor Bilharz. All surgical procedures and post-surgical care were performed in compliance with the National Research Council’s Guide for the Care Use of Laboratory Animals. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Ain Shams University.

[0132] Rats were randomly divided into three groups (n=6). The hemostatic efficacy was evaluated using the liver laceration model26. After anesthesia with intraperitoneal injection of Ketamine 80-100 mg / kg / Xylazine 10—12.5 mg / kg, the liver was exposed via an anterior abdominal subcostal incision. Then, the left lateral lobe was gently externalized, and a pre-weighed aseptic gauze (WO) was placed behind the lobe. A 10-mm transverse incision was made on the left lobe with a scalpel blade to mimic hemorrhage. Then, the sponge was quickly applied to the wound. Bleeding was observed, and the gauze was replaced every 1 minute. No bleeding within 5 min. was considered hemostatic, and the hemostatic time was recorded. The incision was closed, and the animal was left to recover. The blood loss was calculated using the following equation: Blood loss Wt-WO.

[0133] Then, at the end of the experiment, Rats were anesthetized with Ketamine / Xylazine (ketamine 80-100 mg / kg, xylazine 10-12.5 mg / kg IP). Blood was collected by cardiac puncture, and tissue samples were collected for biochemical and histological analysis.

[0134] Biochemical assays. Blood was collected by cardiac puncture, at the end of the experiment, rats were anesthetized and 1 ml of blood was drawn. The collected samples were used to assess the concentrations of liver enzymes Aspartate and Alanine aminotransferases (AST and ALT), and the concentrations of serum urea and blood creatinine were measured by clinical chemistry autoanalyzer Mindray BS-240, China.27

[0135] Histological assessment. At the end of the 14 days, the rat tissues were dissected and fixed in 4% paraformaldehyde for 24 hours. Skin, liver, and kidney samples were trimmed off, washed, and dehydrated in ascending grades of alcohol. The dehydrated specimens were then cleared in xylene, embedded in paraffin blocks, and sectioned at 4-6 pm thick. The obtained tissue sections were deparaffinized using xylol and stained using hematoxylin and eosin (H&E) and Masson’s Trichrome stain for histopathological examination using a light microscope (Olympus xc30. Tokyo. Japan with CellSence Dimensions software)28.

[0136] Water absorption rate and swelling rate of the fabricated foam. By calculating the swelling percentage, the inventors were able to further characterize the ability of the developed foam to potentially fill a void space upon implantation and its capacity to absorb blood as shown by Table 2 below.

[0137] By calculating changes in volume, it was determined that Gelatin / Starch / CMC foam swelled approximately 15x, whereas Cutanplast® exhibited a significantly lower swelling of only 2x. This indicates that Gelatin / Starch- CMC foam has a faster initial absorption rate, making it more efficient in controlling bleeding during the critical early stages of a surgical procedure. After 10 minutes, ColiSorB® continued to exhibit superior performance, with a swelling capacity of 77x, while Cutanplast® reached a swelling capacity of 31x. These results indicate that ColiSorB® possesses a higher absorption capacity, allowing it to absorb a greater volume of water compared to Cutanplast®, which displayed limited absorption ability (Figure 1).

[0138] The Gelatin / Starch / CMC swelling result indicates superior capacity to absorb blood as compared to the commercial Cutanplast® that displayed limited absorption ability.

[0139] SEM Morphology and Porosity Analysis

[0140] Cutanplast® was found to display a consistent network structure with various ranges of pore sizes. On the other hand, Gelatin / Starch / CMC foam exhibited an interconnected network structure of gelatin and demonstrated a highly porous structure (Figure 2).

[0141] The porosity of both Gelatin / Starch / CMC foam and Cutanplast® was evaluated. It was observed that the Cutanplast® sponge demonstrated larger pore sizes compared to the Gelatin / Starch / CMC foam. The pore size varied between 100 and 300 pm, with a mean pore size of 210 and 243 for ColiSorB® and Gelatin / Starch / CMC foam, respectively. Most importantly, Cutanplast® exhibited a porosity of 42%, while Gelatin / Starch / CMC foam displayed a higher porosity of 78%

[0142] Mechanical Analysis. The result of the mechanical evaluation demonstrated that Gelatin / Starch / CMC exhibited higher tensile strength as compared to Cutanplast®. The break stress value of 0.155 N / mm2suggests that Gelatin / Starch / CMC is capable of withstanding higher amounts of force or tension before breaking or tearing. This is a positive attribute as it indicates that the Gelatin / Starch / CMC sponge is less likely to rupture during its application compared to Cutanplast®, which has a lower break stress value of 0.105 N / mm2. Additionally, the optimized unique crosslinking that allowed enhanced mechanical integrity resulted in optimal mechanical strength that allowed for maintaining an integral structure of the sponge while allowing maximum expansion for enhanced swelling ability and blood absorption (Figure 3).

[0143] Contact angle measurements. The wettability of biomaterial surfaces is a significant characteristic that can influence absorption ability, proliferation, migration, and viability. Evaluating the water contact angle is a common method to assess wettability. It helps determine their surface interaction with water.

[0144] For Gelatin / Starch / CMC, at 0 seconds, contact angles were between 107° and 108°, while after 5 seconds, the contact angles were between 76.1° and 71.6° and after 10 seconds contact angle was equal to 0°. For the Cutanplast® surface, contact angles measured were 112°, 108° and 89° after 10 seconds. The observation of the water contact angle on the Gelatin / Starch / CMC revealed that the water droplet was quickly absorbed by the sponge, resulting in the absence of a contact angle within a mere 10 seconds. This indicates that the Gelatin / Starch / CMC has a high level of hydrophilicity, allowing it to rapidly and effectively absorb water. In comparison, the Cutanplast® demonstrated a contact angle, which indicates a hydrophobic surface. This results in slower water absorption which requires 12 minutes for complete absorption of the water droplet (Figure 4).

