MESH-BASED IN SITU CROSS-LINKABLE COMPOSITIONS

Abstract

A mesh-based composition comprising a cross-linkable protein or polypeptide and one or more cross-linking materials according to at least some embodiments. The mesh-based composition may comprise a self-adhering surgical mesh which requires no additional fixation while at the same time minimizes adhesions. The mesh may be in the form of a composite mesh featuring a foamed composition of the cross-linkable protein and a non-toxic material that induces cross-linking of the cross-linkable protein. The composite mesh may be in a sheet-like form.

Claims

1. A mesh-based composition comprising a mesh and a foamed coating, said foamed coating comprising one or more cross-linkable proteins or polypeptides and one or more cross-linking materials, wherein at least a portion of said mesh is coated with said foamed coating to form a coated mesh composition that is: self-adhering requires no additional fixation, and is capable of minimizing tissue adhesions upon application, and wherein the foamed coating extends beyond the mesh such that a total area of the coating is larger than a total area of the mesh.

2. (canceled)

3. The composition of claim 1, wherein the mesh-based composition is in a sheet-like form.

4. The composition of claim 1, wherein the cross-linkable protein or polypeptide comprises gelatin.

5. The composition of claim 4, wherein the gelatin is foamed to form a gelatin foam.

6. The composition of claim 5, wherein the gelatin foam is in a density range of 1 to 100 mg/cm.sup.3.

7. The composition of claim 5, wherein said gelatin foam comprises dried or lyophilized gelatin.

8. The composition of claim 7, wherein prior to foaming, the concentration of the gelatin solution is between 0.1% and 30% w/w.

9. The composition of claim 8, wherein prior to foaming, a concentration of a gelatin solution that is foamed is between 1% and 20% w/w.

10. The composition of claim 9, wherein prior to foaming, the concentration of the gelatin solution is between 5% and 15% w/w.

11. The composition of claim 4, wherein said one or more cross-linking materials comprise transglutaminase.

12. The composition of claim 1, wherein the mesh-based composition comprises an incorporated surgical mesh.

13. The composition of claim 1, comprising two sections, one of which is composed of the mesh enclosed within the coating and another of which contains the coating alone, without mesh within it.

14. The composition of claim 1, further comprising a non-sticky, protective backing.

15. The composition of claim 14, wherein the backing comprises one or more cellulose ether derivatives, and/or crosslinked gelatin.

16. The composition of claim 15, wherein said backing comprises HPMC (hydroxypropyl methylcellulose), HPC (hydroxypropyl cellulose), HEC (hydroxyethyl cellulose) or EC (ethyl cellulose).

17. The composition of claim 1, comprising a non-adhesive backing layer comprising a water-erodable, film-forming pharmaceutically acceptable polymer.

18. The composition of claim 14, wherein said backing remains for up to 1 month following implantation.

19. The composition of claim 1, wherein said one or more cross-linkable proteins comprises gelatin and further comprises a cross-linkable material selected from the group consisting of aminated polyethylene glycol (PEG), aminated polyvinyl alcohol (PVA), alginate and chitosan.

20. The composition of claim 1, wherein only the mesh remains after one month following implantation.

21. The composition of claim 1, wherein the mesh comprises a composite mesh comprising a non-foamed composition of the cross-linkable proteins or polypeptides and said one or more cross-linking materials.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0105] In the drawings:

[0106] FIG. 1 shows a top view of the device design, including a section of adhesive covered mesh and a section of only adhesive surrounding the mesh-adhesive section.

[0107] FIG. 2A shows a side view of the device portraying an optional placement of the mesh within the adhesive, in a way that the adhesive surrounds the mesh in all directions, while FIG. 2B shows an exploded view of the layers.

[0108] FIG. 3 shows the measurement of relative tackiness of the various backing and composition combinations with an Instron.

[0109] FIG. 4 shows an exemplary device implanted in vivo. An embedded surgical hernia mesh is strongly fixated to a peritoneal tissue.

[0110] FIG. 5 shows an optional and non-limiting method of preparation of the composition.

[0111] FIG. 6 shows some optional dimensions, minimum and maximum, for various mesh based composition shapes. See FIG. 2A for a description on how the dimensions given relate to the product.

[0112] FIG. 7 shows the effect of pH on mTG activity by viscometer test. 9% gelatin in pH 5.5 (native), 4 and 3.5 were mixed with 40 u/ml enzyme solution at 37° c. (2:1)

[0113] FIG. 8 shows a schematic view of the aeration and mixing system;

[0114] FIG. 9 shows an SEM image of a Cross-section of a gelatin-mTG dry foam article, prepared by the process described in example 7;

[0115] FIG. 10 shows an SEM image of the adhesive surface of a gelatin-mTG dry foam article, prepared by the process described in example 7.

[0116] FIGS. 11A and 11B show SEM images of articles prepared as described herein: FIG. 11A shows an SEM image of a cross-section of LM-147 article—Mesh in embedded in the middle of the foam, while FIG. 11B shows an SEM image of a cross-section of LM-149 article—Surgical mesh is located at the bottom (surface);

[0117] FIGS. 12A and 12B show SEM images of the bottom of the articles of FIG. 11: FIG. 12A shows an SEM image of the bottom surface of LM-147 article—Surgical mesh is not visible as it is fully covered by foam, while FIG. 12B shows an SEM image of the bottom surface of LM-149 article—Surgical mesh is located at the surface level, not fully covered;

[0118] FIGS. 13A and 13B show SEM images of the articles of FIG. 11, fixated on collagen: FIG. 13A shows an SEM image of LM-149 after fixation on collagen as a tissue simulating substrate (collagen is the bottom layer), while FIG. 13B shows an SEM image of LM-147 after fixation on collagen as a tissue simulating substrate (collagen is the top layer);

[0119] FIG. 14 shows the fixation strength of the different models on collagen, as tested by the lap shear method, 4 minutes after fixation;

[0120] FIG. 15 shows the fixation strength of the different models on swine peritoneum tissue, as tested by the lap shear method;

[0121] FIG. 16 shows representative histopathology images of samples explanted after 7 days from swine;

[0122] FIG. 17 shows a boxplot representing % surface area of Control mesh (bare Surgical Mesh) and Mesh based compositions (Surgical mesh covered with adhesive) groups GF-199 (Crosslinked gelatin), GF-200 (HPMC) covered by adhesions, 14 days after implantation;

[0123] FIG. 18 shows the fixation strength of Gelatin-Alginate mesh based composition (batch No. GF-502) on collagen, as tested by the lap shear method, 24 hours after fixation. GF-502 articles were immersed for 24 hours post application on collagen in either 0.9% saline or 0.9% saline with addition of 50 mM CaCl.sub.2);

[0124] FIG. 19 shows the fixation strength of Gelatin-Chitosan mesh based compositions (batches No. GF-510, GF-511, GF-512) on collagen, as tested by the lap shear method, 24 hours after fixation. The articles were immersed for 24 hours post application on collagen in 0.9% saline.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0125] The present invention is of mesh-based compositions comprising a solution of a cross-linkable protein or polypeptide, and an agent which induces cross-linking of the cross-linkable protein.

[0126] The present invention provides a mesh-based composition comprising a cross-linkable protein or polypeptide and one or more cross-linking materials according to at least some embodiments.

[0127] The mesh optionally and preferably comprises a self-adhering surgical mesh which requires no additional fixation means while at the same time minimizes adhesions. Optionally the mesh may comprise a composite mesh, featuring for example (and without limitation) a foamed composition of the cross-linkable protein and a non-toxic material that induces cross-linking of the cross-linkable material. The composite mesh may optionally be in a sheet-like form.

[0128] According to at least some embodiments, preferably the composite mesh features an incorporated hernia mesh. The structure preferably features two sections, one of which is composed of a mesh enclosed within the adhesive composition and another which contains the adhesive composition alone, without mesh within it. This design allows for a self-fixating adhesion minimizing hernia mesh device, preventing the mesh from shifting, migrating, rolling up its edges, or changing its position without the use of sutures, staples and other additional means of fixation.

[0129] Optionally and preferably, the cross-linkable protein includes gelatin and any gelatin variant or variant protein as described herein. Optionally and preferably, the non-toxic material comprises transglutaminase (TG), which may optionally comprise any type of calcium dependent or independent transglutaminase, which may for example optionally be a calcium-independent microbial transglutaminase (mTG). Without wishing to be limited in any way, among the improved properties of at least some embodiments of the present invention, the compositions of the present invention provide an increased rate of protein cross-linking as compared to background art compositions. Furthermore, the crosslinking reaction of mTG represents a significant improvement over that catalyzed by Factor XIIIa of the blood coagulation system. Unlike Factor XIIIa, the microbial enzyme requires neither thrombin nor calcium for activity.

[0130] Various embodiments of the present invention are described in greater detail below, under section headings which are provided for the sake of clarity only and without any intention of being limiting in any way.

[0131] Gelatin and Transglutaminase

[0132] According to preferred embodiments of the present invention, there is provided a mesh-based composition in which the cross-linking material comprises transglutaminase and the cross-linkable protein comprises gelatin.

[0133] According to a preferred embodiment, transglutaminase is present at a specific activity level of at least about 5 U/mg.

[0134] Suitable gelatin and transglutaminase can be obtained by any of the methods known and available to those skilled in the art. Gelatin may optionally comprise any type of gelatin which comprises protein that is known in the art, preferably including but not limited to gelatin obtained by partial hydrolysis of animal tissue and/or collagen obtained from animal tissue, including but not limited to animal skin, connective tissue (including but not limited to ligaments, cartilage and the like), antlers or horns and the like, and/or bones, and/or fish scales and/or bones or other components; and/or a recombinant gelatin produced using bacterial, yeast, animal, insect, or plant systems or any type of cell culture.

[0135] According to preferred embodiments of the present invention, gelatin from animal origins preferably comprises gelatin from mammalian origins and more preferably comprises one or more of pork skins, pork and cattle bones, or split cattle hides, or any other pig or bovine source. More preferably, such gelatin comprises porcine gelatin since it has a lower rate of anaphylaxis. Gelatin from animal origins may optionally be of type A (Acid Treated) or of type B (Alkaline Treated), though it is preferably type A.

[0136] Preferably, gelatin from animal origins comprises gelatin obtained during the first extraction, which is generally performed at lower temperatures (50-60° C., although this exact temperature range is not necessarily a limitation). Gelatin produced in this manner will be in the range of 250-300 bloom and has a high molecular weight of at least about 95-100 kDa. Preferably, 275-300 bloom gelatin is used.

[0137] A non-limiting example of a producer of such gelatins is PB Gelatins (Tessenderlo Group, Belgium).

[0138] According to some embodiments of the present invention, gelatin from animal origins optionally comprises gelatin from fish. Optionally any type of fish may be used, preferably a cold water variety of fish such as carp, cod, or pike, or tuna. The pH of this gelatin (measured in a 10% solution) preferably ranges from 4-6.

[0139] Cold water fish gelatin forms a solution in water at 10° C. and thus all cold water fish gelatin are considered to be 0 bloom. For the current invention, a high molecular weight cold water fish gelatin is preferably used, more preferably including a molecular weight of at least about 95-100 kDa. This is equivalent to the molecular weight of a 250-300 bloom animal gelatin. A non-limiting example of a producer of such a gelatin is Norland Products (Cranbury, N.J.).

[0140] In a preferred embodiment of the invention, the gelatin is purified to remove salts. This can be accomplished according to previously described techniques. One such technique involves forming a 20% w/v solution of gelatin in water and heating it to 60° C. under stirring. The mixture is then let to stand still overnight. The gel obtained is dialysed against repeated changes of deionized water to eliminate salts, stirred and heated to 50° C. to disaggregate the physical network. The final solution was filtered and freeze-dried. (Crescenzi V, Francescangeli A, Taglienti A. (2002). Biomacromolecules. 3:1384-1391). Alternatively, the gelatin can be desalted by size exclusion column.

[0141] According to some embodiments of the present invention, a recombinant gelatin is used. Recombinant gelatins are currently commercially produced by FibroGen (San Francisco, Calif.). The currently preferred method is using a recombinant yeast system (Pichia Pastoris) to express specified fragments of Type I, alpha1 human sequence collagen.

[0142] In an optional but preferred embodiment of the present invention, recombinant gelatins are fully synthetic molecules, containing no contaminating components from humans or any animals. By “synthetic” it is meant that the gelatin is preferably produced according to a method selected from chemical synthesis, cell free protein synthesis, cell tissue culture, any type of bacterial, insect or yeast culture, or in plants. The use of synthetic gelatins eliminates many of the variables and drawbacks associated with tissue-derived materials, including provoking unwanted immune responses. For example, fish gelatins demonstrate high allergenicity and animal gelatins demonstrate low-moderate allergencity, while recombinant gelatins can have zero allergenicity. In human safety studies, no adverse events related to recombinant gelatin were found.

[0143] Methods of creating recombinant gelatins and the benefits of their use are fully described in U.S. Pat. Nos. 6,413,742 and 6,992,172, which are hereby incorporated by reference as if fully set forth herein.

[0144] Recombinant gelatins can be produced to be highly (99%) purified. Recombinant gelatin production allows for the optional production of gelatins with at least one defined and predetermined characteristic, including but not limited to defined molecular weights, pI (isoelectric point), guaranteed lot-to-lot reproducibility, and the ability to tailor the molecule to match a specific application.

