COLLAGEN WITH SELECTIVE CHARACTERISTICS, COLLAGEN PRODUCTS CONTAINING SAME AND METHODS FOR PRODUCING SAME

Abstract

Disclosed are methods for forming targeted collagen products having an amplified desired characteristic, including the step of adding peptides exhibiting a desired characteristic to collagen to form the targeted collagen product. The adding step is performed so that the collagen is crosslinked to the peptide exhibiting the desired characteristic. Crosslinking of the collagen to the peptide exhibiting the desired characteristic occurs by modification of the peptide to facilitate binding to the collagen. Further disclosed are methods for forming a targeted collagen product lacking an undesired characteristic, including the step of subtracting peptides exhibiting the undesired characteristic from collagen to form the targeted collagen product. Also disclosed are targeted collagen products formed by the disclosed methods.

Claims

1. A method for forming a targeted collagen product having an amplified desired characteristic, comprising: adding peptides exhibiting a desired characteristic to collagen to form the targeted collagen product; wherein the adding step includes adding the peptides exhibiting the desired characteristic to a mixture to modify collagen into the targeted collagen product; wherein the adding step is performed so that the modified collagen is crosslinked to the peptide exhibiting the desired characteristic and wherein the crosslinking of the collagen to the peptide exhibiting the desired characteristic occurs by modification of the peptide to facilitate binding to the collagen.

2. The method of claim 1, wherein the peptide is modified with a thermal-reactive group.

3. The method of claim 2, wherein the thermal-reactive group is selected from N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, carbodiimide (e.g., EDC), maleimide, bromo- or iodo-haloacetyl, pyridyldisulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, or isocyanate.

4. The method of claim 3, wherein the thermal-reactive group is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC).

5. The method of claim 3, wherein the thermal-reactive group is sulfo-NHS.

6. The method of claim 3, wherein the thermal-reactive group is maleimide.

7. The method of claim 1, wherein the peptide is end capped with amino acids bearing a thermal-reactive side group.

8. The method of claim 7, wherein the amino acids (thermal reactive side group) are selected from Lysine (NH2), Cysteine (SH), Aspartic acid and Glutamic acid (COOH), and Asparagine and Glutamine (H2NCO).

9. The method of claim 7, wherein the peptide is a synthetic peptide synthesized with amino acid end caps using a standard peptide synthesizer.

10. The method of claim 1, wherein the peptide is end capped with chemicals bearing more than one of the same thermal-reactive group, or multiple thermal-reactive groups.

11. The method of claim 10, wherein the thermal reactive groups are selected from polyamino (NH.sub.2), polysulfhydryl (SH), and poly carboxyl (COOH).

12. The method of claim 10, wherein the peptide is modified with a polyamino-bearing chemical.

13. The method of claim 12, wherein the polyamino-bearing chemical is selected from propane-1,2,3-triamine, tris(2-aminoethyl)amine, tetraaminomethane, tetra(2-aminoethyl)methane, 1,1,2,2-ethanetetraamine, and 2,2-bis(aminomethyl)propane-1,3-diamine.

14. The method of claim 10, wherein the peptide is modified with a polycarboxyl-bearing chemical.

15. The method of claim 14, wherein the polycarboxyl-bearing chemical is selected from propane-1,2,3-tricarboxylic acid and citric acid.

16. The method of claim 10, wherein the peptide is modified with a polysulfhydryl-bearing chemical.

17. The method of claim 16, wherein the polysulfhydryl-bearing chemical is selected from 2,3-dimercaptopropionic acid and 2,4-dimercaptopentanedioic acid.

18. The method of claim 10, wherein the peptide is modified with a polyamino and carboxyl-bearing chemical.

19. The method of claim 18, wherein the polyamino and polycarboxyl-bearing chemical is selected from 2,2-diaminoacetic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, and 2,4-diamino-pentanedioic acid (two terminal COOH groups and two NH.sub.2 groups).

20. The method of claim 1, wherein the peptide is a synthetic peptide modified after its synthesis.

21. The method of claim 1, wherein the peptide is modified with a photo-reactive group.

22. The method of claim 21, wherein the photo-reactive group is selected from aryl azides, acyl azides, azidoformates, sulfonyl azides, phosphoryl azides, diazoalkanes, diazoketones, diazoacetates, beta-keto-alpha-diazoacetates, aliphatic azo, diazirines, ketenes, photoactivated ketones, dialkyl peroxides, diacyl peroxides, or peroxyesters.

23. The method of claim 22, wherein the photo-reactive group is an aryl ketone.

24. The method of claim 22, wherein the photo-reactive group is a benzophenone or benzophenone derivative.

25. The method of claim 1, wherein the modified collagen is reconstituted collagen.

26. A method for forming a targeted collagen product having an amplified desired characteristic, comprising: adding peptides exhibiting a desired characteristic to collagen to form the targeted collagen product; wherein the adding step includes adding the peptides exhibiting the desired characteristic to a mixture to modify collagen into the targeted collagen product; wherein the adding step is performed so that the modified collagen is crosslinked to the peptide exhibiting the desired characteristic and wherein the crosslinking of the collagen to the peptide exhibiting the desired characteristic occurs by modification of the collagen to facilitate binding to the peptide.

