METHODS OF USE THEREOF

RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 62/490,356, filed April 26, 2017, the entirety of which is incorporated herein by reference for all purposes. FIELD

[0002] The present invention relates to bioadhesive hydrogels comprising: gelatin, an alginate, a water-soluble crosslinking agent, such as carbodiimide (EDC), and fiber. In an exemplary bioadhesive hydrogel, the fiber is a cellulose fiber (CF). Also encompassed herein are methods of using bioadhesive hydrogels described herein for a variety of applications, including, without limitation, as medical sealants for soft tissue repair (adhesives for closure of, e.g., incisions, blood vessels, dura mater, lung, and/or spinal cord), dental applications, and cosmetic applications. Use of bioadhesive hydrogels for at least one of closure of soft tissue sites wherein a tear has occurred, restoration of an intact structure to fractured teeth and/or bone, adherence of dental devices and appliances, and augmentation of cohesiveness of cosmetics to a body part (such as, e.g., skin and/or hair) are also encompassed herein.

BACKGROUND

[0003] Bioadhesive formul alions/hydrogel s offer superior closure properties over conventional suture or stapling approaches in many applications wherein comprehensive sealing is desirable to prevent body fluid or air leakage. For soft tissue repair, bioadhesive hydrogels are used for topical wound closure (e.g., incision closure): closure of ruptured blood vessels (e.g., aortic dissections); closure of lung perforations (pneumothorax); and repair of various spinal cord injuries, Bioadhesive hydrogels may also be used for internal and/or external fixation of medical and dental devices. For hard tissue repair, bioadhesive hydrogels may be used in lieu of traditional nai ling and plating techniques to fix, e.g., fractured bones and teeth.

SUMMARY

[0004] in an aspect, a bioadhesive hydrogel is presented comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel, and fiber, wherein the fiber is a) 0.1-50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000. In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of the bioadhesive hydrogel. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[0005] In an embodiment thereof, the fiber is at least one of natural fiber and a synthetic fiber. In another embodiment thereof, the natural fiber is at least one of a cellulose fiber, a cellulose whisker, collagen, keratin, fibroin, chitin, chitosan, silk, and catgut. In a further embodiment thereof, the natural fiber is at least one of a cellulose fiber, a cellulose whisker, keratin, fibroin, chitosan, silk, and catgut. In a still further embodiment thereof, the natural fiber is a cellulose fiber, a cellulose whisker, collagen, keratin, fibroin, chitin, chitosan, silk, or catgut. In a more particular embodiment thereof, the natural fiber is a cellulose fiber or a cellulose whisker.

[0006] In another embodiment thereof, the natural fiber is coated with a coating comprising a bioactive agent.

[0007] In yet another embodiment thereof, the synthetic fiber is at least one of polyester, an acrylic fiber, nylon, polyethylene, and polypropylene. In a further embodiment thereof, the synthetic fiber comprises or is coated with a bioactive agent.

[0008] In embodiments thereof, wherein the natural fiber is coated with a coating comprising a bioactive agent or the synthetic fiber comprises or is coated with a bioactive agent, the bioactive agent is at least one of a drug, a growth factor, and a hemostatic agent. As described herein, the drug is at least one of an antibiotic, an analgesic drug, an anti-inflammatory agent, and an anesthetic drug; a growth factor is at least one of stromal -derived growth factor (SDF)-al, fibroblast growth factor (FGF)-2, and bone morphogenetic protein (BMP)-7); and a hemostatic agent is at least one of kaolin, tranexamic acid, and montmorillonite (MMT). [0009] Bioadhesive hydrogels may further comprise at least one of tricalcium phosphate, particulate dentin, hydroxyapatite, whitlockite, aceramic filler, and a particulate filler. Exemplary ceramic fillers are BioGlass or BioGlass Morsels, which are useful in applications relating to bone, such as, for example, dental and orthopedic applications. An exemplary particulate filler is a starch.

[00010] Exemplary cross-linking agents include at least one of a carbodiimide-type coupling agent, glyoxal, formaldehyde, glutaraldehyde, polyglutaraldehyde, dextran, citric acid derivatives, microbial transglutaminase, genipin, diphenyl phosphoryl azide (DPPA), isocyanates, epoxides, and polyepoxides.

[00011] In a particular embodiment, a bioadhesive hydrogel comprises 400 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber, wherein the fiber is present at a concentration of at least 10 mg/ml. In a more particular embodiment, a bioadhesive hydrogel comprises 400 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 2 mg/ml to about 100 mg/ml or about 10 mg/ml to about 80 mg/ml or about 20 mg/ml to about 30 mg/ml. In a still more particular embodiment, a bioadhesive hydrogel comprises 400 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 20 mg/ml to about 30 mg/ml, wherein the fiber is a cellulose fiber.

[00012] In a particular embodiment, a bioadhesive hydrogel comprises 500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber, wherein the fiber is present at a concentration of at least 10 mg/ml. In a more particular embodiment, a bioadhesive hydrogel comprises 500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 2 mg/ml to about 100 mg/ml or about 10 mg/ml to about 80 mg/ml or about 20 mg/ml to about 30 mg/ml. In a still more particular embodiment, a bioadhesive hydrogel comprises 500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 20 mg/ml to about 30 mg/ml, wherein the fiber is a cellulose fiber

[00013] In a particular embodiment, a bioadhesive hydrogel comprises 400-500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber, wherein the fiber is present at a concentration of at least 10 mg/ml. In a more particular embodiment, a bioadhesive hydrogel comprises 400-500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 2 mg/ml to about 100 mg/ml or about 10 mg/ml to about 80 mg/ml or about 20 mg/ml to about 30 mg/ml. In a still more particular embodiment, a bioadhesive hydrogel comprises 400- 500 mg/mL gelatin, 10 mg/mL alginate, 20 mg/mL EDC, and fiber in a range of about 20 mg/ml to about 30 mg/ml, wherein the fiber is a cellulose fiber

[00014] In another aspect, a bioadhesive hydrogel is presented comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel, wherein the crosslinking agent is carbodiimide (EDC); and fiber, wherein the fiber is a) 0.1-50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and the fiber is present at a concentration in the bioadhesive hydrogel sufficient to decrease swelling ratio when tested in a viscosity assay at least 6 hours after post-mixing the gelatin, the alginate, the cross- linking agent, and the fiber. In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of the bioadhesive hydrogel. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00015] In another aspect, a bioadhesive hydrogel is presented comprising a gelatin; an alginate; a crosslinking agent is water-soluble carbodiimide (EDC) in an amount sufficient to form a hydrogel; and cellulose fibers.

[00016] In another aspect, a bioadhesive formulation is presented comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and fiber, wherein the fiber is a) 0.1-50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000. In a particular embodiment thereof, the crosslinking agent is water- soluble carbodiimide (EDC). In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of the bioadhesive formulation. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00017] In another aspect, a bioadhesive formulation is presented comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and cellulose fibers. In a particular embodiment thereof, the crosslinking agent is EDC.

[00018] In yet another aspect, a method for promoting dentin-pulp regeneration is presented, comprising: administering to a subject in need thereof a dental implant comprising a bioadhesive hydrogel or a bioadhesive formulation described herein, wherein the dental implant is administered directly to an injured site in the subject's mouth, and wherein administration of the bioadhesive hydrogel or the bioadhesive formulation to the injured site promotes formation of the dentin-pulp complex at the injured site.

[00019] In an embodiment of the method for promoting dentin-pulp regeneration, the bioadhesive hydrogel or the bioadhesive formulation further comprises at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of the dentin-pulp complex. In a particular embodiment thereof, the at least one growth factor is stromal -derived growth factor (SDF)-al , fibroblast growth factor (FGF)-2, and bone morphogenetic protein (BMP -7). In another particular embodiment thereof, the at least one supportive matrix is an enamel matrix derivative. In yet another embodiment, the at least one DPSC is positive for at least one of STRO-1, cluster of differentiation 29 (CD29), CD44, CD90 and CD 146 and is negative for CD31.

[00020] In another aspect, a method for treating a condition of an oral cavity of a subject is presented, wherein the condition comprises damage to at least one of enamel, dentin, pulp, nerve, and periodontal membrane of the oral cavity, comprising: administering to a subject in need thereof a dental implant comprising a bioadhesive hydrogel or a bioadhesive formulation described herein, wherein the dental implant is administered directly to an injured site in the subject's oral cavity, and wherein administration of the bioadhesive hydrogel or the bioadhesive formulation to the injured site promotes regeneration at the injured site, thereby treating the condition. [00021] In an embodiment of the method for treating a condition of an oral cavity of a subject, the bioadhesive hydrogel further comprises at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of the dentin-pulp complex. In a more particular embodiment, the at least one growth factor is stromal-derived growth factor (SDF)-al, fibroblast growth factor (FGF)-2, and bone morphogenetic protein (BMP-7). In another embodiment, the at least one supportive matrix is an enamel matrix derivative. In a further embodiment, the at least one DPSC is positive for at least one of STRO-1, cluster of differentiation 29 (CD29), CD44, CD90 and CD146 and is negative for CD31.

[00022] In another aspect, a method for producing a bioadhesive hydrogel or a bioadhesive formulation is presented, comprising: mixing

a) a gelatin, wherein the concentration of the gelatin ranges from 50 mg/ml to 400 mg/ml; b) an alginate, wherein the concentration of the alginate ranges from 10 mg/ml to 60 mg/ml; and

c) fiber, wherein each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and the concentration of the fiber ranges from 10 mg/ml to 50 mg/ml;

in a sufficient amount of water to produce a colloidal solution;

heating the colloidal solution to a sufficient temperature to dissolve the gelatin, the alginate, and the fiber in the water, thereby producing a solution of dissolved gelatin, alginate, and fiber; and adding a sufficient amount of a cross-linking agent to the solution of dissolved gelatin, alginate, and fiber to produce the bioadhesive hydrogel or the bioadhesive formulation. In a particular embodiment, the cross-linking agent is EDC. In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v of the bioadhesive hydrogel or formulation. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00023] In a particular embodiment of the method for producing a bioadhesive hydrogel or a bioadhesive formulation, the fiber is at least one of a natural fiber and a synthetic fiber. In a still more particular embodiment, the natural fiber is a cellulose fiber or a cellulose whisker. In another embodiment, the synthetic fiber is at least one of polyester, an acrylic fiber, nylon, polyethylene, and polypropylene. In embodiments thereof, wherein the natural fiber is coated with a coating comprising a bioactive agent or the synthetic fiber comprises or is coated with a bioactive agent, the bioactive agent is at least one of a drug, a growth factor, and a hemostatic agent. As described herein, the drug is at least one of an antibiotic, an analgesic drug, an antiinflammatory agent, and an anesthetic drug; a growth factor is at least one of stromal -derived growth factor (SDF)-al, fibroblast growth factor (FGFV2, and bone rnorphogenetic protein (BMP)-7); and a hemostatic agent is at least one of kaolin, tranexamic acid, and montmorillonite (MMT).

[00024] In another aspect, a dental implant comprising a bioadhesive hydrogel or a bioadhesive formulation is presented comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; fiber, wherein the fiber is a) 0.1-50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of a dentin-pulp complex. In a particular embodiment, the cross-linking agent is EDC. In a particular embodiment, the at least one growth factor is stromai-derived growth factor (SDF)-al, fibroblast growth factor (FGF)-2, and bone rnorphogenetic protein (BMP-7). In another embodiment, the at least one supportive matrix is an enamel matrix derivative. In yet another embodiment, the at least one DPSC is positive for at least one of STRO-1, cluster of differentiation 29 (CD29), CD44, CD90 and CD146 and is negative for CD31.

[00025] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%), or 50%> v/v of the bioadhesive hydrogel or formulation. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100. [00026] In a further aspect, a method for promoting dentin-pulp regeneration is presented, comprising: administering to a subject in need thereof a dental implant comprising a bioadhesive hydrogel or a bioadhesive formulation, wherein the dental implant is administered directly to an injured site in the subject's mouth, and wherein the bioadhesive hydrogel or the bioadhesive formulation comprises: a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; fiber, wherein the fiber is a) 0.1-50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of a dentin-pulp complex. In a particular embodiment, the cross-linking agent is EDC. In another particular embodiment, the at least one growth factor is stromal -derived growth factor (SDF)-al, fibroblast growth factor (FGF)-2, and bone morphogenetic protein (BMP-7). In yet another particular embodiment, the at least one supportive matrix is an enamel matrix derivative. In a further embodiment, the at least one DPSC is positive for at least one of STRO-1, cluster of differentiation 29 (CD29), CD44, CD90 and CD146 and is negative for CD31.

[00027] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%), or 50%> v/v of the bioadhesive hydrogel or formulation of the dental implant. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5- 1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00028] In another aspect, a method for repairing a soft tissue injury in a subject is presented, comprising: administering a bioadhesive hydrogel or a bioadhesive formulation to the subject, wherein the bioadhesive hydrogel or the bioadhesive formulation is administered directly to the soft tissue injury, and wherein the bioadhesive hydrogel or the bioadhesive formulation comprises: a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and fiber, wherein the fiber is a) 0.1-50%) v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000. In a particular embodiment, the cross- linking agent is EDC. In an embodiment thereof, the soft tissue injury is an incision site. In a particular embodiment thereof, the soft tissue injury is an injury to at least one of a blood vessel, dura mater, lung, and spine.

[00029] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%), or 50%> v/v of the bioadhesive hydrogel or formulation used in a method to repair a soft tissue injury. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1- 5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5- 100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00030] In a further aspect, a bioadhesive hydrogel or bioadhesive formulation is presented for use in treating a condition of the oral cavity, wherein the condition comprises damage to at least one of enamel, dentin, pulp, nerve, and periodontal membrane of the oral cavity, wherein the bioadhesive hydrogel or the bioadhesive formulation comprises a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and fiber, wherein the fiber is a) 0.1-50%> v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of a dentin-pulp complex. In a particular embodiment thereof, the cross-linking agent is EDC.