[0145] An example of tailoring a molecule to match a specific application has been previously described wherein a gelatin was created to be highly hydrophilic (Werten M W T, et al. (2001). Protein Engineering. 14 (6): 447-454). Optionally and preferably a gelatin according to the present invention comprises a gelatin having at least one adjusted, tailored or predetermined characteristic.

[0146] The gelatin employed in the device can be a gelatin complex or any gelatin, or a derivative or metabolite thereof, or a gelatin produced according to a single process or a plurality of processes. For example, the gelatin may optionally comprise gelatin type A or gelatin type B, or a combination thereof.

[0147] The transglutaminase may optionally comprise any plant, animal, or microbe derived transglutaminase, preferably other than blood derived Factor XIII. Preferably, microbial transglutaminase (mTG) derived from Streptoverticillium mobaraensis is used.

[0148] The transglutaminase may optionally be in a composition comprising at least one other substance, such as a stabilizer or filler for example. Non-limiting examples of such materials include maltodextrin, hydrolyzed skim milk protein or any other protein substance, sodium chloride, safflower oil, trisodium phosphate, sodium caseinate or lactose, or a combination thereof.

[0149] Transglutaminase features a negative temperature coefficient. Over the temperature range of the transglutaminase activity, it takes a shorter time to react at higher temperatures and longer amount of time to start functioning at lower temperatures. The following table 1 shows different reaction times at different temperatures comparing the same reaction grade as the reaction at 50° C., pH 6.0 that occurs in 10 minutes:

[0150] Table 1 showing reaction temperatures of transglutaminase.

TABLE-US-00001 Temperature 5° C. 15° C. 20° C. 30° C. 40° C. Time (minutes) 240 105 70 35 20

[0151] Non-limiting examples of commercially available transglutaminase products include those produced by Ajinomoto Co. (Kawasaki, Japan). A preferred example of such a product from this company is the Activa TG-TI (In Europe: Activa WM)—Ingredients: mTG and maltodextrin; Activity: 81-135 U/g of Activa. Other non-limiting examples of suitable products from this company include Activa TG-FP (ingredients: hydrolyzed skim milk protein, mTG; activity: 34-65 U/g of Activa TG-FP); Activa TG-GS (ingredients: sodium chloride, gelatin, trisodium phosphate, maltodextrin, mTG, and safflower oil (processing aid); activity: 47-82 U/g of Activa TG-GS); Active TG-RM (In Europe: Activa EB)—ingredients: sodium caseinate, maltodextrin, and mTG; activity: 34-65 U/g of Activa; Activa MP (ingredients: mTG, Lactose and Maltodextrin; activity: 78-126 U/g of Activa).

[0152] Other non-limiting examples of commercially available transglutaminase products include those produced by Yiming Biological Products Co. (Jiangsu, China). A preferred example of such a product from this company is the TG-B (ingredients: 1% mTG, 99% co-protein; activity: 80-130 U/g of TG-B). Other non-limiting examples of suitable products from this company include TG-A (ingredients: 0.5% mTG, 99.5% co-protein; activity: 40-65 U/g of TG-A).

[0153] For both examples, preferred transglutaminase products are those with the highest specific activity and simplest co-ingredients, as they are believed (without wishing to be limited by a single hypothesis) to have the best reactivity upon application and a lower potential for undesired side effects.

[0154] In another embodiment, a transglutaminase may optionally be extracted from Streptoverticillium Baldaccii or a Streptomyces Hygroscopicus strain to produce enzyme variants that have been shown to function optimally at lower temperatures (approximately 37° C. and 37°−45° C., respectively) (Negus SS. A Novel Microbial Transglutaminase Derived from Streptoverticillium Baldaccii. PhD Thesis. School of Biomolecular and Biomedical Science. Griffith University, Queensland, Australia and Cui L et al. Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. 2007: 105 (2). p. 612-618). Higher specific activity at lower temperatures is desirable for achieving faster and stronger cross linking of the gelatin under ambient conditions.

[0155] According to some embodiments, transglutaminase can be used in the form of any of the above described compositions, optionally including any of the commercially available mixtures that include transglutaminase.

[0156] In another embodiment, any of the above transglutaminase mixtures may optionally be purified by means of gel filtration, cation-exchange chromatography, hollow fiber filtration, or tangential flow filtration to remove their carrier proteins and/or carbohydrates. Some of these methods have been previously described (Bertoni F, Barbani N, Giusti P, Ciardelli G. Transglutaminase reactivity with gelatine: perspective applications in tissue engineering. Biotechnol Lett (2006) 28:697-702) (Broderick E P, et al. Enzymatic Stabilization of Gelatin-Based Scaffolds J Biomed Mater Res 72B: 37-42, 2005). The filter pore size used for filtration is preferably approximately 10 kDA.

[0157] Preferably, the transglutaminase is purified in a process that includes cation-exchange chromatography, hydrophobic chromatography, and ultrafiltration, as described more fully in for example U.S. Pat. No. 8,367,388, filed on Jun. 18, 2009, owned in common with the present application and having at least some inventors in common with the present application.

[0158] Regardless, the activity of transglutaminase is preferably measured prior to use and/or manufacture of a composition according to the present invention with a transglutaminase reactivity assay. Such an assay may optionally include but is not limited to the Hydroxamate Method, Nessler's Assay, a Colorimetric Assay, or any other assay of transglutaminase activity (see for example Folk J E, Cole P W. Transglutaminase: mechanistic features of the active site as determined by kinetic and inhibitor studies. Biochim Biophys Acta. 1966; 122:244-64; or the Nessler Assay as described in: Bertoni F, Barbani N, Giusti P, Ciardelli G. Transglutaminase reactivity with gelatine: perspective applications in tissue engineering. Biotechnol Lett (2006) 28:697-702).

[0159] In general, the purity and/or quality of the gelatin and/or the transglutaminase for use in the device (tissue adhesive, hemostatic or sealing product) composition will be of an appropriate purity known to one of ordinary skill in the relevant art to lead to efficacy and stability of the protein.

Enzyme Purification and Concentration

[0160] According to some embodiments of the present invention, transglutaminase solutions undergo one-stage or multiple-stage purification to perform one or more of 1) removing fermentation residue from the transglutaminase mixture; 2) concentrating the amount of active translglutaminase in a transglutaminase solution; 3) further purifying the transglutaminse solution from carrier proteins or carbohydrates; 4) lowering the endotoxin level of the transglutaminase solution; and/or 5) removing all microbes from the transglutaminase solution, effectively sterilizing the solution; all without wishing to be limited to a closed list.

[0161] According to some embodiments, the solution of cross-linking material is filtered prior to mixing with the cross-linkable protein of polypeptide.

[0162] In an embodiment of the present invention, the filtration process first uses coarse filtration, sometimes known as clarification, to remove large blocks of fermentation residue. Non-limiting examples of such coarse filtration features a pore size above 0.22 μm, such as for example from about 0.45 μm pore size filtration, optionally including about 0.65 μm pore size filtration.

[0163] According to another embodiment of the present invention, the solution of cross-linking material is optionally and preferably passed through a filter of pore size of below 0.22 μm in a secondary filtration process after coarse filtration, for example to reduce the bioburden of the material below 10 colony forming units (CFU) per gram and make it appropriate for medical use. Preferably, the bioburden is practically eliminated to achieve a sterility assurance level (SAL) of less than about 10.sup.−2 and more preferably less than about 10.sup.−3, where SAL is a term used in microbiology to describe the probability of a single unit being non-sterile after it has been subjected to a sterilization process.

[0164] According to another embodiment of the present invention, either tangential flow or hollow fiber ultra-filtration techniques are used after such a secondary filtration stage, not only to purify the solution of cross-linking material by removal of carrier carbohydrates and proteins, but also to concentrate the solution. Preferred pore sizes for use with this invention are those with pore sizes larger than the size of the components of the cross-linking composition.

[0165] In an embodiment, the crosslinking material is mTG and the pore size is in the range of 10-50 kDa. In a more preferred embodiment, the crosslinking material is mTG and the pore sizes are in the range of 10-30 kDa.

[0166] According to another embodiment, one or more size exclusion chromatography steps is used to selectively separate the crosslinking material from surrounding substances.

[0167] According to another embodiment, one or more hydrophobic or hydrophilic interaction chromatography steps is used to selectively separate the crosslinking material from surrounding substances.

[0168] According to another embodiment of the present invention, the crosslinking material is a protein and one or more ion exchange chromatography steps is used to preferentially bind the crosslinking protein, thereby purifying it from the surrounding materials.

[0169] According to a more preferred embodiment, the crosslinking protein is mTG and one or cation exchange chromatography steps is used to purify the mTG.

[0170] In a preferred embodiment, the cation exchange resin is a sepharose resin.

[0171] According to another preferred embodiment, purification reduces the endotoxin level of the crosslinking material to <5 endotoxin units (EU) per gram.

[0172] According to another preferred embodiment, the crosslinking material is mTG and purification results in an mTG composition wherein the specific activity is greater than 20 enzyme units per milligram and preferably greater than 25 units per milligram.

[0173] According to another preferred embodiment, the crosslinking material is mTG and purification results in electrophoretic purity of at least 95% and preferably of at least 98%.

[0174] An mTG purification process, as a non-limiting example, is described herein that purifies a food-grade mTG product to produce an mTG composition with specific activity >25 enzyme units per milligram, >95% electrophoretic purity, <5 endotoxin units per gram, and <10 CFU/g.

[0175] As described above, mTG concentration is also a preferred parameter for some embodiments of the composition of the present invention. The above purification processes may also result in more concentrated mTG material. In addition to cross-linking gelatin more rapidly than non-concentrated mTG solutions, concentrated mTG solutions formed gels that were more elastic, more adhesive, and more transparent compared to the non-concentrated controls.

[0176] One or more supplements can also be contained in the tissue adhesive, hemostatic or sealing product, e.g., drugs such as growth factors, polyclonal and monoclonal antibodies and other compounds. Illustrative examples of such supplements include, but are not limited to: antibiotics, such as tetracycline and ciprofloxacin, amoxicillin, and metronidazole; anticoagulants, such as activated protein C, heparin, prostracyclin (PGI2), prostaglandins, leukotrienes, antitransglutaminase III, ADPase, and plasminogen activator; steroids, such as dexamethasone, inhibitors of prostacyclin, prostaglandins, leukotrienes and/or kinins to inhibit inflammation; cardiovascular drugs, such as calcium channel blockers, vasodilators and vasoconstrictors; chemoattractants; local anesthetics such as bupivacaine; and antiproliferative/antitumor drugs such as 5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such as gangcyclovir, zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine and antibodies to viral components or gene products; cytokines, such as alpha- or beta- or gamma-Interferon, alpha- or beta-tumor necrosis factor, and interleukins; colony stimulating factors; erythropoietin; antifungals, such as diflucan, ketaconizole and nystatin; antiparasitic agents, such as pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsin and alpha-1-antichymotrypsin; anesthetics, such as bupivacaine; analgesics; antiseptics; and hormones. Other illustrative supplements include, but are not limited to: vitamins and other nutritional supplements; glycoproteins; fibronectin; peptides and proteins; carbohydrates (both simple and/or complex); proteoglycans; antiangiogenins; antigens; lipids or liposomes; and oligonucleotides (sense and/or antisense DNA and/or RNA).

Illustrative Compositions

[0177] The above described cross-linking substrates and cross-linking materials may optionally be combined with one or more additional materials to form various compositions according to the present invention, for use with a patch as described herein. According to some embodiments, the adhesive material optionally and preferably comprises: (i) gelatin; (ii) a transglutaminase. More preferably, the gelatin and transglutaminase are provided in sufficient quantities to be useful as a tissue adhesive, sealing, or hemostatic agent.

[0178] In addition, one or more supplements can also be contained in the tissue adhesive, sealing, or hemostatic product, e.g., drugs such as growth factors, polyclonal and monoclonal antibodies and other compounds. Illustrative examples of such supplements include, but are not limited to: antibiotics, such as tetracycline and ciprofloxacin, amoxicillin, and metronidazole; anticoagulants, such as activated protein C, heparin, prostracyclin (PGI2), prostaglandins, leukotrienes, antitransglutaminase III, ADPase, and plasminogen activator; steroids, such as dexamethasone, inhibitors of prostacyclin, prostaglandins, leukotrienes and/or kinins to inhibit inflammation; cardiovascular drugs, such as calcium channel blockers, vasodilators and vasoconstrictors; chemoattractants; local anesthetics such as bupivacaine; and antiproliferative/antitumor drugs such as 5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such as gangcyclovir, zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine and antibodies to viral components or gene products; cytokines, such as alpha- or beta- or gamma-Interferon, alpha- or beta-tumor necrosis factor, and interleukins; colony stimulating factors; erythropoietin; antifungals, such as diflucan, ketaconizole and nystatin; antiparasitic agents, such as pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsin and alpha-1-antichymotrypsin; anesthetics, such as bupivacaine; analgesics; antiseptics; and hormones. Other illustrative supplements include, but are not limited to: vitamins and other nutritional supplements; glycoproteins; fibronectin; peptides and proteins; carbohydrates (both simple and/or complex); proteoglycans; antiangiogenins; antigens; lipids or liposomes; and oligonucleotides (sense and/or antisense DNA and/or RNA).