[0145] Degradation test. The degradation percentage of the Gelatin / Starch / CMC sponge was assessed in phosphate buffer saline incubated with shaking at 37°C using the commercial Cutanplast® as the positive control. The Gelatin / Starch / CMC samples demonstrated a fast degradation rate where 59% of the whole weight of the sponge was degraded by day 3. Complete degradation of the Gelatin / Starch / CMC sponge was achieved by the third day 3 (Figure 5). On the other hand, the commercial Cutanplast® samples showed slower degradation, where 50% were degraded by day 5 and complete degradation was achieved after 8 days (Figure 5). Such results demonstrate the high degradation rate of the developed ColiSorB® sponge as compared to Cutanplast® In vitro Assessment

[0146] Blood Uptake Ratio Test. The blood uptake ratio for Gelatin / Starch / CMC and Cutanplast was measured after three 3 minutes on blood-sponge surface interaction. Gelatin / Starch / CMC demonstrated a strong blood-absorbing capability showing a 2.94X higher uptake ratio than Cutanplast (Figure. 6b). Additionally, Gelatin / Starch / CMC showed a higher ability to retain its original sponge structure after blood uptake and slight pressure application with forceps. Whereas Cutanplast® demonstrated lower mechanical stability than Gelatin / Starch / CMC as it loses its (Icmxlcmxlcm) shape over time (Figure.6a), thus resulting in a lower ability to absorb and maintain blood within its matrix.

[0147] Blood Clotting Time Test. The blood clotting time with Gelatin / Starch / CMC was significantly lower (4 minutes) as compared to 5.8 minutes with Cutanplast®.

[0148] Blood Clotting Time Index. The whole Blood Coagulation test measures the coagulation capability of hemostatic materials, where lower values of the Blood Clotting Index (BCI) are an indication for enhancing the coagulation process. BCI values for Gelatin / Starch / CMC and Cutanplast® were measured compared to the negative control (no sponge). Gelatin / Starch / CMC exhibited the lowest clotting index, almost 53% lower than the control, while Cutanplast® was only 34% lower than the control. (Figure. 6d).

[0149] Prothrombin Time Analysis. Coagulation was assessed using Prothrombin time (PT) in vitro to evaluate the Gelatin / Starch / CMC sponge and the Cutanplast® for their plasma coagulation activity compared to the negative control. Gelatin / Starch / CMC had a much lower PT time than both the control and Cutanplast® by 0.5 seconds and 2.85 seconds, respectively (Figure. 6e). Activated partial prothrombin time (aPTT) was significantly higher in Gelatin / Starch / CMC and Cutanplast® treated samples compared to the untreated control.

[0150] In Vitro Hemolysis Test. Gelatin / Starch / CMC and Cutanplast® were compared to negative and positive controls that had no sponge samples. The Gelatin / Starch / CMC and Cutanplast® showed no significant hemolytic activity, and both were lower than the positive control and higher than the negative control (Figure 7).

[0151] Cytocompatibility Assessment. In vitro cell viability assessment was conducted using an MTT cytotoxicity assay. The results showed that Gelatin / Starch / CMC did not significantly affect the cell viability of L929 cells as compared to the control and Cutanplast®. The cell viability of L929 cells with Gelatin / Starch / CMC was 98%, while with Cutanplast®, the viability was 80% (Figure 8).

[0152] In vivo Assessment of Hemostasis

[0153] Blood Loss. Blood loss was measured in vivo in a liver laceration injury model in the rat. Gelatin / Starch / CMC and Cutanplast® were assessed in comparison with the negative control. Gelatin / Starch / CMC showed the lowest blood loss (100 mg) as compared to the untreated control and Cutanplast®, with 450 mg and 300 mg amount of blood loss, respectively (Figure. 9a). Gelatin / Starch / CMC demonstrated significantly lower blood loss than the untreated control by 78% while Cutanplast® was lower than the control by only 33%.

[0154] Blood Clotting Time. Gelatin / Starch / CMC and Cutanplast® sponges (1cm x 1cm x 1cm) were placed on the liver incision, and time was recorded until the bleeding stopped. Gelatin / Starch / CMC exhibited enhanced clotting ability and had the highest blood clotting rate, where the time to hemostasis was only 40 seconds as compared to the Cutanplast®, which achieved hemostasis in 90 seconds. Therefore, Gelatin / Starch / CMC exhibited a significantly higher rate of blood clotting compared to Cutanplast® and untreated control samples (figure. 9b).

[0155] In vivo Assessment

[0156] Assessment of Liver tissue. The liver tissue section of the untreated control showed dense fibrous connective tissue proliferation at the line of incision with moderate inflammatory cell infiltration, mainly lymphocytes and macrophages with few numbers of polymorphonuclear cells. The underlying hepatocytes showed mild swelling with granular cytoplasm. Examination of the liver treated with Gelatin / Starch / CMC revealed fibrous connective tissue in addition to vacuolar degeneration of the hepatocytes and a few numbers of apoptotic bodies. The liver tissue section of Cutanplast® treated liver showed a relatively thin layer of fibrous tissue mixed with inflammatory cells and embedded sponge material (Figure 10).