27. The method of claim 26, wherein the collagen is modified with a thermal-reactive group.

28. The method of claim 27 wherein the thermal-reactive group is selected from N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, carbodiimide (e.g., EDC), maleimide, bromo- or iodo-haloacetyl, pyridyldisulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, or isocyanate.

29. The method of claim 28, wherein the thermal-reactive group is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC).

30. The method of claim 28, wherein the thermal-reactive group is sulfo-NHS.

31. The method of claim 28, wherein the thermal-reactive group is maleimide.

32. The method of claim 26, wherein the collagen is modified with a photo-reactive group.

33. The method of claim 32, wherein the photo-reactive group is selected from aryl azides, acyl azides, azidoformates, sulfonyl azides, phosphoryl azides, diazoalkanes, diazoketones, diazoacetates, beta-keto-alpha-diazoacetates, aliphatic azo, diazirines, ketenes, photoactivated ketones, dialkyl peroxides, diacyl peroxides, or peroxyesters.

34. The method of claim 33, wherein the photo-reactive group is an aryl ketone.

35. The method of claim 34, wherein the photo-reactive group is a benzophenone or benzophenone derivative.

36. The method of claim 35, wherein the photo-reactive group is multiple benzophenones or benzophenone derivatives.

37. The method of claim 36, wherein the reagent bearing multiple benzophenones is tetrakis (4-benzoylbenzyl ether) of pentaerythritol (TBBE).

38. The method of claim 26, wherein the peptide is end capped with amino acids bearing a thermal-reactive side group.

39. The method of claim 38, wherein the amino acids (thermal reactive side group) are selected from Lysine (NH2), Cysteine (SH), Aspartic acid and Glutamic acid (COOH), and Asparagine and Glutamine (H2NCO).

40. The method of claim 39, wherein the peptide is a synthetic peptide synthesized with amino acid end caps using a standard peptide synthesizer.

41. The method of claim 26, wherein the modified collagen is reconstituted collagen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Embodiments of the invention are further described but are in no way limited by the following drawings.

[0187] FIG. 1 is a flow chart of a method according to a first embodiment of the present invention;

[0188] FIG. 2 is a flow chart of a method according to a second embodiment of the present invention;

[0189] FIG. 3 is a flow chart of a method according to a third embodiment of the present invention;

[0190] FIG. 4 is a flow chart of a method according to a fourth embodiment of the present invention.

[0191] FIG. 5 is a flow chart of the method according to fifth embodiment of the present invention;

[0192] FIG. 6 is a schematic view of a multilayer collagen product according to an embodiment of the present invention;

[0193] FIG. 7 is a is a schematic view of a hybrid medical product including a substrate and a collagen layer according to an embodiment of the present invention;

[0194] FIG. 8 is a flow chart of a method according to a sixth embodiment of the present invention;

[0195] FIG. 9 is a flow chart of a method according to a seventh embodiment of the present invention;

[0196] FIG. 10 is a flow chart of a method according to an eighth embodiment of the present invention;

[0197] FIG. 11 is a flow chart of a method according to a ninth embodiment of the present invention;

[0198] FIG. 12 is a list of amino acid functional groups frequently targeted for conjugation;

[0199] FIG. 13 is a list of primary amine reactive chemical groups;

[0200] FIG. 14 is the reaction scheme for NHS ester-mediated coupling of carboxylic acid to a primary amine of a biomolecule;

[0201] FIG. 15 is the reaction scheme for imidoester coupling to a primary amine of a biomolecule;

[0202] FIG. 16 is the reaction scheme for carbodiimide-mediated coupling of carboxylic acid to a primary amine of a biomolecule;

[0203] FIG. 17 is the reaction scheme for carbodiimide-mediated coupling of carboxylic acid to a primary amine of a biomolecule in conjunction with sulfo-NHS;

[0204] FIG. 18 is the reaction scheme for maleimide coupling to a sulfhydryl of a biomolecule;

[0205] FIG. 19 is the reaction scheme for iodoacetyl coupling to a sulfhydryl of a biomolecule;

[0206] FIG. 20 is the reaction scheme for pyridyl disulfide coupling to a sulfhydryl of a biomolecule; and

[0207] FIG. 21A shows the aryl ketone coupling mechanism;

[0208] FIG. 21B is the reaction scheme for aryl ketone coupling to a substrate having abstractable hydrogen;

[0209] FIG. 22 shows the chemical structure of the reagent tetrakis (4-benzoylbenzyl ether) of pentaerythritol (TBBE), which includes four latent reactive benzophenone groups;

[0210] FIG. 23 shows Arginine and Aspartic acid amino acids bearing guanidino and carboxyl side chains, respectively, in the RGD peptide;

[0211] FIG. 24 shows the structures of various polyamino-bearing chemicals;

[0212] FIG. 25 shows the structures of various polycarboxyl-bearing chemicals;

[0213] FIG. 26 shows the structures of various polysulfhydryl-bearing chemicals; and

[0214] FIG. 27 shows the structures of various polyamino- and carboxyl-bearing chemicals.

