Collagen with selective characteristics, collagen products containing the same, and methods for producing the same

Abstract

To provide methods for forming a targeted collagen product having amplified desired characteristics or reduced undesired characteristics.SOLUTION: Disclosed is a method for forming a targeted collagen product having an amplified desired characteristic, comprising providing synthesized collagen mimetic peptides or collagen-hybridized peptides, and adding synthesized peptides exhibiting a desired characteristic to collagen to form the targeted collagen product.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to (1) U.S. Provisional Patent Application No. 63 / 079,187, filed September 16, 2020; (2) U.S. Provisional Patent Application No. 63 / 093,554, filed October 19, 2020; and (3) U.S. Provisional Patent Application No. 63 / 149,068, filed February 12, 2021, all three of which are incorporated by reference in their entireties herein.

[0002] The present invention relates to customizable collagen intended for application to various tissues for a variety of purposes, including biological tissue repair. More particularly, the present invention relates to methods for amplifying or decreasing specific properties of collagen for targeted use, such as soft tissue repair or bone repair. The present invention also relates to engineered collagen constructs, including layered targeted collagen products / constructs, methods for making same, methods for using 3D printing of biomolecules including customizable collagen, and collagen constructs formed by such methods. [Background technology]

[0003] Collagen is a naturally occurring protein found in humans and animals. Collagen tissue is often procured from donors and used for a variety of medical applications, ranging from cosmetic surgery to bone repair to wound healing. Prokaryotic collagen may also be obtained from genetically engineered microorganisms and genetically engineered plants.

[0004] Collagen can be treated and / or processed in many different ways, such as strengthening, reconstituting, thickening, crosslinking, combining with other biological materials, etc. Thus, a variety of collagen products can be produced for different medical applications.

[0005] Some embodiments of the present invention generally relate to rapid prototyping systems, particularly 3D printing systems for making collagen-based medical and dental devices, such as dental bone grafts, dental membranes, stents, punctal plugs, ocular collagen onlays and inlays, contact lenses, orthopedic bone applications, spinal applications, neuro applications, and skin applications. More particularly, the present invention relates to building medical devices as three-dimensional objects from many of the material systems and novel resin systems of the present invention using inkjet, fused deposition modeling (FDM), selective laser sintering (SLS), stereolithography (SLA), digital light processing (DLP), bioprinting, or combinations thereof. Inkjet printing systems eject material through an inkjet printhead to form a 3D object that cools, polymerizes, and hardens upon exposure to light. FDM extrudes thermoplastic resin material through a nozzle to build a 3D object. SLS uses a laser as a power source to sinter powdered material to form a solid object. SLA uses a laser beam to trace the shape of each layer and harden the photosensitive resin in a vat (reservoir or bath). DLP systems use a Digital Light Processor (DLP) projector to project successive voxel planes into liquid resin and build three-dimensional objects by hardening the liquid resin. Bioprinting is a layer-by-layer process in which a biological matrix is ​​printed with or without cells. The object then serves as a matrix or scaffold for growing cellularized tissue. In general, rapid prototyping refers to traditional manufacturing processes used to produce parts that are built on a layer-by-layer basis using layers of hardening material. According to this technique, the manufactured part is considered as a series of separate cross-sectional areas that, when assembled, make up a three-dimensional structure. Building a part layer-by-layer is very different from traditional machining techniques in which metal or plastic pieces are cut and drilled to produce the desired shape. In rapid prototyping techniques, parts are created directly from computer-aided design (CAD) or other digital images.Software is used to slice the digital image into thin cross-sectional layers. Parts are then constructed by placing layers of plastic or other hardening material on top of each other. There are various techniques that can be used to combine the layers of structural material. Some techniques may require a final curing step to fully harden the layers of material. Some techniques, such as powder bed inkjet printing or FDM, may require the application of a sealer to form a dense 3D object. Additional milling may be added to some techniques.

[0006] Inkjet printing technology is a rapid prototyping method that can be used to manufacture three-dimensional objects. In one well-known inkjet printing method developed at Massachusetts Institute of Technology, described in U.S. Patent No. 5,204,055 to Sachs et al., incorporated herein by reference in its entirety, a printer head is used to eject a binder material onto layers of powder particles in a powder bed. The powder layers correspond to digitally superimposed sections of the object to be produced. The binder fuses the powder particles together in selected areas. This results in a fused cross-sectional segment of the object being formed on the platform. The process is repeated with each new layer until the desired object is obtained. In the final step, a laser beam scans the object, sintering and fusing the powder layers as required. In another inkjet printing process described in U.S. Patent Nos. 5,506,607 and 5,740,051 to Sanders, a low melting thermoplastic material is ejected through one inkjet print head to form the three-dimensional object. A second inkjet printer head ejects a wax material or other support material to form the support for the three-dimensional object. After the object is generated, the wax support is removed and the object is finished as needed. MultiJet printers, such as the high-quality PolyJet and MultiJet 3D printing processes, use ultraviolet light to crosslink the photopolymer. However, instead of scanning a laser to harden the layers, the printer jets spray tiny droplets of photopolymer (similar to the ink in an inkjet printer) into the shape of the first layer. UV lamps attached to the printer head crosslink the polymer, fixing the shape of the layer. The build platform then lowers one layer thickness and further material is deposited directly on top of the previous layer. Triple Jet Technology (PolyJet), used in the Stratasys Objet 500 Connex3, is the most advanced method of PolyJet 3D printing. This technology allows for the mixing of three colors by performing precision printing of three materials.