Mesh-Based Composition and Structure

[0179] According to at least some embodiments, the present invention is of a temporary resorbable anti adhesive non-stick backing. It is designed to protect medical devices from sticking to gloves, surgical tools and internal organs.

[0180] Examples are pads, foams, bandages used for hemostasis or sealing purposes. Other examples are coated surgical meshes. Some of these devices contain or are coated with an adhesive layer that fixes the device by adhering it to a tissue. If the same adhesive is present on the side of the device that is facing away from the tissue it might become tacky and stick to gloves, surgical tools and internal organs. In addition, if the coating layers are not tacky by themselves, during the surgery they could still stick to gloves, surgical tools and internal organs after the latter become moist or wet by contact with peritoneal fluids, blood or saline.

[0181] In another scenario, during device handling, the coating might unintentionally touch a moist tissue such as the intestines and stick to it before the surgeon has the opportunity to position the device in the desired location. The non-stick backing is designed to prevent such an occurrence.

[0182] The device backing may be composed of any polymeric material, of natural, semi-synthetic or synthetic nature, that is soluble in water to some extent such as polysaccharides, proteins etc.

[0183] The backing is required for the short duration in which there is a risk of the device coating sticking to unwanted surfaces or tissues, for example during handling of the device or during the surgery. Once the device has been placed and secured at the desired location the backing is no longer required, and therefore was designed to dissolve quickly. The backing can be made of cellulose ether derivatives, such as HPMC (hydroxypropyl methylcellulose) or HPC (hydroxypropyl cellulose), HEC (hydroxyethyl cellulose) or EC (ethyl cellulose). The backing can be also made from crosslinked gelatin (enzymatic, physical or chemical crosslinking).

[0184] According to at least some embodiments of the present invention, there is provided a patch, comprising a gelatin layer and a reinforcing back layer, wherein said gelatin layer comprises gelatin and an enzyme integrated into a carrier selected from a group consisting of: HPC (hydroxypropyl cellulose), HPMC (hydroxypropyl methylcellulose), carboxymethyl cellulose, hydroxylethyl cellulose, ethylcellulose, PVP (polyvinyl pyrrolidone), PVA (polyvinyl alcohol), PEG (polyethylene glycol), PEI (polyethyleneimine), starch, microcrystalline cellulose, oxidized cellulose.

[0185] The molecular weight of the polymer may optionally be selected in order to determine the performance of the backing. Without wishing to be limited by a single hypothesis, it is believed that small molecular weight results in faster dissolution of the polymer in moist/wet conditions-thus resulting in development of tackiness in the backing itself. The larger the molecular weight, the less soluble the polymer is and as a result the tendency for tackiness is reduced.

[0186] The non-adhesive backing layer may comprise a water-erodable, film-forming pharmaceutically acceptable polymer such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethylmethyl cellulose, polyvinylalcohol, polyethylene glycol, polyethylene oxide, ethylene oxide-propylene oxide copolymers, collagen and derivatives, gelatin, albumin, polyaminoacids and derivatives, polyphosphazenes, polysaccharides and derivatives, chitin and chitosan, alone or in combination. The backing layer component may or may not be crosslinked depending on the desired erosion kinetics.

[0187] In one embodiment, the preferred backing layer component comprises Hydroxypropyl Methylcellulose (HPMC). Preferably, in the case of hydroxyethyl cellulose, the average molecular weight (Mw estimated from intrinsic viscosity measurements) is in the range 2×104 to 1.2×106, and more preferably in the range 2.5×105 to 1×106.

[0188] In another embodiment, it is possible to mix two HPMC polymers differ in MW in order to achieve the desired mechanical properties or film thickness. For example, using a 0.5-2% of HPMC (1×10{circumflex over ( )}6 Da) mixed with 1-4% of HPMC (4×10{circumflex over ( )}5 Da) is contemplated within at least some embodiments of the present invention.

[0189] The erosion kinetics of the backing layer may optionally be altered in many different ways in order to modify the residence time.

[0190] Optionally, such alteration may be performed with a combination which comprises hydroxypropyl methylcellulose and an alkylcellulose such as ethylcellulose. Such a combination comprises a film-forming amount of alkylcellulose, hydroxypropyl methylcellulose, and a suitable solvent. Advantageously, the characteristics of the film formed from the gel may be modified depending upon the ratio of hydroxypropyl methylcellulose to alkylcellulose. Such modifiable characteristics advantageously include the kinetics of erodability.

[0191] Typically, the ratio of hydroxypropyl methylcellulose to alkylcellulose is that necessary to form a suitable film. This ratio may vary based on the other components and the type of alkylcellulose. However, if ethylcellulose is employed then the ratio of hydroxypropyl cellulose to ethyl cellulose is usually from about 1000:1 to about 3:1, preferably from about 200:1 to about 4:1, more preferably from about 200:1 to about 8:1. Typically, as the ratio of hydroxypropyl cellulose to alkylcellulose increases, the water erodability increases, i.e., the films are more readily washed away. Thus, the ethylcellulose is a component which acts to adjust the kinetics of erodability of the device.

[0192] According to another embodiment the backing layer is designed to remain in place for a longer duration, e.g. up to 1 month following implantation, where it may reduce adhesions of the mesh or the adhesive layer to the visceral organs. In this case the backing layer may only dissolve very slowly, this can be achieved by crosslinking the polymer which comprise the backing.

[0193] Crosslinking the film-forming polymer may also optionally be performed to affect its properties. Crosslinking agents known in the art are appropriate for use in the invention and may include glyoxal, propylene glycol, glycerol, dihydroxy-polyethylene glycol of different sizes, butylene glycol, and combinations thereof. The amount of crosslinking agent used may vary, depending on the particular polymers and crosslinking agent but usually should not exceed 5% molar equivalent of the polymeric material, and preferably comprises 0 to 3% molar equivalent of the polymeric material.

[0194] Gelatin backing can be crosslinked by methods known to those skilled in the art, and include but are not limited chemical crosslinkers such as formaldehyde, glutaraldehude and EDC, physical methods such has DHT (dehydrothermal) treatment or enzymatic methods, e.g. by mTG of any type, including without limitation mammalian TG, or Factor XIII.

[0195] It is possible to control the mechanical properties of the backing layer by employing excipients which plasticize the film concomitantly. The excipient or plasticizer often improves the mechanical properties of the device and/or modifies the drug release profile or disintegation time. Suitable excipients or plasticizers modifying the erosion behavior of the layer(s) may include alkyl-glycol such as propylene glycol, polyethyleneglycols, oleate, sebacate, stearate or esters of glycerol, phthalate and others. Other suitable plasticizers include esters such as acetyl citrate, amyl oleate, myristyl acetate, butyl oleate and stearate, dibutyl sebacate, phthalate esters such as diethyl, dibutyl, and diethoxy ethyl phthalate and the like, fatty acids such as oleic and stearic acid, fatty alcohols such as cetyl, myristyl, and stearyl alcohol. Moreover, in some instances, a polymer, a pharmaceutical, or solvent residual may act as a plasticizer. One preferable plasticizer is PEG. The MW range is from 200 Da to 1000 Da. Preferably, the concentration of PEG in the film is from 10% w/w to 50% w/w, more preferably from 25-40%. Another preferred plasticizer is glycerol, the concentration of glycerol in the film is from 10% w/w to 50% w/w, more preferably from 25-40% w/w.

[0196] The backing layer can be attached to the adhesive layer, by means of direct application or indirect application.

[0197] By “direct application” various methods may optionally be used, including spreading or spraying the backing in a liquid form on the gelatin layer and drying it. Using non aqueous solvents such as ethanol prevents undesired dissolution of the gelatin layer.

[0198] In yet another example of direct application the backing layer is cast as a film and dried, then the adhesive layer is applied wet on top of the said film and subjected to another round of drying, such as lyophilization.

[0199] In another embodiment, direct application may optionally be performed as follows: the backing layer can be attached to the adhesive layer by taking advantage of the inherent tackiness of polymers after they have been wetted with water, either liquid or steam. While the polymer, either the adhesive layer, backing layer, or both, are wet and/or tacky, the two layers are attached to each other and the resulting bond formed is permanent, even after the water used for activation is eliminated.

[0200] In indirect application, the backing may optionally be prepared separately as a film, e.g. by cast drying, and then attached to the adhesive layer by applying a bonding layer on the film or on the adhesive layer by spraying or spreading. The adhesive layer is then attached to the backing layer where the bonding layer glues them together. The bonding layer can be dried before or after the attachment of the gelatin layer to the backing layer.

[0201] In another embodiment, the attachment may also optionally be performed by wetting the adhesive layer or the bonding layer directly with water or other aqueous liquid such as saline or by incubating in a high humidity environment (such as humidity chamber or by steam).

[0202] The bonding layer is optionally composed of a naturally derived polysaccharide such as maltodextrin, starch, cellulosics (e.g. hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose or blended compositions thereof), or from a synthetic polymer such as PVP. The preferred MW range for PVP is from 1×10{circumflex over ( )}4 Da to 1.3×10{circumflex over ( )}6 Da.

[0203] A plasticizer can be added to increase the moisture content of the bonding layer and thus its tackiness. One preferable plasticizer is PEG. The MW range of PEG is from 200 Da to 1000 Da. Preferably, the concentration of PEG in the bonding layer is from 10% w/w to 50% w/w, more preferably from 25-40%. Another preferred plasticizer is glycerol, such that optionally the concentration of glycerol in the bonding layer is from 10% w/w to 50% w/w, more preferably from 25-40%. Above 50% w/w plasticizer the bonding layer becomes too wet and the backing layer might slide off the gelatin layer and below 10% w/w plasticizer it dries out too quickly.

[0204] Optionally the product features a mesh and is adapted for surgical mesh fixation where mesh can be adhered to an organ surface, tissue surface, or cavity.

[0205] Optionally said mesh comprises any degradable or non degradable material, including without limitation synthetic mesh, biological mesh, or a combination synthetic-biological mesh.

[0206] Optionally the product is adapted for inguinal, femoral, umbilical or incisional ventral hernia repair, or other types of surgical mesh reconstruction.

[0207] Optionally the product is adapted for use with a reduced stapling or suturing procedure.

[0208] Optionally the product is adapted for use with one or more of staples, tacks, or sutures to supplement mesh adhesion.

[0209] Optionally the product is adapted for any of large diaphragmatic hernia repair, for rectopexy (rectal prolapsed) mesh fixation, for reconstruction of a prolapsed vaginal vault, or for other pelvic floor mesh reinforcement operations (gynecology procedures).

[0210] Optionally the product further comprises an additional agent selected from the group consisting of: an antibiotic, an anticoagulant, a steroid, a cardiovascular drug, a local anesthetic, a antiproliferative/antitumor drug, an antiviral, a cytokine, colony stimulating factors; erythropoietin; an antifungal; an antiparasitic agent; anti-inflammatory agents; anesthetics, such as bupivacaine; analgesics; antiseptics; and hormones.

[0211] Optionally the product further comprises an additional agent selected from the group consisting of vitamins and other nutritional supplements; glycoproteins; fibronectin; peptides and proteins; carbohydrates (both simple and/or complex); proteoglycans; antiangiogenins; antigens; lipids or liposomes; and oligonucleotides (sense and/or antisense DNA and/or RNA).

[0212] Optionally said cytokine is selected from the group consisting of alpha- or beta- or gamma-Interferon, alpha- or beta-tumor necrosis factor, and interleukins.

[0213] Optionally said antiviral is selected from the group consisting of gangcyclovir, zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine and antibodies to viral components or gene products. Optionally said anti-tumor drug is selected from the group consisting of 5-fluorouracil (5-FU), taxol and/or taxotere.

[0214] Optionally said cardiovascular drug is selected from the group consisting of calcium channel blockers, vasodilators and vasoconstrictors; chemoattractants.

[0215] Optionally said steroid is selected from the group consisting of dexamethasone, inhibitors of prostacyclin, prostaglandins, leukotrienes and/or kinins to inhibit inflammation.

[0216] Optionally said anticoagulant is selected from the group consisting of activated protein C, heparin, prostracyclin (PGI2), prostaglandins, leukotrienes, antitransglutaminase III, ADPase, and plasminogen activator.

[0217] Optionally said antibiotic is selected from the group consisting of tetracycline, ciprofloxacin, amoxicillin, and metronidazole.

[0218] Optionally the product further comprises a wound healing agent.

[0219] Optionally the product further comprises a hemostatic agent.

[0220] According to at least some embodiments of the present invention, there is provided a method of producing a mesh based composition, comprising: producing a cross-linkable protein matrix, comprising a cross-linkable protein; depositing an enzymatic composition in said protein matrix, wherein said enzymatic composition comprises an enzyme capable of cross-linking said cross-linkable protein; thereby producing the product. In some embodiments, the protein matrix is deposited at a depth of at least 0.5 mm. In other embodiments, the depth of deposition may be lower.

[0221] Optionally said enzyme comprises transglutaminase and said transglutaminase comprises any type of calcium dependent or independent transglutaminase. Optionally said transglutaminase comprises a microbial transglutaminase (mTG).