DETAILED DESCRIPTION OF THE INVENTION

[0215] Reference is made to FIG. 1, which is a flowchart of a first exemplary embodiment of the method (100) according to the present disclosure, as further described below.

[0216] Collagen is procured (110) from an animal (e.g., bovine, porcine, equine, caprine) or a human source for processing, or genetically engineered from microorganisms or genetically engineered cells. Examples of collagen tissue that can be procured for this application are dermis, tendon, peritoneal tissue, pericardium, cartilage. Bone can also be a source of collagen to be further digested and fractionated. The type of collagen can be any type of collagen, for example, type 1, 3, etc. or genetically modified variants. Of course, it is also recognized that collagen can also be derived from genetically engineered microorganisms.

[0217] The collagen is then digested and fractioned (120), to break the collagen down into its constituent fragments, or peptides. Such peptides include, for example, P20 and P35. Commercial preparation is typically accomplished by one of five methods: (1) alkaline hydrolysis; (2) enzymatic hydrolysis (using e.g., pepsin, papain, collagenase, pancreatin, etc.); (3) acid hydrolysis; (4) a hybrid method of chemical/enzyme; (5) synthetically by fermentation. The hydrolyzed collagen is further fractionated by use of ultra-filtration membranes. In various embodiments, the peptides are synthesized on a peptide synthesizer.

[0218] Different peptides have different biological properties that imbue them with desired/desirable attributes/characteristics, such as a higher affinity for therapeutic usage in specific medical applications. In an embodiment, after digestion and fragmentation of the collagen tissue, each of the collagen fractions/peptides is tested (130) for various biological properties, such as, for example, attributes/characteristics that are useful for wound healing, blood clotting, bone formation/osteogenesis, cosmetic application, scaffolds for organ regeneration, use as antioxidants, antibacterial and/or anti-inflammatory activity, and for the delivery of drugs such as insulin and methylene blue, showing lower water absorbency.

[0219] In various embodiments, the collagen is tested by being subjected to an assay. Such an assay may include, for example, the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), as described in H. Yasmin et al., Amplified and selective assay of collagens by enzymatic and fluorescent reactions, Scientific Reports, 4: 4950, May 13, 2014, which is incorporated by reference herein in its entirety. Cellular assays can be used to determine cellular activity, for example osteogenesis, to determine stimulatory activity, osseoinduction or osseoconduction. Another assay could be clotting ability of peptide via a blood assay for wound healing.

[0220] In some embodiments of the method, testing (130) of the collagen fractions/peptides is conducted only once. In other embodiments of the method, such testing may be conducted more than once. In still other embodiments no testing is conducted, e.g., where the biological properties of a collagen fraction/peptide are already known.

[0221] Following the performance of an assay or other testing, and based on the results thereof, or if already known from prior testing, collagen fractions/peptides having a specific desired/desirable property, such as, for example, facilitating wound healing of soft tissue, are identified, isolated (140) and selected for adding to a modified collagen product. In various non-limiting examples described herein, the modified collagen product is a reconstituted collagen product. First, a native source of collagen, such as skin, bone, tendon, or ligament is cleaned, washed, and non-collagenous impurities removed by methods well known in the art (see, e.g., U.S. Pat. No. 5,512,291 and Oneson, et al., J. Am. Leather Chemists Assoc. 65:440-450, 1970, both of which are incorporated by reference herein in their entireties). The fibers obtained from the purification process are then further processed into a reconstituted collagen matrix. The reconstituted collagen matrix can be formed using the following general steps: a) forming an aqueous dispersion containing biopolymeric fibers; b) reconstituting the fibers; c) orienting the reconstituted fibers on a rotating mandrel to form a tubular membrane or forming them into a flat sheet if fabricating a sheet; d) compressing the hydrated fibers to remove excess solution to desired density or thickness; e) drying the fibers; and f) crosslinking the membrane. (see, e.g., U.S. Pat. Nos. 6,391,333, 6,599,524, and 7,807,192, all of which are incorporated by reference herein in their entireties).

[0222] The selected collagen fractions/peptides are added (150) to a mixture to reconstitute collagen into a “targeted collagen” having the amplified desired characteristic of the isolated fractions/peptides, such as a collagen targeted for soft tissue wound healing. In various embodiments, the collagen fractions or synthesized collagen peptide (e.g., CHP or CMP) can be a single-, double- or triple-stranded peptide.

[0223] In one embodiment, the selected fractioned, bioactive peptide(s) are added prior to performing the reconstituted collagen process, and cross-linked into the reconstituted collagen during the process. An alternative to adding the bioactive peptides to the reconstituted collagen would be adding such peptides after processing of the reconstituted collagen so that the bioactive peptides are not covalently linked into the collagen but are able to release, from the reconstituted collagen, over time. In other alternate embodiments, the bio-active peptide(s) are covalently linked to the reconstituted collagen after the collagen is reconstituted. In another embodiment, a bioactive peptide could be removed prior to being reconstituted so that the collagen does not have that bioactive characteristic. In another embodiment, the fraction alone is reconstituted into a medical device. Combinations and variations of these steps are also possible.