[00031] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%), or 50%> v/v of the bioadhesive hydrogel or formulation for use in treating a condition of the oral cavity. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5- 100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100. [00032] In a still further aspect, a bioadhesive hydrogel or bioadhesive formulation is presented for use in promoting dentin-pulp regeneration, wherein the bioadhesive hydrogel or the bioadhesive formulation comprises a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and fiber, wherein the fiber is a) 0. 1 -50% v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1 -5 mm in length and ranges from 0.1 -250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5- 10,000; and at least one of a growth factor, a supportive matrix, and a dental pulp stem cell (DPSC) that promotes formation of a dentin-pulp complex. In a particular embodiment thereof, the cross-linking agent is EDC.

[00033] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%o, or 50%> v/v of the bioadhesive hydrogel or formulation for use in promoting dentin-pulp regeneration. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1 - 5 mm; 2-5 mm; 3 -5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5- 100; 5-250; 5-500; or 5- 1 ,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1 -0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5- 100.

[00034] In another aspect, a serum, lotion, or skin mask is presented comprising a bioadhesive hydrogel comprising a gelatin; an alginate; a crosslinking agent in an amount sufficient to form a hydrogel; and fiber, wherein the fiber is a) 0. 1 -50%> v/v of the bioadhesive hydrogel and b) each fiber ranges from 0.1 -5 mm in length and ranges from 0.1 -250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5- 10,000; and at least one of a growth factor and a vitamin that promotes skin repair. Such serums, lotions, and masked may be used to ameliorate and/or treat dry, cracked skin. In a particular embodiment thereof, the cross- linking agent is EDC.

[00035] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%o, 40%), 45%o, or 50%> v/v of the bioadhesive hydrogel of the serum, lotion, or skin mask. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1 -5 mm; 2-5 mm; 3 -5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150-250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5- 100; 5-250; 5-500; or 5- 1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00036] In another aspect, a kit is presented comprising: a) a first chamber containing a first aqueous solution comprising: a gelatin; an alginate; fiber, wherein the fiber is i) 0.1-50% v/v of the first aqueous solution and ii) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and water; b) a second chamber containing a second solution comprising a cross-linking agent in an amount sufficient to form a bioadhesive hydrogel; and c) user-instructions for mixing the first aqueous solution and the second solution to form the bioadhesive hydrogel.

[00037] In a particular embodiment thereof, the fiber is 5%, 10%, 15%, 20%, 25%, 30%,

35%), 40%), 45%), or 50%> v/v of the first aqueous solution. In another particular embodiment, each fiber ranges from 0.5-5 mm; 1-5 mm; 2-5 mm; 3-5 mm, or 4-5 mm in length and ranges from 5-250 microns; 10-250 microns; 25-250 microns; 50-250 microns; 100-250 microns; 150- 250 microns; or 200-250 microns in diameter. In yet another particular embodiment, the length/diameter ratio (L/d) ranges from 5-100; 5-250; 5-500; or 5-1,000. In another particular embodiment, each fiber ranges from 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and has a L/d of: 5-100.

[00038] In yet another aspect, a device for applying a bioadhesive hydrogel to a subject in need thereof is presented, the device comprising: a) a first chamber containing a first aqueous solution comprising: a gelatin; an alginate; fiber, wherein the fiber is i) 0.1-50%) v/v of the first aqueous solution and ii) each fiber ranges from 0.1-5 mm in length and ranges from 0.1-250 microns in diameter and has a length/diameter ratio (L/d) ranging from 5-10,000; and water; b) a second chamber containing a second solution comprising a cross-linking agent in an amount sufficient to form a bioadhesive hydrogel; wherein the bioadhesive hydrogel is produced upon mixing the first aqueous solution and the second solution, and wherein the bioadhesive hydrogel is in an amount effective to treat the subject in need thereof. In a particular embodiment thereof, the device comprises a syringe. In a more particular embodiment thereof, the device comprises a static mixer, wherein the first aqueous solution and the second solution are mixed.

[00039] The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.

[00040] The figures constitute a part of this specification and include illustrative embodiments of the present invention and illustrate various objects and features thereof. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. In addition, any measurements, specifications and the like shown in the figures are intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[00041] Figure 1 : Effect of the CF concentration on the burst strength of bioadhesive hydrogels. Burst strengths for polymeric hydrogel (control; no fiber) and bioadhesive hydrogels comprising the indicated fiber concentrations are shown. Significant differences are marked with (*).

[00042] Figure 2: Effect of the CF concentration on the bonding strength in lap shear of bioadhesive hydrogels. Bonding strength for polymeric hydrogel (control; no fiber) and bioadhesive hydrogels comprising the indicated fiber concentrations are shown. Significant differences are marked with (*).

[00043] Figure 3 : Effect of the CF concentration on the compression elastic modulus of bioadhesive hydrogels. Compression elastic modulus for polymeric hydrogel (control; no fiber) and bioadhesive hydrogels comprising the indicated fiber concentrations are shown. Significant differences are marked with (*).

[00044] Figure 4: ESEM fractographs of the (a) CFs, (b) a polymeric hydrogel, and (c-d) bioadhesive hydrogel loaded with 50 mg/mL CF.

[00045] Figure 5: Viscosity of polymeric hydrogel (control; no fiber) and bioadhesive hydrogels as affected by the CF concentration. Significant differences are marked with (*).

[00046] Figure 6: Swelling ratio of polymeric hydrogel (control; no fiber; US) and bioadhesive hydrogels as affected by the concentration of CF. (1223 -10, 1223-20, 223-30, 33-40 and 2 ^* -50 mg/mL). Significant differences are marked with (*). [00047] Figure 7: Weight loss of polymeric hydrogel (control; no fiber;ill) and bioadhesive hydrogels as affected by the concentration of CF. (S3 -10, && -20, KS-30, 113-40 and ^* -50 mg/mL). Significant differences are marked with (*).

[00048] Figure 8: Gelation time of polymeric hydrogel (control; no fiber) and bioadhesive hydrogels as affected by the CF concentration. Significant differences are marked with (*)

[00049] Figure 9: Schematic representation of a qualitative model summarizing the effects of CF incorporation on the bioadhesive hydrogel' s properties. The green/red boxes represent a case where the fibers' incorporation leads to an increase/decrease in certain properties, respectively. Grey boxes represent a mixed response.

[00050] Figure 10: Effect of cellulose fiber concentration on the burst strength of a bioadhesive hydrogel comprising 400 mg/ml gelatin, 10 mg/ml alginate, and 20 mg/ml EDC (40: 1 :2 ratio of gelatin:alginate:EDC) is depicted, at the indicated concentrations of the hemostatic agent kaolin.

[00051] Figure 11 : Effect of the kaolin concentration on the burst strength of a bioadhesive hydrogel comprising 400 mg/ml gelatin, 10 mg/ml alginate, and 20 mg/ml EDC (40: 1 :2 ratio of gelatin:alginate:EDC) is depicted, at the indicated concentrations of cellulose fibers. Burst strength for polymeric hydrogel (control; no fiber) indicated as histogram bar labeled 0 mg/ml CF.

[00052] Figure 12: Double barrel syringe comprising a polymeric solution and a cross- linker solution.

DESCRIPTION

[00053] Among those benefits and improvements that have been disclosed, other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention which are intended to be illustrative, and not restrictive.

[00054] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases "in one embodiment" and "in some embodiments" as used herein do not necessarily refer to the same embodiment(s), though it may. Furthermore, the phrases "in another embodiment" and "in some other embodiments" as used herein do not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[00055] In addition, as used herein, the term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of "a," "an," and "the" include plural references. The meaning of "in" includes "in" and "on."

[00056] As used herein, the term "polymeric hydrogel" refers to a gel comprising a network of polymer chains in which water is typically the dispersion medium.

[00057] As used herein, the term "bioadhesive hydrogel" refers to a polymeric hydrogel into which fibers have been incorporated. In a particular embodiment, the fiber is homogenously dispersed in a continuous medium of the bioadhesive hydrogel.

[00058] In a particular embodiment, a bioadhesive hydrogel has a dry density of 0.7-1.3 grams/cm ³ . In another particular embodiment, a bioadhesive hydrogel has a wet density of 0.9- 1.7 grams/cm ³ .

[00059] In a particular embodiment, a bioadhesive hydrogel composition/formulation is a non-foamable bioadhesive hydrogel composition/formulation.

[00060] In a particular embodiment, a bioadhesive hydrogel comprises at least one of a hydrophilic fiber such as, for example, at least one of a cellulose fiber, a cellulose whisker, collagen, keratin, fibroin, chitin, chitosan, silk, and catgut. In a more particular embodiment, a bioadhesive hydrogel comprises at least one of a hydrophilic fiber such as, for example, at least one of a cellulose fiber, a cellulose whisker, keratin, fibroin, chitosan, silk, and catgut. In a still more particular embodiment, a bioadhesive hydrogel comprises at least one of a hydrophilic fiber such as, for example, at least one of a cellulose fiber or a cellulose whisker.

[00061] In a particular embodiment, most short fibers are in the range of 0.1-5 mm length and 0.1-250 microns in diameter and have a length/diameter ratio (L/d) of 5-10,000. In a particular embodiment wherein the fibers are nanowhiskers, the nanowhiskers (e.g., cellulose nanowhiskers) are in the range of 1-2 microns (0.001-0.002 mm) length and 0.01-0.05 microns in diameter and have a L/d of 20-200. Collagen fibers described herein are in the range of 100-500 microns (0.1-0.5 mm) in length and 5-20 microns in diameter and have a L/d of: 5-100.

[00062] The present invention relates to bioadhesive formulations and bioadhesive hydrogels. In some embodiments, natural cellulose fibers were selected for enhancement of polymeric hydrogel properties. In some embodiments, polymeric hydrogels and formulations thereof based on a combination of gelatin and alginate crosslinked with water-soluble carbodiimide were used as a generic formulation. In some embodiments, the polymeric hydrogels and the cellulose fibers showed high affinity which resulted in an increase in the viscosity and in the burst strength of bioadhesive hydrogels generated therefrom. In some embodiments, the subject bioadhesive hydrogels can be used for surgical sealant applications due to improvements in physical properties of the bioadhesive hydrogels relative to polymeric hydrogels, e.g., an increase in the cohesive strength of bioadhesive hydrogels and a decrease in the swelling ratio in bioadhesive hydrogels relative to polymeric hydrogels.

[00063] In some embodiments, the bioadhesive hydrogel comprises at least one fiber, wherein the at least one fiber comprises: cellulose, silk, polyglycolic acid, polylactic acid, polycaprolactone, polydioxanone, copolymers of lactic acid and glycolic acid, polyamide, polyethylene terephthalate, polyethylete, polypropylene, or any combination thereof. In some embodiments, the bioadhesive hydrogel comprises cellulose fibers. In some embodiments, the cellulose fibers are natural. In some embodiments, the cellulose fibers are synthetic.

[00064] In some embodiments, the bioadhesive hydrogel is loaded with at least one fiber. In some embodiments, the bioadhesive hydrogel is loaded with at least one drug-eluting fiber. In some embodiments, the bioadhesive hydrogel is loaded with at least one fiber and at least one drug molecule, wherein the at least one fiber and the at least one drug molecule are located separately in the bioadhesive hydrogel. In some embodiments, the bioadhesive hydrogel is loaded with at least one fiber and a hemostatic agent, wherein the at least one fiber and the hemostatic agent are located separately in the polymeric hydrogel.

[00065] In some embodiments, requirements for polymeric hydrogels for bioadhesive and sealant applications are first of all biocompatibility, as well as other mechanical and physical properties such as the burst strength, tensile strength, shear strength, curing time, viscosity, swelling degree and degradation. Methods for testing each of these mechanical and physical properties are described in, e.g., M. Mehdizadeh, J. Yang, Macromol. Biosci. 2013, 13, 271-288, and A. H. Landrock, S. Ebnesajjad, Adhesives technology handbook. Editor, William Andrew, 2008, both of which references are herein incorporated in their entireties.

[00066] As used herein, and is known in the art, the term "gelatin" describes a water- soluble protein that can form gel under certain conditions. Gelatin is typically obtained by heat dissolution at acidic or alkaline and partially hydrolyzing conditions of collagen. Type A gelatin is obtained by acidic process and has a high density of amino groups causing a positive charge. Type B gelatin is obtained by alkaline process and has high density of carboxyl groups causing negative charge. There are different sources for collagen such as animal skin and bone, which afford a variety of gelatin forms with a range of physical and chemical properties. Typically, gelatin contains eighteen amino acids that are linked in partially ordered fashion; glycine or alanine is about a third to half of the residues, proline or hydroxyproline are about one fourth and the remaining forth include acidic or basic amino acid residues. Typically, in order to dissolve gelatin in water it is necessary to reach a temperature of at least 35 °C by heating or stirring and adding hot water, depending on the source of gelatin used. Moderate heating enhances solubility and severe heating may cause aggregation or partial hydrolysis of gelatin. The viscosity of gelatin varies with type, concentration, time and temperature. Acid processed gelatin has slightly greater intrinsic viscosity compared to alkali processed gelatin. Gelatin is relatively cheap, it is biocompatible with negligible immunologic problems, and it is biodegradable. "Bloom" is a test to measure the strength of a gel or gelatin. The test determines the weight (in grams) needed by a probe (normally with a diameter of 0.5 inch) to deflect the surface of the gel 4 mm without breaking it. The result is expressed in Bloom grades or Bloom number, and it is typically between 30 and 300 Bloom. To perform the Bloom test on gelatin, a 6.67 % gelatin solution is kept for 17-18 hours at 10 °C prior to being tested.