[0222] Optionally said cross-linkable protein comprises gelatin in the form of a gelatin solution, comprising mixing said gelatin solution in a mixer at a rate to form a foamed solution, drying said foamed solution to form a dried solution and combining said dried solution with said enzyme.

[0223] Optionally said mixing said gelatin solution comprises mixing said gelatin solution in a mixer with pressurized air, at a mixing rate and air pressure so as to foam the solution; wherein said method further comprises lyophilizing the foamed gelatin solution to form a lyophilized porous layer of gelatin.

[0224] Optionally said rate is from 100 RPM to 10,000 RPM. Optionally said rate is from 1000 RPM to 6000 RPM.

[0225] Optionally said rate is from 0.1 cm.sup.3/second to 10,000 cm.sup.3/second per volume of foam.

[0226] Optionally said cross-linkable protein comprises gelatin in the form of a gelatin solution, comprising mixing said gelatin solution with a chemical foaming agent so as to foam the solution; wherein said method further comprises drying the foamed gelatin solution to a dried porous layer of gelatin.

[0227] Optionally said chemical foaming agent comprises sodium bicarbonate and wherein the mixture of the gelatin solution and the sodium bicarbonate has a pH below 7.

[0228] Optionally said cross-linkable protein comprises gelatin in the form of a gelatin solution, comprising freeze-drying in a temperature and pressure range, optionally with aeration so as to foam the solution and form a dried porous layer of gelatin.

[0229] Another method of creating a porous sponge-like structure featuring a dry mixture of gelatin-enzyme is using the freeze-drying method but optionally without such aeration.

[0230] In this method, first, the homogenous gelatin solution is cooled rapidly, transforming it to a bi-phase solid which contains a gelatin rich continuous phase and an ice phase concentrated in secluded spots. Second, by reducing pressure in the freeze-drying chamber, the water in the solid undergoes sublimation, leaving pores in place where the ice phase was concentrated before.

[0231] Without wishing to be limited to a single range, in the freeze-drying process, it was shown that a porous flexible dry foam can be obtained with the same ratio of gelatin to mTG enzyme (80 U/g gelatin), but without physical aeration. The density of the dry foam is comparable to the one prepared by physical aeration, for example with N.sub.2 gas. Accordingly, adhesion forces tested in-vitro were also comparable.

[0232] Optionally the method further comprises producing a gelatin layer by mixing a gelatin solution with said enzyme, said enzyme comprising transglutaminase, to form a foamed gelatin solution; wherein said method further comprises lyophilizing the foamed gelatin solution to form a lyophilized foamed gelatin solution and adding said lyophilized foamed gelatin solution to said product.

[0233] Optionally said transglutaminase is added to said gelatin solution prior to said mixing or during said mixing. Optionally said transglutaminase is added to said gelatin solution through continuous streaming during mixing.

[0234] Optionally the method further comprises cooling said foamed gelatin solution before said lyophilizing is performed. Optionally the method further comprises foaming a gelatin solution to form a foamed gelatin solution; drying the foamed gelatin solution to form said dried foamed gelatin solution; and adding said transglutaminase in a solution to said dried foamed gelatin solution to form an enzyme containing foam.

[0235] Optionally said transglutaminase to said dried solution comprises one or more of spraying an enzyme solution onto dry gelatin matrix surface; injecting an enzyme solution into the gelatin matrix through needles or matrix of needles; submersing dry gelatin matrix into an enzyme-containing solvent mixture; and/or dispensing enzyme-containing solvent mixture onto dry gelatin matrix.

[0236] Optionally the method further comprises drying said enzyme containing foam.

[0237] Optionally said drying said enzyme containing foam comprises one or more of air drying, vacuum drying, lyophilization and/or heat drying.

[0238] Optionally said drying occurs at a plurality of temperatures ranging from −40 C to 65 C. Optionally drying occurs at a temperature of up to 30 C. Optionally said drying occurs at a temperature of up to 20 C. Optionally said drying occurs at a plurality of temperatures ranging from 0 C to 20 C.

[0239] Optionally the mesh based composition comprises a plurality of gelatin layers and wherein optionally each of said gelatin layers has a different density of gelatin. Optionally at least one gelatin layer comprises a percentage of gelatin in solution of from about 1% w/w to about 15% w/w. Optionally at least one gelatin layer comprises a percentage of gelatin of from about 2.5% w/w to about 10% w/w. Optionally at least one gelatin layer comprises a percentage of gelatin of at least about 5% w/w.

[0240] Optionally at least one gelatin layer comprises a lubricant. Optionally said lubricant comprises glycerol. Optionally said glycerol is present in an amount of from 0.1% to 10%. Optionally said glycerol is present in an amount of from 2% to 6%.

[0241] According to at least some embodiments of the present invention, there is provided a mesh based composition comprising a cross-linkable porous protein matrix and a non blood-derived enzyme which induces cross-linking of the cross-linkable protein, wherein matrix density is in range of 5-100 mg/cm.sup.3. Optionally said density is in a range of 40-70 mg/cm.sup.3. Optionally the product has a total moisture content of less than 30%, a total moisture content of less than 20% or a total moisture content of less than 10%. Optionally such a mesh-based composition is useful for a variety of applications, including without limitation as a hemostatic dressing, tissue adhesive or wound closure.

[0242] Optionally a ratio of enzyme to matrix is from 0.05 to 5 mg enzyme/cm.sup.3 matrix. Optionally said ratio is 0.5 to 2.5 mg enzyme/cm.sup.3 matrix.

[0243] Optionally the product is sterilized to a sterility assurance level of 10{circumflex over ( )}6 through exposure to electron beam radiation. Optionally the radiation dosage is in the range of 10-50 kGy. Optionally the radiation dosage is in the range of 20-40 kGy.

[0244] Optionally the product is sterilized to a sterility assurance level of 10-6 through exposure to ethylene oxide gas.

[0245] Optionally the product further comprises a radioprotectant selected from the group consisting of Ascorbate, Benzyl alcohol, Benzyl benzoate, Butylated Hydroxyanisole (BHA), Chlorobutanol, Cysteine, Mannitol, Methyl paraben, Niacinamide, Phenol, Propylene glycol, Propyl gallate, Propyl paraben, Sodium bisulfate, Sodium metabisulfite, Sodium salicylate, Sodium thiosulfate, Tocopherol, Trehalose.

[0246] Optionally the product further comprises a buffer optionally selected from the group including Sodium Acetate, HEPES, Sodium Citrate, Sodium Benzoate.

[0247] Optionally the product further comprises one or more foaming stabilizers, optionally selected from the group consisting of Ionic surfactants (i.e. SDS), Hydroxyl Propyl Methyl Cellulose, Hyaluronic Acid, Glycine, Dextran.

[0248] Optionally a plurality of discrete enzyme-containing protein matrix segments together form a single product. Optionally each segment is of diameter in range of 0.1 to 10 cm. Optionally each segment is of diameter in range of 1-5 cm.

[0249] Table 2 showing synthetic surgical meshes that can be used as a component it the product described above

TABLE-US-00002 Commercial Mesh Name Material Manufacturer  1. VICRYL ™ Woven Mesh Polygiactin 910 Ethicon (Polyglycolic Acid) (Somerville, NJ)  2. PROLENE ™ 3D Patch Polypropylene Ethicon Polypropylene Mesh  3. PROLENE ™ Polypropylene Ethcon Polypropylene Mesh  4. PROLENE ™ Polypropylene Ethicon Polypropylene Hernia System  5. MERSILENE ™ Polyethylene Terephthaiate Ethicon Polyester Fiber Mesh  6. ULTRAPRO ™ Monocryl Ethicon Partially Absorbable (Poliglecaprone 25) Lightweight Mesh and Polypropylene  7. ULTRAPRO ™ Plug Monocryl Ethicon (Poliglecaprone 25) and Polypropylene  8. ULTRAPRO ™ Monocryl Ethicon Hernia System (Poliglecaprone 25) and Polypropylene  9. PVP ™ Device Oxidized Regenerated Ethicon Cellulose (ORC) and Polypropylene 10. PROCEED ™ Oxidized regenerated Ethicon Surgical Mesh cellulose (ORC) and Polypropylene 11. Parietex ™ Macroporous Polyester, Covidien Composite (POC) Mesh with a Three Dimensional (Mansfield, MA) Weave Material with resorbable collagen film 12. Parietex ™ Macroporous Polyester, Covidien composite open skirt with a three Dimensional (PCO OS) mesh Weave Material with resorbable collagen film 13. Parietex ™ Macroporous Polyester, Covidien Composite (POC) monofilament material Parastomal mesh 14. Parietex ™ Macroporous Polyester, Covidien Composite (POC) with a three Dimensional Hiatal mesh Weave Material with resorbable collagen fifth 15. Parietex ™ Macroporous Polyester, Covidien anatomical mesh 2D weave with 3D weave 16. Parietex ™ Macroporous Polyester Covidien Folding mesh 17. Parietex Easegrip ™ mesh Polyester, with a Covidien combination of two and three Dimensional Weave Material 18. Parietex ™ Monofilament knit, Covidien lightweight macroporous polyester monofilament mesh 19. Parietex ™ Polyester, with both Covidien Flat sheet mesh two and three Dimensional Weave options 20. Surgipro ™ Fiat Polypropylene Covidien Sheet mesh 21. PERM ™ Light Plug Monofilament Davol (Bard) Polypropylene (Warwick, RI) 22. PerFix ™ Plug Monofilament Davol (Bard) Polypropylene 23. Kugel ™ Patch Monofilament Davol (Bard) Polypropylene 24. 3DMax ™ Light Mesh Monofilament Davol (Bard) Polypropylene 25. Bard ™ Soft Mesh Large pore monofilament Davol (Bard) polypropylene 26. Bard ™ Mesh Monofilament Davol (Bard) Polypropylene 27. Bard ™ Visilex ™ Mesh Monofilament Davol (Bard) Polypropylene 28. Ventrio ™ Hernia Patch Monofilament Davol (Bard) Polypropylene and polydioxanone, with Submicronic ePTFE side 29. Composix ™ L/P Mesh Low profile Davol (Bard) polypropylene Bard Soft Mesh and sub-micronic ePTFE side 30. Composix ™ E/X Polypropylene Bard Davol (Bard) Soft Mesh and sub-micronic ePTFE side 31. Composix ™ Self-expanding Davol (Bard) Kugel ™ Patch polypropylene/ePTFE mesh 32. Dulex ™ Mesh Dual-sided ePTFE mesh Davol (Bard) 33. VENTRALEX ™ Self-expanding Davol (Bard) Hernia Patch polypropylene and ePTFE 34. Sepramesh ™ IP Polypropylene mesh with Davol (Bard) Composite a hydrogel safety coating 35. C-QUR ™ V-Patch Polypropylene mesh Atrium with an all natural, (Hudson, NH) pharmaceutical grade Atrium Omega 3 fatty acid 36. C-QUR ™ Mesh Polypropylene mesh Atrium with an all natural, pharmaceutical grade Omega 3 fatty acid 37. C-QUR Lite ™ Mesh Polypropylene mesh Atrium with a thin, 30 day omega 3 fatty acid 38. C-QUR Edge ™ Bloabsorbable Oil Atrium (O3FA) Coated mesh features a reinforced edge design 39. ProLoop ™ Mesh Non-absorbable, lightweight, Atrium pre-formed, three- dimensional plug constructed of knitted rows of monofilament polypropylene with multiple protruding monofilament loops 40. ProLite ™ Mesh Polypropylene Mesh Atrium 41. ProLite ™ Thin wall Atrium Ultra ™ Mesh polypropylene mesh 42. BIO-A ™ Tissue Polyglycolic acid: Gore Medical Reinforcement Trimethylene carbonate (Flagstaff, AZ) (PGA: TMC) fibers form a non-woven web with open, highly interconnected pores 43. DUALMESH ™ Two-surface hernia Gore PLUS Biomaterial repair material with antimicrobial technology 44. DUALMESH ™ ePTFE material that offers Gore Biomaterial two-surface design intended for minimizing tissue attachment along another surface. 45. MYCROMESH ™ Microporous node and Gore Biomaterial fibril structure with regularly spaced macropores. 46. MYCROMESH ™ Includes Gore PLUS Biomaterial antimicrobial technology 47. GORE-TEX ™ Expanded Gore Soft Tissue polytetrafluoroethylene Patch (ePTFE) 48. BIO-A ™ Porous fibrous structure Gore Hernia Plug composed of synthetic copolymer 49. INFINIT ™ Mesh 100% monofilament PIPE, Gore large pore knitted surgical mesh

[0250] Table 3 showing biological surgical meshes

TABLE-US-00003 Commercial Mesh Name Material Manufacturer  1. FLEXHD ™ Acellular Acellular human skin Ethicon Hydrated Dermis  2. Permacol ™ Derived from porcine Covidien Biologic Implant dermal collagen  3. XENMATRIX ™ Non-crosslinked Davol (Bard) Surgical Graft collagen matrix  4. COLLAMEND ™ All-natural Davol (Bard) FM Implants porcine collagen  5. AlloMax ™ All-natural biologic implant Davol (Bard) Surgical Graft derived from human dermal collagen.  6. Biodesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small (Lafayette, IN) Hernia Graft intestinal submucosa  7. Biodesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small Hiatal Hernia Graft intestinal submucosa  8. Biodesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small Inguinal Hernia Graft intestinal submucosa  9. Biodesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small Umbilical Hernia Graft intestinal submucosa 10. Biodesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small Abdominal Lock Graft intestinal submucosa 11. Biadesign ™ Dry, acellular Cook Biotech (Surgisis ™) porcine small 8-Layer Tissue Graft intestinal submucosa 12. Strattice ™ Decellularized LifeCell- Reconstructive porcine skin Genzyme Corp Tissue Matrix (Branchburg, NJ) 13. AlloDerm ™ Decellularized human LifeCell cadaver skin

Copolymers of Crosslinkable Protein/Polypeptide

[0251] According to some embodiments, polyethylene glycol (PEG), also known as poly(ethylene oxide) (PEO) or polyoxyethylene (POE), is added to the protein/polypeptide or crosslinker solution as a copolymer, to improve one or more properties of the composition, for example (and without limitation) to increase the flexibility of the composition or to shield from the body's immune response to the protein-crosslinker composition. PEG is available over a wide range of molecular weights from 300 Da to 10 MDa and may be a liquid or low-melting solid, depending on the molecular weights.