[0157] As shown herein, the gelatin / starch / CMC developed foams are more effective in promoting hemostasis than a gelatin sponge alone. Notably, the gelatin / starch / CMC sponge has a higher absorbency capacity than a gelatin sponge.

[0158] While not being bound to any particular explanation or theory, the inventors consider that the CMC in the sponge provides hemostatic advantages of procoagulant capability, high safety, antibacterial capability, biodegradability, massive source, and low cost. They also consider that it can activate the coagulation factor XII by negatively charged carboxyl groups and concentrate erythrocytes, to help initiate the blood coagulation process. It is also considered that this component may absorb many times its weight in water and forming a gel-like substance that adheres to a wound site. Such a gel-like substance may act as a physical barrier to blood flow and aid in the formation of blood clots by providing a scaffold for platelets and other clotting factors to adhere to. The inventors also consider that the addition of starch may act as molecular sieves that locally dehydrate blood by absorbing its fluids and molecular components, concentrating platelets and other serum proteins on the surface of the scaffolds, which could activate the clotting process. The starch in the sponge may improve the adhesion of the sponge to the wound site and reduce the risk of dislodgement and promote more effective hemostasis.

[0159] Overall, as shown herein, the gelatin / starch / CMC sponge is more hemostatic than a gelatin sponge due to its increased absorbency, enhanced biocompatibility, anti-inflammatory and bacteriostatic properties, and improved adhesion. These properties make it an ideal hemostatic agent for use in surgical and trauma settings, where effective hemostasis is critical for patient outcomes.

[0160] Different blood-sponge interaction tests were performed to assess the efficacy of the Gelatin / Starch- CMC foam. The developed Gelatin / Starch- CMC foam sponge novel crosslinking allowed it to exhibit maximum air entrapment that reached up to 77% of the total sponge volume allowing it to reach 15x its weight absorption capacity after only 30 seconds. Most importantly, it is the interplay of very high porosity and optimal mechanical integrity that allowed the sponge to swell while maintaining mechanical integrity and reach up to 77x absorption capacity at 10 min, unlike Cutanplast® which reached a swelling capacity of only 3 lx its weight at the same time point and demonstrated mechanical collapse not allowing it to swell efficiently and maintain blood absorption. Such results indicate that the Gelatin / Starch- CMC foam can absorb and retain larger amounts of blood. Additionally, the hydrophilic surface of the newly developed Gelatin / Starch- CMC foam contributes to its superior efficiency in absorbing and retaining blood. Most importantly, it allows the Gelatin / Starch- CMC foam to directly absorb blood without the need for a prewetting step that is usually mandated for currently commercially available gelatin sponges, thus saving precious time during surgical operations.

[0161] The strong absorption capacity as well as optimal mechanical property of the developed Gelatin / Starch- CMC foam was reflected in the assessment of the blood intake, clotting time, and blood clotting index. The blood intake of the developed Gelatin / Starch- CMC foam demonstrated its high ability for blood absorption reaching to blood intake of 735ul after 3 min as compared to 250pl for Cutanplast®. Such strong absorption ability is reached due to the hydrophilic nature of the developed sponge and its high porosity as well as its optimal mechanical properties allowing it to maintain a well-structured matrix even after blood absorption for a few minutes. Thus, allowing the sponge to maintain optimal mechanical strength while allowing maximum expansion for enhanced swelling ability and blood absorption.

[0162] Such high absorption capacity leads to a fast concentration of the clotting factors resulting in a shorter clotting time of only 4 minutes as compared to the commercial product Cutanplast®, which exhibited a blood clotting time of 12 minutes. In addition, Gelatin / Starch- CMC foam exhibited the lowest clotting index, almost 53% lower than the control, while Cutanplast® was only 34% lower than the control. This is possibly due to the unique cross-linking of the Gelatin / Starch- CMC foam that allowed maximum air entrapment that reached up to 70% of the total sponge volume. Prothrombin time (PT) measurements showed that the Gelatin / Starch- CMC foam had a lower PT time as compared to the commercial product. Prothrombin time (PT) is an assay that measures the time it takes for the liquid portion (plasma) of the blood to coagulate. PT is correlated with the extrinsic coagulation pathway16>17. Previous studies showed that the gelatin sponge demonstrated the lowest PT values compared to other samples made with either keratin or collagen16. On the other hand, others reported that the difference was not significant.18

[0163] Ideal hemostatic materials should have a hemolysis rate that is less than 5 %.16Assessment of the hemolysis test is essential to assess the biocompatibility of the biomaterials.19The results of the in vitro hemolysis assay of the Gelatin / Starch- CMC foam demonstrated no significant difference in the hemolytic activity as compared to the commercial product Cutanplast®. Yan et al. reported that gelatin sponges showed the lowest hemolysis rate when compared with keratin and chitosan sponges.16

[0164] In vitro, cytotoxicity assessment using mouse fibroblasts (L929) showed that the Gelatin / Starch- CMC foam enhanced cell viability compared to the commercial product Cutanplast®. Ding et al. showed that gelatin hydrogel showed no cytotoxicity on both human fetal hepatocytes and mouse embryonic fibroblast cells.5The hemolysis and cytotoxicity results confirm the safety of the developed Gelatin / Starch- CMC foam.

