阅读说明：本技术 交联的蛋白质泡沫及其使用多用途细胞支架的方法 (Cross-linked protein foams and methods of using the same ) 是由 I·阿塔 S·Y·S·哲利 于 2018-10-04 设计创作，主要内容包括：在一个实施例中,本发明提供了组合物,其中所述组合物是多孔支架,其中所述支架的孔为1至500微米,所述组合物包含：a)选自胶原和明胶的可交联蛋白质；b)诱导可交联蛋白质交联的交联剂；和c)液体。(In one embodiment, the present invention provides a composition, wherein the composition is a porous scaffold, wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising: a) a cross-linkable protein selected from the group consisting of collagen and gelatin; b) a cross-linking agent that induces cross-linking of the cross-linkable protein; and c) a liquid.)

1. A composition of matter, a method of making,

wherein the composition is a porous scaffold and the composition is,

wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising:

a cross-linkable protein selected from the group consisting of collagen and gelatin,

a cross-linking agent that induces cross-linking of the cross-linkable protein; and

a liquid.

2. The composition of claim 1, wherein the liquid is a physiological buffer.

3. The composition of claim 1, wherein the composition is a foam.

4. The composition according to claim 1, wherein the crosslinkable protein is introduced into the composition as a micronized protein powder having an average particle size of 5 to 200 microns.

5. The composition of claim 1, wherein the cross-linkable protein comprises 200 to 300 bloom gelatin.

6. The composition according to claim 1, wherein the crosslinkable gelatin is present in the composition in the range of 0.5% w/w to 25% w/w.

7. The composition of claim 1, wherein the cross-linking agent is transglutaminase.

8. The composition of claim 1, wherein the cross-linking agent is purified transglutaminase.

9. The composition of claim 1, wherein the cross-linking agent is microbial transglutaminase.

10. The composition of claim 1, wherein the composition is formed in situ in the patient at a site in the patient in need of treatment for a skin defect.

11. The composition of claim 1, wherein the composition is formed prior to introducing the composition into the patient at a site in the patient in need of treatment of a skin defect.

12. The composition of claim 1, wherein the composition is formed prior to introducing the composition into the patient at a site in the patient in need of skin collagen regeneration.

13. The composition of claim 1, having a sufficient amount of crosslinkable protein such that when a porous scaffold is formed, the formed porous scaffold results in a young's modulus of about 0.5-10 KPa.

14. The composition of claim 1, having a sufficient amount of crosslinkable protein such that when a porous scaffold is formed, the formed porous scaffold has an elongation less than 5 times its original length.

15. The composition of claim 1, having a sufficient amount of crosslinker such that when the porous scaffold is formed, the formed porous scaffold results in a young's modulus of about 0.5-10 KPa.

16. The composition of claim 1, having a sufficient amount of crosslinker such that when the porous scaffold is formed, the formed porous scaffold has an elongation less than 5 times its original length.

17. A composition, comprising:

a cross-linkable protein selected from the group consisting of collagen and gelatin;

a cross-linking agent that induces cross-linking of the cross-linkable protein;

a liquid; and

the content of the hyaluronic acid is shown in the specification,

wherein the composition is a porous scaffold having a pore size of 1 to 500 microns.

18. A composition, comprising:

a cross-linkable gelatin;

wherein the cross-linkable gelatin is a micronized gelatin powder having a particle size of 5 to 200 microns,

wherein the crosslinkable gelatin is 200 to 300 bloom,

wherein the crosslinkable gelatin is in the range of 0.5% w/w to 25% w/w, transglutaminase inducing crosslinking of the crosslinkable gelatin;

wherein the transglutaminase is in the range of 0.0001% w/w to 2% w/w, and a liquid,

wherein the composition is a porous scaffold having a pore size of 1 to 500 microns.

19. The composition of claim 18, wherein the liquid is a physiological buffer.

20. The composition of claim 18, wherein the composition is a foam.

21. The composition of claim 18, wherein the composition is formed in situ in the patient at a site in the patient in need of treatment for a skin defect.

22. The composition of claim 18, wherein the composition is formed prior to introducing the composition into the patient at a site in the patient in need of treatment of a skin defect.

23. The composition of claim 18, wherein the composition is formed prior to introducing the composition into the patient at a site in the patient in need of skin collagen regeneration.

24. The composition of claim 18, having a sufficient amount of cross-linkable gelatin such that when the porous scaffold is formed, the formed porous scaffold results in a young's modulus of about 0.5-10 KPa.

25. The composition of claim 18, having a sufficient amount of cross-linkable gelatin such that when the porous scaffold is formed, the formed porous scaffold has an elongation less than 5 times its original length.

26. The composition of claim 18, having a sufficient amount of transglutaminase such that when the porous scaffold is formed, the formed porous scaffold results in a young's modulus of about 0.5-10 KPa.

27. The composition of claim 18, having a sufficient amount of transglutaminase such that when the porous scaffold is formed, the formed porous scaffold has an elongation less than 5 times its original length.

28. A method of treating a skin defect or strengthening collagen in skin tissue in a patient in need thereof, comprising:

introducing the composition of claim 18 into a patient at the site of a tissue defect in an amount sufficient to treat skin;

wherein the composition adheres to the tissue at the defect site.

29. A method of treating a skin defect or supplementing skin collagen or activating skin fibroblasts to produce new collagen fibers in a patient in need thereof, comprising:

forming a composition according to claim 18 at a site of a tissue defect in a patient in an amount sufficient to treat skin;

wherein the composition adheres to tissue at the site of defect; and is

Wherein the composition is configured to promote fibroblast attachment and collagen synthesis.

30. The method of claim 28 or 29, wherein the composition has a sufficient amount of the cross-linkable gelatin and the transglutaminase such that when a porous scaffold is formed, the formed porous scaffold results in a young's modulus of about 0.5-10 KPa.

31. The method of claim 28 or 29, wherein the composition has a sufficient amount of the cross-linkable gelatin and the transglutaminase such that when a porous scaffold is formed, the formed porous scaffold has an elongation that is less than 5 times its original length.

32. The method of claim 28 or 29, wherein the composition induces the production of endogenous collagen by a tissue at the site of application.

33. The method of claim 28 or 29, wherein the composition induces production of endogenous procollagen in the tissue at the site of application.

34. The method of claim 28 or 29 wherein the composition induces production of endogenous type I collagen in a tissue at the site of application.

35. The method of claim 28 or 29, wherein the composition induces the production of endogenous collagen by a tissue at the site of application, and

wherein at least 80% of the composition degrades after 3 months.

Technical Field

The present invention relates to improved cross-linking compositions comprising a cross-linkable protein and a non-toxic material that induces cross-linking of the cross-linkable protein. The composition is intended for triggering biological processes of tissue regeneration.

Background

Biomaterials that can form gels in situ can be used in a variety of applications, for example, injectable matrices for controlled delivery of cells and/or drugs, injectable scaffolds for tissue engineering, or adhesives that bind tissue or seal gas or liquid leaks in physiological environments.

Disclosure of Invention

In one embodiment, the present invention provides a composition

Wherein the composition is a porous scaffold and the composition is,

wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising:

a) a cross-linkable protein selected from the group consisting of collagen and gelatin;

b) a cross-linking agent that induces cross-linking of the cross-linkable protein; and

c) a liquid.

In one embodiment, the liquid is a physiological buffer.

In one embodiment, the composition is a foam.

In one embodiment, the cross-linkable protein is introduced into the composition as a micronized protein powder having an average particle size of 5 to 200 microns.

In one embodiment, the cross-linkable protein comprises 200 to 300 bloom gelatin.

In one embodiment, the crosslinkable gelatin is present in the composition in a range of 0.5% w/w to 25% w/w.

In one embodiment, the crosslinking agent is transglutaminase.

In one embodiment, the transglutaminase is present in the composition in a range of 0.0001% w/w to 2% w/w.

In one embodiment, the present invention provides a composition comprising:

a) a cross-linkable gelatin;

b) transglutaminase inducing cross-linking of the cross-linkable gelatin; and

c) the liquid is a mixture of a liquid and a gas,

wherein the composition is a porous scaffold having a pore size of 1 to 500 microns,

wherein the cross-linkable gelatin is introduced into the composition as a micronized gelatin powder having a particle size of 5 to 200 microns,

wherein the crosslinkable gelatin is 200 to 300 bloom,

wherein the crosslinkable gelatin is present in the composition in the range of 0.5% w/w to 30% w/w, and

wherein the transglutaminase is present in the composition in the range of 0.0001% w/w to 2% w/w.

In one embodiment, the liquid is a physiological buffer.

In one embodiment, the composition is formed in situ in the patient at the site of the patient in need of treatment of the tissue defect.

In one embodiment, the composition is formed prior to introducing the composition into the patient at a site where the patient is in need of treatment of a tissue defect.

In one embodiment, the present invention provides a method, wherein the method treats a tissue defect or disease in a patient in need thereof, comprising:

a) introducing the composition into the patient at the site of the tissue defect in an amount sufficient to treat the tissue defect or disease;

wherein the composition adheres to the tissue at the defect site.

In one embodiment, the present invention provides a method, wherein the method treats a tissue defect or disease in a patient in need thereof, comprising:

a) forming a composition at a site of the tissue defect in the patient in an amount sufficient to treat the tissue defect or disease;

wherein the composition adheres to the tissue at the defect site.

In one embodiment, the tissue defect is a wound.

In one embodiment, the tissue is damaged and requires regeneration.

In one embodiment, the tissue defect is a bone defect.

In one embodiment, the composition induces regeneration of bone in the patient.

In one embodiment, at least one cell type infiltrates into the composition and grows therein.

In one embodiment, the at least one cell type is a cell type selected from the group consisting of: pancreatic stem cells, intestinal secretory cells, bone cells, liver cells, tendon cells, muscle cells, blood cells, chondrocytes, epithelial cells, endothelial cells, neurons, embryonic stem cells, mesenchymal stem cells, autologous bone marrow-derived mesenchymal stem cells, progenitor cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, bone system stem cells, chondrocyte-lineage stem cells, epithelial stem cells, and hepatic stem cells.

In one embodiment, the invention provides a method, wherein the method stimulates fibroblasts to produce new collagen in a patient, comprising:

a) forming a composition in the patient in an amount sufficient to induce fibroblast stimulation,

b) wherein the composition is configured to promote fibroblast stimulation and collagen synthesis, wherein the fibroblast attachment and collagen synthesis induces tissue (skin) regeneration, and

c) wherein the composition is a porous scaffold and the composition is,

d) wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising:

i. a cross-linkable protein selected from the group consisting of collagen and gelatin;

a crosslinking agent that induces crosslinking of the crosslinkable protein; and

a liquid.

In one embodiment, the gelatin is present in the composition in the range of 3% w/w to 30% w/w after dilution with a liquid.

In one embodiment, the gelatin is present in the composition in the range of 8% w/w to 25% w/w after dilution with a liquid.

In one embodiment, the gelatin is present in the composition in the range of 8% w/w to 20% w/w after dilution with a liquid.

Drawings

Figure 1 is a graph showing the particle size distribution of micronized proteins according to some embodiments of the present invention.

Fig. 2 is a photograph of an apparatus for forming a composition of the present invention, according to some embodiments of the present invention.

Fig. 3A is a photograph showing cells in treated tissue, according to some embodiments of the invention.

Fig. 3B is a photograph showing cells in untreated tissue, according to some embodiments of the invention.

Fig. 4 is a photograph showing cells seeded in a scaffold composition according to some embodiments of the invention.

Figure 5 is a photograph of collagen deposition analysis according to some embodiments of the present invention.

Fig. 6A-6E are photographs of compositions of the invention, according to some embodiments of the invention.

Fig. 7A-D are photographs of compositions of the invention, according to some embodiments of the invention.

Fig. 8 is a photomicrograph of a composition according to some embodiments of the invention.

Fig. 9 is a photograph of a jet milling apparatus according to some embodiments of the present invention.

Fig. 10 shows viability of cells seeded in scaffold compositions according to some embodiments of the invention.

Fig. 11A and 11B are photographs of compositions of the invention, according to some embodiments of the invention.

Fig. 12 is a photograph of a composition of the present invention, according to some embodiments of the present invention.

Fig. 13A-D are photographs of compositions of the invention, according to some embodiments of the invention.

Fig. 14 is a photograph of a composition of the present invention, according to some embodiments of the present invention.

Detailed Description

For clarity of disclosure and not limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the invention.

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

In addition, as used herein, the term "or" is an inclusive "or" operator, and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, the meaning of "a", "an", and "the" includes plural referents throughout the specification. The meaning of "in … …" includes "in … …" and "on … …".

As used herein, "gelatin" is obtained by partial hydrolysis of animal tissue or collagen obtained from animal tissue, wherein the animal tissue is selected from the group consisting of animal skin, connective tissue, antlers, horns, bones, fish scales, and recombinant gelatin produced using bacterial, yeast, animal, insect or plant systems (including, by way of non-limiting example, tobacco), or any type of cell culture, or any combination thereof.

As used herein, "bloom" is defined as the weight in grams required to press a plunger of one-half inch diameter into a gelatin solution containing 6% solids, which gels at 10DC for 17 hours, for 4 mm.

As used herein, "carrier" refers to a polymer, protein, polysaccharide, or any other constituent that binds, covalently or non-covalently, a crosslinking enzyme prior to or during a crosslinking reaction.

As used herein, "copolymer" refers to a constituent of a matrix that can participate in a crosslinking reaction and is not typically the primary constituent of the matrix. The copolymer is typically not covalently bound to the enzyme or to a protein base of the matrix material, e.g., the matrix. Non-limiting examples of copolymers are polysaccharides such as dextran, and/or cellulosic polymers such as carboxymethyl cellulose.

As used herein, "crosslinking enzyme" refers to at least one enzyme (such as, but not limited to, 1 enzyme, 2 enzymes, 3 enzymes, 4 enzymes, 5 enzymes, etc.) that can crosslink substrate groups on a polymer strand directly (such as, but not limited to, transglutaminase amidation) or indirectly (such as, but not limited to, quinone or free radical formation) to form a matrix such as, but not limited to, a hydrogel.

As used herein, "diffusion" or "mobility" refers to random molecular motion of, for example, but not limited to, enzymes and/or other molecules (such as, but not limited to, any protein, hydrogen, or substrate) in solution, which may result from brownian motion.