[0007] Fused Deposition Modeling (FDM) technology was first developed and implemented by Scott Crump, founder of Stratasys Ltd., in the 1980s. The advantage of this technology is that all parts printed with FDM can be made of high-performance, engineering-grade thermoplastics. FDM is the only 3D printing technology that builds parts with production-grade thermoplastics, and the prints have excellent mechanical, thermal, and chemical qualities. 3D printers that use FDM technology build objects layer by layer from the bottom up by heating and extruding a thermoplastic filament. In addition to the thermoplastic, the printer can also extrude support materials. The printer then heats the thermoplastic to its melting point and extrudes it from the nozzle onto the build platform. To support the top layer, the printer may place a special material underneath that can melt after the print is complete. Once the thin layer of plastic bonds to the layer below it, it cools and hardens. Once the layer is finished, the base is lowered to begin building the next layer. The technology is considered easy to use and environmentally friendly. Various types of thermoplastic resins can be used to print dental objects.

[0008] Selective laser sintering (SLS) is a technique that uses a laser as a power source to form solid 3D objects. The technique was developed in the 1980s by Carl Deckard, a student at the University of Texas, and Joe Beaman, his professor. The main difference between SLS and SLA is that it uses a vat of powdered material instead of liquid resin as stereolithography does. Unlike other additive manufacturing processes such as stereolithography (SLA) and fused deposition modeling (FDM), SLS does not require the use of support structures because the object being printed is always surrounded by unsintered powder. The wide variety of materials that can be used with this type of 3D printer makes this technique very popular for 3D printing customized products. SLS requires the use of a high-powered laser, which makes the printer very expensive. Dental objects made with this process require extensive surface finishing.

[0009] The SLA 3D printing method was patented in 1986 by Charles Hull, co-founder of 3D Systems, Inc., and converts liquid plastic into solid 3D objects. SLA 3D printers work with excess liquid resin, which is hardened by irradiation to form a solid object. The parts built are usually smooth in surface, but the quality varies depending on the quality of the SLA equipment used. After the plastic has hardened, the printer platform is lowered (top-down printers) or raised (bottom-up printers) a fraction of a millimeter in the tank, and the next layer is lasered until the print is complete. Once all layers are printed, the object is rinsed with solvent and placed in an oven to harden and finish the process.

[0010] Digital Light Processing is another 3D printing process that is very similar to stereolithography. DLP technology was created in 1987 by Larry Hornbeck at Texas Instruments and gained a lot of popularity in projector manufacturing. It uses digital micromirrors arranged on a semiconductor chip. 3D inkjet, DLP and SLA all work with photopolymers. The difference between SLA and DLP processes is the different light source. The DLP method projects successive voxel planes into liquid resin, which is then cured. The material used for printing is liquid resin in a transparent resin container. The resin hardens rapidly by irradiating it with light. The printing speed is impressive, especially with Carbon3D's CLIP (Continuous Liquid Interface Production) technology. A layer of hardened material can be created in a few seconds with such a printer. Once a layer is finished, it moves up and work on the next layer begins. CLIP technology balances light and oxygen, eliminating the mechanical steps and layers of the standard DLP process, making it possible to produce commercial quality objects at high speeds.

[0011] Bioprinting involves the printing of one or more cells, a matrix, and nutrients, known as bioink, into a printer cartridge and deposited using a medical scan of the patient. When the bioprinted pre-tissue is transferred to an incubator, this cell-based pre-tissue matures into a tissue. The matrix can also be printed without cells and then subsequently populated with cells in-vivo or ex-vivo.

[0012] 3D bioprinting for the production of biological constructs typically involves distributing cells onto a biocompatible scaffold using a sequential layer-by-layer approach to generate tissue-like three-dimensional structures. Given that all tissues in the body are naturally composed of different cell types, many techniques for printing these cells vary in their ability to ensure the stability and viability of the cells during the manufacturing process. Some of the methods used for 3D bioprinting of cells include some of the printing techniques mentioned above as well as extrusion printing onto a support gel.

[0013] One aspect of the invention relates to the use of 3D printing to digitally engineer dental and medical devices and ECM and cell scaffolds that are primarily composed of collagen, engineered collagen, and / or collagen-based peptides. To generate polymerizable peptides, collagen is enzymatically digested and then the peptides are modified with radiation-polymerizable functional groups. These modified peptides are then compounded with initiators, crosslinkers, solvents, and / or other additives to create the desired design input for a particular dental or medical application. This compound can then be 3D printed. In another process, collagen is digested to create peptides and other peptides are added or removed to generate the desired design input customized for a particular dental or medical application. These newly formulated peptides are then modified with radiation-polymerizable functional groups. These modified peptides are then compounded with initiators, crosslinkers, solvents, and / or other additives to create the desired design input for a particular dental or medical application. This compound can then be 3D printed. Another example involves extruding collagen that has been modified to be soluble in a solvent and, optionally, modified with functional group chemistry that allows for energy-driven post-processing. To enhance the biological processing of collagen, the extrudable collagen can also include other collagen-based peptides, such as collagen mimetic peptides ("CMP") and collagen hybridizing peptides ("CHP"). To enhance the healing process, the extrudable collagen can also include other bioactive-based peptides and growth factors.