[0252] Different forms of chemically-modified PEG are also available, depending on the initiator used for the polymerization process, the most common of which is a monofunctional methyl ether PEG (methoxypoly(ethylene glycol)). PEGs are also available with different geometries. Branched PEGs have 3 to 10 PEG chains emanating from a central core group. Star PEGs have 10-100 PEG chains emanating from a central core group. Comb PEGs have multiple PEG chains normally grafted to a polymer backbone. All of these types of PEGs should be considered useful in the present invention.

[0253] PEGs can be added to either the protein or crosslinker components of a protein-crosslinker composition. Preferentially, PEG is added at a dry weight ratio between 20:1 to 1:1, protein:PEG. PEG can be added to the protein component or crosslinker component through modification of the protein or crosslinker and/or modification of the PEG molecules. One example of such modification is the process known as PEGylation. PEGylation is the act of covalently coupling a PEG structure to another larger molecule. This process can be performed on either the protein or crosslinker molecules.

[0254] The gelatin PEGylation embodiment has, among its many advantages and without wishing to be limiting, the advantage that the PEG is part of the protein chain, therefore inducing changes in properties of the protein surface including but not limited to charge and hydrophilicity, as well as steric effects that are due to its bulkiness. The embodiment is described for example in U.S. Pat. No. 8,367,388, filed on Jun. 18, 2009, owned in common with the present application and having at least one inventor in common with the present application, which is hereby incorporated by reference as if fully set forth herein. As a result, the covalently attached PEG can have profound effects on intermolecular interactions between protein chains and in turn on physical gelation and crosslinker dependent crosslinking as well as on the mechanical properties of gels prepared by these methods.

[0255] The PEG molecules used in PEGylation are usually activated, meaning they react spontaneously with functional groups on the target protein. A non limiting example of PEGylation is using NHS ester derivatives of PEG. These activated PEG molecules react with primary amines on proteins to form amide bonds with the release of N-hydroxy-succinimide (NHS).

[0256] Other ways in which a protein can be modified is by reacting the primary amines found inside chains of lysine and at the amino termini of the protein chains. The modification may be by alkylation, succinylation, carbamylation, or by any other method of protein modification.

[0257] In a preferred embodiment, the crosslinkable protein/polypeptide is first reacted with activated PEG to create PEGylated protein. The PEGylated protein is purified from excess unreacted PEG and other reaction products by methods such as, but not limited to, dialysis, ultrafiltration, and gel filtration chromatography. The PEGylated protein can then be reacted with a crosslinker to form a crosslinked gel.

[0258] PEGs can also optionally be added through the use of PEG amine as a substrate for a crosslinker that targets amine groups. The crosslinker crosslinks the PEG molecule through its terminal amine group to crosslinker substrates on the protein molecule, thus competing with the natural amine groups on the protein.

[0259] PEG amines comprise PEG that has been bound to amine-functional groups. These are commercially available in all types of PEG geometries. Sources of amine-functional PEG products include NOF (Japan), Nanocs (New York, N.Y.) and Pierce Biotechnology (Rockford, Ill.).

[0260] In all approaches of incorporating PEG, the number of natural substrates available for crosslinking is reduced, resulting in reduced cross-linking. This may affect the mechanical properties of the crosslinked gel, for example optionally allowing it to become less rigid and more flexible. In addition, and without wishing to be limited by a single hypothesis, the PEG molecule itself may act as a plasticizer and further contribute to the flexibility of the resulting gel.

[0261] According to a preferred embodiment, the PEG amine comprises active lysine amino acids.

[0262] According to another embodiment, of the present invention, Polyvinyl Alcohol (PVA) is added to a gelatin or mTG solution as a copolymer to increase the flexibility or adhesiveness of a protein-crosslinker composition. PVA is a water-soluble synthetic polymer with high tensile strength and flexibility. In a high humidity environment, such as inside the body, PVA will absorb water. The water, which acts as a plasticizer, can then reduce the tensile strength of the PVA, but increase its elongation.

[0263] According to some embodiments, the copolymer comprises PVA-amine. When the amine-targeting crosslinker is added to the solution, both the protein and PVA-amine will act as substrates and a protein-PVA copolymer will be formed with better flexibility than a comparable cross-linked protein polymer.

[0264] A non-limiting example of a process that can be used for producing amine functional derivatives of poly (vinyl alcohol) is described in U.S. Pat. No. 6,107,401.

[0265] Another non-limiting example of a process that can be used for producing an amine copolymer of PVA is described in U.S. Pat. No. 4,931,501 where poly(vinyl alcohol) is reacted with an amino-aldehyde dialkyl acetal.

[0266] A process of synthesizing amine-modified poly(vinyl alcohol)s by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution has also previously been described (Wittman M, et al. Biophysical and Transfection Studies of an Amine-Modified Poly(vinyl alcohol) for Gene Delivery. Bioconjugate Chem., 16 (6), 1390-1398, 2005), as another non-limiting example.

[0267] According to some embodiments of the present invention, there is provided a composition comprising gelatin, transglutaminase and a calcium crosslinkable alginate matrix. Optionally said calcium crosslinkable alginate matrix is added in a weight ratio of between 1 to 30% weight per weight according to the weight of the gelatin, and preferably in the ratio of 5 to 20%.

[0268] Under the pH used in an exemplary embodiment (pH 3.8), a precipitate was formed. When such a precipitate is formed, it may optionally be dissolved by addition of a suitable salt in a suitable amount, such as NaCl for example. In this non-limiting example and without wishing to be limited by a single hypothesis, alginate may have precipitated alone, and/or gelatin and alginate may have formed a polyelectrolyte complex which precipitated from the solution. As noted previously, it was found out that addition of 12 gr/L or 0.2M NaCl dissolved the precipitate and clarified the solution.

[0269] Optionally gelatin, transglutaminase, calcium crosslinkable alginate matrix, a suitable salt such as NaCl in a suitable amount, optionally with additional additives, may be dissolved homogenically in solution. Mesh based compositions may be prepared in the same methods described above from said solution.

[0270] According to some embodiments of the present invention, there is provided a composition comprising gelatin, transglutaminase and Chitosan.

[0271] Optionally said Chitosan is added in a weight ratio of between 1 to 100% to the gelatin weight, more preferably in the ratio of 20 to 100%.

Surfactants

[0272] According to some embodiments of the present invention, one or more biocompatible surfactants are added to the solution of cross-linkable protein or polypeptide, for example in order to reduce the surface tension of that solution.

[0273] Surfactants are wetting agents that lower the surface tension of a liquid, allowing easier spreading, and lower the interfacial tension between two liquids. Lower surface tension facilitates easier handling of a solution of a cross-linkable peptide as it is easier to pass through an applicator, and easier to mix with a solution of a cross-linking material. Surfactants can also lower the viscosity of the solution. Additionally, lowering the surface tension of a gelatin solution has great utility when a gelatin solution is lyophilized either alone or together with a mTG solution, as it can prevent the formation of a film on the top layer of the dried gelatin. Such a film inhibits the reconstitution of lyophilized gelatin into a homogenous solution.

[0274] Non-limiting examples of biocompatible surfactants useful in context of the present invention are polysorbate 20 (Tween™ 20), polyoxyethyleneglycol dodecyl ether (Brij™ 35), polyoxyethylene-polyoxypropylene block copolymer (Pluronic™ F-68), sodium lauryl sulfate (SLS) or sodium dodecyl sulfate (SDS), sodium laureth sulfate or sodium lauryl ether sulfate (SLES), poloxamers or poloxamines, alkyl polyglucosides, fatty alcohols, fatty acid salts, cocamide monoethanolamine, and cocamide diethanolamine.

[0275] Surfactants may be used also as plasticizers. Tween80 for example has been shown to reduce the glass transition point (T.sub.g) of several hydrophilic polymers. The presence of the smaller molecules of Tween80 within the polymer were thought to dilute and weaken the cohesive interactions between the polymers chains. This reduced the friction and entanglement by increasing the free volume in the polymer matrix. (Ghebremeskel et al, 2006, International Journal of Pharmaceutics 328:119-129).

[0276] In a preferred embodiment of the present invention, one or more surfactants are used as a plasticizer to improve the elasticity of the crosslinked composition, particularly as it stiffens over time.

[0277] In another optional embodiment, one or more surfactants are combined with another plasticizer from the plasticizers listed above as relevant to the present invention. Rodriguez et al (Food Research International 39 (2006) 840-6) demonstrated a synergistic effect between a plasticizer (glycerol) and surfactants (Tween20, Span 80, Lecithin) on increasing the elasticity of non-crosslinked dry gelatin films.

[0278] Preferentially, surfactants are added to a gelatin solution at a weight ratio of 0.1-5% of the dry weight of gelatin in the solution. Alternatively, surfactants are added to a gelatin solution at a concentration approximately equal to the critical micelle concentration (CMC) of that particular surfactant in solution. The CMC of each surfactant varies and is dependant on the ionic concentration of the solution into which the surfactant is dissolved.

Configurations of Lyophilized Product

[0279] In an embodiment of the present invention, a dried composition is formed wherein the dry crosslinker material is thoroughly dispersed through a lyophilized composition of cross-linkable protein or polypeptide.

[0280] In an embodiment of the present invention, a dried or frozen composition is formed wherein the cross-linkable protein or polypeptide is thoroughly mixed with the non-toxic cross-linker to form a homogenous solution and the temperature of the solutions is reduced immediately to prevent completion of the cross-linking process. The mixed composition is then either frozen or frozen and dried to form a novel, uniform composition.

[0281] In another embodiment, the protein is gelatin and a non-crosslinked gelatin foam is lyophilized prior to dispersal of crosslinker throughout such a porous foam.

[0282] In another embodiment, dry crosslinker material is added to the gelatin foam such that the crosslinker does not dissolve into the foam (ie no crosslinking activity is observed prior to lyophilization).

[0283] It was surprisingly found that a reconstitutable foam could optionally be formed from a gelatin solution that was sufficiently stabile so as to allow for the lyophilization of the gelatin in foam form without the addition of any stabilizing or crosslinking agents.

[0284] In a preferred, illustrative embodiment of the present invention for forming such a foam, a gelatin solution is prepared and held at a temperature where it is in liquid form. The gelatin solution is then subjected to an extended and preferably continuous foaming process while it is cooled to a temperature below its sol-gel transition point.

[0285] The concentration of gelatin solution is preferably in the range of 0.5%-20% w/w, more preferably 5-10% w/w.

[0286] The initial temperature of the gelatin solution is 30° C.-70° C., preferably 30° C.-50° C., and more preferably 35° C.-45° C. The environmental temperature during the foaming process is 0° C.-25° C., preferably 15° C.-25° C. Non-limiting examples of foaming processes include stirring, mixing, blending, and injection of a gas.

[0287] Preferably, the foaming process includes stirring or mixing.

[0288] One or more foaming techniques may optionally be used in the foaming process. Alternatively, one foaming technique may optionally be used multiple times under different conditions: for example, gentle stirring to generate a low level of foam following by vigorous stirring to achieve maximal aeration in the gelatin foam.

[0289] In an optional embodiment, upon the completion of foaming, the gelatin foam is preferably transferred to a vessel that had been cooled to a temperature lower than the temperature of the gelatin foam upon completion of the foaming process.

[0290] In another embodiment, the gelatin foam is optionally and preferably rapidly cooled immediately upon completion of foaming process. A non-limiting example of rapid cooling is exposing gelatin foam to liquid nitrogen immediately after the foaming process.

[0291] In a preferred embodiment, the dry gelatin foam contains less than about 15% moisture. In a more preferred embodiment, the dry gelatin foam contains less than about 10% moisture.

[0292] In another embodiment, the gelatin foam is optionally not further stabilized by cooling or other method upon the completion of foaming such that the foam partially collapses resulting in the formation of a denser layer of gelatin foam on the bottom of the foam. For example, optionally a period of time elapses before such stabilization is performed, for example and without limitation up to 10 minutes. If stabilization comprises cooling, optionally such cooling is delayed by between 2 to 10 minutes after foaming.

[0293] In an embodiment of the above, the denser layer optionally comprises less than about 50% of the thickness of the lyophilized gelatin composition, preferably less than about 35%, and more preferably less than about 20%.