[00067] The term "biodegradable" and any adjective, conjugation and declination thereof as used herein, refers to a characteristic of a material to undergo chemical and/or physical transformation from a detectable solid, semi-solid, gel, mucus or otherwise a localized form, to a delocalized and/or undetectable form such as any soluble, washable, volatile, absorbable and/or resorbable breakdown products or metabolites thereof. A biodegradable material undergoes such transformation at physiological conditions due to the action of chemical, biological and/or physical factors, such as, for example, innate chemical bond lability, enzymatic breakdown processes, melting, dissolution and any combination thereof. [00068] In some embodiments, alternatives to gelatin may include non-animal gel sources such as agar-agar (a complex carbohydrate harvested from seaweed), carrageenan (a complex carbohydrate harvested from seaweed), pectin (a colloidal carbohydrate that occur in ripe fruit and vegetables), konjaka (a colloidal carbohydrate extracted from plants of the genus Amorphophallus), guar gum (guaran, a type of galactomannan extracted from cluster beans of the genus Cyamopsis tetragonolobus) and various combinations thereof with or without gelatin.

[00069] As used herein, and is known in the art, the term "alginate" describes an anionic polysaccharide. Alginate, which is also referred to herein and in the art as alginic acid, is a block copolymer composed of β-D mannuronic acid monomers (M blocks) and a-L guluronic acid (G blocks), with different forms of alginate having different ratio of M/G. The term "alginate", as used herein, encompasses various M/G ratio. M/G ratio varies according to the species, source and harvest season of the algae/plant.

[00070] In some embodiments, the alginate has an M/G ratio that ranges from 0.3 to 4, from 0.7 to 3, or from 1 to 2. In other embodiments, the M/G ratio is 0.7, 0.9, 1, 1.3, 1.5, 1.7, 1.9, 2, 2.3, 2.5, 2.7, 3, 3.5 or 4.

[00071] Alginate is known to form a viscous gum by binding water (capable of absorbing

200-300 times its own weight in water).

[00072] Alginate undergoes reversible gelation in aqueous solution under mild conditions through interactions with divalent cations that bind between G-blocks of adjacent alginate chains creating ionic inter chain bridges. Since alginate is generally anionic polymer with carboxyl end, it is known and used as a good mucoadhesive agent.

[00073] Naturally occurring alginate is typically produced in marine brown algae (e.g., Macrocystis pyrifera, Ascophyllum nodosum and Laminarid) and soil bacteria (Pseudomonas and Azotobacter). Synthetically prepared alginates are also contemplated.

[00074] As used herein, a "bioadhesive" refers to a material that can bond tissues together when applied to their surfaces and prevent separation by transferring the applied loads from one tissue to another.

[00075] As used herein, a "surgical sealant" refers to a material that when applied, can prevent leakage of fluids by creating a sealing barrier on the wounded area. A sealant must be able to withstand pressure and an adhesive must be able to withstand shear stress. [00076] As used herein, the terms "localized" or "site-specific" are used interchangeably to mean delivery of a bioactive agent within a limited area, as opposed to a "systemic" delivery throughout the body of a subject or patient. Systemic therapy sometimes results in a therapeutically ineffective level of an antibiotic, drug or the like, at the site of interest (wound, infection and the like). Systemic treatment can also be associated with different degrees of toxicity. Furthermore, the expense involved in acquiring large amounts of a bioactive agent used for systemic treatment may restrict therapy for some subjects or patients. By comparison, localized delivery of bioactive agent can reduce systemic side effects by using a fraction of the systemic dose of the bioactive agent (e.g., antibiotic) to combat a particular disorder.

[00077] As used herein, the terms "sustained release" or "long term release or delivery" are used interchangeably herein to mean that the expected delivery of a bioactive agent from a bioadhesive hydrogel described herein is prolonged relative to that expected based solely upon diffusion kinetics. Typically, delivery will be at least a day or more, and may extend to weeks or months. Long term release may be achieved by any of a number of mechanisms. The bioactive agent may be added to the bioadhesive hydrogel as a solid. The bioactive agent may be added in solution in a carrier or hydrating agent which has a higher rate of diffusion than that of the bioadhesive hydrogel so that upon diffusion of the carrier or hydrating agent from the hydrogel, the bioactive agent is precipitated within the hydrogel. Ethanol is an example of such a carrier. The bioactive agent may be precipitated into the bioadhesive hydrogel from a supersaturated solution. The bioactive agent may be added in such a mass as to exceed the volume which would be soluble in the bioadhesive hydrogel. The bioactive agent may be added as an emulsion or dispersion, for example, in a lipid or oil-based carrier. Release of the bioactive agent from the bioadhesive hydrogel may also be delayed because of specific physical or biochemical interactions with the bioadhesive hydrogel.

[00078] As used herein, the term "wound" refers to any damage to any tissue in a living organism. The tissue may be an internal tissue, such as the stomach lining or a bone, or an external tissue, such as the skin. As such, a wound may include, but is not limited to, a gastrointestinal tract ulcer, a broken bone, a neoplasia, and cut or abraded skin. A wound may be in a soft tissue, such as the spleen, or in a hard tissue, such as bone. The wound may have been caused by any agent, including traumatic injury, infection or surgical intervention. [00079] In some embodiments, the present invention is a composition comprising: 1-15 wt

% cellulose fibers (CF), 60-95 wt % gelatin, 1-25 wt% alginate, and 1-20 wt% crosslinking agent. In some embodiments, the composition comprises 1-13 wt% CF. In some embodiments, the composition comprises 1-11 wt% CF. In some embodiments, the composition comprises 1-9 wt% CF. In some embodiments, the composition comprises 1-7 wt% CF. In some embodiments, the composition comprises 1-5 wt% CF. In some embodiments, the composition comprises 1-3 wt% CF.

[00080] In some embodiments, the composition comprises 3-15 wt% CF. In some embodiments, the composition comprises 5-15 wt% CF. In some embodiments, the composition comprises 7-15 wt% CF. In some embodiments, the composition comprises 9-15 wt% CF. In some embodiments, the composition comprises 11-15 wt% CF. In some embodiments, the composition comprises 13-15 wt% CF. In some embodiments, the composition comprises 3-13 wt% CF. In some embodiments, the composition comprises 5-11 wt% CF. In some embodiments, the composition comprises 7-9 wt% CF.

[00081] In some embodiments, the composition comprises 65-95 wt% gelatin. In some embodiments, the composition comprises 70-95 wt% gelatin. In some embodiments, the composition comprises 75-95 wt% gelatin. In some embodiments, the composition comprises 80-95 wt% gelatin. In some embodiments, the composition comprises 85-95 wt% gelatin. In some embodiments, the composition comprises 90-95 wt% gelatin.

[00082] In some embodiments, the composition comprises 60-90 wt% gelatin. In some embodiments, the composition comprises 60-85 wt% gelatin. In some embodiments, the composition comprises 60-80 wt% gelatin. In some embodiments, the composition comprises 60-75 wt% gelatin. In some embodiments, the composition comprises 60-70 wt% gelatin. In some embodiments, the composition comprises 60-65 wt% gelatin. In some embodiments, the composition comprises 65-90 wt% gelatin. In some embodiments, the composition comprises 70-85 wt% gelatin. In some embodiments, the composition comprises 75-80 wt% gelatin.

[00083] In some embodiments, the composition comprises 1-20 wt% alginate. In some embodiments, the composition comprises 1-15 wt% alginate. In some embodiments, the composition comprises 1-10 wt% alginate. In some embodiments, the composition comprises 1-5 wt% alginate. [00084] In some embodiments, the composition comprises 5-25 wt% alginate. In some embodiments, the composition comprises 10-25 wt% alginate. In some embodiments, the composition comprises 15-25 wt% alginate. In some embodiments, the composition comprises 20-25 wt% alginate. In some embodiments, the composition comprises 5-20 wt% alginate. In some embodiments, the composition comprises 10-15 wt% alginate.

[00085] In some embodiments, the composition comprises 1-15 wt% crosslinking agent.

In some embodiments, the composition comprises 1-10 wt% crosslinking agent. In some embodiments, the composition comprises 1-5 wt% crosslinking agent.

[00086] In some embodiments, the composition comprises 5-20 wt% crosslinking agent.

In some embodiments, the composition comprises 10-20 wt% crosslinking agent. In some embodiments, the composition comprises 15-20 wt% crosslinking agent. In some embodiments, the composition comprises 5-15 wt% crosslinking agent. In some embodiments, the composition comprises 10-15 wt% crosslinking agent. In some embodiments, the composition comprises 5-10 wt% crosslinking agent.

[00087] In some embodiments, the crosslinking agent is water-soluble carbodiimide

(EDC).

[00088] In some embodiments, the polymeric solution (gelatin-alginate aqueous solution before adding the crosslinker solution) has a room temperature viscosity from 0.02 Pa-sec to 50 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 1 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 2 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 3 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 4 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 5 Pa-sec to 10 Pa- sec. In some embodiments, the polymeric solution has a room temperature viscosity from 6 Pa- sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 7 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 8 Pa-sec to 10 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 9 Pa-sec to 10 Pa-sec. [00089] In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 9 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 8 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 7 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 6 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 5 Pa- sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa- sec to 4 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 3 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 2 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 0.5 Pa-sec to 1 Pa-sec.

[00090] In some embodiments, the polymeric solution has a room temperature viscosity from 1 Pa-sec to 9 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 2 Pa-sec to 8 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 3 Pa-sec to 7 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 4 Pa-sec to 6 Pa-sec. In some embodiments, the polymeric solution has a room temperature viscosity from 4 Pa-sec to 5 Pa-sec.

[00091] In some embodiments, the formulation comprises 400 mg/mL gelatin, 10 mg/mL alginate and 20 mg/mL EDC (400-10-20 mg/mL gelatin-alginate-EDC) and exhibits a low cytotoxicity. In an embodiment thereof, the fiber concentration range is 2- 100 mg/ml. The effect of CF was studied in concentrations of 10-50 mg/mL. Crosslinking agent (EDC) solution was added to the bioadhesive formulation just prior to use.

[00092] In the context of some embodiments of the present invention, alginate can be used in a high-viscosity (HV) form, exhibiting more than 2 Pa-sec, or low-viscosity (LV) form, exhibiting 0.1-0.3 Pa-sec. As demonstrated in the Examples section hereinbelow, use of the LV/HV alginate forms adds another parameter with which to fine-tune bioadhesive formulations/hydrogels presented herein.

[00093] The bioadhesive hydrogel may further comprise a clay mineral, which is typically added to the formulation of gelatin/alginate mixture as a dry powder or a suspension of solid particles. Clay minerals include, without limitation, Kaolinite (Kaolin, Al ₂ Si205(OH) ₄ ), Montmorillonite (MMT, (Na,Ca)o.33(Al,Mg) ₂ Si ₄ Oio(OH) ₂ -«H ₂ 0), Halloysite (Al ₂ Si ₂ 0 ₅ (OH) ₄ ), Illite ((K,H ₃ 0)(Al,Mg,Fe)2(Si,Al) ₄ Oio[(OH) ₂ ,(H ₂ 0)]),

Vermiculite ((MgFe,Al) ₃ (Al,Si) ₄ Oi ₀ (OH) ₂ 4H ₂ 0), Talc (Mg ₃ Si ₄ Oi ₀ (OH) ₂ ),

Sepiolite (Mg ₄ Si ₆ Oi ₅ (OH) ₂ -6H ₂ 0), Palygorskite (Attapulgit, (Mg,Al) ₂ Si ₄ Oi ₀ (OH) 4(H ₂ 0)) and Pyrophyllite (Al ₂ Si ₄ Oi ₀ (OH) ₂ ).

[00094] Kaolinite (Kaolin) and Montmorillonite (MMT) are both layered silicates

(phyllosilicates, or clay minerals) having a general form of parallel sheets of silicate tetrahedra of Si ₂ 0 ₅ (aka 2:5 ratio) sandwiching a layer on another oxide such as alumina, and bonded by hydrogen bonds via water or hydroxyl groups. While kaolin is a 1 : 1 clay, meaning the silica and alumina layers alternate at a 1 : 1 ratio, MMT is a 2: 1 clay, meaning that it has 2 tetrahedral silica sheets sandwiching a central octahedral oxide sheet. It has been observed that kaolin does not expand when wetted, while MMT has a remarkable wet expansion or swelling capacity. These structural differences may explain the difference in performance of the composite gelatin- alginate bioadhesive sealant formulations.

[00095] As discussed hereinabove, while kaolin is a well-known and widely used as a coagulant or coagulation-promoting agent, e.g., a hemostatic agent (e.g., but not limited to, kaolin, montmorillonite (MMT), tranexamic acid, etc.), in commercially available medical products, MMT is rarely used for such purposes. As demonstrated in the Examples section below, MMT was found to be advantageous in the context of embodiments of the present invention; for example, by conferring a higher burst strength to bioadhesive hydrogels compared to those comprising kaolin. Hence, according to embodiments of the present invention, the bioadhesive hydrogel comprises MMT.

[00096] The term "coupling agent", as used herein, refers to a reagent that can catalyze or form a bond between two or more functional groups intra-molecularly, inter-molecularly or both. Coupling agents are widely used to increase polymeric networks and promote crosslinking between polymeric chains, hence, in the context of some embodiments of the present invention, the coupling agent is such that can promote crosslinking between polymeric chains; or such that can promote crosslinking between amino functional groups and carboxylic functional groups, or between other chemically compatible functional groups of polymeric chains; or is such that can promote crosslinking between gelatin and alginate. In some embodiments of the present invention the term "coupling agent" may be replaced with the term "crosslinking agent". In some embodiments, one of the polymers serves as the coupling agent and acts as a crosslinking polymer.