[0014] 3D printing is often referred to as "rapid prototyping". The present invention includes methods for utilizing 3D printing to create final manufactured products. Some embodiments of the present invention are directed to collagen amplification in dental or medical devices by concentrating, diluting, adding or removing CMP and CHP. The devices can be manufactured by molding or 3D printing. Summary of the Invention

[0015] Provided herein are methods for forming targeted collagen products that have enhanced desirable properties or reduced undesirable properties.

[0016] In one exemplary embodiment, a method for forming a target collagen product having an amplified desired property includes adding a peptide exhibiting the desired property to collagen to form the target collagen product.

[0017] In another exemplary embodiment, a method for forming a target collagen product lacking undesirable properties comprises removing peptides that exhibit the undesirable properties from collagen to form the target collagen product.

[0018] Further aspects of the invention include:

[0019] 1. A method for forming a target collagen product having an amplified desired property, comprising the steps of procuring collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the desired property; isolating peptides that exhibit the desired property; and adding the peptides that exhibit the desired property to a mixture to remodel the collagen into the target collagen product.

[0020] 2. The method of aspect 1 above, wherein the obtaining step comprises obtaining the collagen from a eukaryote or a genetically modified prokaryote.

[0021] 3. The method of claim 1, wherein the digesting step comprises subjecting the procured collagen to an enzyme.

[0022] 4. The method of claim 3, wherein the enzyme is selected from the group consisting of collagenase, pepsin, papain, and pancreatin.

[0023] 5. The method of aspect 1 above, wherein the desirable properties of the isolated peptide have uses selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, cardiovascular repair, endothelial repair, breast repair, antioxidant, antibacterial, anti-inflammatory activity, and for drug delivery.

[0024] 6a. The method of embodiment 1 above, wherein the target collagen product is a device.

[0025] 6b. The method of embodiment 1 above, wherein the adding step comprises adding one or more amplifying peptides to the mixture to reconstitute collagen.

[0026] 7. The method of embodiment 6b above, wherein the adding step comprises covalently linking the amplifying peptide to the reconstituted collagen.

[0027] 8. The method of embodiment 6b above, wherein the adding step comprises non-covalently linking the amplifying peptide to the reconstituted collagen.

[0028] 9. The method of embodiment 8 above, wherein the non-covalently linked amplifying peptide is released over a period of time.

[0029] 10. A method for forming a target collagen product lacking undesirable properties, the method comprising the steps of: obtaining collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the undesirable properties; isolating peptides that exhibit the undesirable properties; and removing peptides that exhibit the undesirable properties from the collagen mixture; and reconstituting the collagen mixture to form the target collagen product.

[0030] 11. A method for forming a target collagen product having an amplified desired property, comprising the steps of procuring collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the desired property; isolating peptides that exhibit the desired property; and adding the peptides that exhibit the desired property to intact collagen to form the target collagen product.

[0031] 12. The method of claim 11, wherein the obtaining step comprises obtaining the collagen from a eukaryote or a genetically modified prokaryote.

[0032] 13. The method of claim 11, wherein the step of digesting comprises subjecting the procured collagen to an enzyme.

[0033] 14. The method of claim 13, wherein the enzyme is selected from the group consisting of collagenase and pepsin.

[0034] 15. The method of aspect 11 above, wherein the desirable properties of the isolated peptide have uses selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, endothelial repair.

[0035] 16. The method of aspect 11 above, wherein the adding step comprises adding one or more amplifying peptides to the mixture to reconstitute collagen.

[0036] 17. The method of embodiment 16 above, wherein the adding step comprises covalently linking the amplifying peptide to the intact collagen.

[0037] 18. The method of embodiment 16 above, wherein the adding step comprises non-covalently linking the amplifying peptide to the intact collagen.

[0038] 19. The method of embodiment 18 above, wherein the non-covalently linked amplifying peptide is released over a period of time.

[0039] 20. A method for forming a target collagen product lacking undesirable properties, comprising the steps of: procuring collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the undesirable properties; isolating peptides that exhibit the undesirable properties; and removing peptides that exhibit the undesirable properties from intact collagen to form the target collagen product.

[0040] 21. A method for forming a target collagen product with desired properties, either amplified or de-amplified, comprising genetically engineering cells to produce modified collagen.

[0041] 22. The method of claim 21, wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, and a plant cell.

[0042] 23. A method for forming a target collagen product having an amplified desired property, comprising the steps of procuring collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the desired property; isolating peptides that exhibit the desired property; and adding the peptides that exhibit the desired property to a mixture to reconstitute the collagen into the target collagen product, wherein the reconstituted protein allows for control of the release profile of the bioactive protein or peptide.

[0043] 24. A method for forming a target collagen product lacking undesirable properties, comprising the steps of: procuring collagen; digesting the collagen to obtain peptides therefrom; testing the peptides for the undesirable properties; isolating peptides that exhibit the undesirable properties; and removing peptides that exhibit the undesirable properties from the collagen mixture; and reconstituting the collagen mixture to form the target collagen product, such that the reconstituted protein controls the release profile of the bioactive protein or peptide.