[0294] Density as used herein refers to an increase in the weight of gelatin per volume of lyophilized composition. Such an increase can optionally be as little as 5% but is preferably greater than about 10% and more preferably greater than about 20%.

[0295] Without wishing to be limited to a single hypothesis or to a closed list, it is believed that such a dense layer of gelatin foam provides mechanical strength to the lyophilized gelatin composition without affecting the reconstitution profile of the top part of the dry composition.

[0296] Mesh-Based Composition Preparation Methods

[0297] Different suitable preparation methods may optionally be used for preparation of the mesh-based composition. Various exemplary stages are given below, which may optionally be differently configured and recombined. Each such exemplary stage may optionally be performed according to different methods, each of which may optionally be combined with any other preparation method as described herein. The stages are given in the order in which they are optionally performed.

[0298] Backing (non-adhesive component) application may optionally comprise blade coating, i.e. a gap controller which extrudes the material, thus determining the wet thickness of the applied coating. Additionally or alternatively, such backing application may optionally comprise one or more of air knife coating, spraying, silk printing or any other relevant printing process, spin coating, dip coating, curtain coating, solution casting, any other suitable method for coating layers of polymer dispersions/solutions, and/or melt extrusion (no solvent involved, only heating the polymer).

[0299] Creating the porous structure for the adhesive matrix may optionally comprise (physical) gas foaming, which involves introducing pressurized inert gas (e.g. nitrogen) to the gelatin solution. Additionally or alternatively, such creation of the porous structure may optionally comprise one or more of (physical) emulsion freeze drying, (chemical) solvent casting/particulate leaching, (physical) high pressure processing with supercritical gas, e.g. co.sub.2, (physical and/or chemical) gas foaming/particulate leaching, e.g. with dispersed ammonium bicarbonate salt particles, thermally induced phase separation, electrospinning, and/or rapid prototyping (for example with a 3D printer).

[0300] Next Mixing gelatin and enzyme is performed, for example after gelatin solution foaming, using static mixers (mixing element is rotating by the movement of the material through it). Enzyme solution is introduced after gelatin solution is foamed. Various methods may optionally be performed, such as for example mixing gelatin and enzyme together in the same solution as first step, for example and without limitation mixing in a form of an oil in water or water in oil emulsion and/or mixing encapsulated enzyme micro-particulates as a dispersion in a gelatin continuous phase. In another exemplary method, the enzyme is introduced in a later stage, for example and without limitation deploying enzyme dry particulates over the pre-dried gelatin matrix, and/or layer by layer application of alternating gelatin and enzyme layers.

[0301] Next adhesive layer (gelatin-enzyme foamed solution) application is performed, for example with blade coating, i.e. a gap controller which extrudes the material, thus determining the wet thickness of the applied coating. Other exemplary methods include without limitation air knife coating, spraying, silk printing or any other relevant printing process, spin coating, dip coating, curtain coating, solution casting, a suitable method for coating layers of polymer dispersions/solutions, injection molding and/or molding (casting).

[0302] Polypropylene mesh positioning may optionally be performed in various ways. For example and without limitation, optionally adhesive layer application is performed in two steps, in which the polypropylene mesh is positioned over the first adhesive layer, then second adhesive layer covers it. Optionally the mesh is positioned before or after foam application.

[0303] Attachment of adhesive and backing layers may optionally be performed through direct application of the adhesive foamed solution over the already dry backing. Additionally or alternatively, such attachment may optionally performed through one or more of drying the two films separately and then attaching them through a suitable method, for example and without limitation by using an adhesive (film transfer method) or by a lamination process. Optionally the backing is applied on top of the already dry adhesive layer, for example and without limitation by air knife coating, spraying, silk printing or any other relevant printing process, spin coating, curtain coating, any other methods known for coating layers of polymer dispersions/solution, and/or hot-melt extrusion.

[0304] Drying of the adhesive layer may optionally be performed through lyophilization (freeze-drying) at −20° C. Such drying may optionally also be performed, additionally or alternatively through one or more of hot air drying, vacuum drying, or by keeping the product frozen until application.

[0305] Mesh Device Dimensions

[0306] The mesh-based composition may also optionally be described as a mesh device with particular dimensions. Non-limiting examples of such dimensions are provided below and in FIG. 6.

[0307] The dimensions may optionally vary for example according to the shape of the device, for example as a rectangle, optionally including a square, or as a rounded shape, optionally include an ellipse or a circle. The diameter (or distance from edge to edge for a non-rounded shape) may optionally be in a range of from 0.5 cm to 60 cm. Non-limiting examples are given as 1 cm and 50 cm in FIG. 6. However, optionally these dimensions only relate to the mesh size but not the size of the total device. For such an implementation, the size of the total device is given below.

[0308] The area of the mesh in cm squared optionally ranges from 0.5 cm.sup.2 to 3600 cm.sup.2. Non-limiting examples of the area of the mesh are given as 0.8 cm.sup.2 for a 1 square cm device if rounded, or 1 cm.sup.2 if not rounded. The area of the mesh is not necessarily the total area of the device according to those implementations in which the above dimensions for the device do not include the margin for example and/or otherwise do not relate to the total dimensions of the device.

[0309] The margin size is the section of the device that extends beyond the mesh, which may optionally comprise an adhesive for example. The margin size may optionally be in a range of from 0.01 to 10 cm. Non-limiting examples are given as 0.1 cm and 5 cm in FIG. 6. The margin size as a percentage of the diameter or edge to edge distance optionally ranges from 0.1% to 10%.

[0310] The area of the margin is optionally from 0.0001 cm.sup.2 to 1200 cm.sup.2, but is preferably from 0.1 cm.sup.2 to 900 cm.sup.2 for round shaped devices, and 0.1 cm.sup.2 to 1100 cm.sup.2 for square shaped devices.

[0311] The area of the total device, when different from the dimensions given above, optionally ranges from 0.5 cm.sup.2 to 3000 cm.sup.2 for round shaped devices, and from 0.5 cm.sup.2 to 4000 cm.sup.2 for square shaped devices.

[0312] The margin area as a percentage of the mesh area optionally ranges from 0.1 cm.sup.2 to 14,000 cm.sup.2. The margin area as a percentage of the total device area optionally ranges from 0.1% to 100%.

[0313] The dimensions of the thickness of different parameters of the device, according to the symbols given in FIG. 2A, optionally range for d1 (adhesive layer thickness from edge of device to mesh) (μm) from 0 to 10000; for d2 (adhesive layer thickness from mesh to bonding layer) (μm) from 0 to 10000; for d3 (thickness of foam enclosed within the mesh) (μm) from 100 to 2500; for d4 (thickness of backing) (μm) from 1 to 2000; and for d5 (thickness of bonding layer) (μm) from 1 to 100. The total thickness (μm) ranges from 101 to 24600.

[0314] The ratios (as a percentage) of the dimensions of the thickness of different parameters of the device optionally range for the % of backing thickness to foam thickness from 0.004 to 2000.000; and for the % of backing to total thickness, from 0.004 to 1980.198.

[0315] The foam density (g/ml) optionally ranges from 0.001 to 0.1.

[0316] Optionally all dimensions, percentages and other parameter values described herein may be increased or decreased by 10%, and are still considered to be within the present invention as described herein.

EXAMPLES

[0317] Reference is now made to the following examples, which together with the above description, illustrate some embodiments of the invention in a non limiting fashion.

Example 1

[0318] As shown herein, various exemplary implementations of the structure featuring the mesh-based composition are possible and are considered to fall within the present invention.

[0319] FIG. 1 shows a top view of the device design, including a section of adhesive covered mesh and a section of only adhesive surrounding the mesh-adhesive section.

[0320] FIG. 2A shows a side view of the device portraying an optional placement of the mesh within the adhesive, in a way that the adhesive surrounds the mesh in all directions. FIG. 2B shows an exploded view of the layers.

[0321] A non-limiting example of mesh materials and the associated composition are provided in Table 4 below.

TABLE-US-00004 TABLE 4 Materials Mesh-based structure Mesh Material PP monofilament Foam Density 0.28 ± 0.2 g/ml Adhesive Gelatin + mTG Margin size Width: 1 cm Area: 50.27 cm.sup.2 22% Backing and HPMC based backing layer bonding layer and PVP based bonding layer Device thickness 1.3 mm

[0322] The adhesive may optionally be composed as described in U.S. Pat. No. 8,961,544 B2, hereby incorporated by reference as if fully set forth herein, owned in common with the present application and having at least some inventors in common with the present application. Other materials and parameters as given above are as examples only, without any intention of being limiting.

Example 2

[0323] The tackiness of various substrates to moist latex glove was tested using a vertical electromechanocal testing system (Instron, UK). Two types of backing were used, crosslinked (CL) gelatin and HPMC. The backings covered a layer of a non-crosslinked foamed gelatin. A flat attachment was connected to the Instron head to which a latex glove was attached tightly. The latex glove was dipped in 0.9% saline prior to attachment to the Instron. A sample of foamed adhesive and backing was connected to the bottom flat plate with double sided tape. The Instron head was compressed perpendicularly, until a force of 0.3-0.9 N was achieved. The force was held for a second and extension was performed while extension load was measured by the 50 N load cell.

[0324] The data clearly shows that the backing decreases the tackiness of the foamed gelatin layer, and is not different than the tackiness between latex gloves to itself. The results are shown in FIG. 3, which shows the measurement of relative tackiness of the various backing and composition combinations.

Example 3

[0325] Backings were prepared as films by casting, and after drying were cut into 1 cm wide strips and placed into the holders of the vertical electromechanocal testing system (Instron, UK). The film length was 5 cm. Backing was set on bottom and upper holders, where the bottom holder was fixed, and the upper holder was connected to 50 N load cell. After this, tensile extension of the sample was performed. Extension rate was set to 0.5 mm/s, while tensile stress and strain were measured.

[0326] The data shows that the HPMC backing becomes more elastic when plasticizers are added and that this effect is concentration dependent. PEG400 is a better plasticizer than sorbitol, when same amounts are compared. The results are shown in Table 5 below.

TABLE-US-00005 TABLE 5 effect of plasticizer on elasticity Backing composition % plasticizer % elongation 1% HPMC 0   1.11 1% HPMC + 0.1% PEG400 9.1 3.49 1% HPMC + 0.3% PEG400 23   9.74 1% HPMC + 0.1% sorbitol 9.1 1.87 1% HPMC + 0.3% sorbitol 23   4.57

Example 4

[0327] This example relates to the surrounding adhesive margin section (A in FIG. 1) and its performance in vivo; FIG. 4 shows a lightweight mesh fixated to peritoneal tissue in vivo using the gelatin-mTG adhesive matrix. This image shows a mesh based device that was inserted laparoscopically into place and applied to the porcine peritoneum. The size of the mesh applied is 15×15 cm of surgical mesh surrounded by adhesive and covered by a backing layer (HPMC (k100)+HPMC (k4)) and a bonding layer composed of Plasdone C17), plus 1 cm margins on the periphery which include only adhesive and backing (connected with the bonding layer), without mesh. These margins can be seen in the image as having a slightly different color than the center of the device and the tissue surrounding the surgical site. In this implantation, the mesh successfully adhered to the tissue due to the adhesive layer and the backing prevented any tackiness to surgical tools.

Example 5

[0328] This Example relates to non-limiting, exemplary methods of preparation of mesh based compositions.

[0329] As shown on the left-hand branch of FIG. 5, in blue, the adhesive composition is optionally prepared as follows. The gelatin solution is optionally foamed first with gas, preferably an inert gas such as nitrogen. Next the foamed solution is mixed with the enzyme solution. This adhesive composition is then optionally combined with the non-adhesive component, such as HPMC for example, by applying the adhesive layer on the non-adhesive layer, and then embedding the mesh. This mesh-composition combination is then optionally first frozen at −70 to −80 C, and then freeze dried at −20 to 15 C.

[0330] Table 6 relates to various optional and non-limiting methods of preparation at each stage of the process.