[0224] FIG. 2 illustrates an embodiment of the method (200) wherein a bioactive peptide is removed prior to the collagen being reconstituted (or otherwise modified) so that the collagen does not have that bioactive characteristic. By removing a section of the collagen, the remaining collagen may have a bioactive peptide sequence, thereby actually concentrating that bioactive sequence after reconstituting. The method includes several of the same steps of the method (100) shown in FIG. 1 and discussed above, including procuring collagen (210) and digesting and fractioning the collagen (220) to yield peptides. The fractioned collagen peptides are then tested (235) for undesired properties/characteristics, and peptides having such undesired properties/characteristics are then isolated (240) and subtracted from a collagen mixture (255). The collagen mixture is then reconstituted (260) to form the targeted collagen product. In an alternate embodiment, the collagen mixture is not reconstituted, and the targeted collagen product is formed upon completion of the subtracting step (255).

[0225] FIG. 3 illustrates another embodiment of the method (300) that includes the step (350) of adding fractioned collagen peptides with desired properties/characteristics to a mixture to reconstitute (or otherwise modify) collagen into a targeted collagen product. In various embodiments, the method (300) may include additional steps, such as those illustrated in FIG. 1 and discussed above in connection with its associated method (100).

[0226] FIG. 4 illustrates still another embodiment of the method (400) that includes the step (455) of subtracting fractioned collagen peptides with undesired properties/characteristics from a mixture to form a targeted collagen product. In various embodiments, the method (400) may include additional steps, such as those illustrated in FIG. 2 and discussed above in connection with its associated method (200).

[0227] Also disclosed herein are collagen layers and surface coatings (the term “layers” being used herein to include both layers and surface coatings), wherein the collagen has been imbued with selective characteristics for various medical applications (as discussed in the embodiments above). Also disclosed herein are multi-layer collagen products, layered collagen/substrate products and methods for forming same.

[0228] In various embodiments of the disclosed invention, collagen is layered such that each layer contains reconstructed collagen that is different in properties and/or amino-acid content. The layers could contain more or less amino acid sequences that become useful at different stages of a biological process; wound healing process or bone repair process. The layers could contain amino acid sequences that allow collagen fibers to reconstitute at faster or slower rates when processing so that each layer has a distinct amount of collagen fiber so that each layer has different physical properties or biological activities.

[0229] In various embodiments, the layers of collagen are impregnated with active pharmaceutical ingredients (APIs), such as growth factors, bioactive peptides, Steroids, Antibiotics, Oncology, GI, Cardiovascular, Renal, AntiVirals, RNA(s), CNS, Neuromuscular and the like APIs which can be released into or onto the diseased or damaged part of a patient's area following application or implantation.

[0230] Reference is now made to FIG. 5, which is a flowchart of another exemplary embodiment of a method (500) according to the present disclosure, as further described below. The method (500) includes several of the same steps of the method (100) shown in FIG. 1 and discussed above, including procuring collagen (510), digesting and fractioning the collagen (520) to yield peptides, testing each of the collagen fractions/peptides (530) for various biological properties, isolating (140) the collagen fractions/peptides having a specific desired/desirable property, and adding the selected collagen fractions/peptides (550) to a mixture to reconstitute (or otherwise modify) collagen into a “targeted collagen” having the amplified desired characteristic of the isolated fractions/peptides, such as a collagen targeted for soft tissue wound healing. Synthetically produced peptides (i.e., CHPs and/or CMPs) may alternatively be used in various embodiments.

[0231] In some embodiments of the method, the testing (530) of the collagen fractions/peptides is conducted only once. In other embodiments of the method, such testing may be conducted more than once. In still other embodiments no testing is conducted, e.g., where the biological properties of a collagen fraction/peptide are already known.

[0232] In one embodiment, the selected fractioned, bioactive peptide(s) are added prior to performing the reconstituted collagen process, and cross-linked into the reconstituted collagen during the process. An alternative to adding the bioactive peptides to the reconstituted collagen would be adding such peptides after processing of the reconstituted collagen so that the bioactive peptides are not covalently linked into the collagen but are able to release, from the reconstituted collagen, over time. In other alternate embodiments, the bioactive peptide(s) are covalently linked to the reconstituted collagen after the collagen is reconstituted. In other embodiments, the bioactive peptide(s) are added to collagen that has not been reconstituted. In another embodiment, a bioactive peptide could be removed prior to being reconstituted so that the collagen does not have that bioactive characteristic. In another embodiment, a portion of the collagen is removed to enhance the concentration of the remaining bioactive sites. This portion is then reconstituted to form a device. Combinations and variations of these steps are also possible.