As defined herein, "diffusion coefficient" or "diffusivity" specifies a term that quantifies the degree of diffusion of a single type of molecule under a particular condition. Specifically, the diffusion coefficient or diffusivity is a proportional relationship, i.e., a constant between the molar flux due to molecular diffusion and the gradient in species concentration (or driving force for diffusion). The measurement method is performed by measuring the elution of the enzyme from the hydrogel, i.e. the rate and/or the total amount of enzyme eluted by the hydrogel.

As defined herein, "hydrodynamic volume" refers to the molecular weight of a protein or enzyme that can generally be measured using size exclusion chromatography. The hydrodynamic volume of a constituent refers to the diameter and/or volume that the constituent takes when it moves in liquid form.

As defined herein, "matrix" refers to a composition of crosslinked material. Generally, when matrix-forming materials are crosslinked, compositions comprising these materials transition from a liquid state to a gel state, thereby forming a "gel," hydrogel, "or" gelled composition.

As used herein, "molecular weight," abbreviated "MW," refers to the absolute weight in daltons or kilodaltons of a protein or polymer. For example, the MW of a pegylated protein (such as, but not limited to, a protein to which one or more PEG (polyethylene glycol) molecules have been coupled) is the sum of the MW of all its constituent components.

As used herein, "patient" refers to any animal or human in need of treatment according to the methods of the present invention.

As defined herein, "perceived volume" or "effective volume" refers to the effective hydrodynamic volume of the cross-linking enzyme inside the cross-linked matrix. The perceived volume may be increased by covalent or non-covalent binding of the enzyme to another molecule, carrier, polymer, protein, polysaccharide and others, either before or during the cross-linking reaction.

As used herein, "polymer" refers to a natural, synthetic, or semi-synthetic molecule containing a repeatable unit.

As defined herein, "reduced mobility" refers to slower molecular movement or smaller diffusion coefficient of a protein or enzyme in solution or inside a hydrogel, and can be measured by elution rate.

As defined herein, "size" refers to the molecular weight or hydrodynamic volume or perceived volume of a molecule.

As defined herein, "grinding" refers to milling a material by any of the following methods: jet milling, rotational/vortex milling, ball milling, high pressure homogenization and microfluidization, spray drying, recrystallization, emulsion-solvent extraction, and methods using supercritical fluids such as supercritical solution Rapid Expansion (RESS).

"jet milling" refers to a micronization process by rotational or vortex milling.

Cross-linkable proteins

According to at least some embodiments with respect to the method and/or matrix, the at least one substrate polymer comprises a substrate polymer selected from the group consisting of: natural crosslinkable polymers, partially denatured polymers that can be crosslinked by enzymes, and polymers containing functional groups or peptides that can be crosslinked by unmodified or modified enzymes. Optionally, the at least one substrate polymer comprises gelatin, collagen, casein or albumin, or a modified polymer, and wherein the modified enzyme molecule comprises transglutaminase and/or oxidase, modified transglutaminase and/or modified oxidase. Optionally, the at least one substrate polymer comprises gelatin selected from gelatin obtained by partial hydrolysis of animal tissue or collagen obtained from animal tissue, wherein the animal tissue is selected from animal skin, connective tissue, antlers, horns, bones, fish scales, and recombinant gelatin produced using bacteria, yeast, animal, insect or plant systems, or any type of cell culture, or any combination thereof. Optionally, the gelatin is of mammalian or fish origin. Optionally, the gelatin is type a (acid treated) or type B (base treated). Optionally, the gelatin is 250-. In some embodiments, the gelatin has an average molecular weight of 75 to 150 kda.

In some embodiments, synthetic or partially synthetic polymers having one or more suitable functional groups may also serve as crosslinkable substrates for any of the enzymes described herein. In another embodiment of the invention, a combination of enzymes is used. In another embodiment of the invention, a combination of cross-linking agents is used, not necessarily just enzymes.

In some embodiments, the crosslinkable polymer comprises at least one RGD (Arg-Gly-Asp) motif. In some embodiments, at least one RGD motif promotes cell attachment to the compositions of the invention.

In some embodiments, the crosslinkable polymer comprises a heterogeneous distribution of RGD motifs, which can act as a scaffold for cells, independent of cell type or motility state, making it a versatile cell scaffold.

Thus, the present invention provides crosslinkable polymers with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides non-recombinant cross-linkable polypeptides with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides gelatin with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides non-recombinant gelatin with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides micronized gelatin with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides a porous scaffold, such as gelatin foam, with improved cell attachment and motor compatibility through versatile display of the RGD motif.

Thus, the present invention provides a cross-linked porous scaffold with improved cell attachment and motor compatibility through versatile display of the RGD motif, e.g. a gelatin foam with at least 3% w/w gelatin.

Thus, the present invention provides a cross-linked porous scaffold with improved cell attachment and motor compatibility through versatile display of the RGD motif, e.g. gelatin foam with 105-25% w/w gelatin.

Thus, the present invention provides any of the compositions described with the addition of fibrin.

As defined herein, a "polymer strand" or "polymer chain" refers to a substrate polymer for enzymatic crosslinking, which, according to at least some embodiments of the present invention, belongs to one of the following categories (as non-limiting examples only):

1) any polymer having substrate groups which are naturally cross-linkable by an enzyme and which are themselves naturally cross-linkable by an enzyme. For example, in the case of transglutaminase, this includes proteins or polypeptides that can be naturally cross-linked by enzymes, such as gelatin, collagen and casein.

2) Polymers containing substrate groups that can be cross-linked by enzymes, but cannot be cross-linked naturally by enzymes due to their structure. In this case, the polymer structure must be modified before enzymatic crosslinking. For example, in the case of transglutaminase, this includes proteins such as albumin or lactoglobulin, which are not natural substrates for the enzyme, as they have a globular structure that hinders the entry of the enzyme. These can be prepared into a substrate by partially denaturing the protein using a reducing agent, a denaturing agent, or heat.

3) A natural or synthetic polymer which is not a substrate for enzymatic cross-linking but which has been modified with a peptide or functional group which is a substrate for an enzyme, thus rendering the modified polymer cross-linkable by the enzyme. Non-limiting examples of such polymers include any suitable type of protein, which may for example comprise gelatin as described above. Gelatin may include any type of gelatin comprising proteins known in the art, including but not limited to gelatin obtained by partial hydrolysis of 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 bone, and/or fish scales and/or bone or other components, or collagen obtained from animal tissue; and/or recombinant gelatin produced using bacteria, yeast, animal, insect, or plant systems, or any type of cell culture, or any combination thereof.

According to some embodiments of the invention, gelatin from animal origin may include one or more of gelatin from mammalian origin, such as but not limited to porcine skin, porcine bone and bovine bone, or segmented bovine skin, or any other porcine or bovine source, or any combination thereof. In some embodiments, the gelatin may comprise porcine gelatin because of its lower allergic reaction rate. Gelatin from animal origin may optionally be of type a (acid treated) or type B (base treated), although it may be of type a.

In some embodiments, gelatin from animal origin comprises gelatin obtained during a first extraction, which is typically performed at a lower temperature (50-60 ℃, although this exact temperature range is not necessarily limiting). The gelatin produced in this manner is in the range of 250-. In some embodiments, 275-. A non-limiting example of a producer of such gelatin is PBGelatins (Tessenderlo Group, Belgium).

According to some embodiments of the invention, the gelatin from animal origin may comprise gelatin from fish. In some embodiments, any type of fish may be used, such as cold water species of fish, such as carp, cod, or canine or tuna. In some embodiments, the pH (measured in a 10% w/w solution) of the fish gelatin may range from pH 4 to pH 6.

In some embodiments, the cold water fish gelatin forms a solution in water at 10 ℃. In some embodiments, the cold water fish gelatin is 0 bloom. In some embodiments, high molecular weight cold water fish gelatin may be used, which includes an average molecular weight of at least about 95-115kDa (where the cold water fish gelatin is comparable to the molecular weight of 250-300 bloom animal gelatin). In some embodiments, the cold water fish gelatin undergoes thermally reversible gelation at lower temperatures than animal gelatin due to the reduced amounts of proline and hydroxyproline. A non-limiting example of a producer of such gelatin is Norland Products (Cranbury, NJ).

In some embodiments of the invention, low endotoxin activity (endotoxin) gelatin is used to form the gelatin solution component of the gelatin matrix composition. In some embodiments, the low endotoxin activity gelatin is obtained from a commercial supplier such as Gelita^TM(Eberbach, germany) is commercially available. As used herein, a low endotoxin activity gelatin is defined as a gelatin having less than 1000 endotoxin activity units (EU)/gram. In some embodiments, gelatin having an endotoxin activity of less than 500 EU/gram is used.

In some embodiments, when producing a material for contact with the spine or brain, gelatin is used having an endotoxin activity of less than 100 EU/gram (e.g., between 1-100 EU/gram). In some embodiments, gelatin having less than 50EU/g is used. In some embodiments, gelatin with an endotoxin activity of less than 10EU/g may be used.

According to some embodiments of the invention, type I, type II or any other type of hydrolyzed or non-hydrolyzed collagen replaces gelatin because the proteinaceous material is cross-linked. Various types of collagen have demonstrated the ability to form thermally stable mTG cross-linked gels. For example, as in the publication "Characterisation of a microbial Transglutamase cross-linked type II collagen scaffold"; PMID 16846344.

According to some embodiments of the invention, recombinant human gelatin is used. In some embodiments, the recombinant human gelatin is obtained from a supplier such as fibrigen^TMIn some embodiments, recombinant human gelatin may be at least about 90% pure (e.g., 90.01-100%), in some embodiments, recombinant gelatin may be at least about 95% pure (e.g., 95.01-100%), in some embodiments, recombinant gelatin may be non-gelling at 10 ℃, and thus considered to be 0 bloom.

In some embodiments, the cross-linkable protein may comprise gelatin, but may also or alternatively comprise another type of protein. According to some embodiments of the invention, the protein is also a substrate for transglutaminase. In some embodiments, the substrate of transglutaminase can include collagen, or other synthetic polymer sequences independently having the property of forming a bioadhesive, or polymers that have been modified with a transglutaminase-specific substrate that increases the ability of the material to be cross-linked by transglutaminase. The composition may also include fibrin.

Non-limiting examples of such peptides are described in U.S. patent nos. 5,428,014 and 5,939,385, which are incorporated herein by reference, which describe biocompatible, bioadhesive, transglutaminase-crosslinkable polypeptides, as fully set forth herein, wherein the transglutaminase catalyzes an acyl transfer reaction between the γ -carboxamido group of the protein-bound glutaminyl residue and the-amino group of the L ys residue, resulting in the formation of an 8- (γ -glutamyl) lysine isopeptide bond.

In some embodiments, the compounds of the present invention are substantially dry gelatins configured to be rapidly hydrated with warm or cold liquids (e.g., without limitation, liquids between 4 ℃ to 37 ℃) to form a porous scaffold, gel, or foam, wherein the porous scaffold, gel, or foam is further configured to be shaped and molded into any cavity, ex vivo or in vivo, a body cavity, on a wound, on an organ, or any combination thereof. In some embodiments, the non-crosslinked gelatin is reacted/mixed with the crosslinking agent after hydration/reconstitution of the non-crosslinked gelatin and the crosslinking agent, wherein the mixing of the non-crosslinked gelatin and the crosslinking agent results in the formation of a stable, insoluble, non-thermoreversible gel, foam, or porous scaffold.

As used herein, the effect of particle size on solubility constant can be quantified as follows:

wherein K_AIs related to the solubility constant of solute particles of molar surface area A_A→0Is about the solubility constant of a substance whose molar surface area tends to zero (i.e., when the particles are large), γ is the surface tension of the solute particles in the solvent, and Am is the molar surface area of the solute (in m)²In mol), R is the universal gas constant, and T is the absolute temperature.

Typical techniques for preparing micron-sized particles of drugs and proteins are mechanical comminution (e.g., by crushing, milling, and grinding) of previously formed larger particles. In some embodiments of the method of the present invention, the grinding is accomplished by mortar and in other preferred embodiments in jet milling. Figure 1 shows the particle size distribution after micronization by jet milling.

In some embodiments, the methods of the invention include preparing a rapidly dissolving dried protein that is not a cross-linked protein (such as, but not limited to, gelatin). In some embodiments, the gelatin is prepared by jet milling to achieve a particle size of 2 to 250 microns. In some embodiments, the particle size is between 5 and 130 microns. In some embodiments, the particle size is between 10 and 80 microns. In some embodiments, the particle size is between 10 and 70 microns. In some embodiments, the particle size is between 10 and 60 microns. In some embodiments, the particle size is between 10 and 50 microns. In some embodiments, the particle size is between 10 and 40 microns. In some embodiments, the particle size is between 10 and 30 microns. In some embodiments, the particle size is between 10 and 20 microns. In some embodiments, the particle size is between 2 and 10 microns. In some embodiments, the particle size is between 10 and 100 microns. In some embodiments, the particle size is between 20 and 100 microns. In some embodiments, the particle size is between 30 and 100 microns. In some embodiments, the particle size is between 40 and 100 microns. In some embodiments, the particle size is between 50 and 100 microns. In some embodiments, the particle size is between 60 and 100 microns. In some embodiments, the particle size is between 70 and 100 microns. In some embodiments, the particle size is between 80 and 100 microns. In some embodiments, the particle size is between 90 and 100 microns. In some embodiments, the particle size is between 5 and 50 microns. In some embodiments, the particle size is between 10 and 20 microns. In some embodiments, the particle size is between 10 and 15 microns. In some embodiments, the particle size is between 15 and 20 microns. In some embodiments, the particle size is between 12 and 18 microns.

In some embodiments, the process of the present invention comprises jet milling, wherein the jet milling results in the preparation of the surface of each gelatin particle (crystal) for rapid dissolution in a liquid (e.g., without limitation, dissolution within 0.01 seconds to 60 seconds) (i.e., the resulting jet milled particles surprisingly exhibit increased moisture absorption characteristics compared to non-jet milled raw materials). In some embodiments, the jet-milled particles can be mixed with a crosslinking agent to result in the formation of a thermally stable and tissue-adherent hydrogel or foam. In some embodiments, a porous scaffold, gel/foam may be placed within a body cavity and/or between layers of tissue of a human or animal. In some embodiments, the present invention may be used in various medical applications. In some embodiments, the invention may be used as a cell scaffold that exhibits improved exposure and accessibility of integrin attachment sites (e.g., RGD motif).