[0044] 25. A method for forming a target collagen product having an amplified desired property, comprising adding a peptide exhibiting a desired property to a mixture to reconstitute collagen into the target collagen product.

[0045] 26. The method of embodiment 25 above, further comprising the step of procuring collagen.

[0046] 27. The method according to aspect 25 above, further comprising the step of digesting collagen to obtain peptides therefrom.

[0047] 28. The method of embodiment 25 above, further comprising testing the peptide for desirable properties.

[0048] 29. The method of embodiment 25 above, further comprising the step of isolating peptides exhibiting desirable properties.

[0049] 30. A method for forming a target collagen product lacking undesirable properties, comprising removing peptides exhibiting the undesirable properties from a collagen mixture to form the target collagen product.

[0050] 31. The method of embodiment 30 above, further comprising the step of procuring collagen.

[0051] 32. The method according to aspect 30 above, further comprising the step of digesting collagen and obtaining peptides therefrom.

[0052] 33. The method of embodiment 30 above, further comprising testing the peptide for undesirable properties.

[0053] 34. The method of embodiment 30 above, further comprising the step of isolating peptides that exhibit undesirable properties.

[0054] 35. The method of embodiment 30 above, further comprising the step of reconstituting the collagen mixture.

[0055] 36. A method for forming a layered target collagen product having at least one amplified selected property, the method comprising: forming a first target collagen product having peptides exhibiting at least one selected amplified property; preparing a first collagen layer comprising the first target collagen product; and overlaying the first collagen layer on a second layer.

[0056] 37. The method of embodiment 36 above, wherein the preparing step comprises adding a first target collagen product to a collagen layer to form a first collagen layer.

[0057] 38. The method of embodiment 36 above, wherein the preparing step comprises forming the first target collagen product as a first collagen layer.

[0058] 39. The method of embodiment 36 above, wherein the second layer is a second collagen layer.

[0059] 40. The method of embodiment 39 above, wherein the second collagen layer comprises a second target collagen product.

[0060] 41. The method of aspect 40 above, wherein the at least one selected amplification characteristic includes a first selected amplification characteristic at a first target collagen product and the first collagen layer, and a second selected amplification characteristic at a second target collagen product and the second collagen layer; and the first selected amplification characteristic is different from the second selected amplification characteristic.

[0061] 42. The method of embodiment 36 above, wherein the second layer is a substrate.

[0062] 43. The method of claim 42, wherein the substrate does not contain collagen.

[0063] 44. A layered target collagen product comprising: a first collagen layer having a first target collagen product including a first amplification property; and a second collagen layer overlying the first collagen layer and having a second target collagen product including a second amplification property, the first amplification property being different from the second amplification property.

[0064] 45. The product of aspect 44 above, wherein the first amplifying property and the second amplifying property have an application selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, endothelial repair.

[0065] 46. ​​A layered target collagen product comprising a substrate and a collagen layer overlying said substrate, said layer having a target collagen product comprising at least one amplifying property.

[0066] 47. The product of embodiment 46 above, wherein the substrate comprises unmodified collagen.

[0067] 48. A method for forming a layered target collagen product lacking at least one selected undesirable property, the method comprising the steps of forming a first target collagen product by removing peptides from collagen that exhibit at least one selected undesirable property; preparing a first collagen layer comprising the first target collagen product; and overlaying the first collagen layer onto a second layer.

[0068] 49. The method of embodiment 48 above, wherein the preparing step comprises adding a first target collagen product to the collagen layer to form the first collagen layer.

[0069] 50. The method of embodiment 48 above, wherein the preparing step comprises forming the first target collagen product as a first collagen layer.

[0070] 51. The method of embodiment 48 above, wherein the second layer is a second collagen layer.

[0071] 52. The method of embodiment 51 above, wherein the second collagen layer comprises a second target collagen product.

[0072] 53. The method of aspect 52 above, wherein the at least one selected undesirable characteristic includes a first selected undesirable characteristic of the first target collagen product and the first collagen layer, and a second selected undesirable characteristic of the second target collagen product and the second collagen layer; and the first selected undesirable characteristic is different from the second selected undesirable characteristic.

[0073] 54. The method of embodiment 48, above, wherein the second layer is a substrate.

[0074] 55. The method of claim 54, wherein the substrate does not contain collagen.

[0075] 56. A layered target collagen product comprising: a first collagen layer having a first target collagen product lacking a first selected undesirable property; and a second collagen layer overlying the first collagen layer and having a second target collagen product lacking a second selected undesirable property, wherein the first selected undesirable property is different from the second selected undesirable property.

[0076] 57. The product of aspect 56 above, wherein the first selected undesirable property and the second selected undesirable property have an application selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, endothelial repair.

[0077] 58. A layered target collagen product comprising a substrate and a collagen layer overlying said substrate, the target collagen product having a defect in at least one selected undesirable characteristic.

[0078] 59. The product of embodiment 58 above, wherein the substrate comprises unmodified collagen.

[0079] 60. A method for forming a target collagen product having an amplified desired property, comprising: adding a collagen-based peptide exhibiting the desired property to a collagen mixture containing collagen; and 3D printing the collagen mixture to form the target collagen product.

[0080] 61. The method of claim 60, wherein the collagen mixture comprises cells and growth medium.