[0233] In various embodiments, after the collagen product is formed according to the above steps (either reconstituted or not), it may be formed as or added to one or more layers (560). This layer(s) formation step or layering steps (560) enable the collagen amplification to be engineered in a layered way. In other words, the selected fractioned, bioactive peptide(s) is added to collagen to form the targeted collagen, which is then formed as one or more layers to facilitate control of a biologic process.

[0234] In some embodiments, two or more of the targeted collagen product layers are overlaid on each other to form a layered collagen product, such as a laminate. Reference is made to FIG. 6, which illustrates a layered collagen product CP having three collagen layers, CL1, CL2 and CL3. In various embodiments, the different targeted collagen layers CL1, CL2 and CL3 are engineered to have different bioactive properties. As a non-limiting example, the different collagen layers may be “programmed” to have different release times, e.g., for medications, proteins or other bioactive substance impregnated or selectively absorbed and released from its biological environment within one or more of the layers

[0235] In other embodiments, the targeted collagen layer is used to coat or contact a substrate or other surface that is not a targeted collagen layer. Reference is made to FIG. 7, which illustrates a layered hybrid product CP having a substrate S and a collagen layer CL. Whereas the collagen layer CL constitutes or includes the targeted collagen product formed according to the methods described herein, the substrate S may be untreated collagen, or any other biocompatible material. As a non-limiting example, the foregoing method can be used to form a collagen layer CL that has been amplified with a selected peptide that binds bone morphogenic proteins (BMPs) to cause osteoblast proliferation, and the substrate S is the outer surface of an orthopedic implant on which the collagen layer CL has been deposited In a further embodiment, a surface coating (not shown) is formed on the collagen layer CL opposite the implant surface/substrate S. The surface coating is designed to bind a high concentration of alpha2beta1 at the surface to recruit osteoblasts from a patient's surrounding tissue. Once recruited onto the surface coating, the osteoblasts migrate into the underlying amplified collagen layer CL, the peptide of which binds BMP to cause the recruited osteoblasts to proliferate. In various embodiments, the substrate S includes collagen that has not been modified according to the methods disclosed herein (i.e., unmodified collagen).

[0236] In other embodiments of the method described above, a bioactive peptide is removed from (as opposed to added to) the collagen, so that the resulting collagen product does not have that bioactive characteristic. The collagen may be reconstituted or not reconstituted.

[0237] Targeted collagen product containing the specific collagen fractions/peptides is thus designed for specific medical applications, and provided in or formed as a collagen layer. For example, targeted collagen product layers may be engineered in this manner to address blood clotting, bone formation/osteogenesis, breast repair or wound healing. A programmed multilayer collagen product can be created with different specific collagen fractions/peptides in the different collagen layers, such that the layers have different bioactive properties and functions, as described above.

[0238] Further, it is recognized that a layered targeted collagen product may be formed from the process descried above, as well as any one or more additional processes known in the art.

[0239] Reference is now made to FIG. 8, which is a flowchart of another exemplary embodiment of a method (600) according to the present disclosure, as further described below. The method (600) includes several of the same steps of the method (100) shown in FIG. 1 and discussed above, including procuring collagen (610), digesting and fractioning the collagen (620) to yield peptides, testing each of the collagen fractions/peptides (630) for various biological properties, and isolating (640) the collagen fractions/peptides having a specific desired/desirable property.

[0240] In some embodiments of the method, testing (630) of the collagen fractions/peptides is conducted only once. In other embodiments of the method, such testing may be conducted more than once. In still other embodiments no testing is conducted, e.g., where the biological properties of a collagen fraction/peptide are already known.

[0241] Known collagen mimic peptides (CMPs), single, double and triple helical CMPs, can be synthetically made via a peptide synthesis. Such CMPs can also be added to the reconstituted (or otherwise modified) collagen matrix in various embodiments.

[0242] The selected collagen fractions/peptides are added (665) to a 3D printable collagen mixture, and the collagen mixture is then 3D printed (670) to form a “targeted collagen” having the amplified desired characteristic of the isolated fractions/peptides, such as a collagen targeted for soft tissue wound healing.

[0243] In one embodiment, the selected fractioned, bioactive peptide(s) are added prior to performing the 3D printing collagen process, and cross-linked into the collagen during the printing process. One alternative to adding the bioactive peptides to the printed collagen is adding such peptides after processing of the printed collagen so that the bioactive peptides are not covalently linked into the collagen but are able to release, from the reconstituted (or otherwise modified) collagen, over time. In other alternate embodiments, the bio-active peptide(s) are covalently linked to the printed collagen after or while the collagen is printed. In another embodiment, a bioactive peptide could be removed prior to being printed so that the collagen does not have that bioactive characteristic. Combinations and variations of these steps are also possible.

[0244] FIG. 9 illustrates another embodiment of a method (700) wherein a bioactive peptide is removed prior to being 3D printed so that the collagen does not have that bioactive characteristic. The method includes several of the same steps of the method (200) shown in FIG. 2 and discussed above, including procuring collagen (710), digesting and fractioning the collagen (720) to yield peptides, testing each of the collagen fractions/peptides (735) for various undesired biological properties, isolating (740) the collagen fractions/peptides having the undesired property, and subtracting (755) the collagen fractions/peptides having the undesired property from the collagen mixture. The collagen mixture is then 3D printed (770) to form the targeted collagen product.