[0165] In vivo experiments showed that the Gelatin / Starch- CMC foam resulted in the lowest volume of blood loss and had the best blood clotting rate. For instance, an investigation of in vivo coagulation showed that Gelatin / Starch- CMC foam promoted hemostasis in a rat liver laceration model and significantly reduced the amount of blood loss (100 mg with Gelatin / Starch- CMC foam as compared to 300 mg with Cutanplast®). A previous report showed that wound healing using gelatin sponges was associated with enhanced hemostasis and a high level of patient satisfaction 20

[0166] As shown herein, the newly developed gelatin based sponge, ColiSorB® is a biocompatible sponge that helped to minimize post-operative bleeding and complications. ColiSorB® can enhance early hemostasis through serving as a matrix that stimulates healing of soft tissue, hence accelerating post-operative wound healing and improving treatment outcome for patients undergoing molar extraction.

[0167] Citations relevant to Example 1.

[0168] 1. Nepal, A., Tran, H. D. N., Nguyen, N.-T., & Ta, H. T. (2023). Advances in haemostatic sponges: Characteristics and the underlying mechanisms for rapid haemostasis. Bioactive Materials, 27, 231-256. https: / / doi.Org / 10.1016 / j.bioactmat.2023.04.008

[0169] 2. Vulliamy, P., Thaventhiran, A. J., & Davenport, R. A. (2019). What’s new for trauma haemorrhage management? British Journal of Hospital Medicine, 80(5), 268-273. https: / / doi.Org / 10.12968 / hmed.2019.80.5.268

[0170] 3. Johnson, A. B., & Burns, B. (2023). Hemorrhage. In StatPearls. StatPearls Publishing. http: / / www.ncbi.nlm.nih.gov / books / NBK542273 /

[0171] 4. Zhang, K., Li, J., Wang, Y., Mu, Y., Sun, X., Su, C., Dong, Y ., Pang, J., Huang, L.,

[0172] Chen, X., & Feng, C. (2020). Hydroxybutyl chitosan / diatom-biosilica composite sponge for hemorrhage control. Carbohydrate Polymers, 236, 116051. https: / / doi.org / ! 0.1016 / j .carbpol.2020. 116051

[0173] 5. Ding, R., Wei, X., Liu, Y., Wang, Y., Xing, Z., Wang, L., Liu, H, & Fan, Y. (2023).

[0174] Epidermal growth factor-loaded microspheres / hydrogel composite for instant hemostasis and liver regeneration. Smart Materials in Medicine, 4, 173-182. https: / / doi.Org / 10.1016 / j.smaim.2022.09.006

[0175] 6. Dobson, G. P. (2020). Trauma of major surgery: A global problem that is not going away. International Journal of Surgery, 81, 47-54. https: / / doi.Org / 10.1016 / j.ijsu.2020.07.017 7. Vyas, K. S., & Saha, S. P. (2013). Comparison of hemostatic agents used in vascular surgery. Expert Opinion on Biological Therapy, 13(12), 1663-1672. https: / / doi.org / 10.1517 / 14712598.2013.848193

[0176] 8. Chaudhry, R., Usama, S. M., & Babiker, H. M. (2023). Physiology, Coagulation Pathways. In StatPearls. StatPearls Publishing. http: / / www.ncbi.nlm.nih.gov / books / NBK482253 /

[0177] 9. Cheng, J., Liu, J., Li, M., Liu, Z., Wang, X., Zhang, L., & Wang, Z. (2021). Hydrogel- Based Biomaterials Engineered from Natural-Derived Polysaccharides and Proteins for Hemostasis and Wound Healing. Frontiers in Bioengineering and Biotechnology, 9. https: / / www.frontiersin.org / articles / 10.3389 / fbioe.2021.780187

[0178] 10. Paternd, V. A., Bisin, A., & Addis, A. (2020). Comparison of the efficacy of five standard topical hemostats: A study in porcine liver and spleen models of surgical bleeding. BMC Surgery, 20(1), 215. https: / / doi.org / 10.1186 / sl2893-020-00874-w

[0179] 11. Ranjbar, J., Koosha, M., Chi, H, Ghasemi, A., Zare, F., Abdollahifar, M. A., Darvishi, M., & Li, T. (2021). Novel chitosan / gelatin / oxidized cellulose sponges as absorbable hemostatic agents. Cellulose, 28(6), 3663-3675. https: / / doi.org / 10.1007 / sl0570-021-03699-9

[0180] 12. Schreiber, M. A., & Neveleff, D. J. (2011). Achieving Hemostasis With Topical Hemostats: Making Clinically and Economically Appropriate Decisions in the Surgical and Trauma Settings. AORN Journal, 94(5), S1-S20. https: / / doi.Org / 10.1016 / j.aorn.2011.09.018

[0181] 13. Davenport, R., Manson, J., De’Ath, H., Platton, S., Coates, A., Allard, S., Hart, D., Pearse, R., Pasi, K. J., MacCallum, P., Stanworth, S., & Brohi, K. (2011). Functional definition and characterization of acute traumatic coagulopathy. Critical Care Medicine, 39(12), 2652. https : / / doi . org / 10.1097 / C CM. ObO 13 e3182281 af5

[0182] 14. Raza, I., Davenport, R., Rourke, C., Platton, S., Manson, J., Spoors, C., Khan, S., De’ATH, H. D , Allard, S., Hart, D. P., Pasi, K. J., Hunt, B. J., Stanworth, S., MacCALLUM, P. K., & Brohi, K. (2013). The incidence and magnitude of fibrinolytic activation in trauma patients. Journal of Thrombosis and Haemostasis, 11(2), 307-314. https: / / doi.org / 10.l l l l / jth.12078 15. Chan, L. W., Kim, C. H., Wang, X , Pun, S. H , White, N. J., & Kim, T. H. (2016). PolySTAT -modified chitosan gauzes for improved hemostasis in external hemorrhage. Acta Biomaterialia, 31, 178-185. https: / / doi.Org / 10.1016 / j.actbio.2015. l l.017