[0081] 62. The method of claim 60, wherein the 3D printing step comprises an operation selected from the group consisting of DLP, SLA, extrusion, bioprinting, and inkjet printing.

[0082] 63. The method of embodiment 60 above, further comprising the step of post-processing the target collagen product.

[0083] 64. The method of embodiment 63 above, wherein the post-treating step comprises adding cells to the target collagen product.

[0084] 65. The method of embodiment 63 above, wherein the post-treating step comprises adding a growth medium to the target collagen product.

[0085] 66. The method of embodiment 63 above, wherein the post-treating step comprises adding a growth factor to the target collagen product.

[0086] 67. The method of embodiment 63 above, wherein the post-processing step comprises extracting residual molecules from the target collagen product.

[0087] 68. The method of embodiment 63 above, wherein the post-treating step comprises UV curing the target collagen product.

[0088] 69. The method of embodiment 63 above, wherein the post-treating step comprises heat-treating the target collagen product.

[0089] 70. The method of embodiment 63 above, wherein the post-treating step comprises crosslinking the target collagen product.

[0090] 71. The method of aspect 60 above, wherein the desirable properties of the isolated peptide have uses selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, cardiovascular repair, endothelial repair, breast repair, antioxidant, antibacterial, anti-inflammatory activity, and for drug delivery.

[0091] 72. The method of embodiment 60 above, wherein the adding step comprises adding one or more amplifying peptides to the collagen mixture.

[0092] 73. The method of embodiment 72 above, wherein the adding step comprises covalently linking one or more amplifying peptides to the collagen mixture.

[0093] 74. The method of embodiment 72 above, wherein the adding step comprises non-covalently linking one or more amplifying peptides to the collagen mixture.

[0094] 75. The method of embodiment 74 above, wherein the one or more non-covalently linked amplifying peptides are released over a period of time.

[0095] 76. The method of embodiment 60 above, further comprising modifying collagen of the collagen mixture.

[0096] 77. The method of embodiment 76, wherein the modifying step is carried out using one or more from the group consisting of a free radical, a condensation reaction, and a ring-opening polymerization group.

[0097] 78. The method of embodiment 60 above, wherein the collagen of the collagen mixture is soluble collagen.

[0098] 79. The method of embodiment 60 above, wherein the collagen of the collagen mixture is insoluble collagen.

[0099] 80. The method of embodiment 60 above, wherein the adding step includes adding one or more minerals to the collagen mixture.

[0100] 81. The method of embodiment 80 above, wherein at least one mineral is hydroxyapatite or a hydroxyapatite-like material.

[0101] 82. A method for forming a target collagen product lacking undesirable properties, comprising the steps of removing collagen-based peptides exhibiting the undesirable properties from a collagen mixture containing collagen; and 3D printing the collagen mixture to form the target collagen product.

[0102] 83. The method of claim 82, wherein the 3D printing step comprises an operation selected from the group consisting of DLP, SLA, extrusion, bioprinting, and inkjet printing.

[0103] 84. The method of embodiment 82 above, further comprising the step of post-processing the target collagen product.

[0104] 85. The method of embodiment 84 above, wherein the post-treating step comprises adding cells to the target collagen product.

[0105] 86. The method of embodiment 84 above, wherein the post-treating step comprises adding growth medium to the target collagen product.

[0106] 87. The method of embodiment 84 above, wherein the post-treating step comprises adding a growth factor to the target collagen product.

[0107] 88. The method of embodiment 84 above, wherein the post-processing step comprises extracting residual molecules from the target collagen product.

[0108] 89. The method of embodiment 84 above, wherein the post-treating step comprises UV curing the target collagen product.

[0109] 90. The method of embodiment 84 above, wherein the post-treating step comprises heat-treating the target collagen product.

[0110] 91. The method of embodiment 84 above, wherein the post-treating step comprises crosslinking the target collagen product.

[0111] 92. The method of embodiment 82 above, further comprising modifying collagen of the collagen mixture.

[0112] 93. The method of embodiment 92 above, wherein the modifying step is carried out using one or more from the group consisting of a free radical, a condensation reaction, and a ring-opening polymerization group.

[0113] 94. The method of embodiment 82 above, wherein the collagen of the collagen mixture is soluble collagen.

[0114] 95. The method of embodiment 82 above, wherein the collagen of the collagen mixture is insoluble collagen.

[0115] 96. The method of embodiment 94 above, wherein at least one mineral is hydroxyapatite or a hydroxyapatite-like material.

[0116] 97. A method for forming a target collagen product having an amplified desired property, comprising: adding a collagen-based peptide exhibiting the desired property to collagen; and 3D printing the collagen to form the target collagen product.

[0117] 98. A method for forming a target collagen product lacking undesirable properties, comprising the steps of removing peptides that exhibit the undesirable properties from collagen; and 3D printing the collagen to form the target collagen product.

[0118] 99. A method for forming a target collagen product having an amplified desired property, comprising: genetically engineering cells to produce engineered collagen; and 3D printing the engineered collagen to form the target collagen product.

[0119] 100. The method of embodiment 99 above, wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, and a plant cell.

[0120] 101. The method of any one of aspects 60, 82, 97, or 98 above, further comprising the step of procuring collagen.