[00097] By "chemically compatible" it is meant that two or more types of functional groups can react with one another so as to form a bond.

[00098] Exemplary functional groups which are typically present in gelatins and alginates include, but are not limited to, amines (mostly primary amines - H ₂ ), carboxyls (-C0 ₂ H), sulfhydryls and hydroxyls (-SH and -OH respectively), and carbonyls (-COH aldehydes and - CO- ketones).

[00099] Primary amines occur at the N-terminus of polypeptide chains (called the alpha- amine), at the side chain of lysine (Lys, K) residues (the epsilon-amine), as found in gelatin, as well as in various naturally occurring polysaccharides and aminoglycosides. Because of its positive charge at physiologic conditions, primary amines are usually outward-facing (i.e., found on the outer surface) of proteins and other macromolecules; thus, they are usually accessible for conjugation.

[000100] Carboxyls occur at the C-terminus of polypeptide chain, at the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E), as well as in naturally occurring aminoglycosides and polysaccharides such as alginate. Like primary amines, carboxyls are usually on the surface of large polymeric compounds such as proteins and polysaccharides.

[000101] Sulfhydryls and hydroxyls occur in the side chain of cysteine (Cys, C) and serine, (Ser, S) respectively. Hydroxyls are abundant in polysaccharides and aminoglycosides.

[000102] Carbonyls as ketones or aldehydes can be form in glycoproteins, glycosides and polysaccharides by various oxidizing processes, synthetic and/or natural.

[000103] According to some embodiments of the present invention, the coupling agent can be selected according to the type of functional groups and the nature of the crosslinking bond that can be formed therebetween. For example, carboxyl coupling directly to an amine can be afforded using a carbodiimide type coupling agent, such as EDC; amines may be coupled to carboxyls, carbonyls and other reactive functional groups by N-hydroxysuccinimide esters ( HS-esters), imidoester, PFP-ester or hydroxymethyl phosphine; sulfhydryls may be coupled to carboxyls, carbonyls, amines and other reactive functional groups by maleimide, haloacetyl (bromo- or iodo-), pyridyldisulfide and vinyl sulfone; aldehydes as in oxidized carbohydrates, may be coupled to other reactive functional groups with hydrazide; and hydroxyl may be coupled to carboxyls, carbonyls, amines and other reactive functional groups with isocyanate.

[000104] Hence, suitable coupling agents that can be used in some embodiments of the present invention include, but are not limited to, carbodiimides, NHS-esters, imidoesters, PFP- esters or hydroxymethyl phosphines.

[000105] A carbodiimide is a complete crosslinker that facilitates the direct coupling (conjugation) of carboxyls to primary amines. Thus, unlike other reagents, carbodiimide is a zero-length crosslinker; it does not become part of the final crosslink between the coupled molecules. Because peptides, proteins, polysaccharides and aminoglycosides contain multiple carboxyls and amines, direct carbodiimide-mediated coupling/crosslinking usually causes random polymerization of polypeptides.

[000106] EDC, or N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, is a widely used carbodiimide-type coupling agent and crosslinker which enables the condensation between carboxyl and amino groups to form amide bonds and the byproduct urea. Once reacted with amine/hydroxyl reactants, EDC is not present in the structure of the coupled product; hence its biocompatibility and biodegradability are not an issue in the context of the present embodiments. As a gelatin molecule exhibits both carboxyl and amino groups, this type of polymer may undergo intermolecular crosslinking by EDC.

[000107] Alternatives for carbodiimide-type coupling agent, according to some embodiments of the present invention, include without limitation, glyoxal, formaldehyde, glutaraldehyde, polyglutaraldehyde, dextran, citric acid derivatives, microbial transglutaminase, genipin, diphenyl phosphoryl azide (DPP A), isocyanates such as hexamethylene diisocyanate (HMDC), epoxides (such as ethylene glycol diglycidyl ether), polyepoxides (such as glycerol polyglucidyl ether, sorbitol polyglycidyl ether, and polyethylene glycol diglycidyl ether).

[000108] In some embodiments, the coupling agent is used up during the coupling reaction, and produces a urea derivative as a byproduct of the coupling reaction between amine and carboxyl groups. The nature of the urea derivative is determined by the nature of the coupling agent used.

[000109] According to some embodiments of the present invention, various coupling and crosslinking agents may be combined or used as additives in any given bioadhesive formulation based on gelatin and alginate and a coupling agent, so as to further promote the crosslinking reaction. In a representative example, NHS-esters are added to a carbodiimide-type coupling agent such as EDC.

[000110] The addition of NHS to the crosslinking reaction of EDC affords an NHS- activated carboxylic acid group, which is less susceptible to hydrolysis and prevents rearrangements. On the other hand, at high concentration NHS can react with the EDC and compete with the crosslinking reaction, thereby reducing the effective amount of EDC for crosslinking. Hence, reagents such as NHS are referred to herein a crosslinking promoting agents.

[000111] By adding various agents that promote the coupling reaction, and, in the context of the present invention, promote the formation of crosslinks in the forming bioadhesive hydrogel, it is intended to increase the crosslinking efficiency and/or reduce the amount of coupling agent needed to form a hydrogel the exhibits the desired characteristics, as discussed hereinabove. Hence, such agents are referred to herein as "crosslinking promoting agents". The amount of a crosslinking promoting agent is given as weight/volume per weight/volume percent (w/v / w/v), i.e. relative to the amount of the coupling agent, and according to some embodiments of the present invention, this amount ranges from about 1 % to 100 %, or from 1 % to 200 % weight/volume per weight/volume percent.

[000112] Representative examples of crosslinking promoting agents include, without limitation, sulfo-NHS, HOBt, HOAt, HBtU, HCtU, HAtU, TBtU, PyBOP, DIC pentafluorophenol and the likes.

[000113] According to some embodiments of the present invention, a combination of a crosslinking agent and a crosslinking promoting agent in the bioadhesive formulations presented herein, affords bioadhesive hydrogels with improved bonding strength. Furthermore, the combination of a crosslinking agent such as EDC and a crosslinking promoting agent such as N- hydroxysuccinimide (NHS), allows for a significant reduction in the amount of EDC in the bioadhesive hydrogel. A reduction of the amount of EDC is beneficial due to the medical safety and cytotoxicity implications of using EDC.

[000114] In some embodiments, the amount of the crosslinking promoting agent may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 100, 150, 200 %, including any value between 1 and 200 % relative to the amount of the coupling agent, or can be even higher. In some embodiments of the present invention, the amount of the crosslinking promoting agent is 5, 10, 15, 20, 30 or 40 % relative to the amount of the coupling agent, including any value from 5 to 40.

[000115] When used in tandem, EDC and NHS afford stronger bonding bioadhesive hydrogels, even when relatively low concentration of EDC is used. An exemplary formulation comprises an amount of EDC which is 10 mg/ml and an amount of NHS which is 10 % relative to the amount of the EDC.

[000116] Various additional and optional additives may be added to the bioadhesive hydrogel in order to modify its pre-curing characteristics, such as for example, viscosity modifiers for improved application and spread confinement, penetration enhancers, and colorants or fluorescent agents for allowing tracking during application and follow-up. Various additives may be added to the bioadhesive hydrogel in order to modify its post-curing characteristics, namely additives that affect the characteristics of the resulting hydrogel, such as for example, additional coupling/crosslinking agents, calcium ions and ions of other earth-metals, which act as gelling agents by virtue of being crosslinkers for various alginate species, plasticizes, hardeners, softeners, fillers and other agents for modifying the flexural modulus of the hydrogel, and additives that affect the release rate, penetration and absorption of a bioactive agent, when present in the bioadhesive hydrogel, as discussed in more detail hereinbelow.

[000117] In some embodiments, the present invention comprises a kit, wherein the kit includes a composition comprising: 2-12% cellulose fibers (CF), 90-95% gelatin, 1-3% alginate, and 3-5% crosslinking agent, and a device configured to apply the composition to a subject.

[000118] Drug-eluting bioadhesive formulations:

[000119] According to some embodiments of the present invention, a bioadhesive hydrogel as described herein further comprises one or more bioactive agent(s). In some embodiments, such a formulation is designed to afford a drug-eluting bioadhesive hydrogel upon curing. In other words, bioadhesive hydrogels that include a bioactive agent, cure to form a drug-eluting bioadhesive hydrogel in which the bioactive agent is incorporated. In some embodiments, such drug-eluting bioadhesive hydrogels are formed such that the bioactive agent is released therefrom upon contacting a physiological medium. Thus, the bioadhesive hydrogel, according to some embodiments of the present invention, can be used for various bioadhesion applications, as discussed herein, while at the same time serving as a reservoir and vehicle for delivering a bioactive agent. [000120] It is noted herein that while the incorporation of a bioactive agent in the bioadhesive hydrogel may affect the characteristics thereof, the bioadhesive hydrogel is designed to possess desired properties presented hereinabove while adding the capacity of eluting bioactive agent(s) as discussed hereinbelow.

[000121] The term "incorporated", as used in the context of a bioactive agent and a bioadhesive hydrogel, according to some embodiments of the present invention, is used synonymously with terms such as "sequestered", "loaded", "encapsulated", "associated with", "charged" and any inflection of these terms, all of which are used interchangeably to describe the presence of the bioactive agent, as defined hereinbelow, within the bioadhesive hydrogel. A sequestered bioactive agent can elute or be released from the bioadhesive hydrogel via, for example, diffusion, dissolution, elution, extraction, leaching, as a result of any or combination of wetting, swelling, dissolution, chemical breakdown, degradation, biodegradation, enzymatic decomposition and other processes that affect the bioadhesive hydrogel. A bioactive agent may also elute from the bioadhesive hydrogel without any significant change to the bioadhesive hydrogel structure, or with partial change.

[000122] As used herein, the phrase "bioactive agent" describes a molecule, compound, complex, adduct and/or composite that exerts one or more biological and/or pharmaceutical activities. The bioactive agent can thus be used, for example, to relieve pain, prevent inflammation, prevent and/or reduce and/or eradicate an infection, promote wound healing, promote tissue regeneration, effect tumor/metastasis eradication/suppression, effect local immune-system suppression, and/or to prevent, ameliorate or treat various medical conditions.

[000123] "Bioactive agents", "pharmaceutically active agents", "pharmaceutically active materials", "pharmaceuticals", "therapeutic active agents", "biologically active agents", "therapeutic agents", "medicine", "medicament", "drugs" and other related terms may be used herein interchangeably, and all of which are meant to be encompassed by the term "bioactive agent".

[000124] The term "bioactive agent" in the context of the present invention also includes diagnostic agents, including, for example, chromogenic, fluorescent, luminescent, phosphorescent agents used for marking, tracing, imaging and identifying various biological elements such as small and macromolecules, cells, tissue and organs; as well as radioactive materials which can serve for both radiotherapy and tracing, for destroying harmful tissues such as tumors/metastases in the local area, or to inhibit growth of healthy tissues, such as in current stent applications; or as biomarkers for use in nuclear medicine and radio-imaging.

[000125] Bioactive agents useful in accordance with the present invention may be used singly or in combination, namely more than one type of bioactive agents may be used together in one bioadhesive formulation, and therefore be released simultaneously from the bioadhesive hydrogel.

[000126] In some embodiments, the concentration of a bioactive agent in the formulation ranges from 0.1 percent weight per volume to 10 percent weight per volume of the total volume of said formulation, and even more in some embodiments. Higher and lower values of the content of the bioactive agent are also contemplated, depending on the nature of the bioactive agent used and the intended use of the bioadhesive hydrogel.

[000127] When using the term "bioactive agent" in the context of releasing or eluting a bioactive agent, it is meant that the bioactive agent is substantially active upon its release.

[000128] As discussed hereinbelow, the bioactive agent may have an influence on the bioadhesive hydrogel by virtue of its own reactivity with one or more of the bioadhesive hydrogel components, or by virtue of its chemical and/or physical properties perse. It is therefore noted that in general, the bioactive agent is selected suitable for being incorporated into the bioadhesive hydrogel such that it can elute from the bioadhesive hydrogel in the intended effective amount and release rate, while allowing the pre-curing bioadhesive formulation to exhibit desired properties, as discussed herein, and while allowing a bioadhesive hydrogel to cure that exhibits the desired properties, as discussed herein. For example, any agent that interferes with the coupling and crosslinking reaction is excluded from the scope of the invention. For example, bioactive agents exhibiting a carboxylic group or a primary amine group may react with a coupling agent which is selected for its reactivity towards such functional groups. In such cases, in order to maintain desirable characteristics of the resulting hydrogel, some adjustments may be introduced to the bioadhesive formulation in terms of the type of ingredients and their concentrations.

[000129] A bioactive agent, according to some embodiments of the present invention, can be, for example, a macro-biomolecule or a small, organic molecule. [000130] According to some embodiments of the present invention, the bioactive agent is a non-proteinous substance, namely a substance possessing no more than four amino acid residues in its structure.

[000131] According to some embodiments of the present invention, the bioactive agent is a non-carbohydrate substance, namely a substance possessing no more than four sugar (aminoglycoside inclusive) moieties in its structure.

[000132] According to some embodiments of the present invention, the bioactive agent is substantially devoid of one or more of the following functional groups: a carboxyl, a primary amine, a hydroxyl, a sulfhydroxyl and an aldehyde.