[0183] 16. Yan, R.-R., Xue, D , Su, C., Xu, Y ., Gong, J.-S., Liu, Y.-L., Jiang, M., Geng, Y ., Lv, G.-Z., Xu, Z.-H., & Shi, J.-S. (2022). A keratin / chitosan sponge with excellent hemostatic performance for uncontrolled bleeding. Colloids and Surfaces B: Biointerfaces, 218, 112770. http s : / / doi . org / 10.1016 / j . col surfb .2022.112770

[0184] 17. Yang, R., & Moosavi, L. (2023). Prothrombin Time. In StatPearls. StatPearls Publishing. http: / / www.ncbi.nlm.nih.gov / books / NBK544269 /

[0185] 18. Lan, G., Lu, B., Wang, T., Wang, L., Chen, J., Yu, K., Liu, L, Dai, F., & Wu, D. (2015). Chitosan / gelatin composite sponge is an absorbable surgical hemostatic agent. Colloids and Surfaces B: Biointerfaces, 136, 1026-1034. https: / / doi.Org / 10.1016 / j.colsurfb.2015.10.039

[0186] 19. Fan, X., Xue, R., Li, N., Yang, Q., Yu, C., Dong, Y., Li, Y., Tang, K., & Wan, G.

[0187] (2022). A rapid hemostasis for incompressible wounds by microwave-assisting construction of cellulose sponge with chitosan. Materials & Design, 221, 111009. https: / / doi.Org / 10.1016 / j.matdes.2022.l 11009

[0188] 20. Rullan, P. P., Vallbona, C., Rullan, J. M., Mansbridge, J. N., & Morhenn, V. B. (2011). Use of gelatin sponges in Mohs micrographic surgery defects and staged melanoma excisions: A novel approach to secondary wound healing. Journal of Drugs in Dermatology: JDD, 10(1), 68- 73.

[0189] 21. Li, J., Wu, X., Wu, Y., Tang, Z , Sun, X., Pan, M„ Chen, Y., Li, J., Xiao, R„ Wang, Z.,

[0190] & Liu, H. (2017). Porous chitosan microspheres for application as quick in vitro and in vivo hemostat. Materials Science and Engineering: C, 77, 411-419. https: / / doi.Org / 10.1016 / j.msec.2017.03.276

[0191] 22. Yang, G., Xiao, Z., Long, H., Ma, K., Zhang, J., Ren, X., & Zhang, J. (2018). Assessment of the characteristics and biocompatibility of gelatin sponge scaffolds prepared by various crosslinking methods. Scientific Reports, 8(1), 1616. https: / / doi.org / 10.1038 / s41598-018- 20006-y 23. Kuo, Z.-K., Lai, P.-L., Toh, E. K.-W., Weng, C.-H , Tseng, H -W , Chang, P -Z , Chen, C.-C., & Cheng, C.-M. (2016). Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge. Scientific Reports, 6(1), 32884. https: / / doi.org / 10.1038 / srep32884

[0192] 24. Jahan, N., Ibne Mahbub, M. S., Lee, B.-T., & Bae, S. H. (2023). In Vivo and In Vitro Investigation of a Novel Gelatin / Sodium Polyacrylate Composite Hemostatic Sponge for Topical Bleeding. Journal of Functional Biomaterials, 14(5), Article 5. https: / / doi.org / 10.3390 / jfbl4050265

[0193] 25. Shakiba-Marani, R., & Ehtesabi, H. (2023). A flexible and hemostatic chitosan, polyvinyl alcohol, carbon dot nanocomposite sponge for wound dressing application. International Journal of Biological Macromolecules, 224, 831-839. https: / / doi.Org / 10.1016 / j.ijbiomac.2022.10.169

[0194] 26. Mohammed, B. M., Monroe, D. M., & Gailani, D. (2020). Mouse Models of Hemostasis. Platelets, 31(4), 417-422. https: / / doi.org / 10.1080 / 09537104.2020.1719056

[0195] 27. Khalil, E. A., Swelim, H., El-Tantawi, H., Bakr, A. F., & Abdellatif, A. (2022). Characterization, cytotoxicity and antioxidant activity of sea urchins (Diadema savignyi) and jellyfish (Aurelia aurita) extracts. The Egyptian Journal of Aquatic Research, 48(4), 343-348. https: / / doi.Org / 10.1016 / j.ejar.2022.05.005

[0196] 28. Bancroft, J. D., & Layton, C. (2019). 12 — Connective and other mesenchymal tissues with their stains. In S. K. Suvama, C. Layton, & J. D. Bancroft (Eds.), Bancroft’s Theory and Practice of Histological Techniques (Eighth Edition) (Eighth Edition, pp. 153-175). Elsevier. https: / / doi.org / 10.1016 / B978-0-7020-6864-5.00012-8

[0197] 29. Park, H.-J., Kim, H.-G., Wang, J.-H, Choi, M.-K., Han, J.-M., Lee, J.-S., & Son, C -

[0198] G. (2016). Comparison of TGF-P, PDGF, and CTGF in hepatic fibrosis models using DMN, CC14, and TAA. Drug and Chemical Toxicology, 39(1), 111-118. https: / / doi.org / 10.3109 / 01480545.2Q15.1052143.