[0121] 102. The method of any one of aspects 60, 82, 97, or 98 above, further comprising the step of digesting collagen to obtain collagen-based peptides therefrom.

[0122] 103. The method of aspect 102 above, further comprising testing the collagen-based peptide for desirable or undesirable properties.

[0123] 104. The method of any one of aspects 60, 82, 97 or 98 above, further comprising the step of isolating peptides exhibiting desirable or undesirable properties.

[0124] 105. The method of any one of aspects 60, 82, 97, or 98, above, further comprising the step of adding one or more growth factors to the collagen mixture.

[0125] 106. A method for forming a layered target collagen product having at least one amplified selected property, the method comprising: forming a first target collagen product having peptides exhibiting at least one selected amplified property; preparing a first collagen layer comprising the first target collagen product; and overlaying the first collagen layer on a second layer.

[0126] 107. The method of embodiment 106, above, wherein the preparing step comprises adding a first target collagen product to a collagen layer to form a first collagen layer.

[0127] 108. The method of embodiment 106, above, wherein the preparing step includes forming the first target collagen product as a first collagen layer.

[0128] 109. The method of embodiment 106 above, wherein the second layer is a second collagen layer comprising a second target collagen product.

[0129] 110. The method of aspect 109 above, wherein the at least one selected amplification characteristic includes a first selected amplification characteristic at a first target collagen product and the first collagen layer, and a second selected amplification characteristic at a second target collagen product and the second collagen layer; and the first selected amplification characteristic is different from the second selected amplification characteristic.

[0130] 111. The method of embodiment 106, above, wherein the second layer is a collagen-free substrate.

[0131] 112. A layered target collagen product comprising a first collagen layer having a first target collagen product including a first amplifying property, and a second layer overlying the first collagen layer.

[0132] 113. The layered target collagen product of embodiment 112 above, wherein the second layer is a second collagen layer having a second target collagen product including a second amplification property; and the first amplification property is different from the second amplification property.

[0133] 114. The layered targeted collagen product of aspect 113 above, wherein the first amplifying property and the second amplifying property have an application selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, endothelial repair.

[0134] 115. The layered targeted collagen product of embodiment 112 above, wherein the second layer is a substrate.

[0135] 116. The layered targeted collagen product of embodiment 115 above, wherein the substrate comprises unmodified collagen.

[0136] 117. A method for forming a layered target collagen product lacking at least one selected undesirable property, the method comprising the steps of forming a first target collagen product by removing peptides exhibiting at least one selected undesirable property from collagen; preparing a first collagen layer comprising the first target collagen product; and overlaying the first collagen layer onto a second layer.

[0137] 118. The method of embodiment 117, above, wherein the preparing step comprises adding a first target collagen product to the collagen layer to form the first collagen layer.

[0138] 119. The method of embodiment 117, above, wherein the preparing step includes forming the first target collagen product as a first collagen layer.

[0139] 120. The method of embodiment 117 above, wherein the second layer is a second collagen layer comprising a second target collagen product.

[0140] 121. The method of aspect 120 above, wherein the at least one selected undesirable characteristic includes a first selected undesirable characteristic in the first target collagen product and the first collagen layer, and a second selected undesirable characteristic in the second target collagen product and the second collagen layer; and the first selected undesirable characteristic is different from the second selected undesirable characteristic.

[0141] 122. The method of embodiment 117, above, wherein the second layer is a collagen-free substrate.

[0142] 123. A layered targeted collagen product comprising a first collagen layer having a first targeted collagen product deficient in a first selected undesirable property, and a second layer overlying the first collagen layer.

[0143] 124. The layered targeted collagen product of embodiment 123 above, wherein the second layer is a second collagen layer having a second targeted collagen product lacking a second selected undesirable property; and the first selected undesirable property is different from the second selected undesirable property.

[0144] 125. The product of aspect 124 above, wherein the first selected undesirable property and the second selected undesirable property have an application selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, endothelial repair.

[0145] 126. The layered targeted collagen product of embodiment 123 above, wherein the second layer is a substrate.

[0146] 127. The layered targeted collagen product of embodiment 126 above, wherein the substrate comprises unmodified collagen.

[0147] 128. A method for forming a target collagen product having an amplified desired property, comprising the step of adding a peptide exhibiting the desired property to collagen to form the target collagen product.

[0148] 129. The method of embodiment 128 above, further comprising the step of digesting collagen to obtain peptides therefrom exhibiting desirable properties.

[0149] 130. The method of embodiment 129 above, further comprising the step of procuring collagen for the digestion step, wherein the procuring step comprises obtaining collagen from a eukaryote or a genetically modified prokaryote.

[0150] 131. The method of embodiment 129 above, further comprising testing the peptide for desirable properties.

[0151] 132. The method of embodiment 131 above, further comprising the step of isolating peptides exhibiting desirable properties.

[0152] 133. The method of embodiment 128, above, wherein the adding step comprises adding a peptide exhibiting desirable properties to the mixture to reconstitute collagen into the target collagen product.

[0153] 134. The method of embodiment 133 above, wherein the step of adding reconstituted collagen is carried out to control the release profile of peptides exhibiting desirable properties.

[0154] 135. The method of embodiment 128, above, wherein the adding step comprises adding a peptide exhibiting the desired properties to the intact collagen.