In some embodiments, the gelatin is prepared by milling to a small particle size to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution between 2-200 microns. In some embodiments, the gelatin is prepared by milling to a small particle size to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution between 2 and 100 microns. In some embodiments, the gelatin is prepared by milling to a small particle size to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution between 2 and 50 microns. In some embodiments, the gelatin is prepared by grinding to a small particle size to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution between 50 and 150 microns. In some embodiments, the gelatin is prepared by milling to a small particle size to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution between 100 and 150 microns. In some embodiments, the gelatin is prepared by jet milling to result in an increased cross-linking distribution, wherein the prepared gelatin is characterized by having a micron size distribution. In some embodiments, the pre-crosslinked gelatin may be (1) lyophilized, then (2) jet milled to produce a dry powdered gelatin (i.e., "high bloom gelatin"), wherein the dry powdered gelatin may be dissolved in a solution at a temperature, for example, but not limited to, between 5 ℃ and 37 ℃ over a time period equal to or less than 120 seconds (e.g., between 0.01 seconds and 120 seconds) to produce a solution.

In one embodiment, the grinding apparatus and method are as disclosed in U.S. Pat. No.5,855,326, which is hereby incorporated by reference in its entirety, in another embodiment, the grinding apparatus is as disclosed in U.S. Pat. No. 6,789,756, which is hereby incorporated by reference in its entirety, an example of such a grinding apparatus is the SuperFine Vortex Mill manufactured by SuperFine L td^TM(shown schematically in fig. 9). The entire contents of which are incorporated by referenceU.S. Pat. No.5,855,326, incorporated, assigned to Beliaysky, discloses a rotary grinding chamber for the fine comminution of particulate solid material, the chamber being formed in a housing having a substantially cylindrical shape, said housing having two end faces and a side wall provided with one or more tangential nozzles for injecting a working fluid into the chamber and generating a vortex therein, said chamber comprising means for introducing particulate solid material to be comminuted therein, an axially arranged discharge channel provided in one or both of said end faces, and control means in the form of one or more mechanical elements adapted to interact upon generation of the vortex, the layers thereof moving close to the inner wall of the chamber, allowing the controlled comminution. The operation of the rotating chamber is exemplified in the patents used and U.S. patent No. 6,789,756 to Beliaysky, which is also incorporated by reference in its entirety, discloses an improved vortex mill for grinding substantially particulate solid material comprising one or more working chambers. The mill also includes one or more working fluid inlets and one or more discharge outlets. The one or more working fluid inlets, in conjunction with the one or more discharge ports, promote swirl within the one or more working chambers. There are also one or more feed inlets to provide grinding of the solid material which is discharged from the one or more discharge outlets. In addition, there are devices for inducing controlled disturbances in the flow of the working fluid in the working chamber or chambers, thereby improving the grinding of the solid material in the vortex.

In some embodiments, the methods of the present invention can include using plasma beam energy for increasing the surface hygroscopicity of gelatin particles, wherein the resulting micronized gelatin treated with plasma beam energy has substantially the same characteristics/characteristics as non-plasma beam treated gelatin. In some embodiments, additional substances/compounds for mixing with gelatin, such as, but not limited to, microbial transglutaminase, bone enhancing substances, proteins, and copolymers to be mixed into the final product, or any combination thereof, may be treated with plasma beam energy.

In some embodiments, the compositions of the present invention may be characterized by the hygroscopic granular gelatin powder being configured to dissolve into a flowable gel or foam when mixed with a liquid for a period of time of 0.01 to 120 seconds. In some embodiments, the liquid may be provided as part of a product formulation (i.e., a kit) that is loaded into the syringe on-site by a medical practitioner (e.g., a nurse, physician's assistant, etc.) prior to mixing. In some embodiments, dry gelatin, alone or mixed together with a cross-linking agent, or both gelatin and a cross-linking agent, together with a surgical mesh, may be applied directly to the body and activated by application of a fluid (i.e., saline), or activated by bodily fluids (i.e., for fluids endogenous to the body) when in contact with moist tissue. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 4 and 40 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 4 and 20 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 4 and 15 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 10 and 25 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 25 and 37 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 15 and 25 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 20 and 25 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 10 and 20 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 12 and 18 ℃. In some embodiments, the milled gelatin powder may be hydrated at a temperature between 14 and 19 ℃ (which is a temperature range of an operating room). In some embodiments, the milled gelatin powder may be hydrated at a temperature of about 16 ℃. In some embodiments, the dissolved gelatin of the present invention is configured to maintain a flowable form (i.e., the dissolved gelatin does not immediately pass its liquid-gel transition point, but instead turns back to an inoperable solid) even at temperatures below 37 ℃.

In some embodiments, the dissolved gelatin of the present invention may be delivered through a long needle, catheter or endoscope. In some embodiments, the dissolved gelatin of the present invention has a viscosity when foamed that is less than a confluent gelatin hydrogel of the same composition.

In some embodiments, the methods of the present invention comprise using radiation energy to make/sterilize gelatin, wherein the resulting irradiated gelatin has substantially similar functional properties (e.g., crosslinking ability) as compared to the non-irradiated starting gelatin.

In some embodiments, the methods of the present invention comprise preparing/sterilizing gelatin using radiation energy, wherein the resulting irradiated gelatin has at least 25% functional properties (e.g., crosslinking ability) compared to the non-irradiated starting gelatin. In some embodiments, the methods of the present invention comprise preparing/sterilizing gelatin using radiation energy, wherein the resulting irradiated gelatin has 25% to 100% functional properties (e.g., ability to be cross-linked) compared to the non-irradiated starting gelatin.

In some embodiments, the methods of the present invention comprise mixing the gelatin powder with additional active components such as, but not limited to, stabilizers (such as, but not limited to, EDC (1-ethyl-3- (3- (dimethylaminopropyl) -carbodiimide), NHS (N-hydroxysuccinimide), carbonamide, glutaraldehyde, horseradish peroxidase and/or transglutaminase), wherein the mixed gelatin and stabilizer form a stable porous scaffold, gel or foam, wherein the shape of the porous scaffold, gel or foam is irreversible (i.e., crosslinked due to covalent bonding between gelatin molecules).

In some embodiments, the methods of the invention comprise drying a polymer, such as a protein and/or polypeptide, wherein the protein and/or polypeptide is collagen and/or gelatin and/or any gelatin variant, so as to result in a dried polymer having a substantially faster reconstitution profile in a liquid (such as, but not limited to, 0.01 seconds to 60 seconds reconstitution) compared to typical non-powdered gelatin, including in an environment of a cold or room temperature liquid (such as, but not limited to, a liquid between 5 ℃ to 37 ℃). In some non-limiting exemplary embodiments, the gelatin powder may be characterized as having (1) a substantially longer shelf life (e.g., between 1 month and 36 months), and (2) a substantially more rapid reconstitution/solubility. In some embodiments, the dried polymer is sterilized and the sterilized dried polymer exhibits substantially similar biological function and dissolution/reconstitution characteristics as compared to a substantially similar non-sterilized dried polymer. In some embodiments, the dried polymer is sterilized, and the sterilized dried polymer exhibits at least 25% biological function and dissolution/reconstitution characteristics compared to a substantially similar unsterilized dried polymer. In some embodiments, the dry polymer is sterilized, and the sterilized dry polymer exhibits a biological function and dissolution/reconstitution characteristics of 50% to 100% as compared to a substantially similar non-sterilized dry polymer.

Crosslinking agent

In the illustrative embodiment, non-limiting examples of direct crosslinking enzymes that directly crosslink substrate groups on the polymer strands include transglutaminase and oxidase enzymes. Examples of transglutaminase include microbial transglutaminase (mTG), tissue transglutaminase (tTG), keratinocyte transglutaminase, epidermal transglutaminase, prostate transglutaminase, neuronal transglutaminase, human transglutaminase, and factor XIII. In some embodiments, these enzymes may be from natural or recombinant sources. In some embodiments, the glutamine and lysine amino acids in the polymer strand are substrates for transglutaminase crosslinking.

In the illustrative embodiment, non-limiting examples of oxidases are tyrosinase, laccase, peroxidase, or any combination thereof. In some embodiments, the oxidase crosslinks the polymer by quinone formation (tyrosinase) or free radical formation (laccase, peroxidase). The quinone and the free radical then interact with each other or with other amino acids or phenolic acids to crosslink the polymer. In some embodiments, the crosslinkable substrate for the oxidase can be any protein containing tyrosine or other aromatic amino acids. In some embodiments, the substrate may be a carbohydrate containing a phenolic acid, such as, but not limited to, ferulic acid. In some embodiments, the carbohydrate may be, but is not limited to, arabinoxylan or pectin.

According to some embodiments of the methods of the invention, the transglutaminase solution is subjected to one or more purification stages to perform one or more of the following: 1) removing fermentation residues from the transglutaminase mixture; 2) concentrating an amount of active transglutaminase in a transglutaminase solution; 3) purifying the transglutaminase solution from the carrier protein and/or carbohydrate; 4) reducing the endotoxin level of a transglutaminase solution; 5) removing all microorganisms from the transglutaminase solution, and effectively sterilizing the solution; all of which are not intended to be limited to a closed list or any combination thereof.

According to some embodiments of the invention, the transglutaminase may be a recombinant transglutaminase. Non-limiting examples of such are enzymes expressed in E.coli (E-coli) bacteria.

In some embodiments, the filtration process first uses a coarse filtration, sometimes referred to as clarification, to remove large pieces of fermentation residue that will rapidly clog the finer filtration step, non-limiting examples of such coarse filtration are about 0.45 μm pore size filtration and about 0.65 μm pore size filtration, hi some embodiments, the solution of cross-linked material may be passed through a filter having a pore size of less than 0.22 μm, for example to reduce the bioburden of the material to less than 10 Colony Forming Units (CFU)/gram and make it suitable for medical use.

According to another embodiment of the method of the present invention, tangential flow and/or hollow fiber ultrafiltration techniques are used to purify the solution of cross-linked material by removing carrier carbohydrates and proteins and to concentrate the solution. Pore sizes for use with the present invention are those whose pore size is smaller than the size of the components of the crosslinking composition. In some embodiments, the cross-linked material is mTG and the pore size is in the range of 10-50 kDa. In one embodiment, the cross-linking material is mTG, and the pore size is in the range of 10-30 kDa. In some embodiments, non-limiting commercial examples of this type are uniflux (GE), AKTA Pilot (GE), or AKTA Flux 6 (GE).

In some embodiments, one or more size exclusion chromatography steps are used to selectively separate the cross-linked material from surrounding material (e.g., without limitation, a phenyl sepharose FF column (2.6 x10 cm, Pharmacia Biotech) or, for example, a Sephacryl column (GE)). In some embodiments, one or more hydrophobic and/or hydrophilic interaction chromatography steps are used to selectively separate the cross-linked material from surrounding substances. In some embodiments, the cross-linked material is a protein and one or more ion exchange chromatography steps are used to bind the cross-linked protein, purifying it from surrounding material.

In some embodiments, the cross-linked protein is mTG, and one or more cation exchange chromatography steps are used to purify the mTG. In some embodiments, the cation exchange resin is an agarose resin.

In some embodiments, the purification reduces the endotoxin level of the crosslinked material to less than 5 Endotoxin Units (EU)/gram. In some embodiments, purification reduces the endotoxin level of the crosslinked material to 0.001 to 5 Endotoxin Units (EU)/gram.

In some embodiments, the crosslinking agent is mTG, and purification results in a mTG composition where the specific activity is greater than 20 enzyme units/mg and greater than 25 units/mg. In some embodiments, the cross-linked material is mTG, and purification results in a mTG composition where the specific activity is 10 enzyme units/mg to 35 units/mg. In some embodiments, the cross-linked material is mTG, and purification results in an electrophoretic purity of 95% to 99.9%.

In some embodiments, as non-limiting examples, described herein are mTG purification processes that purify food grade mTG products to produce mTG compositions having specific activity greater than 24 enzyme units/mg, electrophoretic purity greater than 95%, endotoxin units/g less than 5, and CFU/g less than 10. As non-limiting examples, described herein are mTG purification processes that purify food grade mTG products to produce mTG compositions having specific activity of 25-35 enzyme units/mg, electrophoretic purity of 95-99.9%, 0.001 to 5 endotoxin units/g, 0.001<10CFU/g, or any combination thereof. In some embodiments, the purified enzyme of the specification is then lyophilized, with or without additional carbohydrate or stabilizer, and then subjected to terminal sterilization by gamma or electron beam radiation. In some embodiments, the specific activity after terminal sterilization is 5-30 enzyme units/mg. In some embodiments, the specific activity after terminal sterilization is 20-30 enzyme units/mg. In some embodiments, the specific activity after terminal sterilization is 20-25 enzyme units/mg.

In some embodiments, as a non-limiting example, a mTG purification method is described in the publication "purification and Characterization of Novel transflutinase from Bacillus subtilis spores"; PMID 11193401.

In some embodiments, after purification, the mTG may be dried or freeze-dried and then micronized by grinding (of any type) into hygroscopic particles of 5 to 50 microns in a manner similar to that described herein. In some embodiments, after purification, the mTG may be mixed with cellulose ether (HPMC) or trehalose as a stabilizing hydrocolloid.

In some embodiments, transglutaminase can be mixed with maltodextrin. In some embodiments, the maltodextrin is a stabilizer.

In some embodiments, the enriched and/or purified enzyme may be stabilized by adding microparticles comprising mTG and a stabilizing excipient, and further comprising an additive material, such as a stabilizing agent that may aid in stabilization during irradiation (e.g., without limitation, cellulose, sugar, maltodextrin, carotenoids, ascorbic acid, L-tyrosine).

In some embodiments, the modified transglutaminase comprises a modified microbial transglutaminase. In some embodiments, the polymer is modified to allow crosslinking by the modified microbial transglutaminase. In some embodiments, the modified oxidase comprises one or more of tyrosinase, laccase, peroxidase, or any combination thereof. In some embodiments, the matrix further comprises a carbohydrate as at least one substrate polymer, the carbohydrate comprising a phenolic acid for crosslinking by the modified oxidase. In some embodiments, the carbohydrate comprises one or more of arabinoxylan or pectin. In some embodiments, the enzyme molecule is modified by pegylation, and wherein the pegylation provides immunogenic masking by masking the enzyme molecule from the immune system of the host animal receiving the matrix. In some embodiments, the host animal is a human.

Compositions according to some embodiments of the invention

In some embodiments, the present disclosure provides compositions

Wherein the composition is a porous scaffold and the composition is,

wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising:

a) a cross-linkable protein selected from the group consisting of collagen and gelatin;

b) a cross-linking agent that induces cross-linking of the cross-linkable protein; and

c) a liquid.