[0199] EXAMPLE 2

[0200] Randomized Clinical Trial on the Effectiveness of a Novel Dental Hemostatic Sponge Following Dental Extraction This example assesses the efficiency of the developed gelatin / starch / CMC foam disclosed herein and shows that it advantageously enhances post-operative hemostasis in patients undergoing molar extraction(s).

[0201] Methods and Materials. Thirty -four patients undergoing molar extraction were selected for the this study which was conducted as a randomized clinical trial. Post-operative clinical complications, periodontal integrities, and radiographic outcomes were assessed on days 1 and 7 post-operatively.

[0202] Results: The study was conducted on 30 patients in total. Ten patients were assigned to each experimental group. Patients in the control group took the most prolonged time for hemostasis to occur; approximately 6 minutes. The average time of hemostasis was faster with the gelatin / starch / CMC foam / sponge around 1 minute 30 seconds, compared with the commercial sponge Cutanplast, which took an average of 3 minutes for hemostasis to occur; see Fig. 11.

[0203] Assessment of the time taken to achieve full hemostasis post-extraction showed that the developed gelatin / starch / CMC sponge was significantly the fastest to achieve full hemostasis. The average VAS score of the developed gelatin / starch / CMC sponge on day one post-operation was 0, which was significantly lower than the control side (P < 0.05).

[0204] Methods, Study Design and Population. This study was a comparative, randomized controlled clinical trial. The study population included all patients undergoing molar or dental extraction. Healthy patients who visited the Department of Oral & Dental medicine at Cairo University for impacted molar extraction between September 1st 2023 and September 30th 2023 and satisfying the inclusion criteria were selected for the study. A total of 34 patients were recruited for the clinical trial. This study was conducted in full conformance with the ethical principles, including the Research Ethics Committee Faculty of Oral and Dental Medicine Cairo University, and authorized by the Institutional Review Board of Cairo University (IRB No. 26 5 22). Written informed consent was collected from each patient before the surgical procedure.

[0205] The inclusion criteria consisted of the following; patients undergoing a surgical procedure, patients who are 21 years of age or older, patients who were available for a minimum of one postoperative evaluation to be scheduled at the time of the procedure approximately 7 days postsurgery and 2 month after surgery and patients who demonstrated willingness and ability to provide informed consent for participation. The exclusion criteria, on the other hand, consisted of unwillingness of participants to provide informed consent for participation, patients who were taking anticoagulant medications (e.g., aspirin, coumadin, Plavix, etc.) or have discontinued their anticoagulant medications less than 3 days prior to their surgical visit, patients who are pregnant, planning on becoming pregnant during the follow-up period, or actively breastfeeding, patients who have an active or suspected infection at the surgical site, patients who have a known sensitivity or allergy to any component(s) of the hemostatic gelatin sponge, patients who have severe congenital or acquired immunodeficiency, patients who are undergoing bisphosphonate therapy and diabetic patients with unstable blood glucose levels.

[0206] Patients were randomized into three groups. (1) the control group, in which conventional cotton and gauze were applied with biting pressure, (2) first experimental group, in which commercially available hemostatic sponge called Cutanplast® was used, (3) second experimental group, in which the current invention gelatin / starch / CMC; was used.

[0207] Intervention / Surgical Procedure. The molar extractions were performed by the same dentist and under the same conditions in both control and experimental groups. All surgical tools and techniques were the same for all patients. The required recommendations after the oral surgery were verbally explained to the patients by a dental specialist. The surgical procedure was as follows; surgical extractions were performed by the same surgeon with the same techniques under local anesthesia using 2% lidocaine containing 1 : 100,000 epinephrine. Patients were monitored for the appearance of any adverse reactions. After full thickness incision to external oblique ridge and sulcular incision from the distal side of second molar to mid-buccal area of first molar, full thickness muco-periosteal buccal flap was raised and odontomy and ostectomy were performed using low speed round and fissure bur under constant irrigation with sterile saline.

[0208] After the molar extraction was performed, the site was sutured for facilitating wound healing Approximately one to three single sutures were used for the envelope flap distal to the second molar, followed by interdental sutures between the first and second molars. In cases where the use of gelatin sponge was considered unstable, figure eight suturing was performed. All patients were prescribed antibiotics (Huons Amoxicillin Cap. 500 mg tid, Huons, Seoul, Korea), analgesics (Soleton Tab. 80 mg tid, CJ healthcare, Seoul, Korea), and antacid (Famotidine Tab. 20 mg bid, Nelson, Seoul, Korea) for 7 days following the surgery and were given 0.12% chlorhexidine gluconate mouthwash to use every 12 h for 7 days. Patients visited the clinic at 1 week (for suture removal) (day seven).

[0209] Outcome Measures Assessment. The primary outcomes measured were a time of healing, pain score, and facial swelling. The secondary outcomes included frequency of recurrent bleeding, recovery time needed for the wound to seal and patients’ comfort level. In addition, patients were screened for assessment using digital photographs that were taken post-operation on day 1, day 7 and at 6 ± 2 weeks. Day 0 was the same day of the operation and the dentist recorded the time needed to achieve full absorption and hemostasis. Day 1 was one day after the operation and the dentists assessed whether there were any bleeding. Day 7 was seven days after the operation and the dentist reported whether there were any swelling or inflammation at the extraction site. Clinical Evaluation. The clinical evaluation for each patient was done based on three major aspects; pain score, facial swelling and sealing of the wound. Pain score was recorded using the VAS scale from 0 to 10 for pain level, where 0 is equivalent to ‘no pain’ and 10 is equivalent to ‘very painful’. Healing of patient’s wound (sealing of the wound) was assessed by clinical evaluation and digital photographs post-operation on day 1, day 7 and at 6 ± 2 weeks. The scale used for assessment was as follows 1 (significantly getting worse), 2 (neutral), and 3 (significantly improving).