TABLE-US-00006 TABLE 6 process alternatives Stage in process Example method Other possible methods Backing (non-adhesive Blade coating, i.e. a gap 1. Air knife coating. component) application controller which extrudes the 2. Spraying. material, thus determining the 3. Silk printing or any other wet thickness of the applied relevant printing process coating. for that matter. 4. Spin coating. 5. Dip coating. 6. Curtain coating. 7. Solution casting. 8. Any other methods known for coating layers of polymer dispersions/ solutions. 9. Melt extrusion (no solvent involved, only heating the polymer). Creating the (Physical) Gas foaming- 1. (Physical) Emulsion porous structure for Introducing pressurized inert freeze drying. the adhesive matrix gas (e.g. Nitrogen) to the 2. (chemical) Solvent gelatin solution. casting/particulate leaching. 3. (Physical) High pressure processing with supercritical gas, e.g. CO2. 4. (Physical/chemical) Gas foaming/particulate leaching, e.g. with dispersed ammonium bicarbonate salt particles. 5. Thermally induced phase separation. 6. Electrospinning. 7. Rapid prototyping. Mixing gelatin and enzyme. Mixing is performed after 1 Mixing gelatin and gelatin solution foaming, using enzyme together in the static mixers (mixing element same solution as first is rotating by the movement of step, e.g.: the material through it). 1.1 Mixing in a form of an Enzyme solution is introduced oil in water or water after gelatin solution is in oil emulsion. foamed. 1.2 Mixing encapsulated enzyme micro- particulates as a dispersion in a gelatin continuous phase. 2 Introducing enzyme in later steps, e.g.: 2.1 Deploying enzyme dry particulates over the pre-dried gelatin matrix. 2.2 Layer by layer application of alternating gelatin and enzyme layers. Adhesive layer (gelatin- Blade coating, i.e. a gap 1. Air knife coating. enzyme foamed solution) controller which extrudes the 2. Spraying. application material, thus determining the 3. Silk printing or any other wet thickness of the applied relevant printing process coating. for that matter. 4. Spin coating. 5. Dip coating. 6. Curtain coating. 7. Solution casting. 8. Any other methods known for coating layers of polymer dispersions/ solutions. 9. Injection molding. 10. Molding (casting). Polypropylene mesh Adhesive layer application is 1. Positioning the mesh positioning performed in two steps-the before foam application. polypropylene mesh is 2. Positioning the mesh after positioned over the first foam application. adhesive layer, then second adhesive layer covers it. Attachment of adhesive Direct application of the 1. Drying the two films and backing layers adhesive foamed solution over separately and then the already dry backing. attaching them e.g.: 1.1 Using an adhesive (film transfer method). 1.2 By a lamination process. 2. Application of the backing on top of the already dry adhesive layer, by e.g.: 2.2 Air knife coating. 2.3 Spraying. 2.4 Silk printing or any other relevant printing process for that matter. 2.5 Spin coating. 2.6 Curtain coating. 2.7 Any other methods known for coating layers of polymer dispersions/ solutions. 2.8 Hot-Melt extrusion. Drying of the Lyophilization (Freeze-drying) 1. Hot air drying. adhesive layer at −20° C. 2. Vacuum drying. 3. None at all, keeping the product frozen until application.

[0331] FIG. 6 provides some optional dimensions, minimum and maximum, for various patch shapes for patches that are prepared according to the above process. FIG. 1 shows how the dimensions given relate to the product. In particular, the letter “A” shows the section where adhesive is present, while the letter “M” indicates that both the mesh and adhesive are present. The following dimensions are given in FIG. 6 and are explained below:

[0332] d.sub.1—Thickness of adhesive from bottom of device to surgical mesh

[0333] d.sub.2—Thickness of adhesive from surgical mesh to bonding Layer

[0334] d.sub.3—Thickness of surgical mesh

[0335] d.sub.4—Thickness of backing

[0336] d.sub.5—Thickness of bonding layer

Example 6—Inhibition of mTG Enzyme In-Process Using Acetic Acid

[0337] The inhibition of the mTG enzyme throughout the “wet” part of the mesh preparation process through reduction in pH value helps prevent premature cross-linking of the gelatin solution.

[0338] Methods:

[0339] Preparation of gelatin solution—9% w/w, (example for pH 3.8):

[0340] 12285 g purified water was pre-heated to 40-45° c. 1350 g gelatin was added to the vessel gradually, while stirring with coned over-head stirrer.

[0341] After full dissolution of the gelatin, 1365 g 3M acetic acid (360 g 100% glacial acetic acid (J. T Baker) mixed with 1640 g PW) were added to the vessel while stirring for 5 minutes. The pH was tested with pH meter to be 3.8±0.5.

[0342] In the same way, gelatin solutions with pH 5.5, 4.0 and 3.5 were prepared. Purified mTG enzyme solution containing a sodium citrate buffer (preparation described below), 50 U/ml was added to contain 40 U/g gelatin.

[0343] FIG. 7 shows the effect of pH on mTG activity by viscometer test. 9% gelatin in pH 5.5 (native), 4 and 3.5 were mixed with 40 u/ml enzyme solution at 37° c. (2:1). For pH 3.5 (indicated with an *), the test was stopped after 15 minutes as the viscosity didn't rise above 0.1%.

Example 7—Methods for Creation of a Porous Adhesion Layer

[0344] Physical Aeration with Pressurized Gas

[0345] FIG. 8 shows a schematic view of the aeration and mixing system, featuring a hopper into which the gelating solution is placed for aeration. Aeration occurs at the mixing head, followed by movement through temperature controlled tubing. After injection of the mTG solution, static mixing occurs. The material then flows out through a flow manifold.

[0346] Methods:

[0347] Preparation of gelatin solution—9% w/w water, pH=3.8, 15 kg; 12285 g purified water was pre-heated to 40-45° c. 1350 g gelatin was added to the vessel gradually, while stirring with coned over-head stirrer. After full dissolution of the gelatin, 1365 g acetic acid were added to the vessel while stirring for 5 minutes. The pH was tested with pH meter to be 3.8±0.5.

[0348] Preparation of Microbial Transglutaminase Solution

[0349] 56.5 g of concentrated purified mTG solution (885 U/ml) was mixed with 943.5 g 20 mM sodium citrate solution, to yield a diluted mTG solution (50 U/ml).

[0350] Acidified Gelatin solution was pumped to the aeration machine, in which pressurized nitrogen gas was introduced to the pressure chamber to achieve wet foam density of 0.28 g/ml ±0.02.

[0351] Foam flow throughout the system was kept a flow rate of 6 Kg/hr, and its temperature was regulated via a double-jacketed tubing to keep the gelatin solution above its transition temperature, to prevent it from freezing.

[0352] Midway throughout the streamline, enzyme solution at room temperature was introduced through an in-line valve, at a rate of 15 ml/min.

[0353] Static mixing elements inside the tubes (˜2 m long) allowed for adequate mixing of the mTG solution and the gelatin solution foam.

[0354] Mixed gelatin-mTG foam was ejected at the end of the line on top of a PEEK (poly ether ether ketone) flat board, serving as a mold. SurgicalMesh (SurgicalMesh, USA) surgical mesh prosthesis was pre-fixed to the PEEK mold, so that the foam applied on top of the mold covered it entirely.

[0355] Then foam was extruded with a Knife coater to the desired thickness (between 1000 and 1500 micrometers).

[0356] PEEK mold with the foam coated mesh was inserted to −80C refrigerator for freezing. After 1.5 hours the mold was transferred to a lyophilizer (Virtis Genesis EL, SP Scientific) for a freeze-drying process (see Table 7).

TABLE-US-00007 TABLE 7 freeze drying program Temperature, Pressure, Duration of Phase Name Step # ° C. mTorr step, minutes Notes Semi-Auto — −25  800 — This step is done prior to functions lyopilization (and before inserting the samples) in order to keep the shelves in low temperature Freeze, 1 −20  —   1 Condenser Vacuum start permit by condenser and temperature- −60° C. Evacuate Heat start permit by pressure-900 mTorr Primary 1 −20  600  120 Hold Drying 2  0 600  60 Rate (temperature) 3  0 300  120 Hold (Step pressure drop) 4  0 150  60 Rate 5 15 150  60 Rate 6 15  50  60 Rate Secondary 1 15  50 2000 Hold Drying Product — 10 — — temperature Storage — 10 — — temperature Storage — — 100 — Vacuum

[0357] After freeze-drying, mesh articles coated by 1.0-1.5 mm foam containing a dry mixture of gelatin and mTG were obtained, by peeling the foam layer easily off the PEEK molds.

[0358] Results

[0359] FIG. 9 shows an SEM image of a Cross-section of a gelatin-mTG dry foam article, prepared by the process described in example 7;

[0360] FIG. 10 shows an SEM image of the adhesive surface of a gelatin-mTG dry foam article, prepared by the process described in example 7.

Example 8—Freeze-Drying of Non-Eutectic Gelatin in Water Solutions

[0361] Methods:

[0362] Solutions of various concentration of gelatin in water were prepared, by mixing Bloom 275 gelatin particles (Gelita, USA) in DW at 50° C. for 1 hour.

[0363] Solutions were acidified by titration of acetic acid 6M (BioLab) to pH=3.8.

[0364] In the final mixing stage, purified mTG stock solution at a concentration of 50 U/ml was added to the solution in a ratio of 80 U/g gelatin. Solution mixed thoroughly for 1 min. After addition of all materials, solutions contained 2.5% and 1.5% of gelatin in water (w/w).

[0365] Solutions were cooled down and kept over-night at 24.5° C., slightly above the freeze-thaw transition point of the gelatin solutions.

[0366] Vitamesh Blue (Proxy, Ireland) Hernia prosthesis 15 cm round shaped articles were fixed over a flat PEEK boards, serving as molds, which were pre-cooled to 5° C. Acidified gelatin-enzyme mixtures were applied in 1-1.5 mm layers on the flat PEEK board, covering the Vitamesh Blue articles, using a Knife coater.

[0367] The coated boards were inserted to a freezer at −80C for 1.5 hour, and after freezing, inserted to a lyophilizer (Virtis Genesis EL, SP Scientific) for the same freeze drying process as described in Table 7 above.

[0368] Dried foam-coated articles were peeled-off from the PEEK boards prior to inspection.

[0369] Fixation strength in lap shear on collagen method:

[0370] Collagen sheets, cut to a size of 6×7 cm (W×L) were soaked in 0.9% saline until hydrated then heated to 36-37° C. Samples were prepared by cutting whole mesh based composition articles to pieces 5×7 cm (W×L) and covering 2 of the 7 cm with tape for gripping with the test machine. Mesh based composition samples were applied to preheated collagen sheets and allowed 4 minutes of curing in a 37° C. incubator.

[0371] For testing at time zero: samples tested in lap shear using a model 3433 Instron universal testing machine immediately after the 4 minutes curing.

[0372] For testing after 24 hours of immersion in saline: Samples were placed in petri dishes filled with saline then left in a 37° C. incubator for 24 hours. After 24 hours the samples were tested in lap shear using a model 3433 Instron universal testing machine.

[0373] Results

[0374] Table 8 shows the results of the tests. As shown, a desirable gelatin content is at least 2% and preferably at least 2.5%.

TABLE-US-00008 Gelatin concentration 1.5% w/w (according 2.5% w/w (according in solution to example 8) to example 8) Production batch No. GF-491 GF-476 Appearance White foam White foam coating over the coating over the polypropylene mesh polypropylene mesh Feel (qualitative) Very soft, delicate, low Soft and pliable resistance to tearing Thickness 1.4-1.5 mm 1.5-1.6 mm Fixation strength to 2.2 ± 0.3 * 3.0 ± 0.2 collagen after 24 hour immersion in 0.9% saline solution [N/cm] * crosslinked gelatin backing layer ~50 micrometers thick was attached in the back side of the sample for the sake of the measurement.

Example 9—Precise Positioning of the Mesh

[0375] Specific surgical mesh positioning in regard to the gelatin foam, according to at least some embodiments, may increase desirable properties, including with regard to tissue embedding. Surprisingly, the inventors have found that the position of the mesh within the foam coating may reduce the degree of tissue response to the prosthesis at initial stages of the product application on the tissue, without wishing to be limited by a single hypothesis.

[0376] Method:

[0377] In the following examples no bonding layer was used, as the adhesive layer was directly applied over the pre-cured backing layer, bonding them together when the adhesive layer was dried.

[0378] Backing layer was prepared by coating PEEK flat boards:

[0379] Backing solution:1% HPMC K100 (Ashland), 1.7% HPMC K4 (Ashland), 0.9% PEG 400 (Merck)

[0380] Backing casting: The backing solution is poured on the PEEK liner and a knife coater (Doctor Blade) is used in order to adjust its height (1100 m).

[0381] The solution is dried at RT for at least 24 hours.

[0382] Gelatin-mTG foam (9% w/w gelatin in water and acetic acid, pH=3.8, mTG concentration—80 U/g gelatin) was prepared as described above under physical aeration with pressurized gas.

[0383] The following process modifications were implemented for the placement of Vitamesh Blue surgical mesh within the adhesive layer in various locations, shown in Table 9.

TABLE-US-00009 TABLE 9 modifications to test placement of mesh in regard to adhesive layer Surgical mesh d2 + d3 d1 position within (microns, (microns, foam estimated) * estimated) * Preparation process Middle (mesh  ~800 ~400 First wet gelatin-mTG foam layer extruded fully surrounded with a knife coater to 800 microns. Surgical by foam) mesh placed on top of the wet foam layer. Batch No. Second foam layer, covering the surgical LM-147 mesh, was extruded to 1200 microns. (see FIGURE 11A) Surface (mesh ~1200  ~0 Foam layer extruded to 1400 microns. slightly exposed Surgical mesh placed on top of the wet foam to tissue) layer. Tyvek (Dupont, USA) 1073B sheet Batch No. was placed on top of the mesh, covering it. LM-149 Knife coater at 1200 microns was conveyed over the Tyvek sheet, pressing the surgical mesh into the wet foam. Tyvek sheet was removed after freezing, before initiating the freeze-drying process. (see FIGURE 11B) Middle/Surface ~800/~1100 ~400/~100 ** First wet gelatin-mTG foam layer extruded alternating with a knife coater to 800 microns. Surgical stripes pattern mesh placed on top of the wet foam layer. Batch No. Second foam layer, covering the surgical LM-150 mesh, was extruded with a custom-made knife coater with 750/1200 microns alternating gap sections (each section 0.5 cm wide). * corresponding to FIGURE 2A above ** design intended to be middle and surface alternating sections, so that mesh is exposed in the shallow sections. In practice, in these sections the mesh was fully covered by foam.