As used herein, the term "scaffold" refers to a scaffold that has been engineered to cause desired cellular interactions to contribute to the formation of new functional tissue for medical purposes. Cells can be 'seeded' into these structures that are capable of supporting the formation of three-dimensional tissues. The scaffold can mimic the natural extracellular matrix of natural tissues, recapitulate the in vivo environment and allow the cells to influence their own microenvironment. The stent may serve at least one of the following purposes:

1) allowing cells to attach and migrate;

2) deliver and retain cells and biochemical factors;

3) allowing diffusion of vital cell nutrients and expression products; or

4) Exert certain mechanical and biological effects to modify the behavior of the cellular phase.

In some embodiments, the liquid is a physiological buffer.

In some embodiments, the composition is a foam.

In some embodiments, the crosslinkable protein is incorporated into the composition as a micronized protein powder having an average particle size of 5 to 200 microns.

In some embodiments, the crosslinkable protein comprises 200 to 300 bloom of gelatin.

In some embodiments, the crosslinkable gelatin is present in the composition in a range of 0.5% w/w to 25% w/w.

In some embodiments, the crosslinking agent is transglutaminase.

In some embodiments, the transglutaminase is present in the composition in a range of 0.0001% w/w to 2% w/w.

In some embodiments, the present disclosure provides a composition comprising:

a) a cross-linkable gelatin;

b) transglutaminase inducing cross-linking of the cross-linkable gelatin; and

c) the liquid is a mixture of a liquid and a gas,

wherein the composition is a porous scaffold having a pore size of 1 to 500 microns,

wherein the cross-linkable gelatin is introduced into the composition as a micronized gelatin powder having a particle size of 1 to 200 microns,

wherein the crosslinkable gelatin is 200 to 300 bloom,

wherein the crosslinkable gelatin is present in the composition in the range of 0.5% w/w to 25% w/w, and

wherein the transglutaminase is present in the composition in the range of 0.0001% w/w to 2% w/w.

In some embodiments, the liquid is a physiological buffer.

In some embodiments, the composition is formed in situ in the patient at a site in the patient where treatment of the tissue defect is desired.

In some embodiments, the composition is formed prior to introducing the composition into the patient at a site where the patient is in need of treatment of a tissue defect.

In some embodiments, the dried crosslinking agent is transglutaminase. In some embodiments, the dried protein composition is gelatin. In some embodiments, the dry particulate protein does not require a stabilizer. In some embodiments, the powder composition dissolves into a flowable solution in less than 5 minutes. In some embodiments, the powder composition dissolves into a flowable solution in less than 5 minutes. In some embodiments, the powder composition dissolves into a flowable solution in less than 5 minutes at a temperature of less than 37 ℃. In some embodiments, the powder composition dissolves into a flowable solution in less than 1 minute at a temperature of less than 27 ℃. In some embodiments, the powder composition dissolves into a flowable solution in less than 1 minute at a temperature of less than 20 ℃ (which is the standard temperature for operating rooms). In some embodiments, the gelatin composition is stored in a single compartment along with the crosslinker powder. In some embodiments, the gelatin powder and cross-linking agent are mixed with the liquid at a ratio of up to 10ml/1 gram of gelatin. In some embodiments, the gelatin powder and cross-linking agent are mixed with the liquid at a ratio of up to 8ml/1 gram of gelatin. In some embodiments, the gelatin powder and cross-linking agent are mixed with the liquid at a ratio of up to 6ml/1 gram of gelatin. In some embodiments, the gelatin powder and cross-linking agent are mixed with the liquid at a ratio of up to 4ml/1 gram of gelatin. In some embodiments, the gelatin powder and cross-linking agent are mixed with the liquid at a ratio of up to 2ml/1 gram of gelatin. In some embodiments, the powder mixture is pressed into an absorbable or non-absorbable pad to provide a mechanical backing thereto. In some embodiments, the mat is a non-woven oxidized cellulose. In some embodiments, the pressure is pressed into a degradable or non-degradable surgical mesh. In some embodiments, the concentration of the gelatin composition is in the range of 0.5% -25% w/w. In some embodiments, the concentration of the gelatin composition is in the range of 8-20% w/w. In some embodiments, the dry gelatin powder contains less than about 15% moisture. In some embodiments, the dry gelatin powder contains less than about 8% moisture. In some embodiments, the composition has a pH in the range of about 6 to about 7. In some embodiments, the dry crosslinker powder contains less than about 15% moisture. In some embodiments, the dry crosslinker powder contains less than about 8% moisture. In some embodiments, the transglutaminase is calcium independent.

In some embodiments, the protein concentration of the transglutaminase is present in an amount of about 0.0001% to about 2% w/w of the composition, in some embodiments, the transglutaminase is present in an amount of about 0.01% to about 1.35% w/w of the composition, in some embodiments, the concentration of the transglutaminase is in the range of about 1 to about 180 enzyme units (U/m L) of the total composition.

In some embodiments, if the enzyme and gelatin are in solution, the ratio of the enzyme composition to the gelatin composition is about 1:1 to 1:5 v/v. In some embodiments, if the enzyme and gelatin are in solid dry form, the ratio of purified enzyme composition to gelatin composition is about 1:100 to 1:500 w/w.

In some embodiments, the composition further comprises a plasticizer selected from the group consisting of acetylated polyethylene glycol, alkyl citrate, glyceryl monostearate, a polyoxyethylene, or a combination thereof, in some embodiments, the gelatin is of type a (acid treated) or type B (alkali treated), in some embodiments, the gelatin comprises a high molecular weight gelatin of at least about 250 bloom, or an equivalent thereof, in some embodiments, the composition further comprises a surfactant, in some embodiments, the surfactant is selected from polysorbate 20(tween. tm.20), polyoxyethylene glycol lauryl ether (brij. tm.35), polyoxyethylene-polyoxypropylene block copolymer (pluronic. tm.f-68), sodium lauryl sulfate (S L S) or sodium lauryl sulfate (SDS), sodium laureth sulfate or sodium lauryl ether sulfate (S L ES), poloxamer or poloxamine, alkyl polyglucoside, fatty alcohol, fatty acid salts, cocamide monoethanolamine, cocamide diethanolamine, or any combination thereof.

In some embodiments, the composition of the present invention is a crosslinkable composition comprising a milled lyophilized gelatin composition and a dried transglutaminase composition, wherein the dried transglutaminase composition is well dispersed throughout the milled lyophilized gelatin composition.

In some embodiments, the composition of the present invention is a crosslinkable composition comprising gelatin and a crosslinking agent, wherein the crosslinking agent reacts with the gelatin upon mixing to form a biodegradable stable porous scaffold. In some embodiments, the porous scaffold remains flexible on the tissue for at least two weeks. In some embodiments, the transglutaminase is a modified enzyme molecule, the modified enzyme molecule having a modification that changes the perceived volume of the enzyme molecule in the cross-linked matrix when the matrix is formed by cross-linking of the polymer. In some embodiments, the gelatin has an endotoxin content of 1200i.u./g or less. In some embodiments, the gelatin is jet milled by using a pressure drop of less than 10 bar. In some embodiments, the gelatin is jet milled by using a pressure drop of less than 5 bar. In some embodiments, gelatin jet milling is by using less than 5m^A3/min of the required gas flow. In some embodiments, gelatin jet milling is by using less than 2m^A3/min of the required gas flow. In some embodiments, the composition further comprises barium, iodine, other radiopaque substances, or combinations thereof.

In some embodiments, the methods of the present invention comprise preparing/sterilizing a gelatin and crosslinker composition using radiant energy, wherein the resulting irradiated composition has at least 25% functional properties (e.g., crosslinking ability, enzyme specific activity) compared to the non-irradiated starting composition. In some embodiments, the methods of the present invention comprise preparing/sterilizing a gelatin-crosslinker composition using radiant energy, wherein the resulting irradiated gelatin has a functional property (e.g., crosslinking ability) of 25% to 100% as compared to the non-irradiated starting composition.

In some embodiments, the methods of the present invention comprise using ethylene oxide to prepare/sterilize gelatin and crosslinker compositions, wherein the resulting treatment composition has at least 25% functional properties (e.g., crosslinking ability) as compared to the untreated, unsterilized starting gelatin. In some embodiments, the methods of the present disclosure include the use of ethylene oxide to prepare/sterilize compositions, wherein the resulting treatment composition has 25-100% functional properties (e.g., crosslinking ability) as compared to the untreated, unsterilized starting composition.

In some embodiments, the compositions of the present invention are used for at least one purpose selected from the group consisting of: scaffolds for cells, tissue remodelling agents, bulking agents, dermal fillers, bone cements, tissue fillers, lung volume reducing compositions, surgical sealants, bioadhesives, fistula repair compositions, haemostats, surgical meshes, compositions for sustained release of bioactive agents.

In some embodiments, at least one cell type infiltrates into the composition and grows therein.

In some embodiments, the at least one cell type is a cell type selected from the group consisting of: pancreatic stem cells, intestinal secretory cells, bone cells, liver cells, tendon cells, muscle cells, blood cells, chondrocytes, epithelial cells, endothelial cells, neurons, embryonic stem cells, mesenchymal stem cells, autologous bone marrow-derived mesenchymal stem cells, progenitor cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, bone system stem cells, chondrocyte-lineage stem cells, epithelial stem cells, and hepatic stem cells.

In some embodiments, the composition isolates the infiltrated at least one cell type from the immune system of the patient.

In some embodiments, the composition is a foam. Without intending to be bound by any particular theory, once crosslinked (e.g., by mTG), the porous scaffold can remain stable in vivo and induce tissue ingrowth and regeneration. Alternatively, the porous scaffold may act as a three-dimensional support scaffold for the cells. The cross-linked porous scaffold has improved cell attachment and motor compatibility.

In some embodiments, the cross-linkable protein is mixed with a cross-linking agent. In some embodiments, the cross-linkable protein and the cross-linking agent are dry powders, and the dry powders are mixed and then solubilized by a physiological fluid, buffer, or cell support medium. In some embodiments, the crosslinking agent crosslinks the protein only when both the crosslinkable protein and the crosslinking agent are dissolved in a physiological buffer.

In some embodiments, the medical material of the present invention generally includes a medium component, a salt (buffer component) required for cell culture, in addition to the above components. In addition, when implanted, it may also contain tissue in the graft unit that promotes regeneration (growth factor). Useful growth factors such as fibroblast growth factor (FGF: acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and the like, Keratinocyte Growth Factor (KGF)), epithelial cell growth factor (EGF), Nerve Growth Factor (NGF), Transforming Growth Factor (TGF), platelet-derived growth factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), bone morphogenetic protein (BMP: BMP-2, BMP-3, BMP-7 and the like) can be exemplified. These growth factors can be used by appropriate selection according to the type of tissue used for propagation purposes. Specifically, for example, epidermal growth factors in the case of the purpose of epidermal cell regeneration and in the case of the purpose of dermal regeneration, each fibroblast growth factor may be used. Specifically, for example, bone morphogenetic proteins in the case of bone cell regeneration purposes and in the case of dermal regeneration purposes, each fibroblast growth factor can be used. If it is preferable when it is considered, it may be used in combination with two or more kinds of growth factors.

In some embodiments, the dry powder may be packaged in a syringe or any container. In some embodiments, the dry powder may be sterilized by radiation or ethylene oxide (ETO).

In some embodiments, the cross-linkable protein is mixed and the cross-linking agent is combined with at least one other agent selected from the group consisting of: stabilizers (such as, but not limited to, EDC (1-ethyl-3- (3- (dimethylaminopropyl) -carbodiimide), NHS (N-hydroxysuccinimide), carbonamides, glutaraldehyde, horseradish peroxidase, growth factors, therapeutics, and hormones.

In some embodiments, the dry powder has reduced leaching and/or interactions when mixed as a dry powder. Referring to fig. 2, in some embodiments, the dry powders are separately dissolved in physiological buffer and mixed by pushing the dissolved components from one interconnected syringe to another. However, any mixing method, such as stirring, may suffice. Alternatively, the dry powders are mixed together and the mixture is then dissolved in physiological buffer for activation.

In some embodiments, cross-linking of the protein results in a porous scaffold. In some embodiments, the porous scaffold is thermally stable. In some embodiments, the porous scaffold is adhesive. Without intending to be bound by any particular theory, the crosslinking is irreversible due to the covalent bonds that form crosslinks between protein molecules. In some embodiments, the porous scaffold is structurally similar to the extracellular matrix of mammalian tissue, can often be processed under mild conditions, and can be delivered in a minimally invasive manner.

Crosslinking may occur outside or inside the patient's body. Thus, in some embodiments, crosslinking occurs in situ at a site in the patient. Alternatively, the porous scaffold is formed ex vivo and then introduced into the patient.

In some embodiments, the hydrogels of the present invention, when not yet cured, can be passed through a needle once delivered to the tissue in liquid form; the adhesive is stabilized into a stable and consolidated physical formation (i.e., unified individual particles of at least 0.05ml volume).

The results of example 4 indicate that by reducing the particle size below d (0.5) ═ 15 microns, gelatin dissolves well, but reacts slower with microbial transglutaminase. In some embodiments of the present invention, it is desirable to provide a rapidly dissolving powder that is not rapidly stable upon hydration. They require passage of long needles and allow a sufficiently long working time for the surgeon. In some embodiments, such formulations may be achieved by controlling the gelatin particle size between 1-15 microns. In some embodiments, such formulations may be achieved with gelatin particle sizes between 8-14 microns.

In some embodiments, the composition comprises a porous scaffold that is configured (1) to be stable in situ or ex vivo, and (2) to conform to a desired shape or body cavity, resulting in the formation of a biocompatible sealant or scaffold that is configured to allow for the ingrowth of cells and tissues.

In some embodiments, the stable porous scaffold structure is tissue conductive and may be used for the following medical uses: tissue remodelling agent, bulking agent, tissue bulking agent or tissue printing (e.g. 3D tissue printing). Powdering and/or reconstituting of gelatin into a cross-linked porous scaffold (particularly by transglutaminase) greatly improves cell attachment and motility. The heterogeneous and rich display of integrin recognition motifs (e.g., RGD) on gelatin porous scaffolds and the accessibility of cells to such motifs enhances their function as scaffolds.