[0210] Data Collection. Data was collected on day 0, day 1 and day 7 post operation. The time of healing, pain score, and swelling reporting. Data were collected at baseline and at follow-up visits one week after the initial procedure.

[0211] Results. Ten patients were assigned to each experimental group. Patients in the control group took the most prolonged time for hemostasis to occur; approximately 6 minutes. The average time of hemostasis was faster with the developed gelatin / starch / CMC sponge, around 1 minute 30 seconds, compared with the commercial sponge, which took an average of 3 minutes for hemostasis to occur; see TIG. 1 1. The average VAS score on day one was much lower in the experimental group who were assigned ColiSorB® compared to the negative control with VAS scores 1 and 5, respectively. The experimental group assigned the commercial sponge showed a slightly higher VAS score; 2, than the developed gelatin / starch / CMC group VAS score: 1, on day one post-operation. On day seven post-operation, there was a general decrease in the VAS scores in all three groups with ColiSorB® showing the lowestVAS score: 0, compared to the control and commercial sponge group: 3 as shown in FIG. 12.

[0212] Clinical assessment of facial swelling showed that the control group patients experienced facial swelling on day one post-operation, whereas patients in the other two experimental groups; the developed gelatin / starch / CMC and Commercial Sponge, did not experience facial swelling nor on day one or day seven post-operation, see Table 1.

[0213] Example 2. Table 1. Assessment of Clinical Complications

[0214] One day one post-operation, sealing of the wound was significantly improved for both experimental groups; the developed gelatin / starch / CMC and the Commercial Sponge, with no significant difference between the two groups. On the other hand, sealing of the wound on day one post-operation in the control group was getting worse. The difference in the sealing of the wound score between day one and day seven was not significant for both experimental groups but was higher for the control group; from 1.5 on day one to 2.5 on day seven as shown by Example 2, Table 1 and FIG. 13 which shows the sealing the wound for the negative control group, the commercial sponge group, and the developed gelatin / starch / CMC group.

[0215] EXAMPLE 3

[0216] In vivo evaluation of the hemostatic ability of the newly developed Gelatin 'starch / CMC foam in large animals

[0217] The hemostatic effects of Gelatin / starch / CMC foam and Cutanplast® hemostatic sponges were observed before and after their application to the bleeding site.

[0218] Methods / Surgical Procedure for Liver Puncture Injury. Punctures were created at a depth of 3mm within the liver tissue. A time interval of 3 seconds was allowed to assess the bleeding score. gelatin / starch / CMC foam and Cutanplast® foam were applied to the bleeding site. Slight pressure was applied to the bleeding site to allow for complete and unified sponge surface area around the lesion. Coagulation capability was measured in relevance to the time each foam took to achieve complete blood clotting and arrest bleeding at the lesion. Blood Loss Assessment. Upon Lesion creation, a pre-weighted gauze (gO) was used to apply pressure to the sponge-lesion site for 3 minutes. The gauze was then removed and reweighted (gl) as shown in Table.1. Blood loss was measured proportional to the bleeding score on a scale 0-4, with 4 denoting the highest bleeding rate achieved, while 0 is the lowest one. Blood loss was measured before and after sponge application.

[0219] Clotting Time Assessment. Upon foam application to the lesion, the time taken to achieve complete hemostasis and blood clotting time was recorded.

[0220] Example 3, Table 1. Parameter efficacy of Colisorb and Cutanplast hemostatic sponges applied surgically on dogs.

[0221] In terms of blood loss, gelatin / starch / CMC foam showed a significantly higher ability to prevent blood loss and enhanced blood containment around the bleeding site which finally led to a bleeding score of almost Omg. When compared to Cutanplast®, significant blood leakage surrounding the bleeding site was observed (Figure 1), which led to a rebleeding effect and continuous blood loss with bleeding score of 1-1.5. The developed Gelatin / starch / CMC foam was 4.9x better than Cutanplast® for eliminating blood loss at the bleeding site. This also leads to a higher blood uptake ratio for gelatin / starch / CMC foam that is almost 1.5x higher than Cutanplast®. The developed gelatin / starch / CMC foam was the best clotting agent by being almost 1 94x faster than Cutanplast® in terms of clotting achievement that is within 30 sec from sponge application to the bleeding site.

[0222] Terminology. Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0223] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0224] It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0225] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0226] Any numerical range recited herein is intended to include all sub-ranges and values subsumed therein. Where a range of values is provided, it is to be understood that each intervening value between an upper and lower limit of the range and any other stated or intervening value in that stated range is encompassed within the disclosure. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included.

[0227] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology. As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features. Commercially available products identified by trademark or tradename such as CUTANPLAST® are typically those cataloged or commercially available as of the filing date of this application.

[0228] The description and specific examples, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested.

[0229] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.

[0230] The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references and does not constitute an admission as to the accuracy of the content of such references.

Claims

CLAIMS1. A foam comprising: gelatin, starch, and carboxymethylcellulose (CMC); wherein, optionally, the foam comprises gelatin, starch, and carboxymethylcellulose (CMC); wherein, optionally, the foam comprises 0.1-0.5 wt.% starch, 1 to 10 wt.% gelatin, and 0.1-0.5 wt.% CMC; that are preferentially cross linked to form a foam.

