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. 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.

2. The method of claim 1, further comprising the step of digesting the collagen to derive the peptides exhibiting the desired characteristic therefrom.

3. The method of claim 1, further comprising the step of procuring the collagen for the digesting step, and wherein the procuring step includes deriving collagen from the group consisting of eukaryotes, genetically modified prokaryotes and genetically modified plant cells.

4. The method of claim 2, further comprising the step of testing the peptides for the desired characteristic.

5. The method of claim 4, further comprising the step of isolating the peptides exhibiting the desired characteristic.

6. The method of claim 1, wherein the adding step includes adding the peptides exhibiting the desired characteristic to a mixture to reconstitute collagen into the targeted collagen product.

7. The method of claim 6, wherein the adding step is performed so that the reconstituted collagen can control the release profile of the peptide exhibiting the desired characteristic.

8. The method of claim 6, wherein the adding step is performed so that the reconstituted collagen is crosslinked to the peptide exhibiting the desired characteristic.

9. The method of claim 1, wherein the adding step includes adding the peptides exhibiting the desired characteristic to intact collagen.

10. The method of claim 1 wherein the desired characteristic of the peptides has an application selected from the group consisting of wound healing, osteogenesis, blood clotting, organ scaffolds, dura repair, nerve repair, ocular repair, epithelial repair, and cardio-vascular repair, breast repair, and endothelial repair.

11. A method for forming a targeted collagen product lacking an undesired characteristic, comprising: subtracting peptides exhibiting the undesired characteristic from collagen to form the targeted collagen product.

12. The method of claim 11, further comprising the step of digesting the collagen to derive the peptides exhibiting the undesired characteristic therefrom.

13. The method of claim 12, further comprising the step of testing the peptides for the undesired characteristic.

14. The method of claim 13, further comprising the step of isolating the peptides exhibiting the undesired characteristic.

15. The method of claim 11, wherein the subtracting step includes subtracting the peptides exhibiting the undesired characteristic to a mixture to reconstitute collagen into the targeted collagen product.

16. The method of claim 11, wherein the subtracting step includes subtracting the peptides exhibiting the undesired characteristic and reconstituting the remaining collagen that contains a desired characteristic thereby concentrating that desired characteristic.

17. The method of claim 11, wherein the subtracting step includes subtracting the peptide exhibiting the undesired characteristic to intact collagen.

18. The method of claim 10, wherein the peptide facilitates the binding to the collagen of cell types desirable for the application.

19. The method of claim 10, wherein the cell binding to the collagen occurs through the peptide and complementary cell integrins selected from α1β1, α2β1, α10β1 and α11β1.

20. The method of claim 18, wherein the desirable cell types are selected from osteoblasts, fibroblasts, epithelial cells and stem cells.

21. The method of claim 18, wherein the peptide is selected from the group consisting of RGD, GFOGER, DGEA, SVVYGLR, KRSR, FHRRIKA, P15, P20, or P35.

22. The method of claim 11, wherein the undesired characteristic is promotion of excessive thrombosis, inflammation, or osteolysis.

23. The method of claim 11, wherein the peptide facilitates the binding of cell types which are undesirable above maximum threshold concentrations.

24. The method of claim 23, wherein the cell types are selected from inflammatory cells, osteoclasts, and platelets.

25. The method of claim 24, wherein the inflammatory cells are selected from neutrophils, monocytes and lymphocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Embodiments of the invention are further described but are in no way limited by the following drawings.

[0166] FIG. 1 is a flow chart of a method according to a first embodiment of the present invention;

[0167] FIG. 2 is a flow chart of a method according to a second embodiment of the present invention;

[0168] FIG. 3 is a flow chart of a method according to a third embodiment of the present invention;

[0169] FIG. 4 is a flow chart of a method according to a fourth embodiment of the present invention.

[0170] FIG. 5 is a flow chart of the method according to fifth embodiment of the present invention;

[0171] FIG. 6 is a schematic view of a multilayer collagen product according to an embodiment of the present invention;

[0172] 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;

[0173] FIG. 8 is a flow chart of a method according to a sixth embodiment of the present invention;

[0174] FIG. 9 is a flow chart of a method according to a seventh embodiment of the present invention;

[0175] FIG. 10 is a flow chart of a method according to an eighth embodiment of the present invention; and

[0176] FIG. 11 is a flow chart of a method according to a ninth embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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 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).

[0184] 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.

[0185] 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.

[0186] FIG. 2 illustrates an embodiment of the method (200) wherein a bioactive peptide is removed prior to being reconstituted 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).

[0187] 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 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).

[0188] 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).

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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

[0197] 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).

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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 collagen matrix in various embodiments.

[0204] 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.

[0205] 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 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.

[0206] 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.

[0207] 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).

[0208] 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).

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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. In another embodiment, the amplification peptide can be formulated with a biodegradable carrier so that it can release over time.

[0213] In other embodiments, the amplification peptide is added to or subtracted from the reconstituted 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] All cited references are incorporated by reference herein.

[0219] Although embodiments have been disclosed, the invention is not limited thereby.