In tissue printing embodiments, dry powder may be applied by a printer in combination with living cells to construct precise 2D or 3D structures of cells adhered to each other by powdered or reconstituted glues. For example, such 3D structures may contain, as an example, some endothelial cells for forming blood vessels, which may be printed in the stent in order that they provide a blood supply to the internal cells once the stent is implanted.

In some embodiments, the porous scaffold has a density, wherein the density is directly related to degradation and/or cell ingrowth kinetics of the porous scaffold. In some embodiments, the additional bioactive substance can also affect cell ingrowth kinetics (i.e., increase cell ingrowth kinetics) by, for example, but not limited to, an increase of 10% to 300%. In some embodiments, the suspended bioactive substances and/or cells and/or Platelet Rich Plasma (PRP) and/or growth factors and/or bone morphogenic proteins, are substantially retained in the target areas of the body (e.g., without limitation, where the patient does not need to keep the treated body area fixed for unreasonable long periods of time as in normal practice, or multiple injections with factors).

In some embodiments, the foam is initially a closed cell foam having a modulus of elasticity (young's) between 0.1-11KPa, wherein the modulus of elasticity can be varied by varying the concentration of protein, cross-linking agent and/or physiological buffer, and/or by varying the bloom of gelatin used therein.

In some embodiments, the protein foam has an initial elasticity (young) modulus between 1-11 KPa. In some embodiments, the protein foam has an initial elasticity (young) modulus of between 2-10 KPa. In some embodiments, the initial (i.e., about 30 minutes after crosslinking) elongation of the stable foam is from 0.1 to 1 times the original starting length. In some embodiments, the stable foam has an initial elongation of 0.1 to 1.5 times the original starting length. In some embodiments, the stable foam has an initial elongation of 0.1 to 2 times the original starting length. In some embodiments, the stable foam has an initial elongation of 0.1 to 3 times the original starting length. In some embodiments, the stable foam has an initial elongation of 0.1 to 4 times the original starting length. In some embodiments, the stable foam has an initial elongation of 0.1 to 5 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1 to 2 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1 to 3 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1 to 4 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1 to 5 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1.5 to 2 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1.5 to 3 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1.5 to 4 times the original starting length. In some embodiments, the stable foam has an initial elongation of 1.5 to 5 times the original starting length. In some embodiments, the stable foam has an initial elongation of 2 to 3 times the original starting length. In some embodiments, the stable foam has an initial elongation of 2 to 4 times the original starting length. In some embodiments, the stable foam has an initial elongation of 2 to 5 times the original starting length. In some embodiments, the stable foam has an initial elongation of 3 to 4 times the original starting length. In some embodiments, the stable foam has an initial elongation of 3 to 5 times the original starting length.

An example showing mechanical measurements of a foam comprising gelatin as the protein and mTG as the cross-linker is shown in the table of example 5.

In some embodiments, the pore size of the porous scaffold is 1 to 500 microns in diameter. In some embodiments, the pore size of the porous scaffold is 2 to 400 microns in diameter. In some embodiments, the pore size of the porous scaffold is 2 to 300 microns in diameter. In some embodiments, the pore size of the porous scaffold is 2 to 200 microns in diameter.

In some embodiments, the pore size of the porous scaffold is 2 to 50 microns in diameter. In some embodiments, the pore size of the porous scaffold is 2 to 10 microns in diameter.

In some embodiments, the pore size of the porous scaffold is 10 to 500 microns in diameter. In some embodiments, the pore size of the porous scaffold is 50 to 400 microns in diameter. In some embodiments, the pore size of the porous scaffold is 100 to 400 microns in diameter. In some embodiments, the pore size of the porous scaffold is 200 to 400 microns in diameter. In some embodiments, the pore size of the porous scaffold is 300 to 400 microns in diameter.

Referring to fig. 8, in some embodiments, the pore size of the foam is 1 to 500 microns in diameter. In some embodiments, the pore size of the foam is 2 to 400 microns in diameter. In some embodiments, the pore size of the foam is 2 to 300 microns in diameter. In some embodiments, the pore size of the foam is 2 to 200 microns in diameter.

In some embodiments, the pore size of the foam is 1 to 50 microns in diameter. In some embodiments, the pore size of the foam is 2 to 10 microns in diameter.

In some embodiments, the pore size of the foam is 10 to 500 microns in diameter. In some embodiments, the pore size of the foam is 50 to 400 microns in diameter. In some embodiments, the pore size of the foam is 100 to 400 microns in diameter. In some embodiments, the pore size of the foam is 200 to 400 microns in diameter. In some embodiments, the pore size of the foam is 300 to 400 microns in diameter.

In some embodiments, an anti-foaming agent, such as, but not limited to, polydimethylsiloxane, polysorbate, and the like, may be added to achieve a denser foam, wherein the denser foam may have a shear modulus of 5KPa to 15 KPa.

Without intending to be limited by any particular theory, in some embodiments, the addition of the biocompatible detergent and/or surfactant results in disruption that causes foaming, wherein the disruption is introduced before the foam stabilizes and the reconstituted foam is formed as a flowable gel. In some embodiments, the flowable gel can be mixed with the crosslinker solution (e.g., manually, mechanically, or by simultaneously pushing through a static mixer) to create a homogeneous stabilized gel. In some embodiments, a confluent (i.e., as opposed to a foam) stabilized gel may be subject to degradation over extended periods of time in vivo.

In some embodiments, the composition may include at least one surfactant. As used herein, "surfactant" refers to a compound that reduces the surface tension of water. In some embodiments, the surfactant may be an ionic surfactant, such as sodium lauryl sulfate and caprylic acid; or neutral surfactants such as polyoxyethylene ethers, polyoxyethylene esters, and polyoxyethylene sorbitan.

In some embodiments, dextrin (dextran) may be added to the composition for reducing the rate of degradation kinetics in vivo.

In some non-limiting exemplary embodiments, cell ingrowth can be even further enhanced by the addition of sulfated glycosaminoglycans (GAGs), such as chondroitin sulfate. In some embodiments, GAGs may be added as a copolymer powder in a dry formulation of the powder, chemically bonded to the protein/crosslinker (i.e., mTG), any other component prior to the jet milling stage of preparation, or any combination thereof. In some embodiments, GAG concentration can be from 0.5% w/w to 10% w/w of the composition.

Without intending to be bound by any particular theory, cells within a porous scaffold may involve matrix interactions in three dimensions, similar to their experience in the fibrous environment of the native extracellular matrix. A more natural cell diffusion occurring in three dimensions is achieved compared to 2D scaffolds or less suitable scaffold materials.

Without intending to be bound by any particular theory, the cell shape achieved in the three-dimensional porous scaffold may modify gene expression, protein translation, and sequential function.

In some embodiments, the cells are blended into the porous scaffold at the time of preparation (mixed with the hydration liquid) or subsequently into the porous scaffold where they retain their native 3D architecture. Thus, without intending to be limited by any particular theory, in some embodiments, the scaffold not only preserves the natural 3D shape of individual cells, it also acts to aggregate cells together in a more natural manner. This results in the formation of tissue-like structures and intercellular interactions that are more representative of normal tissue function.

In some embodiments utilizing gelatin, the elastic properties of crosslinked gelatin, as well as the abundance of integrin attachment sites (cell binding sites on the polymer), allow for cellular ingrowth, proliferation and lead to regeneration of physiological tissue, and enable various tissue engineering applications. In some embodiments, crosslinked gelatin can be used to generate tissue-like structures in vitro with multiple cell types. Different types of cells can be grouped together in a 3D co-culture model as a mixed population, or as discrete layers of different cell types. According to some embodiments, gelatin and a cross-linking agent such as mTG may be mixed with the cell culture medium; the cell culture medium contains, but is not limited to, the following: glucose, stabilized glutamine (alanyl-glutamine), penicilliumElemental and CaCl₂·H₂O, iron nitrate (Fe (NO)₃)3-9H₂O), potassium chloride (KCl), MgSO₄·7H₂O, sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium phosphate (NaH)₂PO₄·H₂O), D-glucose, phenol red, L-alanyl-L-glutamine, L-arginine-Hcl, L-cystine, glycine, L-histidine HCl-H₂O, L-isoleucine, L-leucine, L-lysine-Hcl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-calcium pantothenate, choline chloride, folic acid, isoinositol, nicotinamide, pyridol Hcl, riboflavin, thiamine HCl, and fetal bovine serum.

Mixing with the cell culture medium did not compromise the cross-linking of the porous scaffold.

According to some embodiments, gelatin and mTG dry powder may be mixed with cell culture media (containing, but not limited to, glucose, stabilized glutamine (alanyl-glutamine), penicillin, CaCl₂·H₂O, iron nitrate (Fe (NO)₃)3-9H₂O), potassium chloride (KCl), MgSO₄·7H₂O, sodium chloride (NaCl), sodium bicarbonate (NaHCO)₃) Sodium phosphate (NaH)₂PO₄·H₂O), D-glucose, phenol red, L-alanyl-L-glutamine, L-arginine-Hcl, L-cystine, glycine, L-histidine HCl-H₂O, L-isoleucine, L-leucine, L-lysine-Hcl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-calcium pantothenate, choline chloride, folic acid, isoinositol, nicotinamide, pyridol Hcl, riboflavin, thiamine HCl, and fetal bovine serum)Forming the optimal scaffold seeded with cells. An exemplary embodiment showing cell viability in the cross-linked gelatin porous scaffold of the present invention is detailed in example 19.

In some embodiments, the methods of the invention may include modifying the perceived volume of the enzyme molecules in the cross-linked matrix to be formed. In some embodiments, the modified perceived volume is determined as a function of the degree of cross-linking of the polymer forming the matrix, such that a reduced degree of cross-linking compared to the degree of cross-linking with the unmodified enzyme molecule is indicative of an increased perceived volume. In some embodiments, one method of increasing the perceived volume of an enzyme molecule is to increase the size and/or hydrodynamic volume of the molecule by covalent or non-covalent attachment of at least one molecule or moiety to the enzyme molecule. In some embodiments, the methods of the invention include the use of a modified enzyme.

In some embodiments, the method of increasing the perceived volume is by modifying the electrostatic charge of the enzyme molecules such that their net charge is of opposite polarity to the net charge on the polymer or copolymer chain. In one embodiment, increasing the sensing volume can be achieved by changing the isoelectric point (pi) of the enzyme.

According to some embodiments of the compositions of the present invention, there is provided a cross-linked porous scaffold comprising a substrate polymer cross-linked by a modified enzyme molecule, wherein the modified enzyme molecule has a modification that alters the perceived volume of the enzyme molecule when the matrix is formed by cross-linking of the polymer. In some embodiments, the modified enzyme molecule has a modification that increases the actual size of the modified enzyme molecule. In some embodiments, the modified enzyme molecule has a modification that increases the hydrodynamic volume of the modified enzyme molecule. In some embodiments, the modified enzyme molecule has a modification that modifies the electrostatic charge of the modified enzyme molecule to have an opposite sign to the net charge of the substrate polymer by changing the isoelectric point (pi) of the modified enzyme as compared to the unmodified enzyme. In some embodiments, the modification is to the s-amino group of the lysine of the enzyme by a process selected from the group consisting of: succinylation (with succinic anhydride), acetylation (with acetic anhydride), carbamylation (with cyanate), reductive alkylation (aldehyde), and treatment with maleic anhydride. In some embodiments, one or more carboxylic acid-containing side chains directed to the enzyme are modified to reduce the number of negative charges.

In some embodiments, the modification comprises covalent or non-covalent attachment of at least one molecule or moiety to the modified enzyme molecule. In some embodiments, the modification comprises covalent attachment of a modifying molecule to the modified enzyme molecule. In some embodiments, the modified enzyme molecule has a reduced diffusion rate and a reduced crosslinking rate compared to the unmodified enzyme, but has at least a similar measured enzyme activity (e.g., without limitation, about 20% to 100% activity compared to the unmodified enzyme) compared to the unmodified enzyme.

In some embodiments, the reduced crosslinking rate is at least 10% of the unmodified enzyme crosslinking rate. In some embodiments, the reduced crosslinking rate is 10% to 40% of the unmodified enzyme crosslinking rate.

In some embodiments, the modifying molecule comprises a carrier or a polymer. In some embodiments, the polymer comprises a synthetic polymer, a cellulosic polymer, a protein, a polysaccharide, or any combination thereof. In some embodiments, the cellulosic polymer comprises one or more of carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, methyl cellulose, or any combination thereof. In some embodiments, the polysaccharide comprises one or more of dextran, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronic acid, a starch derivative, or any combination thereof.

In some embodiments of the compositions of the present invention, the modifying molecule comprises PEG (polyethylene glycol). In some embodiments, the PEG comprises a PEG derivative. In some embodiments, the PEG derivative comprises an activated PEG. In some embodiments, the activated PEG comprises one or more of the following: methoxy PEG (mPEG), derivatives thereof, mPEG-NHS, succinimide (NHS) esters of mPEG (mPEG-succinate-NHS), mPEG-glutarate-NHS, mPEG-valerate-NHS, mPEG-carbonate-NHS, mPEG-carboxymethyl-NHS, mPEG-propionate-NHS, mPEG-carboxypentyl-NHS), mPEG-nitrophenylcarbonate, mPEG-propionaldehyde, mPEG-tosylate, mPEG-carbonylimidazole, mPEG-isocyanate, mPEG-epoxide, or combinations thereof. In some embodiments, the activated PEG reacts with an amine or thiol group on the enzyme. In some embodiments, the molar ratio of activated PEG to lysine residues of the activated enzyme is in the range of 0.5 to 25. In some embodiments, the activated PEG is monofunctional, heterobifunctional, homobifunctional, or multifunctional. In some embodiments, the activated PEG is a branched PEG or a multi-arm PEG. In some embodiments, the activated PEG has a size ranging from 1000 to 40,000 daltons.

In some embodiments, the porous scaffold further comprises a copolymer that is not covalently bound to the enzyme or substrate polymer. In some embodiments, the copolymer comprises a polysaccharide or a cellulosic polymer. In some embodiments, the polysaccharide comprises dextran, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronic acid, a starch derivative, or any combination thereof. In some embodiments, the cellulosic polymer comprises carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, methyl cellulose.

In some embodiments, the modified enzyme molecule is modified by cross-linking the modified enzyme molecule with a plurality of other enzyme molecules to form an aggregate of the plurality of cross-linked enzyme molecules. In some embodiments, the modification of the enzyme molecule affects at least one characteristic of the substrate. In some embodiments, the at least one property is selected from tensile strength, stiffness, degree of crosslinking of the base polymer, viscosity, elasticity, flexibility, strain at break, stress at break, poisson's ratio, degree of swelling, and young's modulus, or a combination thereof.