[000133] The term "macro-biomolecules" as used herein, refers to a polymeric biochemical substance, or biopolymers, that occur naturally in living organisms. Amino acids and nucleic acids are some of the most important building blocks of polymeric macro-biomolecules, therefore macro-biomolecules are typically comprised of one or more chains of polymerized amino acids, polymerized nucleic acids, polymerized saccharides, polymerized lipids and combinations thereof. Macromolecules may comprise a complex of several macromolecular subunits which may be covalently or non-covalently attached to one another. Hence, a ribosome, a cell organelle and even an intact virus can be regarded as a macro-biomolecule.

[000134] A macro-biomolecule, as used herein, has a molecular weight higher than 1000 dalton (Da), and can be higher than 3000 Da, higher than 5000 Da, higher than 10 kDa and even higher than 50 KDa.

[000135] Representative examples of macro-biomolecules, which can be beneficially incorporated in the bioadhesive hydrogels described herein include, without limitation, peptides, polypeptides, proteins, enzymes, antibodies, oligonucleotides and labeled oligonucleotides, nucleic acid constructs, DNA, RNA, antisense, polysaccharides, viruses and any combination thereof, as well as cells, including intact cells or other sub-cellular components and cell fragments.

[000136] As used herein, the phrase "small organic molecule" or "small organic compound" refers to small compounds which consist primarily of carbon and hydrogen, along with nitrogen, oxygen, phosphorus and sulfur and other elements at a lower rate of occurrence. In the context of the present invention, the term "small" with respect to a compound, agent or molecule, refers to a molecular weight lower than about 1000 grams per mole. Hence, a small organic molecule has a molecular weight lower than 1000 Da, lower than 500 Da, lower than 300 Da, or lower than 100 Da.

[000137] Representative examples of small organic molecules, that can be beneficially incorporated in the bioadhesive hydrogel described herein include, without limitation, angiogenesis-promoters, cytokines, chemokines, chemo-attractants, chemo-repellants, drugs, agonists, amino acids, antagonists, anti-histamines, antibiotics, antigens, antidepressants, antihypertensive agents, analgesic and anesthetic agents, anti-inflammatory agents, antioxidants, anti-proliferative agents, immunosuppressive agents, clotting factors, osseointegration agents, anti-viral agents, chemotherapeutic agents, co-factors, fatty acids, growth factors, haptens, hormones, inhibitors, ligands, saccharides, radioisotopes, radiopharmaceuticals, steroids, toxins, vitamins, minerals and any combination thereof.

[000138] Representative examples of bioactive agents suitable for use in the context of the present embodiments include, without limitation, analgesic, anesthetic agents, antibiotics, antitumor and chemotherapy agents, agonists and antagonists agents, amino acids, angiogenesis- promoters, anorexics, anti-allergics, anti-arthritics, anti-asthmatic agents, antibodies, anticholinergics, anti-convulsants, anti-depressants, anti-diabetic agents, anti-diarrheals, anti-fungals, antigens, antihistamines, anti-hypertensive agents, anti-inflammatory agents, anti-migraine agents, anti-emetics anti-neoplastics, anti -oxidants, anti -parkinsonism drugs, anti-proliferative agents, anti-protozoal agents, anti-pruritics, anti-psychotics, anti-pyretics, antisense nucleic acid constructs, anti-spasmodics, anti-viral agents, bile acids, calcium channel blockers, cardiovascular preparations, cells, central nervous system stimulants, chemo-attractants, chemokines, chemo-repellants, chemotherapeutic agents, cholesterol, co-factors, contraceptives, cytokines, decongestants, diuretics, DNA, Drugs and therapeutic agents, enzyme inhibitors, enzymes, fatty acids, glycolipids, growth factors, growth hormones, haemostatic and anti- hemorrhagic agents, haptens, hormone inhibitors, hormones, hypnotics, immunoactive agents, immunosuppressive agents, inhibitors and ligands, labeled oligonucleotides, microbicides, muscle relaxants, nucleic acid constructs, oligonucleotides, parasympatholytics, peptides, peripheral and cerebral vasodilators, phospholipids, polysaccharides, proteins, psychostimulants, radioisotopes, radiopharmaceuticals, receptor agonists, RNA, saccharides, saponins, sedatives, small organic molecules, spermicides, steroids, sympathomimetics, toxins, tranquilizers, vaccines, vasodilating agents, viral components, viral vectors, viruses, vitamins, and any combination thereof.

[000139] The bioactive agent may be selected to achieve either a local or a systemic response. The bioactive agent may be any prophylactic agent or therapeutic agent suitable for various topical, enteral and parenteral types of administration routes including, but not limited to sub- or trans-cutaneous, intradermal transdermal, transmucosal, intramuscular administration and mucosal administration.

[000140] One class of bioactive agents which can be encapsulated in the bioadhesive hydrogel, according to some embodiments of the present invention, comprises analgesic agents that alleviate pain e.g. NSAIDs, COX-2 inhibitors, opiates and morphinomimetics.

[000141] Another class of bioactive agents which can be incorporated in the bioadhesive hydrogel, according to some embodiments of the present invention, is the class of anesthetic agents. Another class of bioactive agents which can be incorporated in the bioadhesive hydrogel, according to some embodiments of the present invention, is the class of therapeutic agents that promote angiogenesis. Non-limiting examples include growth factors, cytokines, chemokines, steroids, and cell survival and proliferation agents.

[000142] Another class of bioactive agents which can be incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, especially in certain embodiments wherein tissue regeneration is desirable, and application involving implantable devices and tissue healing, are cytokines, chemokines and related factors.

[000143] Non-limiting examples of immunosuppressive drugs or agents, commonly referred to herein as immunosuppressants, include glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins and other immunosuppressants.

[000144] Non-limiting examples of haemostatic agents include kaolin, smectite and tranexamic acid.

[000145] It is noted herein that kaolin is an exemplary bioactive agent which has a limited solubility in the bioadhesive formulation, and is therefore added in the form of a dry powder, and thus also acts, at least to some extent, as a filler in the bioadhesive formulation/hydrogel. This dual function, bioactive agent and filler, may characterize any additive or bioactive agent which is encompassed by embodiments of the present invention and is contemplated therewith. [000146] Additional bioactive agents which can be beneficially incorporated in the bioadhesive hydrogel, according to some embodiments of the present invention, include cytotoxic factors or cell cycle inhibitors and other agents useful for interfering with cell proliferation.

[000147] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include genetic therapeutic agents and proteins, such as ribozymes, anti-sense polynucelotides and polynucleotides coding for a specific product (including recombinant nucleic acids) such as genomic DNA, cDNA, or RNA. The polynucleotide can be provided in "naked" form or in connection with vector systems that enhances uptake and expression of polynucleotides. These can include DNA compacting agents (such as histones), non-infectious vectors (such as plasmids, lipids, liposomes, cationic polymers and cationic lipids) and viral vectors such as viruses and virus-like particles (i.e., synthetic particles made to act like viruses). The vector may further have attached peptide targeting sequences, anti-sense nucleic acids (DNA and RNA), and DNA chimeras which include gene sequences encoding for ferry proteins such as membrane translocating sequences ("MTS"), tRNA or rRNA to replace defective or deficient endogenous molecules and herpes simplex virus-1 ("VP22").

[000148] Additional bioactive agents which can be beneficially incorporated in the bioadhesive hydrogel, according to some embodiments of the present invention, include gene delivery agents, which may be either endogenously or exogenously controlled.

[000149] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include the family of bone morphogenic proteins ("BMP's") as dimers, homodimers, heterodimers, or combinations thereof, alone or together with other molecules. Alternatively or, in addition, molecules capable of inducing an upstream or downstream effect of a BMP can be provided. Such molecules include any of the hedgehog proteins or nucleic acids encoding these proteins.

[000150] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include chemotherapeutic agents. Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include antibiotic agents. [000151] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include antiviral agents comprising nucleoside phosphonates and other nucleoside analogs, AICAR (5- amino-4-imidazolecarboxamide ribonucleotide) analogs, glycolytic pathway inhibitors, glycerides, anionic polymers, and the like.

[000152] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include viral and non-viral vectors.

[000153] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include steroidal anti-inflammatory drugs.

[000154] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include antioxidants.

[000155] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include vitamins.

[000156] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include hormones.

[000157] Additional bioactive agents which can be beneficially incorporated into the bioadhesive hydrogel, according to some embodiments of the present invention, include cells of human origin (autologous or allogeneic), including stem cells, or from an animal source (xenogeneic), which can be genetically engineered if desired to deliver proteins of interest.

[000158] Uses of the bioadhesive hydrogels and formulations

[000159] In general, bioadhesive hydrogels and formulations presented herein can be used in the manufacturing of a product intended for adhering to and/or bonding objects, at least one of which is a biological object.

[000160] According to some embodiments, bioadhesive hydrogels including a bioactive agent or not, and/or kits comprising the same, are identified for use in adhering a biological object. In some embodiments, bioadhesive hydrogels or a kit comprising same is identified for use in sealing a rupture to soft tissue in a biological object. In some embodiments, the bioadhesive hydrogels or kits comprising same are identified for use in bonding at least two objects to one another, wherein at least one of the objects is a biological object.

[000161] According to some embodiments of the present invention, a bioadhesive hydrogel is used topically, namely the bioadhesive hydrogel is used to adhere an object to the skin, to bond the edges of a lesion, to fix a skin graft, or seal a rupture in the skin.

[000162] Alternatively, the bioadhesive hydrogel is used internally to adhere to internal organs and serves to adhere an object to an internal organ, to bond the edges of a lesion in an internal organ, to fix a graft to an internal organ, or seal a rupture in an internal organ.

[000163] For buccal applications, the bioadhesive hydrogel, according to some embodiments of the present invention, adheres to the oral mucosa within seconds and remains adhered until fully eroded (biodegraded), without the need for a backing layer. The bioadhesive hydrogels presented herein combine high biocompatibility with flexibility in application.

[000164] For ocular applications, the bioadhesive hydrogel, according to some embodiments of the present invention, adheres rapidly to the ocular mucosa and remains in place until fully eroded.

[000165] For intra-nasal applications, the bioadhesive hydrogel, according to some embodiments of the present invention, adheres immediately to the nasal mucosa and remains in place until fully eroded. Drug-releasing bioadhesive hydrogels, according to some embodiments of the present invention, offer high drug loading capacity and nasal residence and release time to maximize drug efficacy.

[000166] For vaginal applications, the bioadhesive hydrogel, according to some embodiments of the present invention, adhered to the vaginal mucosa within seconds and can remain adhered for several days until fully eroded. Drug-releasing bioadhesive hydrogels, according to some embodiments of the present invention, offer a safe effective administration and a desired systemic effect.

[000167] Thus, the phrase "biological object", as used herein, refers to any viable/live part of an animal or plant, including a single live animal specimen. A live or viable biological object or tissue is defined as any major or minor part of a plant or animal that is still viable or alive and substantially kept in a physiological environment in order to stay viable or alive. Non-limiting examples of biological objects include any plant or animal, viable tissue samples, skin tissue, bone tissue, connective tissue, muscle tissue, nervous tissue and epithelial tissue. Also encompassed are edges of incisions made in an organ, such as skin, muscle, internal organ in any bodily site of an organism.

[000168] Inanimate objects are objects which cannot be revived, grafted, proliferate or otherwise show any signs of life as defined medically, and include objects of synthetic and/or biological origins. These include, for example, patches, bone-replacement parts, pace makers, ports and vents and any other medical device that required affixing and immobilization to a viable biological object as defined herein.

[000169] According to some embodiments of the present invention, inanimate biological objects can be made partially or entirely from animal or plant materials and products, or partially or entirely from synthetic substances. While the bioadhesive formulation/hydrogel is designed for use in or on viable biological objects, it is noted herein that is can be used effectively to bond biologic or synthetic inanimate objects like any adhesion agent or glue.

[000170] It is noted herein that the term "object" is meant to encompass one or more parts or portions of the same object, thus closing an incision by bonding the two sides of the incision in a tissue or an organ, by using the herein-described formulation, can be regarded as either bonding one object (the tissue or organ) or two objects (the two sides of the incision).

[000171] Bioadhesive hydrogel s can be used in many surgical procedures including, without limitation, corneal perforations, episiotomy, caesarian cases, cleft tip, skin and bone grafting, tendon repair; hernia, thyroid surgery, periodontal surgery, gingivectomy, dental implants, oral ulcerations, gastric varices, wounds of internal organs such as liver and pancreas, and attachment and immobilization of external and internal medical devices.

[000172] According to some embodiments, the drug-eluting hydrogel, resulting from a bioadhesive formulation which incorporates a bioactive agent, is used solely for its drug-eluting and drug-delivery faculties regardless of its bioadhesive hydrogel faculty. Such a hydrogel can serve, for example, as a drug depot, and can be adhered to an organ or tissue where the release of the drug is beneficial (without bonding thereto another object).

[000173] Polymer-cellulose fiber composite systems

[000174] In some embodiments, the present invention is a composite bioadhesive hydrogel reinforced by the integration of cellulose fibers (CF) in order to combine high mechanical strength with biocompatibility. In some embodiments, integration of CF into the polymeric hydrogel increased the cohesiveness of the bioadhesive hydrogel by reinforcement of the polymeric hydrogel. In some embodiments, a bioadhesive formulation/hydrogel based on a combination of gelatin and alginate crosslinked with a water-soluble carbodiimide (EDC) was used as a formulation for this study ("gelatin-alginate-EDC" or "Gel-Alg"). In some embodiments, this gelatin-alginate-EDC bioadhesive hydrogel has improved tissue adherence and improved ex vivo bonding strength and high biocompatibility.