[0245] FIG. 10 illustrates another embodiment of a method (800) that includes the step (850) of adding fractioned collagen peptides with desired properties/characteristics to a mixture. The collagen mixture is then 3D printed (870) to form a targeted collagen product. In various embodiments, the method (800) may include additional steps, such as those illustrated in FIG. 8 and discussed above in connection with its associated method (600).

[0246] FIG. 11 illustrates still another embodiment of a method (900) that includes the step (955) of subtracting fractioned collagen peptides with undesired properties/characteristics from a mixture to form a targeted collagen product. The collagen mixture is then 3D printed (970) to form the targeted collagen product. In various embodiments, the method (900) may include additional steps, such as those illustrated in FIG. 9 and discussed above in connection with its associated method (700).

[0247] In various embodiments, the bioactive peptide(s), once determined, can be added to or subtracted from the collagen via genetic modification. The DNA or RNA can be modified via various CRISPR technologies, as well as zinc-finger nucleases (ZFN), or transcription activator-like endonucleases (TALENS). Once the modified collagen is synthesized within the cell, the modified collagen can be isolated via conventional means. In various embodiments, a bioactive peptide is removed from (rather than added to) the genetic modification.

[0248] Targeted collagen product containing the specific collagen fractions/peptides is thus designed for specific medical applications. For example, targeted collagen product may be engineered in this manner to address blood clotting, bone formation/osteogenesis, breast repair, or wound healing.

[0249] In some embodiments, the peptides with the desired characteristic can also be isolated and can also be added to or subtracted from intact collagen. For example, the blood clotting characteristic may be added or concentrated from a target collagen product if such product is intended to be used for a hemostasis product. The collagen is intact in an embodiment.

[0250] In various embodiments, the amplification peptide can be covalently, ionically, hydrogen bonded or through hydrophobic association linked into the collagen and/or allowed to release over time from the collagen.

[0251] In some embodiments, the amplification peptide can be modified with a thermal or photo-reactive group or groups capable of covalently linking to the collagen. In other embodiments, the collagen can be modified with a thermal or photo-reactive group or groups capable of covalently linking to the amplification peptide. Such covalent linking is referred to as bioconjugation, which can occur between polymers or metals and biomolecules, or between two biomolecules. This includes surface conjugation of peptides to biopolymers such as collagen.

[0252] Collagens comprise polymers of nineteen different amino acids, of which some bear side chains with reactive chemical moieties, including hydroxyl (—OH), primary amine (—NH2), sulfhydryl (—SH), and carboxyl (—COOH) (see Gauza-Wlodarczyk M, Kubisz L, Wlodarczyk D. Amino acid composition in determination of collagen origin and assessment of physical factors effects. Int J Biol Macromol. 2017 November; 104(Pt A):987-991).] These functional chemical groups present in the collagen molecule readily react with chemical cross-linkers. Collagen can readily be modified with peptides of the current invention by reacting the crosslinker first with the collagen, then with the peptide, or vice versa. In the latter case, the peptide is first derivatized with reactive groups that target the respective complimentary chemical moieties on the collagen molecule. This can be accomplished by various methods, including wet chemical and photochemical methods.

[0253] Wet chemical methods useful for the present invention are well described in the Thermo Scientific Crosslinking Technical Handbook (Thermo Fisher Scientific Inc.: Waltham, Mass., 2012, available from https://tools.thermofisher.com/content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf. Accessed Jul. 28, 2022, incorporated herein by reference). FIG. 12 shows chemical moieties frequently targeted for bioconjugation; Table 1 lists common chemical crosslinkers reactive with such moieties.

TABLE-US-00001 TABLE 1 Popular crosslinker reactive groups for protein conjugation (Table 1 of Thermo Scientific Crosslinking Technical Handbook.) Target functional Reactivity class group Reactive chemical group Amine-reactive —NH2 NHS ester Imidoester Pentafluorophenyl ester Hydroxymethyl phosphine Carboxyl-to-amine —COOH Carbodiimide (e.g., EDC) reactive Sulfhydryl-reactive —SH Maleimide Haloacetyl (Bromo- or Iodo-) Pyridyldisulfide Thiosulfonate Vinylsulfone Aldehyde-reactive —CHO Hydrazide i.e., oxidized sugars Alkoxyamine (carbonyls) Photo-reactive i.e., random Diazirine nonselective, random Aryl azide insertion Hydroxyl (nonaqueous)- —OH Isocyanate reactive Azide-reactive —N3 Phosphine

[0254] Amine-reactive chemical groups target the primary amines at the N-terminus of the polypeptide chain (alpha-amine), as well as the side chain of lysine residues (epsilon-amine). Due to positive charge at physiologic conditions, primary amines normally locate at the protein surface and thus are accessible for bioconjugation without denaturation. FIG. 13 shows chemical groups that react with primary amines; those most commonly used are NHS esters and imidoesters. FIGS. 14 and 15 show the respective general crosslinking reaction schemes for these reagents.