[0155] 136. The method of embodiment 128 above, wherein the desirable properties of the peptide have uses selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, breast repair, and cardiovascular repair, and endothelial repair.

[0156] 137. A method for forming a target collagen product lacking undesirable properties, the method comprising removing peptides that exhibit the undesirable properties from collagen to form the target collagen product.

[0157] 138. The method of embodiment 137 above, further comprising the step of digesting collagen to obtain peptides therefrom that exhibit undesirable properties.

[0158] 139. The method of embodiment 138 above, further comprising the step of testing the peptide for undesirable properties.

[0159] 140. The method of embodiment 139 above, further comprising the step of isolating peptides that exhibit undesirable properties.

[0160] 141. The method of embodiment 137 above, wherein the removing step comprises removing peptides exhibiting undesirable properties from the mixture and reconstituting the collagen into the target collagen product.

[0161] 142. The method of embodiment 137 above, wherein the removing step comprises removing peptides exhibiting undesirable properties from intact collagen.

[0162] 143. The method of any one of the above aspects, further comprising adding CHP or CMP peptides to amplify collagen, wherein the CHP or CMP peptides are synthetic.

[0163] 144. The method of embodiment 143, wherein the CHP or CMP peptide is selected from the group consisting of a single-chain peptide, a double-chain peptide, and a triple-chain peptide.

[0164] 145. The method of embodiment 144, wherein the CHP or CMP peptide is modified with a polymerizable or reactive group, thereby capable of binding to collagen or modified collagen.

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

[0166] [Figure 1] FIG. 2 shows a flowchart of a method according to a first embodiment of the present invention. [Diagram 2] FIG. 4 shows a flowchart of a method according to a second embodiment of the present invention. [Diagram 3] FIG. 6 shows a flowchart of a method according to a third embodiment of the present invention. [Figure 4] FIG. 10 shows a flowchart of a method according to a fourth embodiment of the present invention. [Diagram 5] FIG. 10 shows a flowchart of a method according to a fifth embodiment of the present invention. [Figure 6]FIG. 1 shows a schematic diagram of a multi-layered collagen product, according to one embodiment of the present invention. [Figure 7] FIG. 1 shows a schematic diagram of a hybrid medical product comprising a substrate and a collagen layer, according to one embodiment of the present invention. [Figure 8] FIG. 13 shows a flowchart of a method according to a sixth embodiment of the present invention. [Figure 9] FIG. 13 shows a flowchart of a method according to a seventh embodiment of the present invention. [Figure 10] FIG. 13 shows a flowchart of a method according to an eighth embodiment of the present invention. [Figure 11] FIG. 13 shows a flowchart of a method according to a ninth embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0167] As will be further explained below, please refer to FIG. 1, which is a flow chart of a first exemplary embodiment of a method (100) according to the present disclosure.

[0168] Collagen can be procured from animal (e.g., bovine, porcine, equine, caprine) or human sources for processing (110) or genetically engineered from microorganisms or genetically engineered cells. Examples of collagenous tissues that can be procured for this application include dermis, tendons, peritoneal tissue, pericardium, and cartilage. Bone can also be a source of collagen that is further digested and fractionated. The type of collagen can be any type of collagen, e.g., type 1, type 3, etc., or genetically engineered variants. Of course, it is recognized that collagen can also be obtained from genetically engineered microorganisms.

[0169] Collagen is digested and fractionated (120) to break it 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 (e.g., using pepsin, papain, collagenase, pancreatin, etc.); (3) acid hydrolysis; (4) hybrid chemical-enzymatic methods; and (5) synthesis by fermentation. The hydrolyzed collagen is further fractionated using ultrafiltration membranes. In various embodiments, peptides are synthesized in a peptide synthesizer.

[0170] Different peptides have different biological characteristics that confer desirable / desired attributes / properties, such as higher affinity for therapeutic use in a particular medical application. In one embodiment, after digestion and fragmentation of collagen tissue, each of the collagen fractions / peptides is tested for various biological characteristics (130), such as attributes / properties useful for wound healing, blood clotting, bone formation / osteogenesis, cosmetic applications, scaffolds for organ regeneration, use as antioxidants, antimicrobial and / or anti-inflammatory activity, and delivery of drugs such as insulin and methylene blue, and shows lower water absorption.

[0171] In various embodiments, collagen is tested by subjecting it to an assay. Such assays include assaying total collagen in biological samples using a novel fluorescent reagent, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), as described, for example, in H. Yasmin et al., Amplified and selective collagen assay by enzymatic and fluorescent reactions, Scientific Reports, 4:4950, May 13, 2014, which is incorporated herein by reference in its entirety. Cellular assays can be used to determine cellular activity, such as bone formation, to determine stimulatory activity, osteoinduction or osteoconduction. Another assay can be the clotting ability of the peptide by blood assay for wound healing.

[0172] In some embodiments of the method, testing of the collagen fraction / peptides (130) is performed only once. In other embodiments of the method, such testing may be performed more than once. In still other embodiments, for example, where the biological characteristics of the collagen fraction / peptide are already known, testing is not performed.