[000175] In some embodiments, CF can be derived from plant or bacterial origin. In some embodiments, CF can be extracted from agricultural residue cell walls by simple mechanical methods (such as high shear) or by a combined chemo-mechanical method which includes a chemical pretreatment, depending on the cellulose type. In some embodiments, bacterial cellulose has high strength, high modulus and can be engineered structurally and chemically at nano, micro and macro scales, which is described in I. Siro, D. Plackett, Cellulose 2010, 77, 459- 494, which is herein incorporated by reference in its entirety. In some embodiments, the fibers' quality and mechanical properties depend on the fibers' size, maturity and the processing methods adopted for extraction. In some embodiments, the density, electrical resistance, ultimate tensile strength and initial modulus are related to the internal structure and chemical composition of the cellulose fiber. In some embodiments, the properties of a fiber-reinforced composite polymeric hydrogel depend on a combination of the properties of the CF and those of the hydrogel, since the hydrogel transfers the external loads to the fibers, and keeps them in place.

[000176] In some embodiments, varying the concentration of the fiber and cross-linking agent may affect the density of the final hydrogel and vary the setting times. The setting time property and density of bioadhesive formulations/hydrogels described herein may be tailored to maximize the effectiveness of a bioadhesive hydrogel formed thereby as an adhesive agent and/or delivery vehicle. Because of this effect one can alter the time between the mixing of the bioadhesive formulation and the setting of the bioadhesive hydrogel produced therefrom. One can engineer a bioadhesive formulation to, for example, flow freely into deep crevices in a wound, permitting it to fill the wound completely before the bioadhesive hydrogel sets. Alternatively, one can engineer a bioadhesive formulation to set quickly, so as to prevent it from exiting a wound site, particularly if the wound is leaking fluid under pressure (i.e., blood, lymph, intercellular fluid, etc). Bioadhesive formulations/hydrogels having such tunable properties would be ideally suited to treating wounds due to severe trauma, such as those resulting from accidents, assaults, and battlefield injuries. The setting time property of bioadhesive formulations described herein also pertains to preventing bioadhesive hydrogels formed thereby from clogging delivery devices with long passages e.g., catheters, endoscopes, etc.). This point is relevant when administering bioadhesive formulations/hydrogels to sites in the body that can be accessed only via surgical means. The setting time property of bioadhesive formulations described herein is also important for maintaining insoluble bioactive agents in suspension and thereby preventing them from settling in the applicator or in the tissue site.

[000177] For bioadhesive formulations/hydrogels comprising a growth factor, the formulations/hydrogels may further contain an inhibiting compound and/or potentiating compound, wherein the inhibiting compound inhibits the activities of the hydrogel that interfere with any of the biological activities of the growth factor and the potentiating compound potentiates, mediates, or enhances any of the biological activities of the growth factor. The concentration of the inhibiting and/or potentiating compound used for such indications is that which is effective for achieving the inhibition, potentiation, mediation or enhancement. Also applicable in this embodiment are regulatory compounds which simultaneously exhibit an inhibiting effect on exogenous factors, while at the same time potentiate, mediate or enhance the effect of a growth factor in a bioadhesive hydrogel described herein.

[000178] In addition to growth factors, bioadhesive formulations/hydrogels described herein may comprise drugs, polyclonal and/or monoclonal antibodies, oligonucleotides and other compounds, including, but not limited to, demineralized bone matrix (DBM), BMPs, osteogenic or cartilage inducing compositions. Such supplements accelerate wound healing, combat infection, neoplasia, and/or other disease processes, mediate or enhance the activity of growth factors in the matrix, and/or interfere with matrix components which inhibit the activities growth factors in the matrix. DBM, for example, is a source of osteoinductive proteins known as bone morphogenetic proteins (BMP) and growth factors which modulate the proliferation of progenitor bone cells.

[000179] Drugs may include, but are not limited to: antimicrobial compositions, including antibiotics, such as tetracycline, ciprofloxacin, and the like; anti-mycogenic compositions; antivirals, such as gangcyclovir, zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine, and the like, as well as antibodies to viral components or gene products; anti-fungals, such as diflucan, ketaconizole, nystatin, and the like; and anti-parasitic agents, such as pentamidine, and the like. Drugs may further include anti-inflammatory agents, such as a- 1 -anti -trypsin, α-1-antichymotrypsin, and the like; cytokines and interferons, such as a- or β- or γ-interferon, a- or β-tumor necrosis factor, and the like, and interleukins.

[000180] An effective concentration of cytotoxin or cell proliferation inhibiting composition may also be delivered by bioadhesive hydrogels described herein. An effective concentration at least one cytotoxin or cell proliferation inhibiting composition is added to the bioadhesive hydrogel, which upon delivery may act as an alkylating agent, enzyme inhibitor, proliferation inhibitor, lytic agent, DNA synthesis inhibitor, membrane permeability modifier, DNA intercalator, metabolite, mustard derivative, protein production inhibitor, ribosome inhibitor, inducer of apoptosis, angiogenesis inhibitor, neurotoxin, and the like. In a particular aspect, the cytotoxin or cell proliferation inhibiting composition delivered by the bioadhesive hydrogel may include, for example, 5-fluorouracil (5-FU), taxol and/or taxotere, actinomycin D, adriamycin, azaribine, bleomycin, busulfan, butyric acid, carmustine, chlorambucil, cisplatin, cytarabine, cytarabine, dacarbazine, estrogen, hormone analogs, insulins, hydoxyurea, L- asparaginase, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin C, prednisilone, prednisone, procarbazine, steroids, streptozotocin, testosterone, thioguanine, thiotepa, tributyrin, vinblastine, vincristine, gentamycin, carboplatin, cyclophosphamide, ifosphamide, maphosphamide, retinoic acid, ricin, diphtheria toxoid, venoms, antistatin or other plasminogen derivatives, and functionally equivalent analogs thereof; colony stimulating factors; erythropoietin;; steroids; anesthetics; analgesics; and hormones. The above-mentioned drugs may be used to treat, reverse, or prevent neoplasias, cell hyperproliferation. Neurotoxins, including antibiotics having neurotoxic effects such as gentamycin, may also be used to treat specific disorders, such as Meneir's disease. One or more of the above-mentioned cytotoxins or cell proliferation inhibiting compositions may be advantageously combined in the bioadhesive hydrogel with any of above-referenced analgesics, anti-microbial compositions, antiinflammatory compounds, antibodies, anti-coagulants, anti-proliferatives, cytokines, cytotoxins, chemotherapeutic drugs, growth factors, interferons, hormones, hydroxyapatite, lipids, oligonucleotides, osteoinducers, polymers, polysaccharides, proteoglycans, polypeptides, protease inhibitors, proteins (including plasma proteins), steroids, vasoconstrictors, vasodilators, vitamins, minerals, stabilizers, and the like. [000181] Other compounds which may be added to a bioadhesive hydrogel include, but are not limited to: vitamins and other nutritional supplements; hormones; glycoproteins; fibronectin; peptides and proteins; carbohydrates (both simple and/or complex); proteoglycans; anti- angiogenins; antigens; oligonucleotides (sense and/or antisense DNA and/or RNA); BMPs; DBM; osteogenic and cartilage inducing compositions; antibodies (for example, to infectious agents, tumors, drugs or hormones); and gene therapy reagents; anticoagulants; hormones; hydroxyapatite; lipids; polymers; polysaccharides; polypeptides; protease inhibitors; proteins (including plasma proteins), steroids, vasoconstrictors; vasodilators; minerals; stabilizers, and the like.

[000182] General applications of bioadhesive hydrogels and medical sealants

[000183] In some embodiments, the fiber-reinforced hydrogels of the present invention can be used in medical applications, including, e.g., but not limited to, a temporary skin substitute, aorta and heart valve leaflet replacement, small-diameter blood vessel replacement, tissue- engineering scaffold, and drug release, the methods of which are described in J. Fontana, A. De Souza, C. Fontana, I. Torriani, J. Moreschi, B. Gallotti, S. De Souza, G., Narcisco, J. Bichara, L. Farah, Appl. Biochem. Biotechnol. 1990, 24, 253-264; W. K. Czaja, D. J. Young, M. Kawecki, R. M. Brown, Biomacromolecules 2007, 8, 1-12; and H. Lonnberg, L. Fogelstrom, M. A. S. A. Samir, L. Berglund, E. Malmstrom, A. Hult, Eur. Polym. J. 2008, 44, 2991-2997, the contents of which are incorporated herein in their entireties.

[000184] Growth factor-supplemented bioadhesive formulations/hydrogels described herein are useful for promoting the healing of wounds, particularly recalcitrant wounds that are difficult to heal (such as skin ulcers in diabetic individuals and bed sores in bedridden patients), and for delivering growth factors including, but not limited to, angiogenins; endothelins; hepatocyte growth factor and keratinocyte growth factor; fibroblast growth factors, including fibroblast growth factor- 1 (FGF-1), fibroblast growth factor-2 (FGF-2), and fibroblast growth factor-4 (FGF-4); platelet-derived growth factors (PDGF); insulin-binding growth factors (IGF), including insulin-binding growth factor- 1 and insulin-binding growth factor-2; epidermal growth factor (EGF); transforming growth factors (TGFs), including transforming growth factor-a and transforming growth factor-β; cartilage-inducing factors (CIF), including CIP-A and CIP-B; osteoid-inducing factor (OIF); osteogenin and other bone growth factors; bone morphogenetic growth factors (BMP), including BMP-1 and BMP -2; collagen growth factor; heparin-binding growth factors, including heparin-binding growth factor- 1 and heparin-binding growth factor-2; cytokines; interferons; hormones and biologically active derivatives thereof, and providing a medium for prolonged contact between a wound site and the growth factor.

[000185] Growth factor-supplemented bioadhesive formulations/hydrogels described herein may be used to treat burns and other skin wounds and may comprise formulations supplemented with at least one of the following: a growth factor, an antibiotic and/or an analgesic, etc. A growth factor-supplemented bioadhesive formulation/hydrogel may be used to promote engraftment of a natural or artificial graft, such as skin to a skin wound. A growth factor- supplemented bioadhesive formulation/hydrogel may also be used cosmetically, for example, in hair transplants, wherein the formulation/hydrogel may comprise at least one of FGF, EGF, antibiotics, and minoxidil, as well as other compounds. An additional cosmetic use for bioadhesive formulations/hydrogels described herein is to treat wrinkles and scars in lieu of silicone or other compounds commonly used for such purposes. In this embodiment, a bioadhesive formulation/hydrogel may, for example, comprise FGF-1, FGF-4, and/or PDGFs, and fat cells.

[000186] Growth factor-supplemented bioadhesive formulations/hydrogels described herein may be applied to surgical wounds, broken bones or gastric ulcers and other such internal wounds in order to promote sealing, restoration of an intact state, and/or healing thereof. Unsupplemented and growth factor-supplemented bioadhesive formulations/hydrogels described herein may be used to promote integration of a graft, whether artificial or natural tissue, into a subject's body.

Bone Wounds and Their Repair

[000187] Bioactive agents such, for example, DBM and BMPs may be incorporated into bioadhesive formulations/hydrogels described herein and administered directly to a bone lesion (a damaged site in a bone). A bioadhesive hydrogel introduced into a bone lesion serves as a reservoir that releases BMPs and various growth factors in DBM, including BMPs, in a localized and sustained manner and thus promotes regeneration and healing of the damaged bone.

Vascular Prostheses

[000188] Artificial vascular prostheses, frequently made of dacron or olytetrafluoroethylene (PTFE), are used to replace diseased blood vessels in humans and other animals. To maximize patency rates and minimize the thrombogenicity of vascular prostheses, various techniques have been used including seeding of nonautologous endothelial cells onto the prothesis. Various substrates which adhere both to the vascular graft and endothelial cells have been investigated as an intermediate substrate to increase endothelial cell seeding. Drawbacks to this approach include the significant time it takes for a confluent endothelium to be established and the frequency of failure observed in establishing a confluent endothelium at all. The delay in establishing a confluent endothelium results in a high rate of failure due to occlusion of the vascular prosthesis. Additionally, the use of nonautologous cells for seeding the surface of the substrate increases the likelihood of tissue rejection. Coating the artificial vascular prostheses with a bioadhesive hydrogel into which bioactive agents have been incorporated that attract endothelial cells to migrate into and onto the prosthesis, therefore, serves to address the problems associated with occlusion of such prosthesis. It is envisioned that endogenous endothelial cells from the subject into which the prosthesis has been introduced will migrate to and populate the prosthesis in situ and thereby reduce the rate of occlusion and thrombogenicity of such prosthesis, without the potential for tissue rejection.

Angiogenesis

[000189] A bioadhesive hydrogel into which bioactive agents that are angiogenic have been incorporated is envisioned to promote the formation of new blood vessels. Exemplary growth factors having angiogenic activity include heparin binding growth factor- 1 (HBGF-1) and HBGF-2. Such a bioadhesive hydrogel serves as a reservoir that releases angiogenic growth factors, such as HBGF-1 and FIBGF-2, in a localized and sustained manner and thus promotes formation of new blood vessels in a localized fashion in vivo.

Site-Directed, Localized Drug Delivery

[000190] A bioadhesive hydrogel into which bioactive agents have been incorporated is envisioned as an efficacious, site-directed drug delivery system for a variety of medical conditions. Localized drug delivery is needed in the treatment of local infections, such as, for example, sinus infections, wherein systemic administration of antimicrobial agents is sometimes ineffective or causes side effects that cannot be tolerated by a subject being treated. Accordingly, for local infections, such as sinus infections, a bioadhesive hydrogel into which at least one appropriate antibiotic has been incorporated may be administered to the sinus cavity of a patient in need thereof. Appropriate antibiotics may be determined based on susceptibility of the bacterial species responsible for the sinus infection to an antibiotic in question. Those skilled in the art can determine which antibiotics are suitable and whether, for example, a narrow or broad spectrum antibiotic is advisable. In another embodiment, a bioadhesive hydrogel into which at least one anti -fungal agent has been incorporated is administered to a subject with a localized fungal infection. Such infections include, for example, fungal infections of a nail (e.g., toenail or fingernail).