In some embodiments, the degree of modification of the modified enzyme determines the mobility of the modified enzyme in or diffusion from the porous scaffold. In some embodiments, the modification of the modified enzyme reduces the diffusion coefficient of the modified enzyme in a solution of the modified enzyme and the protein, or in a porous scaffold of the modified enzyme and the protein, as compared to a solution of the unmodified enzyme and the protein, or a porous scaffold. In some embodiments, the degree of modification of the modified enzyme determines one or more foam mechanical properties. In some embodiments, the modified enzyme molecule exhibits a greater difference in crosslinking rate in the crosslinked polymer than in solution as compared to the unmodified enzyme molecule.

In some embodiments, the powdered cross-linking agent may be an enzyme and/or modified enzyme that reacts with the powdered polymer. In some embodiments, the powdered polymer may be a protein, such as, but not limited to, gelatin.

In some embodiments, a gel-liquid transition point depressant is selected, such as urea or calcium, as non-limiting examples. Such urea as calcium may be added to the dry formulation in dry powder form. Such additions may increase the injectability of the mixed foam through the fine needle. It also collapses the foam in situ to form a combination of porous scaffold and expanded confluent gel. The higher the concentration of such agents, the less scaffold and more confluent hydrogel is formed. This may be suitable for some applications but not for others.

According to at least some embodiments of the present invention, there is provided a method for controlling the formation of a matrix ("matrix" refers to hydrogel or porous scaffold), comprising modifying an enzyme molecule with a modification that changes the perceived volume of the enzyme molecule in a cross-linked matrix as the matrix is formed; mixing the modified enzyme molecule with at least one substrate polymer that is a substrate for the modified enzyme molecule; and forming a matrix by cross-linking of the at least one substrate polymer via the modified enzyme molecule, wherein forming the matrix is at least partially controlled by the modification of the enzyme molecule. In some embodiments, the modification reduces the rate of crosslinking of the modified enzyme molecule as the degree of crosslinking of the at least one substrate polymer increases. In some embodiments, the modified enzyme molecule and the at least one substrate polymer are mixed in micronized powder form such that as the viscosity of the solution increases, the modification controls the degree of crosslinking of the at least one substrate polymer. In some embodiments, the modification comprises pegylation of the enzyme at a pH ranging from 7 to 9. In some embodiments, the pH of the PEGylation reaction is 7.5-8.5.

Guidance for calculating formulation gelatin dilution ("DG"):

for formulations containing both gelatin and mTG in a maltodextrin carrier:

x g gelatin + Y g maltodextrin + Z g liquid

The dilution of gelatin is equal to DG% w/w ═ 100X/(X + Y + Z)

For example: 1 g gelatin +1 g maltodextrin +4 g liquid 1/6-16.6% w/w.

For the gelatin only formulation:

x g gelatin + Z g liquid

The dilution of gelatin is equal to DG% w/w ═ 100X/(X + Z)

For example: 1 g gelatin +4 g liquid 1/5-20% w/w.

The above calculations assume the following:

1 g mTG ═ 1 g liquid in 1 g gelatin ═ maltodextrin carrier;

1ml liquid to 1 gram liquid; and

the weight of the enzyme is negligible compared to the other components.

Guidance for calculating the enzyme dilution of the formulation ("DE"):

for formulations containing mTG in a maltodextrin carrier:

x g gelatin, Y g maltodextrin, E g enzyme, Z g liquid

Dilution of the enzyme equal to DE% w/w ═ 100E/(X + Y + E + Z)

The above calculation assumes:

1mg enzyme-10 units activity;

1 g gelatin-1 g maltodextrin-1 g liquid; and

1ml liquid-1 g liquid.

For formulations containing mTG without maltodextrin:

x g gelatin, E g enzyme, Z g liquid

Dilution of the enzyme equal to DE% w/w ═ 100E/(X + E + Z)

The above calculation assumes:

1mg enzyme-10 units activity;

1 g gelatin to 1 g liquid, the amount of enzyme being negligible; and

1ml liquid-1 g liquid.

Method of treating a patient in need thereof

In one embodiment, the invention provides a method, wherein the method stimulates fibroblasts to produce new collagen in a patient, comprising:

a) forming a composition in the patient in an amount sufficient to induce fibroblast stimulation,

b) wherein the composition is configured to promote fibroblast stimulation and collagen synthesis, wherein the fibroblast attachment and collagen synthesis induces tissue (skin) regeneration, and

c) wherein the composition is a porous scaffold and the composition is,

d) wherein the pores of the scaffold are from 1 to 500 microns, the composition comprising:

i. a cross-linkable protein selected from the group consisting of collagen and gelatin;

a crosslinking agent that induces crosslinking of the crosslinkable protein; and

a liquid.

In one embodiment, the gelatin is present in the composition in the range of 3% w/w to 30% w/w after dilution with a liquid.

In one embodiment, the gelatin is present in the composition in the range of 8% w/w to 25% w/w after dilution with a liquid.

In one embodiment, the gelatin is present in the composition in the range of 9% w/w to 20% w/w after dilution with a liquid.

According to at least some embodiments, there is provided a composition of a vehicle for topical drug delivery comprising a porous scaffold as described herein. According to at least some embodiments, there is provided a composition for tissue engineering comprising a matrix as described herein, suitable as an injectable porous scaffold. In accordance with at least some embodiments, a method of modifying a composition comprises: providing a modified enzyme having a crosslinkable functional group and a protein having at least one moiety that is crosslinkable by the modified enzyme; and mixing the modified enzyme and the protein, wherein the modified enzyme crosslinks the protein and also crosslinks the protein through a crosslinkable functional group.

According to some embodiments, the composition is used as a vehicle for topical drug delivery. According to some embodiments, the composition is an injectable scaffold or tissue remodelling agent for tissue engineering and repair. In accordance with at least some embodiments, there is provided a composition for a vehicle for the local delivery of lidocaine or other analgesic comprising a porous scaffold as described herein.

As used herein, an "bulking agent" is an injectable substance that fills a space for increasing the volume of tissue. They can be injected under the skin for improved cosmetic effect, injected periurethral to treat urinary incontinence, and injected perianal to treat fecal incontinence.

In some embodiments, the compositions of the present invention are bulking agents that are configured to be non-migratory, durable, degradable, configured to be replaced by soft connective tissue over an extended period of time, or any combination thereof. In some embodiments, migration varies as particle size and number vary. In some embodiments, the compositions provide scaffolds for endogenous or exogenous cells that proliferate and maintain a volume effect over time.

In some embodiments, the composition is an in situ cross-linked gel so as to result in a stable and single consolidated physical formation. In some embodiments, the composition is degradable and can be gradually infiltrated by fibroblasts and immune cells without risk of dislocation and configured to be replaced by tissue.

In some embodiments, the compositions of the present invention are degraded by the body over time. In some embodiments, at least 70% to 100% of the composition degrades after 3 months. In some embodiments, at least 70% to 100% of the composition degrades after 6 months. In some embodiments, at least 75% to 100% of the composition degrades after 3 months. In some embodiments, at least 75% to 100% of the composition degrades after 6 months. In some embodiments, at least 80% to 100% of the composition degrades after 3 months. In some embodiments, at least 80% to 100% of the composition degrades after 6 months. In some embodiments, at least 90% to 100% of the composition degrades after 3 months. In some embodiments, at least 90% to 100% of the composition degrades after 6 months. In some embodiments, at least 70% of the composition degrades after 3 months. In some embodiments, at least 75% of the composition degrades after 3 months. In some embodiments, at least 80% of the composition degrades after 3 months. In some embodiments, at least 85% of the composition degrades after 3 months. In some embodiments, at least 90% of the composition degrades after 3 months. In some embodiments, at least 95% of the composition degrades after 3 months. In some embodiments, at least 100% of the composition degrades after 3 months. In some embodiments, at least 70% of the composition degrades after 4 months. In some embodiments, at least 75% of the composition degrades after 4 months. In some embodiments, at least 80% of the composition degrades after 4 months. In some embodiments, at least 85% of the composition degrades after 4 months. In some embodiments, at least 90% of the composition degrades after 4 months. In some embodiments, at least 95% of the composition degrades after 4 months. In some embodiments, at least 100% of the composition degrades after 4 months. In some embodiments, at least 70% of the composition degrades after 5 months. In some embodiments, at least 75% of the composition degrades after 5 months. In some embodiments, at least 80% of the composition degrades after 5 months. In some embodiments, at least 85% of the composition degrades after 5 months. In some embodiments, at least 90% of the composition degrades after 5 months. In some embodiments, at least 95% of the composition degrades after 5 months. In some embodiments, at least 100% of the composition degrades after 5 months. In some embodiments, at least 70% of the composition degrades after 6 months. In some embodiments, at least 75% of the composition degrades after 6 months. In some embodiments, at least 80% of the composition degrades after 6 months. In some embodiments, at least 85% of the composition degrades after 6 months. In some embodiments, at least 90% of the composition degrades after 6 months. In some embodiments, at least 95% of the composition degrades after 6 months. In some embodiments, at least 100% of the composition degrades after 6 months.

In some embodiments, the composition of the bulking agent may be an in situ cross-linked porous scaffold, foam or gel so as to result in a stable and single consolidated physical formation. In some embodiments, the composition consists of a dry micronized powder of gelatin and a crosslinking agent. In some embodiments, the composition consists of a liquid gelatin and a crosslinking agent (in a preferred embodiment, the crosslinking agent is transglutaminase). In some embodiments, the composition consists of a liquid gelatin and a cross-linking agent, wherein other excipients are used with the gelatin to reduce its natural solid-gel transition point (e.g., urea and calcium) to reduce its viscosity, and wherein the liquid transglutaminase contains excipients to enhance its stability and increase its viscosity.

In some embodiments, the foamed gelatin may be delivered through a long and thin catheter or needle. In some embodiments, the methods of the invention are to deliver a gelatin in situ stable foam through a hollow delivery tube that is at least 10cm long and at most 10French in diameter. In some embodiments, the methods of the invention are to deliver a gelatin in situ stable foam through a hollow delivery tube that is at least 15cm long and at most 6French in diameter. In some embodiments, the methods of the invention are to deliver a gelatin in situ stable foam through a hollow delivery tube that is at least 25cm long and at most 6French in diameter. In some embodiments, the methods of the invention are to deliver a gelatin in situ stable foam through a hollow delivery tube that is at least 30cm long and at most 6French in diameter. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 30 gauge. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 27 gauge. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 25 gauge. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 23 gauge. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 21 gauge. In some embodiments, the foamed gelatin may be delivered through a needle having a diameter of at least 18 gauge.

In some embodiments, the bulking agents of the present invention are configured to remove or ameliorate acne scars and correct wrinkles, elevate existing scars, reform facial contours, or any combination thereof.

In some embodiments, the bulking agent of the present invention is a filler material having the following qualities: (i) physiological-incorporating itself with body tissue; (ii) simple procedure-injectable; (iii) no risk-no complications or adverse effects; (iv) semi-permanent-degradation over time; (v) acting as a scaffold for the ingrowth of cells and a tissue remodelling agent or any combination thereof.

In some embodiments, the compositions of the present invention are fillers, such as intradermal fillers. In some embodiments, the bulking agent may be comprised of gelatin and a transglutaminase stabilizer. In some embodiments, the compositions of the present invention may be used to treat indoor pets, such as but not limited to cats and dogs.

In some embodiments, the invention is a method for stimulating fibroblasts to produce new collagen in patient tissue, wherein an injectable tissue matrix specifically engineered to act on local adhesion of fibroblasts is used. In some embodiments, the injectable tissue matrix is made of a biodegradable cross-linked protein (such as, but not limited to, gelatin) foam that is cross-linked in situ by microbial transglutaminase to form a stable and viscous porous matrix with desirable mechanical properties. Such structures provide optimal mechanical and biological support to stimulate cellular ingrowth and allow proper cross-talk between the matrix and fibroblasts. By mimicking the extracellular matrix of the tissue's natural environment, a neocellular-rich tissue or dermal layer can be grown with the biological and structural properties of young skin. The foam degrades and is eventually replaced by the natural-like architecture. The disclosed tissue matrices provided in one embodiment of the invention allow for growth and proliferation of fibroblasts, secretion of collagen, and stimulation of ECM accumulation in enhanced sites, as the foam described herein is slowly degraded by the body, resulting in biological tissue and/or skin regeneration.

In some embodiments of the invention, the gelatin foam scaffold biomaterial may introduce integrin binding sites into the fibroblast environment. Furthermore, by its favourable mechanical characteristics and its adhesion, together with its abundant RDG (integrin attachment site), it imposes biomechanical signals previously lost to the endogenous ECM into the treatment site. Such biomechanical signaling through the scaffold forms adhesive plaques and stimulates fibroblast collagen production while attenuating MMP production. This allows the gelatin foam scaffold to integrate with the body. It can also transduce any biomechanical signal to its nearby environment, causing a phenotypic shift in neighboring cells. Eventually, the gelatin foam scaffold biomaterial will degrade, but not before the stimulated fibroblasts make new interconnected collagen ECM, to support itself and its neighboring cells, resulting in durable skin regeneration.

In some embodiments of the invention, the gelatin foam scaffold biomaterial may induce collagen production from pre-existing fibroblasts, induce proliferation thereof, and induce fibroblast maturation from mesenchymal lineage cells and fibroblasts. It may also promote cellular infiltration into the foam volume, encouraging fibroblasts to produce ECM within the foam volume. Such processes of foam degradation and ECM production lead to long-term effects in the enhancement site. In other embodiments of the invention, the gelatin foam scaffold material may also induce angiogenesis to support new cell populations and matrix production. These vessels will provide nutrient influx/efflux and promote survival of such changes, thus preserving the effect for a long time. Thus, in some embodiments of the invention, the gelatin foam scaffold biomaterial may support the regeneration of dermal skin afflicted with natural or pathological conditions, depending on where more collagen production is needed.

In some embodiments of the invention, the gelatin foam scaffold biomaterial may induce collagen production and enhance the collagen content of the subcutaneous supportive connective tissue, such as (as a non-limiting example) the septum, which enhances the appearance and texture of the skin.