[0255] Carboxylic acid-reactive chemical groups target the carboxyl C-terminus of the polypeptide chain, as well as the side chains of aspartic acid and glutamic acid. Carboxyl groups also normally locate at the protein surface and are thus accessible for bioconjugation.

[0256] Carbodiimides (EDC and DCC) cause direct conjugation of carboxylates (—COOH) to primary amines (—NH.sub.2). FIG. 16 shows the general reaction scheme for EDC-mediated coupling of carboxylic acid of one biomolecule (e.g., peptide) to a primary amine of another biomolecule (e.g., collagen). FIG. 17 shows the general reaction scheme for EDC coupling in conjunction with sulfo-NHS, which improves reaction efficiency and results in a stable sulfo-NHS derivatized biomolecule that can be stored and used later reacted with a primary amine of a second biomolecule.

[0257] Sulfhydryl-reactive chemical groups target the side chain of cysteine residues. Sometimes disulfide bonds (—S—S—) form between adjacent polypeptide side chains within a protein and must first be reduced to sulfhydryl groups before crosslinking through these groups. Maleimides, haloacetyls and pyridyl disulfide moieties all react with protein sulfhydryl groups. FIGS. 18-20 show the general reaction schemes for maleimide, haloacetyl, and pyridyl disulfide coupling, respectively to a sulfhydryl of a biomolecule. Maleimide chemistry is often used in combination with NHS ester chemistry to produce heterobifunctional crosslinkers that enable controllable, two-step conjugation of peptides and/or proteins.

[0258] In addition to amino acids bearing amino, carboxyl, or sulfhydryl side groups, other chemicals bearing amino, carboxyl, or sulfhydryl groups can also be used to end cap CHP and CMP peptides. Preferably, such chemicals bear a terminal amino or carboxyl group capable of reacting with the C-terminus carboxyl or N-terminus amino group, respectively of the CHP or CMP peptide, and multiple amino, carboxyl, or sulfhydryl side groups, or combinations thereof, to be targeted for bioconjugation with greater efficiency than the unmodified peptide.

[0259] Polyamino-bearing chemicals include propane-1,2,3-triamine (three NH.sub.2 groups), tris(2-aminoethyl)amine (three NH.sub.2 groups), tetraaminomethane (four NH.sub.2 groups), tetra(2-aminoethyl)methane (four NH.sub.2 groups), 1,1,2,2-3 ethanetetraamine (four NH.sub.2 groups), and 2,2-bis(aminomethyl)propane-1,3-diamine (four NH.sub.2 groups).

[0260] Polycarboxyl-bearing chemicals include propane-1,2,3-tricarboxylic acid (three COOH groups) and citric acid (three COOH groups).

[0261] Polysulfhydryl-bearing chemicals include 2,3-dimercaptopropionic acid (one terminal COOH and one terminal SH group, and one additional SH group) and 2,4-dimercaptopentanedioic (two terminal COOH groups and two SH groups)

[0262] Chemicals bearing both polyamino and carboxyl groups include: 2,2-diaminoacetic acid (one terminal COOH and one terminal NH.sub.2 group, with one additional NH.sub.2 group), 2,4-diaminobutyric acid (one terminal COOH and one terminal NH.sub.2 group, with one additional NH.sub.2 group), 2,3-diaminopropionic acid (one terminal COOH and one terminal NH.sub.2 group, with one additional NH.sub.2 group), 2,4-diamino-pentanedioic acid (two terminal COOH groups with two NH.sub.2 groups).

[0263] In addition to wet chemical methods, photochemical methods are also useful for bioconjugation. U.S. Pat. No. 5,563,056, incorporated herein by reference, describes latent reactive groups and the residue functionality of each upon activation as shown in Table 2.

TABLE-US-00002 TABLE 2 Latent Reactive Groups and Their Residues Upon Activation described in U.S. Pat. No. 5,563,056. Latent Reactive Group Residue Functionality aryl azides amine R—NH—R′ acyl azides amide R—CO—NH—R′ azidoformates carbamate R—O—CO—NH—R′ sulfonyl azides sulfonamide R—SO.sub.2 —NH—R′ phosphoryl azides phosphoramide (RO).sub.2 PO—NH—R′ diazoalkanes new C—C bond diazoketones new C—C bond and ketone diazoacetates new C—C bond and ester beta-keto-alpha-diazoacetates new C—C bond and beta-ketoester aliphatic azo new C—C bond diazirines new C—C bond ketenes new C—C bond photoactivated ketones new C—C bond dialkyl peroxides ethers diacyl peroxides esters and new C—C bonds peroxyesters ethers, esters, and new C—C bonds