Controlled Drug Release

[000191] For some clinical applications controlled, localized drug release is desirable. Local implantation of antibiotics in a bioadhesive hydrogel as described herein is, therefore, envisioned for the treatment of wounds, such as open fractures or acute and chronic osteomyelitis. Acute osteomyelitis is a rapidly progressing infection of bone, whereas the chronic form of osteomyelitis results from a long-standing infection of the bone. Acute osteomyelitis can be successfully treated with antibiotics provided that the disease is diagnosed early, while for the chronic form, successful treatment requires debridement of the wound and administration of antibiotics. Antibiotics, an adjunct to thorough debridement, are typically administered systemically. Systemic administration, however, sometimes fails to achieve therapeutically effective doses of antibiotics at the wound site and can, furthermore, cause unacceptable side effects. To address these significant disadvantages of systemic antibiotic administration, administration of a bioadhesive hydrogel comprising an appropriate antibiotic directly to the debridement site is envisioned.

Dental applications of bioadhesive hydrogels and medical sealants

[000192] There are seven major categories of "classic" dental adhesives and sealants: denture bonding agents, pit and fissure sealants, restorative adhesives, cements, orthodontic bonding agents, luting cements (a viscous material placed between a tooth or portion thereof and a prosthesis that hardens to secure the prosthesis to the tooth or portion thereof), tray adhesives and surgical tissue bonding. Adhesives and sealants are projected to achieve the most rapid gains in tissue bonding as more dental professionals realize the benefits of using these materials in various surgical procedures, such as tooth extractions. Pit and fissure sealants will also register above-average gains due to their use in the prevention of cavities.

[000193] Bioadhesive hydrogels described herein may be implemented to advantage in any of the above applications. Bioadhesive formulations/hydrogels (unsupplemented or supplemented) described herein may, for example, be used to advantage to combat some of the challenges/complications associated with certain conditions such as periodontitis. Such challenges/complications include persistent infection, bone resorption, loss of ligaments and premature re-epithelialization of the dental pocket.

[000194] Bioadhesive formulations/hydrogels (unsupplemented or supplemented) described herein may, for example, be used as a denture adhesive hydrogel that stabilizes dentures in a wearer's mouth. Such a denture adhesive hydrogel is desirable if dentures were not properly fitted at the outset or have become loose over time and thus, require stabilization to improve the fit and usability of the dentures, dental plate, or the like.

[000195] Bioadhesive hydrogels described herein may also be used for additional applications for which the properties of a hydrogel are well suited. In that hydrogels, such as those described herein, comprise natural and/or synthetic polymers with high water-absorbing capacity, they may be used in applications relating to regenerative medicine. Indeed, injectable hydrogels have emerged as a promising biomaterial for therapeutic delivery of cells and bioactive molecules for tissue regeneration in dentistry and medicine because of their tunable tissue-like properties, controllability of degradation and release behavior, adaptability in a clinical setting for minimally-invasive surgical procedures, and ability to conform to the three- dimensional (3-D) defect upon gelling.

[000196] In craniofacial and dental tissue engineering, a paradigm shift is taking place from using synthetic implants and tissue grafts to tissue engineering approaches employing biomimetic biomaterial scaffolds, particularly injectable hydrogels integrated with cells and bioactive molecules to regenerate a myriad of tissues including cartilage, bone, nerves, blood vessels and soft tissues (e.g., muscle, subcutaneous fat and skin). Similarly, in regenerative endodontic applications, injectable hydrogels have demonstrated the feasibility of delivering dental pulp stem cells (DPSCs), supporting matrix (e.g., enamel matrix derivative) and growth factors (e.g. stromal -derived growth factor (SDF)-al, fibroblast growth factor (FGF)-2, and bone morphogenetic protein (BMP -7) to support formation of the dentin-pulp complex. Recent advances in tissue engineering have demonstrated that biomaterial scaffolds and bioactive molecules activate endogenous cell migration and tissue repair, thereby underscoring the potential of the 'homing' approach for craniofacial and dentin-pulp regeneration.

[000197] In a particular embodiment, DPSCs are positive for at least one of a mesenchymal antigen (e.g., STRO-1, CD29, CD44, CD90 and CD146) and negative for haematopoietic antigens (e.g., CD31). See, for example, Guo et al. (2013, PloS one 8:e62332); the entire content of which is incorporated herein by reference. Such cells may, for example, be harvested from the developing tooth bud of a mandibular third molar. Such techniques are known in the art; see, for example, Guo et al. (2013, supra), Potdar et al. (2015, World J Stem Cells 7: 839-851), and Tziafas et al. (2010, J Endodontics 36:781-789); the entire content of each of which is incorporated herein by reference. DPSCs can differentiate to form enamel, dentin, blood vessels, dental pulp, and nervous tissues. Accordingly, incorporation of DPSCs into bioadhesive hydrogels described herein generates a dental implant having the capacity to regenerate at least in part damage to a tooth or root thereof.

[000198] When hydrogels are used as scaffolds for bone regeneration it is necessary to add materials that increase the strength and stiffness of the hvdrogel. These can be either fibers or additives such as hydroxyapatite. The bioadhesive hydrogels described herein comprise fibers that confer sufficient strength and stiffness to render them suited for applications directed to bone regeneration,

[000199] In some embodiments, effects of the fiber concentration on the microstructure and on the resulting mechanical and physical properties of a CF-reinforced gelatin-alginate based bioadhesive hydrogel were investigated.

[000200] The phrase "bonding strength" as used herein describes the maximum amount of tensile stress that a pair of bonded objects of given materials can be subjected to before they break apart.

[000201] Materials and Methods

[000202] Materials

[000203] Coldwater fish skin "type A" gelatin (G7041), alginic acid sodium salt (Al l 12), and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich, Rehovot, Israel. CF was purchased from CFF GmbH and Co., Germany.

[000204] Preparation of cellulose fiber-loaded surgical bioadhesive hydrogel

[000205] Preparation of the bioadhesive formulation is based on dissolving CF, gelatin and alginate (Gel-Alg) in double-distilled water, under heating up to 60°C. A selected formulation of 400 mg/mL gelatin, 10 mg/mL alginate and 20 mg/mL EDC (400-10-20 mg/mL gelatin-alginate- EDC) was used as the primary formulation for the study of the fibers' effect. This formulation was found to have low cytotoxicity. In an embodiment thereof, the fiber concentration range is 2- 100 mg/ml. The effect of CF was studied in concentrations of 10-50 mg/mL. Crosslinking agent (EDC) solution was added to the bioadhesive formulation just prior to use. In all experiments, the dual-component bioadhesive formulation/hydrogel was applied using a double-syringe with a static mixer at a 4: 1 volume ratio (Mixpac L-System, Sulzer, Switzerland) which provides consistent mixing of the polymer and crosslinker solutions. The bioadhesive formulation/hydrogel was placed at room temperature of 25±2°C and was left in this environment for approximately ten min, allowing it to reach room temperature prior to application.

[000206] Evaluation of the bioadhesive hydroge s mechanical properties

[000207] Three types of mechanical tests were selected for mechanical evaluation of the novel composite bioadhesive hydrogel, based on relevant standards. The combination of these three methods enables the establishment of a thorough understanding of the bioadhesive hydrogel' s function.

[000208] Burst strength measurements

[000209] The burst strength was tested using a custom-built mechanical burst device following the standard method for Burst Strength of Surgical Sealants ASTM F2392-04. The principle of this test is to measure the maximal pressure at the tissue leakage point that can be held by the bioadhesive hydrogel. A collagen casing (SI Fibran, Spain) with a uniform 3.0 mm diameter hole was used as the tissue substrate. Approximately 0.5 mL of bioadhesive hydrogel was applied to the collagen casing substrate, sealing the defect with a measured thickness of approximately 1 mm. The sample was placed in the test unit and pressure was applied. The pressure at which bioadhesive hydrogel failure occurred was recorded as the maximal burst pressure. A minimum of 10 repetitions were carried out for each formulation.

[000210] Lap shear bonding strength

[000211] A hydrogel should ideally have stiffness and flexibility compared to the wound tissue, in order to resist shear, tension or compression forces. An ex vivo lap shear test is crucial for assessing the adhesive strength of tissue adhesives or sealants on soft tissue.

[000212] Adhesive bonding strength in lap shear was assessed according to ASTM F2255- 05 using a 5500 Instron universal testing machine (Instron Engineering Corp.) in order to investigate the mechanical properties under shear forces that are usually applied to the skin in tissue adhesive applications. Briefly, sheets of collagen casing were cut into 2.5 cm wide strips. An area of 1 cm at the end of each strip was marked to be the overlapping area and the other end of the strip was folded in order to create a thick area that would be easy to grasp. Approximately 60 uL bioadhesive hydrogel was applied to one strip, and the second strip was immediately set over the adherence area. A force of 1.2 N was applied to the bonding area for 15 min immediately following the overlapping, allowing the adhesive to set. The entire procedure was performed at room temperature of 25±2°C. The test specimens were placed in the grips of the testing machine so that the applied load coincided with the long axis of the specimen. The specimen was loaded to failure at a constant cross-head speed of 5 mm/min. 15 specimens were tested for each formulation. For each sample, the maximum force at failure and mode of failure was recorded: whether it was cohesive failure, adhesive failure or failure of the collagen substrate. Only adhesive failures were taken into consideration.

[000213] Compression modulus - bioadhesive hydrogel elasticity

[000214] Cylindrical samples (7.8 mm diameter, 3.4 mm height) were prepared in a silicon mold and analyzed 24 h after casting in order to measure the compression modulus. The compression elastic modulus (Ec) was measured using a 5500 Instron. Cylindrical material samples were preconditioning by 3 cycles of loading/unloading subjected following a ramped compressive displacement at a rate of 0.2 mm/min and maximal strain of 35%. Five specimens were tested for each formulation. The compression modulus (Ec) was calculated as the slope of the linear regression line for data between 5 and 15% of strain.

[000215] Viscosity measurements

[000216] The initial viscosity of the polymeric (Gel-Alg) bioadhesive hydrogel at the moment of application on the tissue is affected mainly by the viscosity of the aqueous Gel-Alg formulation. Viscosity measurements of the polymer compositions were performed using a controlled stress rheometer (model DHR3, TA Instruments Ltd.) fitted with a cone-and-plate geometry (1° cone angle, 40 mm diameter), at a constant temperature of 25°C and a constant shear rate of 10 Hz, in order to investigate the effect of the CF on the bioadhesive hydrogel' s initial viscosity.

[000217] Gelation time

[000218] Crosslinking time, i.e. gelation time, indicates the time required for the bioadhesive formulation/hydrogel to reach the desired state when applied on a wound. Gelation time was determined as the time required for a magnetic bar to stop moving after mixing of the polymer solution with the crosslinker solution. Approximately 1 mL of polymeric hydrogel (i.e., not loaded with CF) or bioadhesive hydrogel (polymeric hydrogel loaded with CF), was poured into a 1.6 cm diameter plate under mixing at 300 rpm with a 1.4 cm magnetic bar at room temperature.

[000219] Swelling and weight loss

[000220] The bioadhesive formulations were poured into 7.0x7.0x3.5 mm ³ silicon molds and after gelation they were carefully removed and dried for 24 h. Subsequently, they were weighed (W,) and immersed in 3 mL PBS (pH 7.0), placed in a static incubator at 37°C and 100% relative humidity for 2, 6, and 24 h. They were then weighed (W _s ) by removing the PBS and blotting using Kimwipes and then dried for 24 h and weighed again (W _j ). The swelling degree and the weight loss were calculated according to the following equations:

[000221] (1) Swelling degree: (W _s - W _f ) I W _f x 100%

[000222] (2) Weight loss: (W, - Wj) I W^ 100%

[000223] 3-4 repetitions were carried out for each formulation at each point of time.

[000224] Scanning electron microscopy

[000225] The microstructure of the bioadhesive hydrogel was investigated in order to characterize the dispersion of the CF in the polymeric hydrogel. For this purpose, 0.5 mL of cubic bioadhesive hydrogel specimens were air-dried in a chemical hood, freeze fractured, and their cross-section was observed using an environmental scanning electron microscope (Quanta 200 FEG ESEM) in a high vacuum mode, with an accelerating voltage of 10 kV.

[000226] Statistical analysis

[000227] All data were processed using the Excel software. Statistical comparison between more than two groups was performed using the ANOVA (with Tukey Kramer post hoc) method via the XLSTAT software. A value of p<0.05 was considered significant. Error bars indicate the standard deviation.

[000228] Results

[000229] The aim of the current study was to investigate the effect of CF incorporation in a gelatin-alginate bioadhesive hydrogel. Selected formulations, based on 400-10-20 (mg/mL) Gel- Alg-EDC were characterized.

[000230] Bioadhesive hydrogel' s mechanical properties [000231] The mechanical properties of the novel composite bioadhesive hydrogels were evaluated by three mechanical tests selected based on relevant standards.

[000232] Burst strength test

[000233] Burst strength measurements, i.e. the maximal pressure that the gelatin-alginate bioadhesive hydrogel can withstand, showed that the burst strength of the basic 400-10-20 (mg/mL) Gel-Alg-EDC formulation is 318±52 mmHg (see, e.g., Fig. 1). A pressure of 200 mmHg is the threshold for surgical sealants in order to be able to withstand the systolic blood pressure. The addition of CF dramatically increased the maximal burst strength. Fibers loaded in the highest concentration of 50 mg/mL increased the bioadhesive hydrogel's burst strength by 80% (565±75 mmHg) compared to the polymeric hydrogel. A significant increase (p<0.05) in the burst strength was observed when the fiber concentration was higher than 10 mg/mL.