In some embodiments of the invention, the powder or gelatin foam scaffold biomaterials of the present invention are reinforced with peroxide to provide a solution for an open cell structure. In some embodiments of the invention, there are advantages to open cell (cellular) foams over closed cell foams. Therefore, it is suggested that mixing a soluble porogen with the powder of the present invention will increase the interconnectivity of the pores. The porogen can be trapped inside the gelatin foam scaffold biomaterial while mixing the porogen powder with the other powders of the present invention. Such porogens are activated by contact with a liquid and degrade into a gaseous form. Air pressure builds up inside each cell to create cracks and connections between adjacent holes. This provides an oxygen-producing scaffold that can alter the progression of a given pathological condition and promote repair processes including, but not limited to, cell proliferation and collagen synthesis.

In some embodiments of the invention, the crosslinked gelatin foam scaffold biomaterial may also be rendered liquid-deprived after complete crosslinking. This may be done by dehydration, heat drying or freeze drying. The resulting foam is a rigid biomaterial that can be used in a variety of applications. When the dry foam is hydrated, it returns to its soft foam form and can act as a scaffold for cells in vivo or in vitro. Application of such a dry form of foam may be, for example and without limitation, drying the foam on an implant (e.g., an orthopaedic implant) to enhance osseointegration with the implant. Another application may be its use for tissue augmentation and cell activation. The dry foam may be micronized into small particles of 50-500 microns so that it may be injected through a needle into tissue, such as dermal tissue.

In some embodiments of the invention, the crosslinking enzyme is purified to remove any manufacturing impurities, and the resulting product is less susceptible to immune response and degradation. As such, its in vivo degradation profile may be slower and allow more time for new collagen accumulation (tissue remodeling). More collagen accumulation translates into enhanced efficacy.

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

Examples of the invention

Example 1: according to some embodiments of the invention, the composition is tested for tensile strength.

Components

(i)0.25 gram ACTIVA WM enzyme preparation (1% microbial transglutaminase from streptoverticillium mobaraense, 99% maltodextrin) (Ajinomoto, Japan)

(ii)0.25 g of Gelita 275 bloom, type A porcine gelatin (Gelita, Sioux City) medical grade low endotoxin, spray milled from Superfine L td to a particle size range of d (0.1) ═ 4.24um, d (0.5) ═ 16.61um, d (0.9) ═ 31.51um

(iii)1ml of saline (+ and liquid additive)

Method of producing a composite material

The powder was sterilized by 9.93 kilogray e-beam manufactured by L-3 Communications sterility was confirmed by Amino L ab L td. (Rechovot, Israel.) testing was performed according to the sterility test of SOP No.50. WI.110-healthcare products.

Tears are not in the glue but are observed when the collagen sheet itself fails, and therefore, tears are not at the glue sections.

The results demonstrate the ability to terminally sterilize micronized powder without significant loss of activity.

Example 2: and (5) carrying out microscopic examination.

The following gelatin refers to gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to particle sizes in the range of d (0.1) 4.24um, d (0.5) 16.61um, d (0.9) 31.51 um. and mTG refers to ACTIVA (Ajinomoto, japan).

gelatin-mTG foams were prepared and visualized under optical microscope at 10x and 40x magnification to evaluate their foam characteristics.

As a result:

it is detected that the bubbles are locked across the body elements in a random manner. The size of the bubbles varies from 1 to 500 microns in diameter.

Example 3: delivery of compositions according to some embodiments of the invention via a needle.

The gelatin referred to below is gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size range of d (0.1) 4.24 μm, d (0.5) 16.61 μm, d (0.9) 31.51 μm.

Gelatin powder was mixed with mTG at a ratio of 1: 1. 0.75 g of the mixture was mixed manually with 2.63ml of water at 26 ℃ to form a foam. Push the foam through an 18 gauge needle (D ═ 1.03 mm); the length is 150 mm.

As a result:

the foam was successfully delivered from the syringe through the needle. The foam was stable after about 3 minutes and remained stable in water at 50 ℃ (meaning that it was thermally irreversible).

Example 4: testing of various compositions according to some embodiments of the present invention.

The following gelatin, referred to as Gelita 275 bloom, type a porcine gelatin (Gelita, Sioux City) medical grade low endotoxin, was jet milled by Superfine L td to a particle size in the following range:

(A)：d(0.1)＝4.24μm，d(0.5)＝16.61μm，d(0.9)＝31.51μm。

(B)：d(0.1)＝4.415μm，d(0.5)＝13.064μm，d(0.9)＝29.621μm。

(C)：d(0.1)＝13.451μm，(C)：d(0.5)＝94.66μm，d(0.9)＝423.785μm。

gelatin powder was mixed with mTG enzyme: ACTIVA (Ajinomoto, Tokyo) and hydration was performed by hand mixing (two syringes connected, one with powder and one with water). The result was a foam, which was tested for its ability to adhere to two collagen sheets, and for thermal reversibility by immersing the article in a water bath at 50 ℃.

Example 5: bonding and characterization of mechanical properties of the gelatin foams crosslinked with transglutaminase.

Gelatin, referred to below, is gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm mTG, referred to below, indicates ACTIVA powder (Ajinomoto, Tokyo) with an activity level of 100U/gram, mTG containing 1% enzyme and 99% maltodextrin, if the specified sterilized powder-gelatin and mTG micronized powder are sterilized using gamma ray 9.38 kilogray (Sorvan, israel).

The rheological parameters of The cross-linked foam were measured using The L loyd Materials Testing L S1 machine (Amerek Test & calibration instruments), 1kN high precision Materials Testing machine to Test The mechanical properties of The biomaterial.the biomaterial was prepared by manual mixing at The ratios specified in The following table and allowed 40 minutes between mixing and testing.it was injected onto a Teflon coated glass plate, between The spacer and The glass lid with a Teflon surface, creating a rectangular shape 3mm thick once stabilized.Standard dog bone formation was punched out of The biomaterial and used for mechanical testing.the tensile Test was performed by moving The upper clamp upward, stretching The biomaterial until failure (complete tear). The maximum load and percent total elongation at maximum force were determined based on The sample width and thickness.Young' S modulus was calculated by selecting two points representing The most linear part of The force graph.the Test speed was 45 mm/min and a 10N serial number sensor (10N 0360) was used.

Burst pressure testing was performed based on ASTM F2392-04, standard Test Method for Surgical sealant Burst Strength (the standard Test Method for Burst Strength of Surgical Sealants.) for each Burst pressure Test, collagen casing (Nitta Casings, n.j.) was used as the base, a 3-mm diameter hole was punched in each casing sample and each sample was clamped within a sample holding manifold.

As a result:

the results described in the table below clearly demonstrate the ability to manufacture biomaterials according to the invention having mechanical properties that are easily controlled to adapt to different needs in the field of tissue engineering or surgical sealing.

L loyd mechanical test:

burst pressure test:

example 6: characterization of nutrient penetration of the cross-linked gelatin foam.

This example demonstrates the ability of the composition according to the invention to allow nutrient circulation by transferring PBS 10 times from one syringe containing a transglutaminase/gelatin composition to another connected syringe, preparing a foam of the following composition by manually mixing the dry components and allowing to crosslink for 15 minutes before adding the coloring medium.4 m L PBS +1 g gelatin and 1 g mtg. biomaterial with a diameter of 21mm and a thickness of 26 mm.red Maimon's food coloring liquid dye is diluted in water, then the biomaterial is immersed in a colored liquid for 24 hours and then cut in half to measure color spread.

Example 7: cells survived after mixing with the gelatin component.

Gelatin, referred to below as Gelita 275 bloom, type a porcine gelatin (Gelita, Sioux City) medical grade low endotoxin, was jet milled from Superfine L td to a particle size range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

The following examples are intended to demonstrate the ability of Normal Human Dermal Fibroblasts (NHDFs) to withstand the shear forces exhibited by the mixing required to produce the composition according to the invention, and to evaluate the optimal method of incorporating cells into the interior of a biomaterial either by direct mixing or using a three-way stopcock. When used as a cell scaffold for tissue engineering purposes, this hybrid approach ensures uniform diffusion and viability of cells within the scaffold volume.

2ml of NHDF cells were separated from a 100mm plate after reaching 80% confluence by treating with trypsin (Trypsin EDTA solution B (0.25%), EDTA (0.05%), with phenol red, Biological Industries, 03-052-1A) for 3-5 minutes, and then counteracting the trypsin with 6m L serum-containing medium (Darbeke modified eagle Medium, high glucose, Biological Industries batch 1530279, supplemented with 10% certified fetal bovine serum, catalog #: 04-001-1A and 1% penicillin-streptomycin solution, Biological Industries, 03-031-1℃) the cells were centrifuged at 1500RPM for 5 minutes and then resuspended in 6ml of pre-warmed medium to give a stock concentration of 360,000NHDF cells/ml.

Three replicates of mixing gelatin powder with cell-containing medium as described in the table below were performed and cell viability was confirmed using trypan blue (0.5%) diluted 1:1 from cell-containing medium and visual inspection at 4, 24 hours post inoculation. In well 1, gelatin and cell-containing medium were mixed directly using a 2 syringe system (male luer lock connected to a female luer lock). In well 2-3, the gelatin was first dissolved using 2.5ml of medium mixed 8 times, then immediately using a three-way stopcock (Elcam Medical), another 1.5ml of medium containing cells was added, and another 2 times of mixing was completed. A gelatin mixture containing cells was seeded on top 6-well plates of tissue culture treated plastic (corning inc., Costar, product #: 3516).

Cell adhesion was readily found 4 hours after seeding, while 24 hours after seeding, the cells appeared to spread completely on the plate, demonstrating that the cells were viable and able to withstand the shear stress experienced during mixing.

Cell viability assay due to shear stress

Example 8: cell viability inside the cross-linked gelatin foam.

Gelatin refers to Gelita 275 bloom, type A porcine gelatin (Gelita, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm, mTG refers to ACTIVA WM powder (Ajinomoto, Tokyo) with an activity level of 100U/gram, mTG contains 1% enzyme and 99% maltodextrin gelatin and mTG micronized powder are sterilized using gamma ray 9.38 kilogray (Sorvan, Israel.) the following medium refers to serum containing medium (Darlaberray modified eagle medium, high glucose, Biological Industries, supplemented with 10% certified bovine serum, catalog # 04-1-04, and penicillin 0311-1% solution of penicillin, Biomycin-1C.

The following examples are intended to demonstrate that fibroblasts embedded or seeded in a composition according to the invention are able to survive and thrive for 7 days or more.

2ml were treated with trypsin (trypsin EDTA solution B, Biological industries, 03-052-1A) for 3-5 minutes, and after reaching 80% confluence, NHDF cells were separated from 100mm plates and then neutralized with serum-containing medium. Cells were centrifuged at 1500RPM for 5 minutes and then resuspended in culture medium to give a stock concentration of 1,000,000 cells/ml.

Group A: a gelatin biomaterial having a composition of 200mg sterilized gelatin mixed with 120mg sterilized mTG was loaded onto a male luer lock syringe and mixed 10 times with 1ml of cell stock medium (darbeke modified eagle medium, high glucose, Biological industries batch 1530279) to produce a biomaterial and injected onto 6-well plates of untreated plastic (Corning inc., Costar, reference #: 3736, batch #: 30015036).

Group B: the same composition of Biological material and cell-free medium mixed, allowed to crosslink for 40 minutes, and in 3ml of medium (Darbeike modified eagle's medium, high glucose, Biological industries batch 1530279, supplemented with 10% certified fetal bovine serum, catalog # 04-001-1A and 1% penicillin-streptomycin solution, Biological industries, 03-031-1C) added 1x10^A6 cells, placed on a shaker at 37 ℃ for 2 hours before moving into an incubator with 5% CO2 and 37 ℃ O/N. This method allows cells to attach to the gelatin scaffold and grow on its periphery rather than being embedded within its volume.

Group C: biological material used as control and containing no cells, but having the same composition as in groups a and B, respectively.

Groups A, B and C were incubated in an incubator with 5% CO2 and 37 degrees Celsius.

Twenty-four hours after inoculation, all groups were cut into four equal parts with a scalpel. Group B biomaterials were transferred to new plastic 6-well plates that were not treated for cell culture to separate test items from cells that were not attached to the biomaterials, and then 10% Alamarblue v/v was added to all wells. Alamarblue reduction was measured 3 and 6 days after cell inoculation. The Alamarblue reaction was allowed to proceed for approximately 24 hours.

As a result:

the results depicted in fig. 10 clearly demonstrate that the cells inside the biomaterial (group a) and on the biomaterial (group B) are viable and are able to reduce Alamarblue at the indicated time points, as shown by the different colouration of the Alamarblue containing medium compared to the control (group C). These results support the following claims: the cross-linked gelatin foam biomaterial can act as a good scaffold for tissue engineering purposes.

Example 9: according to the inventionThe composition of some embodiments of (a).

The gelatin referred to below is gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, and d (0.9) ═ 31.51 μm.

The following mTG refers to ACTIVA WM powder (Ajinomoto, Tokyo) having an activity level of 100U/g. mTG contains 1% enzyme and 99% maltodextrin. The gelatine and mTG micronised powder was sterilised by 9.93 kgy electron beam (Sorvan, israel). The compositions tested comprised: 0.25 grams of gelatin powder +0.25mTG powder in a syringe was mixed with 1ml of sterile saline.

Two 60kg SW pigs were used in this study. The animals were anesthetized and injections of the test composition (0.3-0.5ml foam) were performed under the skin using a needle in the left front leg. After a 90 day follow-up period, animals were euthanized and tissues harvested for microscopic analysis. Tissue samples were fixed in formalin and shipped for slide preparation and hematoxylin and eosin staining.

As a result:

none of the animals has shown any adverse effects. The biomaterial exhibits good resistance and is mostly degraded. Neither necrosis nor cavity formation and migration was recorded.

Fig. 2A shows the positioning of fibroblasts in the treated tissue at 90 days post-injection. As shown in fig. 2A, fibroblasts are filled, spindle-shaped or star-shaped cells with a centrally placed oval or circular nucleus. This is shown by its basophilic staining. Together, these findings suggest that those fibroblasts are activated and are capable of producing new collagen.

Fig. 2B shows fibroblasts in untreated tissue 90 days after injection. As shown in fig. 2B, fibroblasts are thin and flat with a wavy nucleus and their cytoplasmic volume is reduced, suggesting inactive fibroblasts in the maturation stage.

The composition of the invention can stimulate fibroblast activation and new collagen production, suggesting that it can help reconstitute or restore damaged ECM. In the case of skin application, this is expected to make the skin look and feel like younger skin.

Example 10: scanning electron microscopy was used for imaging the morphology of the cells on the scaffold.