[0384] Results:

[0385] FIG. 11A shows an SEM image of a cross-section of LM-147 article—Mesh in embedded in the middle of the foam. FIG. 11B shows an SEM image of a cross-section of LM-149 article—Surgical mesh is located at the bottom (surface). FIG. 12A shows an SEM image of the bottom surface of LM-147 article—Surgical mesh is not visible as it is fully covered by foam. FIG. 12B shows an SEM image of the bottom surface of LM-149 article—Surgical mesh is located at the surface level, not fully covered. FIG. 13A shows an SEM image of LM-149 after fixation on collagen as a tissue simulating substrate (collagen is the bottom layer). FIG. 13B shows an SEM image of LM-147 after fixation on collagen as a tissue simulating substrate (collagen is the top layer).

[0386] FIG. 14 shows the fixation strength of the different models on collagen, as tested by the lap shear method, 4 minutes after fixation. The testing method was performed as described in the section on freeze-drying of non-eutectic gelatin in water solutions.

[0387] FIG. 15 shows the fixation strength of the different models on swine peritoneum tissue, as tested by the lap shear method, different days after implantation (study AT-05-55). Fixation to tissue was tested ex-vivo. Testing method for 0, 3 and 7 days time-points: Explanted Mesh based compositions samples were cut to samples 4×7 cm (W×L), 1 cm of mesh was detached from the tissue on the 7 cm edge for gripping leaving a 4×6 cm effective testing area. Samples were tested in lap shear using an Instron universal testing machine while on one side only tissue was gripped and on the other only mesh was gripped. Day 1 samples were tested by opening the abdominal wall, separating the Mesh based composition for gripping (leaving 4×6 cm testing area) and pulling in lap shear using a force gauge.

[0388] FIG. 16 shows representative histopathology images of samples explanted after 7 days from swine. Left—LM-147, Right—LM-149. The original position of the mesh within the foam affects the amount of new tissue ingrowth surrounding the mesh (higher in LM-149 relative to LM-147).

Example 10—Adhesion Layer—Foam Areal Density

[0389] It was assumed that the porous gelatin structure of the foam is important for fast tissue integration into the surgical mesh. This is because the high effective surface area of the foam increases availability for enzymatic decomposition in the body, enabling fast degradation of the cross-linked gelatin (within days). As the crosslinked gelatin degrades, it makes room for the infiltration and proliferation of new tissue, encapsulating the surgical mesh fibers and fixating it firmly to place. On the other hand, increasing the foam load per area can contribute to the strength of the crosslinked gelatin gel matrix, which is the main load bearing fixation element in the few days after implantation. Therefore, the optimum between fast tissue integration and initial fixation strength of the gel matrix was sought for.

[0390] Method:

[0391] Note—in the following examples no bonding layer was used, as the adhesive layer was directly applied over the pre-cured backing layer, bonding them together when the adhesive layer was dried.

[0392] Gelatin-mTG foam (9% w/w gelatin in water and acetic acid, pH=3.8, mTG concentration—80 U/g gelatin) was prepared as described in the above section on foaming with gas.

[0393] The following process modifications were implemented, as shown in Table 10.

TABLE-US-00010 TABLE 10 Process Modifications Surgical mesh position within d2 + d3 d1 foam (mm) * (microns) * Preparation process Surface (mesh ~0.9-1.2 ~0 Foam layer extruded to 1400 microns. slightly exposed Surgical mesh placed on top of the wet to tissue) foam layer. Tyvek 1073B sheet (Dupont, USA) was placed on top of the mesh, covering it. Knife coater at 1200 microns was conveyed over the Tyvek sheet, pressing the surgical mesh into the wet foam. Tyvek sheet was removed after freezing, before initiating the freeze-drying process. Surface (mesh ~1.3-~1.6 ~0 Foam layer extruded to 2000 microns. slightly exposed Surgical mesh placed on top of the wet to tissue) foam layer. Tyvek 1073B sheet (Dupont, USA) was placed on top of the mesh, covering it. Knife coater at 1200 microns was conveyed over the Tyvek sheet, pressing the surgical mesh into the wet foam. Tyvek sheet was removed after freezing, before initiating the freeze-drying process. * corresponding to FIGURE 2A above

[0394] Results

[0395] Table 11 shows the results of the different foam depth applications, with a greater depth in the left column (2000 microns) and a lower depth in the right column (1400 microns).

TABLE-US-00011 TABLE 11 effect of foam depth Initial extrusion gap (microns) foam 2000 1400 Production LM-272 LM-275 batch No. Thickness 1.35 ± 0.05 mm 1.0 ± 0.1 mm Areal density of 3.4 ± 0.1 1.8 ± 0.2 gelatin-mTG dry foam (mg/cm2) Fixation strength 1.6 ± 0.3 1.2 ± 0.1 to collagen after 24 hour immersion in 0.9% saline solution [N/cm]

Example 11—Backing Alternatives and Adhesion Barrier Properties of Crosslinked Gelatin

[0396] The product contains a surgical mesh embedded within a water-activated adhesive coating. As described in the provisional application, it also contains a backing layer that prevents from the product to be sticky towards its back side (i.e. to the gloves, surgical tools, etc. with which the surgeon applies the product).

[0397] Another important feature, which is required in intraperitoneal mesh implantations, is the prevention of adhesions of the implant to visceral organs, e.g. bowels, liver, spleen and bladder. This property is commonly termed “adhesion barrier” and is known in prior art.

[0398] Two non-adhesive backing versions were examined throughout development:

[0399] The first backing version was HPMC based (hydroxy propyl methyl cellulose K4M 22.3% wt, hydroxy propyl methyl cellulose K100M 44.6% wt, PEG 400 33% wt). The HPMC backing serves only the purpose of prevention of adhesion of the product to surgical tools and wet gloves. It is water-soluble, therefore it dissolves in the fluids existing inside the abdominal cavity within a few hours after implantation. The adhesion barrier properties are coming from the remaining gelatin layer, which covers the surgical mesh completely and has already been fully crosslinked while the HPMC based backing had dissolved away.

[0400] The second backing version was crosslinked gelatin based (Gelatin 70%, mTG—5 U/g gelatin, PEG 400 30% wt). The crosslinked gelatin backing fuses with the in-situ crosslinking adhesive gelatin-mTG layer to become a continuous crosslinked gelatin matrix. During application on tissue it is non-adhesive since it is already crosslinked, therefore preventing sticking in its back side, and it also serves as additional reinforcement to the adhesive, increasing the overall gel mass. Being non-adhesive, it is also an adhesion barrier to visceral organs.

[0401] Methods

[0402] HPMC backing (GF-199):

[0403] Solution: 1% HPMC K100 (Ashland), 0.3% PEG400 (Aldrich), autoclaved.

[0404] Process: Teflon coated trays (12 cm×17 cm and 9.5 cm×9.5 cm) were filled with 60 ml and 26 ml, respectfully, of the solution and allowed to dry at RT.

[0405] Heat cross linked Gelatin backing (GF-200):

[0406] Solution: 1% gelatin 225 type A (Gelita), filtered through 0.2 μm filter.

[0407] Process: ˜60 ml of the solution were cast into 9.5 cm×9.5 cm Teflon coated trays. The solution was allowed to dry at RT, then were incubated in an oven at 160° c. for 2-4 hours.

[0408] Enzyme cross linked gelatin backing (LM-274):

[0409] Solution: 5% gelatin (Gelita), 2.1% PEG 400 (Merck)+5 u purified mTG enzyme (TP1701)/g gelatin (total in sol.)

[0410] Process: The solution is prepared without the enzyme. Before application of the solution on the liner, the enzyme is added, mixed and then the solution applied and set to 1000 μm height using a gap controller (Doctor Blade). The backing is allowed to dry at RT (while cross linking by the enzyme).

[0411] Results

[0412] Fixation strength to collagen after 24 hour immersion in 0.9% saline solution was tested for two models: LM-274 (crosslinked gelatin based backing layer) and LM-216 (HPMC based backing layer). The results are as follows are shown in Table 12.

TABLE-US-00012 TABLE 12 effect of crosslinked gelatin vs HPMC backing Mesh based composition production batch number LM-274 LM-216 Backing layer Crosslinked HPMC gelatin Fixation strength 1.9 ± 0.3 1.0 ± 0.05 to collagen after 24 hours immersion in 0.9% saline solution (N/cm)

[0413] FIG. 17 shows a boxplot representing % surface area of Control mesh (bare Surgical Mesh) and Mesh based composition™ (Surgical mesh covered with adhesive) groups GF-199 (Crosslinked gelatin), GF-200 (HPMC) covered by adhesions, 14 days after implantation. The study was performed with intraperitoneal implantation of the articles in swine. This Example demonstrates that either of HPMC or cross-linked gelatin may optionally be used as backing.

Example 12—Gelatin—Alginate Mesh Based Composition

[0414] Preparation of gelatin/alginate solution

[0415] 300 g purified water was heated to approximately 40° C. 8.35 g gelatin powder (Gelita, Type A gelatin, Bloom 275) was added to the heated water and mixed with a magnetic stirrer until homogenous.

[0416] 98.35 g purified water was heated to approximately 40° C. 1.68 g low viscosity sodium alginate (Sigma Aldrich, cat #A0682) was added to the heated water and mixed with a magnetic stirrer until homogenous.

[0417] The alginate solution was added to the gelatin solution and mixed. As phase separation occurs when alginate and gelatin solutions are mixed, 3.4 ml 6M acetic acid (BioLab, Israel) and 5.7 g NaCl (Merck) were until mixture was at a pH of 3.8 and solution was clear.

[0418] 74.72 g purified water was added to complete to a weight of 490.5 g.

[0419] Preparation of gelatin/alginate coated surgical mesh (Batch #GF-502)

[0420] Purified mTG enzyme stock solution at a concentration of 50 U/ml was added to the acidified gelatin-alginate solution in a ratio of 80 U/g gelatin. Solution mixed thoroughly for 1 min.

[0421] Vitamesh Blue (Proxy, Ireland) Hernia prosthesis 15 cm round shaped articles were fixed over a flat PEEK boards, serving as molds, which were pre-cooled to 5° C. Acidified gelatin-alginate-enzyme mixtures were applied in 1-1.5 mm layers on the flat PEEK board, covering the Vitamesh Blue articles, using a Knife coater.

[0422] The coated boards were inserted to a freezer at −80C for 1.5 hour, and after freezing, inserted to a lyophilizer (Virtis Genesis EL, SP Scientific) for the same freeze drying process as described in Table 7 above.

[0423] Dried foam-coated articles were peeled-off from the PEEK boards prior to inspection.

[0424] Testing fixation strength 1 day post application of gelatin/alginate LifeMesh (GF-502) with a calcium source.

[0425] LifeMesh were cut to samples 5×7 cm (W×L) and 2 cm of the Length were covered with tape, allowing for a place to grip the sample with the testing machine. Collagen sheets were cut to pieces 6×7 cm (W×L) and preheated to 36-37° C., while covered with saline soaked gauze. LifeMesh samples were applied to collagen and allowed 4 minutes to cure in a 37° C. incubator. After curing, samples were submerged in 0.9% saline/0.9% saline containing a calcium source (CaCl.sub.2)). Samples were kept in 37° C. for 24 hours then tested in lap shear using a model 3433 Instron universal testing machine.

[0426] FIG. 18 shows the fixation strength of Gelatin—Alginate mesh based composition (batch No. GF-502) on collagen, as tested by the lap shear method, 24 hours after fixation. GF-502 articles were immersed for 24 hours post application on collagen in either 0.9% saline or 0.9% saline with addition of 50 mM CaCl2.

Example 13—Gelatin—Chitosan Mesh Based Compositions

[0427] Three mixtures of gelatin (Gelita, Type A gelatin, Bloom 275) and low MW chitosan (Aldrich cat #448869) were prepared (see table 13), each with total solids content of 2% w/w, and total weight of 300 g each.

[0428] The mixtures were brought to pH 3.8 with acetic acid, in order to allow dissolution of the chitosan. mTG was added to a final ratio of 80 U per gram gelatin.

TABLE-US-00013 TABLE 13 Gelatin-Chitosan mesh based compositions: Acetic acid Batch no. % chitosan Chitosan (gr) Gelatin (gr) (ml) GF512  5 0.3 5.7 5.4 GF511 10 0.6 5.4 5.7 GF510 20 1.2 4.8 9  

[0429] The solution was cooled down to 16° C., casted on PEEK plates at a layer thickness of 250 μm using a doctor blade. Surgical mesh was placed on the liquid layer and the composition was cooled down at 4° C. Another layer was placed on top of the surgical mesh at a thickness of 1700 μm using a doctor blade. The coated mesh was then frozen at −80° C. and lyophilized.

[0430] The resulting dry coated meshes were tested for fixation strength to collagen as described in Example 12.

[0431] FIG. 19 shows the fixation strength of Gelatin-Chitosan mesh based compositions (batches No. GF-510, GF-511, GF-512) on collagen, as tested by the lap shear method, 24 hours after fixation. The articles were immersed for 24 hours post application on collagen in 0.9% saline.

[0432] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0433] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.