2. The foam of claim 1 that has a density ranging from 5 to 30 mg / cm3, comprises pores ranging in average diameter from 60-300 microns, comprises 0.1-0.5 wt.% starch, 1 to 10 wt.% gelatin, and 0.1-0.5 wt.% CMC; has an absorptive capacity of 20x-70x or more and has a break stress value of 0.12-0.2 N / mm2.

3. The foam of claim 1 or 2 that comprises 0.1-0.5 wt.% starch, 1 to 10 wt.% gelatin, and 0.1-0.5 wt.% CMC.

4. The foam of any one of claims 1-3 that comprises 0.2 wt.% starch, 5 wt.% gelatin, and 0.2 wt.% CMC, wherein said wt.% values may range upward or downward by 1, 2, 5, 10 or 50 wt.%.

5. The foam of any one of claims 1-4, wherein at least 70% of the foam volume is trapped air or gas.

6. The foam any one of claims 1-5 that absorbs 20x-70x of its weight in water, plasma, blood or another fluid.

7. The foam of any one of claims 1-6 that absorbs 60x-70x of its weight in water, plasma or blood or other fluid.

8. The foam of any one of claims 1-7 that absorbs at least 70x of its weight in water, plasma or blood or other fluid.

9. The foam of any one of claims 1 -8 that has a break stress value ranging from 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18 to 0.19 N / mm2.

10. The foam of any one of claims 1-9 in a wet compressed form.

11. The foam of any one of claims 1-10 in a dry compressed form.

12. The foam of any one of claims 1-11 that is made by a process comprising: dissolving the starch in water at an alkaline pH ranging from pH 8 to 14, dissolving gelatin in water, mixing the dissolved starch and dissolved gelatin, adding the CMC to the mixture of dissolved starch and dissolved gelatin until it is dissolved, and then adding glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or another crosslinker and freezing and lyophilizing the resulting mixture, thereby obtaining a foam.

13. A medical product comprising the foam of any one of claims 1-12 in the form of a powder, sheet, pad, or sponge; optionally containing at least one hemostatic drug, an antibiotic, a vitamin, a bleach, an analgesic, or other active ingredient.

14. A medical product comprising the foam of claim 13 in a compressed form or in an uncompressed form.

15. The medical product of claim 13 in a form that can be directly applied to a wound without prewetting.

16. The medical product of claim 13 that is a wound dressing or a part of a single, dual, or multiple layered wound dressing.

17. A method for treating a wound comprising contacting the wound with the foam of any one of claims 1-12 or with a medical product of any one of claims 12-16.

18. The method of claim 17, wherein the wound is accompanied by severe, spontaneous, or chronic bleeding.

19. The method of claim 17, wherein the wound is a puncture wound, laceration, abrasion, or avulsion.

20. The method of claim 17, wherein the wound is a sport injury wound, surgical wound, internal bleeding such as in the gastrointestinal tract or reproductive organs, a burn, a bite or claw wound, a shaving wound, a wound from an vehicular accident, wound from a tool, a gunshot wound, a battlefield wound, wound from a fall, explosion, or other impact, or other traumatic wound, a wound resulting from a medical condition such as from hemophilia, leukemia, liver disease, lupus, Marfan syndrome, meningitis, platelet disorder, vitamin K deficiency, use of a blood thinner, vasculitis or autoimmune condition, prolonged staining such as weightlifting.

21. An absorptive product that is suitable for non-medical use comprising the foam of any one of claims 1-12.

22. A method for making the foam of claim 1 comprising: dissolving the starch in water or another aqueous medium at an alkaline pH ranging from pH 8 to 14, separately dissolving gelatin in water or another aqueous medium, mixing the dissolved starch and dissolved gelatin, adding the CMC to the mixture of dissolved starch and dissolved gelatin until it is dissolved, and then adding glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker and freezing and lyophilizing the resulting mixture, thereby obtaining a foam.

23. The method of claim 22, wherein the starch is dissolved in water or another aqueous medium at a pH ranging from 9.5 to 11 and / or is dissolved at a temperature ranging from >25 to 100°C.

24. The method of claim 22 or 23, wherein the gelatin is dissolved in in water or another aqueous medium at a temperature ranging from >25 to 100°C.

25. The method of any one of claims 22-24, wherein the pH of the mixture of the starch solution and gelatin solution is adjusted to pH 6 to pH 8 prior to the addition of the CMC.

26. The method of any one of claims 22-25, wherein the glutaraldehyde, genepin, polyethylene glycol diglyci dy 1 ether or other crosslinker is added as a solution containing 50 to 200 pl / ml of glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker27. The method of any one of claims 22-26, wherein the mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is stirred at a speed ranging from 1000 to 2000 rpm.

28. The method of any one of claims 22-27, wherein a stirred mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is frozen at its freezing point or at a temperature ranging from -5°C to -25°C.

29. The method of any one of claims 22-28, wherein the stirred mixture of starch, gelatin, CMC and glutaraldehyde, genepin, polyethylene glycol diglycidyl ether or other crosslinker is frozen in a preselected form such as being frozen in a mold, sheet, or other substrate suitable for lyophilization; or alternatively after being lyophilized is shaped or cut into a desired form.

30. A foam comprising: gelatin, starch, and carboxymethylcellulose (CMC); wherein said foam is crosslinked, wherein said foam volume is 70% or more trapped air or gas, wherein said foam has a density of no more than 30 mg / cm3, wherein said foam comprises pores ranging from 180 to 240 microns in average diameter; and wherein said foam preferably comprises 0.1-0.5 wt.% starch, 1 to 10 wt.% gelatin, and 0.1-0.5 wt.% CMC.

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