The following gelatin refers to Gelita 275 bloom, type A porcine gelatin (Gelita, Sieux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.the following mTG refers to ACTIVA WM powder (Ajinomoto, Tokyo) with an activity level of 100U/g.mTG contains 1% enzyme and 99% maltodextrin.gelatin and mTG micronized powder are sterilized using gamma ray 9.38 kilogray (Sorvan, Israel.) gelatin: mTG ratio 1 mTG/10mg gelatin.the following medium refers to medium containing serum (Darbeike modified eagle medium, high glucose, Biological Industries (031), bovine serum supplemented with 10% bovine serum # 04, 10 bovine serum, Biotic catalog # 04-1, 1-1% penicillin-2, Biotic).

2ml were treated with trypsin (trypsin EDTA solution B, Biological industries, 03-052-1A) for 3-5 minutes, and after reaching 80% confluence, NHDF cells were separated from 100mm plates and then neutralized with serum-containing medium. Cells were centrifuged at 1500RPM for 5 minutes and then resuspended in culture medium to give a stock concentration of 1,000,000 cells/ml.

The micronized gelatin and mTG powder mixed in one syringe was mixed with cell-free medium, allowed to crosslink for 40 minutes, and 1x10^6 cells were added to 3ml of medium (Darbeke modified eagle medium, high glucose, Biological Industries batch 1530279, supplemented with 10% certified fetal bovine serum, catalog #: 04-001-1A and 1% penicillin-streptomycin solution, Biological Industries, 03-031-1C), placed on a shaker at 37 ℃ for 2 hours before moving into an incubator with 5% CO2 and 37 ℃ O/N. This method allows cells to attach to the gelatin scaffold and grow on its periphery rather than being embedded within its volume.

After 7 days incubation, a portion of the scaffold was gently cut open. Scaffolds with cells were fixed with 4% paraformaldehyde at 4 ℃ for 24 hours. The scaffold was then dehydrated in a gradient acetone series (30, 50, 70, 80, 90, 95, 100). Followed by drying with balzers CPD 030Critical Point Dryer (CPD). Prior to SEM examination, the samples were coated with a gold spraying instrument.

Fig. 4 shows cells seeded in a scaffold composition according to some embodiments of the invention.

As a result:

it was found that fibroblasts were filled (scaffold) into the surface of the scaffold and its pores. The seeded fibroblasts appear in spindle-shaped morphology rather than flat or round as expected from them in vitro. This morphology can also be described as elongated, representing the phenotype of young fibroblasts. Cells are also capable of forming bonds between themselves. Such results demonstrate that cross-linked gelatin foam can affect fibroblast phenotype, promote new collagen production and tissue regeneration.

Example 11: energy of different dermal fillers for collagen deposition in vivo versus mTG cross-linked gelatin foam And (6) comparing the forces.

Gelatin refers to gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for this study. The animals were anesthetized and the test composition (1.6-2.4ml foam) or commercially available dermal filler injected as follows: 6 skin areas of at least 5x 5cm were selected on the ventral region of the pig. A cannula was inserted inside the midpoint of each area to evenly distribute the different skin enhancing dermal fillers and test compositions of mTG cross-linked gelatin foam.

Injected dermal filler:

commercial dermal fillers were used as directed by the manufacturer (Restylane Vital, SCU L PTRA, radiasesse, Restylane).

Collecting samples: 7mm punch biopsies were taken 30 days, 60 days, 90 days and 150 days post-injection, submerged in 10x volume of 4% paraformaldehyde, and further embedded in paraffin. The blocks were cut into 5 μm sections and stained for hematoxylin and eosin for cell infiltration and tissue response analysis, and masson trichrome for collagen deposition analysis

As a result:

fractional histopathological analysis of different dermal fillers versus mTG cross-linked gelatin foam (group F) at 12 and 30 days post injection.

A semi-quantitative ranking of five grades (0, 1, 2, 3, 4) was used, taking into account the severity of the change (0 ═ no lesion, 1 ═ minimal change, 2 ═ mild change, 3 ═ moderate change, 4 ═ marker change). Lesion formation due to needle trauma, high presence of visible lymphocytes.

All materials showed a response to induce foreign body granulation. Cellular infiltration is composed primarily of macrophages and giant cells, and the material has a degradation pattern characterized by absorption by the giant cells that phagocytose the foreign material. No adverse effects such as necrosis, edema or calcification were shown in any of the materials tested.

Figure 5 shows collagen deposition for different dermal fillers at 30 days post injection compared to group F.

The masson trichrome staining of different dermal fillers and mTG crosslinked gelatin foam scaffold biomaterials clearly showed staining with blue collagen deposits. Collagen deposition in mTG cross-linked gelatin foam is better in terms of quantity and organization. Collagen fibers appear to be organized in parallel, whereas in commercially available dermal fillers, collagen fibers are disorganized.

Fig. 6A-6E illustrate compositions of the invention according to embodiments of the invention.

Images are histopathological sections stained by masson trichrome staining of subdermal tissue treated with the cross-linked gelatin foam scaffold biomaterial. Images were from 12, 30, 60, 90, 150 days post injection. The development of new organization of collagen fibers at the treatment site was confirmed. Mainly the sub-dermal membrane is reinforced by the thicker collagen content. The new collagen was stained blue.

And (4) conclusion:

in a 12 day histological examination, fibroblasts were already abundant in the implantation sites of group F (cross-linked gelatin foam scaffold biomaterial). This phenomenon is not observed in other known fillers.

Twelve days after group F injection, histological examination confirmed a mild grade of inflammatory response, which is equivalent to other commercial materials. It should be noted that minimal higher presence of giant cells, macrophages and lymphocytes is present compared to the commercial product which has been highly purified (this can be explained by the impurities of the test product, which is not yet clinical grade).

Thirty days after group F injection, many collagen fibers were observed in the implantation site. The amount of new collagen deposition after group F injection was significantly higher (scores 2-3) compared to the other test materials (scores 0-1). After group F injection, there was no necrosis, luminal formation or edema, confirming good tissue tolerance.

One hundred and fifty days after group F injection, it was noted that the fibrotic septum at the subcutaneous tissue was reinforced and thickened. The fibers appear wavy and naturally organized, rather than disorganized as in scars or fibrotic tissue, indicating the production of type I collagen following injection of crosslinked gelatin. There was no necrosis, luminal formation or edema.

Example 12: increasing the injectability of the viscous material.

Gelatin refers to gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

As a result:

micronized gelatin powder (larger particles are cut off) is screened to reduce the size of the gelatin particles and to narrow the particle distribution. It was detected that when gelatin powder was selected with a mesh containing a specific range of pore sizes, the initial viscosity of the foam could be adjusted after mixing and thus injection could be performed through different gauge needles. This allows modifying the injectability of the foam, which allows the physician to easily handle fine lines without damaging the patient's skin.

The following table describes the improvement in injectability.

Screening of average pore diameter (μm)	Maximum needle gauge for easy injection
		20	28
50	25
		100	22

Example 13: regulating in vivo functionality of a stent

Gelatin refers to gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for this study. Animals were anesthetized and injections of various gelatins with liquid dilutions of the test compositions were injected and collagen production was assessed 30 days after injection.

As a result:

it was detected that by adjusting the ratio of the components (i.e. liquid, gelatin, mTG) the amount of collagen production could be optimized. The following table describes the amount of collagen produced as a function of the ratio between gelatin and liquid (mtgg enzyme remains constant). A gelatin% w/w of 12.5-8% w/w is likely to be more effective than the highest tested gelatin concentration, e.g. 16.6% w/w.

W/w% of diluted gelatin	Collagen production; the maximum effect%
		16.6	70
12.5	100
		8	100

Figure 11A shows a masson trichrome stained histopathological section of tissue treated with the 8% w/w gelatin crosslinked foam scaffold biomaterial 30 days after injection. The results in fig. 11A demonstrate the development of new organization of collagen fibers at the treatment site. The new collagen was stained blue. Fig. 11B shows hematoxylin and eosin staining of tissue confirming the limited presence of macrophages and giant cells after injection. Residues of invisible material, providing overall good biocompatibility results.

Example 14: scar prevention using biodegradable cross-linked gelatin foam,

gelatin refers to gelata 275 bloom, type a porcine gelatin (gelata, Sioux City) medical grade low endotoxin, jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for this study. The animals were anesthetized and seven surgical incisions were made on each of the left and right sides of the animal's back. The left side was used for clinical observation and the right side for histological analysis. A piece of oval skin was removed. With electrocautery, thermal damage to the wound bed is increased to enhance trauma and possible scarring. Before any intervention, a piece of suture is placed in the middle of the incision to access the incision edges. Two more sutures were placed approximately 1cm above and below the middle of the incision. The suture is placed so that a 1-2mm wound opening is visible and the suture does not close too tightly.

The following table describes the wounds and treatment regimen.

As a result:

all lesions were blindly evaluated by dermatologists who excel in scar treatment. According to his observations, groups 3-6 demonstrated significantly better macroscopic results than the control (group 7) and the early injection (groups 1 and 2).

It was shown that treatment of open wounds with cross-linkable gelatin foam at days 3-20 after surgery is beneficial in preventing scar formation. It was also shown that it was not beneficial to treat the wound with foam on day 0.

Figures 7A-7D show the scars formed by groups 1, 4, 6 and 7, respectively, after 150 days post-wound formation.

Example 16: the cross-linked gelatin composition is mixed with Hyaluronic Acid (HA) without loss of material stability in vivo.

Gelatin, referred to below as gelata 275 bloom, type a porcine gelatin (gelata, sieux City) medical grade low endotoxin, was jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for this study the animals were anesthetized and injection of the test composition (1.5m L dilution) was tested for collagen production, which corresponds to 8% gelatin% w/w with 1U mTG/10mg gelatin before compound injection, 1ml hyaluronic acid (Belotero soft) was added to the syringe and mixed again, the material was injected into the subcutaneous tissue using a 21 gauge needle.

As a result:

fig. 12 shows hematoxylin and eosin stained histopathological sections of tissues demonstrating the presence of macrophages and giant cells after injection. The residue of visible material provided overall good biocompatibility results and showed that the material could be injected with hyaluronic acid without compromising gelatin crosslinking by pH-affected enzymes.

And (4) conclusion: the gelatin-crosslinked composition by microbial transglutaminase can be co-injected with other popular dermal fillers made from HA.

Example 17: cross-linked gelatin was followed 150 days for procollagen type I staining in vivo pig models.

Gelatin, referred to below as gelata 275 bloom, type a porcine gelatin (gelata, sieux City) medical grade low endotoxin, was jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for the study the animals were anesthetized and injection of the test composition (1.5m L dilution) was tested for collagen production, which corresponds to 12.5% gelatin% w/w with 1U mTG/10mg gelatin.material was injected into the 5x 5cm area of subcutaneous tissue using a 21 gauge needle biopsies were taken 15, 30, 90 and 150 days post-injection and stained with the procollagen type I antibody abeam 64409.

As a result:FIGS. 13A-D show procollagen type I cells stained after injection. Procollagen type I cells were stained predominantly at 15 and 30 days post injection. Some staining was also visible 90 days after injection. Staining was not visible 150 days after injection.

And (4) conclusion: mTG crosslinked gelatin compositions are capable of inducing collagen type I production through procollagen type I production. The process peaked around 30 days post-injection and subsided by 150 days post-injection.

Example 18: cross-linked gelatin and purified mTG are able to induce collagen production.

Gelatin referred to below is gelata 275 bloom, type a porcine gelatin (gelata, sieux City) medical grade low endotoxin spray milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm, recombinant mTG grown from 25mM tri-acetate pH 6.5, 300mg/m L maltodextrin "formulation 204" lyophilized purified escherichia coli, or mTG lyophilized purified from 25mM tri-acetate pH 6.5 "formulation 203" native mTG refers to ACTIVA WM powder (Ajinomoto, Tokyo) with an activity level of 100U/g, enzyme activity was measured using the hydroxy acid ester (hydroxyym) assay kit available from zedia GmbH Z009.

The micronized gelatin powder (larger particles cut off) was screened using a 100 micron screen to reduce the size of the gelatin particles and reduce the particle distribution range. The gelatine and mTG micronised powder were sterilised using gamma radiation 9.38 kgy (Sorvan, israel).

One 90kg large white pig, 10 months old, that was crossed with Red Duroc pigs was used for this study the animals were anesthetized and injections of the test composition (1.5m L dilution) were tested for collagen production, corresponding to 12.5 and 14% gelatin% w/w with 1U mTG/10mg gelatin.A 21 gauge needle was used to inject the material into a 5x 5cm area of subcutaneous tissue.a biopsy was taken 30 days after injection.

As a result:

fig. 14 shows masson trichrome staining (left) and hematoxylin and eosin staining (right), showing collagen production. Collagen production and immune response were comparable to those of natural preparations containing Activa-WM mTG.

Example 19: mixing of gelatin, mTG, and urea to achieve slower gel time and denser implant (less gas) A bubble).

Gelatin, referred to below as gelata 275 bloom, type a porcine gelatin (gelata, sieux City) medical grade low endotoxin, was jet milled by Superfine L td to a particle size in the range of d (0.1) ═ 4.24 μm, d (0.5) ═ 16.61 μm, d (0.9) ═ 31.51 μm.

Urea was purchased from Sigma Aldrich.

Solutions containing PBS and 0, 1, 2, 4M urea were prepared.

1.5ml of such solution was loaded into a syringe and connected to another syringe containing a mixture of 1:1 gelatin: mTG (500 mg total) and mixed to produce a foam.

As a result:

after mixing, the foam was incubated at 37 ℃ and the gel time (time until the mixture was stable and non-flowing when turned 90 ℃) and the cross-linking time (time until the gelatin: mTG mixture was stable when inserted into a 55 ℃ water bath) were measured. As the concentration of urea increased, the gel time and the crosslinking time increased (no crosslinking was achieved at 4M urea even after 30 minutes). The results show that the addition of urea can increase the injectability of the foam and improve the working time of the surgeon (especially by means of a fine needle). This is due to the delay in physical gelation time of the hydrated gelatin and inhibition of enzyme activity. It is also noteworthy that urea reduces the amount of air bubbles in the foam and, at high doses, breaks many foams into confluent hydrogels.

Publications cited throughout this document are hereby incorporated by reference in their entirety. While various aspects of the present invention have been described above with reference to examples and preferred embodiments, it is to be understood that the scope of the invention is not to be limited by the foregoing description, but is to be defined by the following claims appropriately interpreted under the doctrine of patent law.