[000234] Bonding strength in lap shear test

[000235] The bonding strength of the bioadhesive hydrogel in lap shear mode (see, e.g., Fig. 2) showed a moderate response to fiber incorporation. In general, no clear correlation was found between the fiber concentration and the bonding strength, contrary to the burst strength tests. The only significant difference was found when the fibers were loaded at a concentration of 30 mg/mL. At this concentration, the bioadhesive hydrogel exhibited a bonding strength of 213±45 kPa, an increase of 45% compared to the polymeric hydrogel.

[000236] Bulk compression modulus

[000237] The elastic modulus (Ec) was measured in a compression procedure on bioadhesive hydrogel cylinders in order to evaluate the cohesive strength of the bioadhesive hydrogel (the crosslinked hydrogel). Contrary to the lap shear and the burst strength tests, this method is not a standard method and is rarely performed in the adhesive field. The elastic modulus was examined in a compression procedure rather than in tension, which is more common, in order to minimize the effect of defects resulting from casting a large sample (such as the standard dog-bone) from a dual component mixture. As shown in Fig. 3, the lowest compression modulus was measured for the polymeric hydrogel. An increase of 40% in the compression modulus was obtained when the fibers were loaded at a concentration of 30 mg/mL. Bioadhesive hydrogels loaded with higher fiber concentrations did not exhibit a significant difference in the compression modulus compared to the polymeric hydrogels. These results seem to indicate that the cohesive strength of the bioadhesive hydrogel increased due to the integration of the fibers, resulting in a fiber-reinforced hydrogel.

[000238] Microstructure

[000239] The bulk cross-section of a polymeric hydrogel (Fig. 4b) and CF morphology and the bulk cross-section of bioadhesive hydrogels were observed using ESEM. The morphologies are presented in Fig. 4. The cellulose fibers (see, e.g., Fig. 4a) were found to have an average length of approximately 300 μπι and a rectangular cross-section. It can be seen (see, e.g., Fig. 4b) that the specimen has a smooth texture, and only a crack caused when fracturing the sample is seen. Fractographs of bioadhesive hydrogels show that the fibers are homogeneously dispersed, and not in clusters, even at the highest concentration of 50 mg/mL (see, e.g., Fig. 4c). Higher magnification (see, e.g., Fig. 4d) shows a smooth and continuous interface between the fibers and the polymeric hydrogel in which they are dispersed, indicating a high affinity interaction between them.

[000240] Viscosity test

[000241] Rheological tests were performed in order to elucidate the effect of a hydrogel's components on the initial viscosity, i.e. before the crosslinking reaction. As seen in Fig. 5, the viscosity increases with the fiber concentration.

[000242] The bioadhesive hydrogel's viscosity increased up to 7.8-fold when loaded with the highest amount of fibers. This wide range of viscosities is suitable for various clinical needs, which are disclosed herein.

[000243] Swelling and weight loss

[000244] The swelling ratio and the weight loss were measured in order to evaluate the effect of the cellulose fiber on the physical properties of the crosslinked hydrogels that result from the microstructure. As can be seen in Fig. 6, the swelling ratio increased with the incubation time. Fiber incorporation did not affect the swelling ratio after incubation for 2 h. However, by 24 h the presence of fiber decreased the swelling ratio by -30% when loaded at the maximal amount of 50 mg/mL. A decrease in swelling ratio was, furthermore, apparent by 6 hours incubation. A significant difference was observed for a cellulose fiber concentration higher than 20 mg/mL. The observed decrease in swelling ratio observed in bioadhesive hydrogels relative to control matched polymeric hydrogels revealed a surprising structural feature of bioadhesive hydrogels described herein. The structural feature of having a decreased swelling ratio, in turn, confers a desirable and unexpected functional feature, namely that a bioadhesive hydrogel introduced into a cavity (e.g., a body cavity, such as, e.g., a bone fissure, dental pit, or wound opening) will exhibit limited swelling. The functional property of limited swelling preserves the bioadhesive hydrogel's properties so a bioadhesive hydrogel can, therefore, best fulfill its intended function. Reduced swelling also minimizes any potential inflammation, irritation, and/or surrounding tissue swelling at a body cavity site at least partially filled by a bioadhesive hydrogel. These structural and functional properties are unexpected and desirable features for various therapeutic applications of bioadhesive hydrogels described herein.

[000245] Similarly, as can be seen in Fig. 7, the cellulose fibers did not affect the weight loss of the bioadhesive hydrogels after 2 h of incubation in PBS. However, after 24 h, a significant increase in weight loss was observed due to integration of the CF. Bioadhesive hydrogels containing 50 mg/mL CF exhibited 57% higher weight loss than control matched polymeric hydrogels.

[000246] Gelation time test

[000247] Clinical applications of bioadhesive hydrogels are wide and diverse. From the aspect of gelation time, i.e. the time necessary for the bioadhesive hydrogel to set and reach the desired strength, the requirements for sealing and adhesion applications are diverse. The gelation time for sealing applications must be in the range of 5-10 sec in order to resist the physiological fluid flux. In contradistinction, the gelation time for adhesion applications needs to be longer in order to enable good positioning and re-positioning of the bioadhesive hydrogel on the tissues.

[000248] As can be seen in Fig. 8, a polymeric hydrogel (non-fiber loaded Gel-Alg formulation) cured in approximately 11 sec. Incorporation of CF was found to accelerate the gelation time to 7 sec when loaded at a fiber concentration of 30 mg/mL. Higher loading of fibers did not result in further acceleration. It is noteworthy that the decrease in gelation time with the incorporation of cellulose fibers is unexpected. Decreased gelation time is desirable in applications such as, e.g., wound repair, wherein it is desirable to close a tissue tear associated with the wound quickly to reduce blood loss and/or additional tissue damage.

[000249] Additional burst strength measurements

[000250] As described hereinabove, the burst strength was tested using a custom-built mechanical burst device following the standard method for Burst Strength of Surgical Sealants ASTM F2392-04. In a particular embodiment, the burst strength (sealing ability) of a bioadhesive hydrogel comprising 400 mg/ml gelatin, 10 mg/ml alginate, and 20 mg/ml EDC is assessed, with and without the addition of the hemostatic agent kaolin. Also shown are the burst strengths observed for polymeric hydrogels comprising 400 mg/ml gelatin, 10 mg/ml alginate, and 20 mg/ml EDC, with and without the addition of the hemostatic agent kaolin. The effect of CF is depicted in Fig. 10, which shows that at concentrations of 20 and 30 mg/ml CF confers increased burst strength (sealing ability) relative to lower concentrations (0 and 10 mg/ml) thereof. The effect of kaolin is depicted in Fig. 1 1, which shows that at concentrations of 5 and 10 mg/ml kaolin confers increased burst strength (sealing ability) relative to lower concentrations (0 and 2.5 mg/ml) thereof. These results show that the burst strength is increased when either of CF or kaolin is increased. Cellulose fiber is, however, more effective than kaolin.

[000251] Discussion of results

[000252] The concept of CF-reinforced hydrogels for bioadhesive hydrogel and sealant applications was investigated herein. The influence of the fibers on the bioadhesive hydrogel's mechanical, physical and structural properties was studied. Natural non-modified CF was used in this study in order to present proof for the concept of enhancement of the bioadhesive hydrogel's properties and thus serve as a basis for future studies investigating a wide range of fibers and fiber treatments that may further enhance the bioadhesive hydrogel's properties. In a particular embodiment, a bioadhesive hydrogel comprising a fiber comprises a hydrophilic fiber. Exemplary hydrophilic fibers are natural fibers such as, for example, cellulose fiber, a cellulose whisker, collagen, keratin, fibroin, chitin, chitosan, silk, and catgut. In a more particular embodiment, hydrophilic fibers are natural fibers limited to the following: of a cellulose fiber, a cellulose whisker, keratin, fibroin, chitosan, silk, and catgut. In a still more particular embodiment, the hydrophilic fiber is a cellulose fiber, a cellulose whisker, collagen, keratin, fibroin, chitin, chitosan, silk, or catgut. In an even more particular embodiment, the hydrophilic fiber is a cellulose fiber or a cellulose whisker.

[000253] The results of the current study clearly show that the addition of fibers (CF) has a significant effect on the mechanical and physical properties of a bioadhesive hydrogel. In most tests, a difference was observed between polymeric hydrogels and bioadhesive hydrogels identical but for the presence of fibers. A qualitative model that summarizes the effect of the fiber incorporation on a bioadhesive hydrogel's parameters is suggested, based on the above findings. A schematic representation of this model is presented in Fig. 9. [000254] As seen in our ESEM observations (see, e.g., Fig. 4), the CF are uniformly dispersed in the gelatin-alginate hydrogel and the latter wraps them and penetrates into the fibers' microspores, which enables a good fiber-polymer hydrogel interaction. The resulting microcomposite structure therefore exhibits enhanced physical and mechanical properties.

[000255] Physical properties

[000256] Another indication for the beneficial interaction between the fibers and the polymeric hydrogel is reflected in the viscosity measurements (see, e.g., Fig. 5, in which a dramatic increase in viscosity was found due to the incorporation of fibers in the bioadhesive hydrogel. Without being bound by theory, an increase in viscosity may result from entanglements of the fibers with themselves, which is reflected in friction and resistance to flow.

[000257] The decrease in the swelling ratio as a result of fiber incorporation is another indication for the reinforcement effect. The bioadhesive hydrogel specimens exhibited a denser hydrogel structure that restricts the expansion ability of the polymer chains and reduces the ability of water to penetrate the hydrogel. Furthermore, the interaction between the CF and the polymers possibly prevents reaction between hydrophilic groups in the polymers and CF with the surrounding medium.

[000258] Mechanical properties

[000259] When there is a strong fiber-polymeric hydrogel interaction, a better reinforcement effect will occur, which would improve the load transition throughout the polymeric hydrogel, thus enabling the composite to withstand higher forces. From a mechanical perspective, the fiber reinforcement effect is evident in the burst strength test (see, e.g., Fig. 1), where the addition of fibers caused a significant increase in the maximal pressure that the bioadhesive hydrogel could withstand. When used as a sealant in the physiological environment, a bioadhesive hydrogel must withstand the blood pressure, which ranges from about 25 mmHg for capillary bleeding to a maximum of 200 mmHg for extreme hypertension. As can be seen from the burst strength results, the gelatin-alginate bioadhesive hydrogel can withstand this pressure in vitro.

[000260] Lap shear tests demonstrated that maximal bonding strength was obtained for a fiber concentration of 30 mg/mL. Higher fiber concentrations led to a decrease in the bonding strength. In the lap shear test, the bioadhesive hydrogel is spread and applied in the form of a thin film, contrary to the burst strength test in which the bioadhesive hydrogel is applied in bulk form with a thickness of 1 mm. The difference in the thickness of the bioadhesive hydrogel layers is the key element for understanding the different effects of the fibers' incorporation on the mechanical properties. The burst strength of an adhesive is a property that reflects mainly the cohesiveness of the bioadhesive hydrogel, whereas the lap shear test reflects both the cohesiveness and the adhesiveness of the bioadhesive hydrogel. However, the latter has a greater effect on the bonding strength due to the thin film geometry. It simulates a situation in which the adhesive binds between two tissues. In this case, the adhered tissues are subjected to shear stress, and so is the adhesive. This kind of situation requires adhesive strength for binding the tissues together and cohesive strength for preventing disintegration of the adhesive hydrogel. The reinforcement mainly affects the cohesive strength of the bioadhesive hydrogel. Thus, the burst strength is influenced by the reinforcement much more due to the bulky specimen. On the other hand, the adhesive strength is less affected by the reinforcement, and the bonding strength measured in the lap shear test was therefore less affected by the reinforcement

[000261] Furthermore, as the fiber concentration increases, the formulation becomes more viscous, and it is therefore more difficult for the bioadhesive hydrogel to penetrate into the microspores of the substrate's surface, and the mechanical interlocking mechanism of adhesion is less effective. Once the fiber concentration reaches a certain critical point, the formulation may be too viscous to be used as an adhesive, despite the formulation's cohesive strength. From that point, there is a decrease in the adhesive strength of the bioadhesive hydrogel. It should be noted that the collagen casing used as the substrate in the lap shear test is less porous than biological tissue, and the adhesion strength in vivo may be higher due to better mechanical interlocking.

[000262] Finally, the fibers' geometry showed a significant effect on the mechanical properties of the bioadhesive hydrogel. Fibers with a high aspect ratio (ratio between length and diameter) are commonly used for reinforcement of materials, whereas fibers with a low aspect ratio (more granular shape) are less effective due to inefficient load transition. The burst strength and viscosity measurements indicate that bioadhesive hydrogels loaded with high aspect ratio fibers can withstand a higher maximal pressure, and are significantly more viscous.

[000263] The incorporation of fibers into the Gel-Alg bioadhesive hydrogel reinforces the polymeric hydrogel into which the fibers are incorporated and results in a composite bioadhesive hydrogel with superior mechanical and physical properties. The inventive bioadhesive hydrogels described herein can be applied as surgical sealants. A variety of currently available fibers may be suitable for integration into polymeric hydrogels for bioadhesive hydrogel applications. The integration of coated fibers for controlled drug release is an effective way to dose antibiotics, agents that confer pain relief (e.g., acetaminophen and non-steroidal anti-inflammatory agents, including ibuprofen, naproxen, and aspirin), hemostatic agents or any other drugs.

[000264] While a number of embodiments of the present invention have been described, it is understood that these embodiments are illustrative only, and not restrictive, and that many modifications may become apparent to those of ordinary skill in the art. Further still, the various steps may be carried out in any desired order (and any desired steps may be added and/or any desired steps may be eliminated).