[0264] FIGS. 21A and 21B illustrate the general photo coupling reaction of aryl ketones using derivatized benzophenone as example (see Simso E J. PhotoLink: a new coating concept, Textile Technology International. 1996; 1:23, available at https://p2infohouse.org/ref/33/32073.pdf). U.S. Pat. No. 5,744,515, incorporated herein by reference, describes the associated coupling mechanism as follows. “Photo-reactive aryl ketones such as acetophenone and benzophenone, or their derivatives, are preferred, since these functional groups, typically, are readily capable of undergoing the activation/inactivation/reactivation cycle described herein. Benzophenone is a particularly preferred photo-reactive group, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes intersystem crossing to the triplet state. The excited triplet state can insert into carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support surface, for example), thus creating a radical pair. Subsequent collapse of the radical pair leads to formation of a new carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is not available for bonding, the ultraviolet light induced excitation of the benzophenone group is reversible and the molecule returns to ground state energy level upon removal of the energy source. Photoactivatable aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency. Hence, photo-reactive aryl ketones are particularly preferred.”

[0265] Particularly useful in this regard is a heterobifunctional crosslinking agent bearing a photo-reactive benzophenone group at one end and an amine-reactive N-oxy-succinimide (NOS) ester group at the other, the ends tethered together via an epsilon aminocaproic acid (EAC) spacer. Such a compound is useful to bind the NOS end to proteins and peptides through their primary amines, this resulting in a photo-derivatized peptide or protein that can be bioconjugated to any material capable of undergoing the free radical reaction with excited benzophenone. Examples of photo-reactive proteins and peptides are described, respectively, in U.S. Pat. Nos. 5,744,515 and 6,121,027, incorporated herein by reference, including fibronectin, laminin, and type IV collagen, and peptides of fibronectin (RGD, C/H-V, C/H-II), laminin (F-9) and type IV collagen (REP-III). The chemical methods described in U.S. Pat. No. 6,121,027, incorporated herein by reference, are particularly useful for preparing photo-reactive peptides of the present invention.

[0266] In the case of photo-derivatized collagen, the NOS end of the crosslinking agent is bound to the collagen molecule through its primary amines, while the benzophenone end remains free to bind peptides of the present invention upon intimate contact and subsequent photo-illumination. In the case of photo-derivatized peptides, the benzophenone end of the crosslinking agent remains free to bind collagen at any of the CH groups of the amino acids composing the collagen, with some CH groups more reactive than others.

[0267] Other photo-crosslinking are also useful for binding the peptides of the current invention to the collagens of the current invention. As first disclosed in U.S. Pat. No. 5,414,075, incorporated herein by reference, restrained multifunctional reagents for surface modification containing multiple latent reactive groups are capable of binding first to a support structure upon activation, then subsequently to a different second molecule upon reactivation. For example, FIG. 22 shows the chemical structure of the reagent tetrakis (4-benzoylbenzyl ether) of pentaerythritol (TBBE), which includes four latent reactive benzophenone groups. These multifunctional reagents readily bind to first the collagen of the present invention, then to the peptides of the current invention.

[0268] In another embodiment, the amplification peptide can be formulated with a biodegradable carrier so that it can release over time.

[0269] In other embodiments, the amplification peptide is added to or subtracted from the reconstituted (or otherwise modified) collagen so that it impacts the pharmacokinetics or release characteristics of a bioactive protein. The collagen can also be genetically modified to impact the release of a bioactive protein or peptides. Examples of bioactive proteins that can be added are BMPs, PDGF, BDNF, EGF, VEGF, NGF, TNF and the like.

[0270] In some embodiments, the targeted collagen with the amplified peptide can be produced though genetically engineered prokaryotes and eukaryotes. This collagen is then further isolated via processing.

[0271] Of course, it is recognized that more than a single desired characteristic may be engineered into or out of the targeted collagen. For example, in skin wound healing, amplification of the collagen via bio-active peptides could be added for clotting, epithelial growth and faster resorption. A clotting test could identify the peptide that induces this process, an epithelial cell assay could identify the bioactive peptide for cell activity and the peptide that is targeted by the enzyme collagenase could be added to increase the collagen absorption.

[0272] In addition to the N-terminus and the carboxyl C-terminus, side chains of amino acids within the CHP or CMP peptides such as Lysine (NH2), Cysteine (SH), Aspartic acid (and Glutamic acid (COOH), and Asparagine and Glutamine (H2NCO) can also be targeted for bioconjugation (FIG. 12). The carboxyl group on Aspartic acid and the guanidino group on Arginine of the Arg-Gly-Asp (RGD) peptide (FIG. 23) are two such examples. Alternatively, these same amino acids can first be used to end cap CHP or CMP peptides, then targeted for bioconjugation through their reactive side chains in order to increase overall peptide reactivity. This can readily be accomplished using a standard peptide synthesizer for production of end capped synthetic CHP or CMP peptides.

[0273] Further, it is recognized that a targeted collagen product may be formed from the process descried above, as well as any one or more additional processes known in the art.

[0274] In general, any combination of disclosed features, components and methods described herein is possible. Steps of a method can be performed in any order that is physically possible.

[0275] All cited references are incorporated by reference herein.

[0276] Although embodiments have been disclosed, the invention is not limited thereby.