[0173] Following performance of the assay or other test, and based on the results, or if already known from prior testing, collagen fractions / peptides having certain desirable / desired characteristics, such as promoting wound healing of soft tissue, are identified, isolated (140) and selected for addition to the reconstituted collagen product. First, a natural source of collagen, such as skin, bone, tendon, or ligament, is cleaned, washed, and freed of non-collagenous impurities 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 herein by reference in their entireties). The fibers resulting from the purification process are further processed into a reconstituted collagen matrix. A reconstituted collagen matrix can be formed using the following general steps: a) forming an aqueous dispersion containing biopolymer 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 sheets are produced; d) compressing the hydrated fibers to remove excess solution and achieve the desired density or thickness; e) drying the fibers; and f) crosslinking the membrane (see, e.g., U.S. Patent Nos. 6,391,333, 6,599,524, and 7,807,192; all of which are incorporated herein by reference in their entireties).

[0174] Selected collagen fractions / peptides are added to the mixture (150) to reconstitute the collagen into a "target collagen" that has the amplified desired properties of the isolated fraction / peptide, such as a collagen targeted to soft tissue wound healing. In various embodiments, the collagen fraction or synthetic collagen peptide (e.g., CHP or CMP) can be a single-chain, double-chain, or triple-chain peptide.

[0175] In one embodiment, the selected fractionated bioactive peptides are added prior to carrying out the reconstituted collagen processing and crosslinked to the reconstituted collagen during processing. As an alternative to adding the bioactive peptides to the reconstituted collagen, such peptides are added after processing of the reconstituted collagen so that the bioactive peptides are released from the reconstituted collagen over time rather than being covalently linked to the collagen. In another alternative embodiment, the bioactive peptides are covalently linked to the reconstituted collagen after the collagen has been reconstituted. In another embodiment, the bioactive peptides can be removed prior to reconstitution so that the collagen does not lose its bioactive properties. In another embodiment, the fractions are reconstituted into the medical device alone. Combinations and variations of these processes are also possible.

[0176] FIG. 2 illustrates an embodiment of the method (200) in which bioactive peptides are removed before reconstitution so that the collagen does not have its bioactive properties. By removing a portion of the collagen, the remaining collagen may have bioactive peptide sequences, thereby effectively enriching the bioactive sequences after reconstitution. The method includes some of the same steps as the method (100) shown in FIG. 1 and discussed above, including sourcing collagen (210), digesting and fractionating the collagen to obtain peptides (220). The fractionated collagen peptides are then tested for undesirable characteristics / properties (235), and peptides having such undesirable characteristics / properties are then isolated (240) and removed (255) from the collagen mixture. The collagen mixture is then reconstituted (260) to form the target collagen product. In another embodiment, the collagen mixture is not reconstituted, and the target collagen product is formed upon completion of the removing step (255).

[0177] 3 illustrates another embodiment of a method (300) that includes a step (350) of adding fractionated collagen peptides having desired characteristics / properties to the mixture to reconstitute collagen into a target collagen product. In various embodiments, the method (300) can include the step of adding as illustrated in FIG 1 and discussed above in connection with its associated method (100).

[0178] 4 illustrates yet another embodiment of a method (400) that includes removing fractionated collagen peptides having undesirable characteristics / properties from the mixture (455) to form a target collagen product. In various embodiments, the method (400) can include an additive step as illustrated in FIG. 2 and discussed above in connection with its associated method (200).

[0179] Also disclosed herein are collagen layers and surface coatings (the term "layer" is used herein to include both layers and surface coatings) in which collagen is endowed with selective properties for various medical applications (as discussed in the embodiments above). Also disclosed herein are multi-layered collagen products, layered collagen / substrate products, and methods of forming the same.

[0180] In various embodiments of the disclosed invention, collagen is layered such that each layer contains reconstituted collagen with different characteristics and / or amino acid content. The layers can contain more or less amino acid sequences that are useful at different stages of a biological process, such as a wound healing process or a bone repair process. The layers can contain amino acid sequences that allow collagen fibers to be reconstituted at a faster or slower rate during processing, such that each layer has a different amount of collagen fibers, such that each layer has different physical characteristics or biological activity.

[0181] In various embodiments, the collagen layer is impregnated with an active pharmaceutical ingredient (API), such as growth factors, bioactive peptides, steroids, antibiotics, oncology, GI, cardiovascular, renal, antiviral, RNA, central nervous system (CNS), neuromuscular, etc., and can release the API into or onto the diseased or damaged area of ​​a patient's area after application or implantation.

[0182] Reference is now made to FIG. 5; as will be further described below, FIG. 5 is a flow chart of another exemplary embodiment of a method (500) according to the present disclosure. The method (500) includes some of the same steps of the method (100) shown in FIG. 1 and discussed above, including procuring collagen (510), digesting and fractionating the collagen (520) to obtain peptides, testing each of the collagen fractions / peptides (530) for various biological characteristics, isolating (140) collagen fractions / peptides having specific desirable / desired characteristics, and adding selected collagen fractions / peptides (550) to the mixture to reconstitute the collagen into a "target collagen" having the amplified desirable properties of the isolated fractions / peptides, e.g., collagen targeted to wound healing of soft tissues. Synthetically generated peptides (i.e., CHP and / or CMP) may alternatively be used in various embodiments.

[0183] In some embodiments of the method, testing of the collagen fraction / peptides (530) is performed only once. In other embodiments of the method, such testing may be performed more than once. In still other embodiments, for example, where the biological characteristics of the collagen fraction / peptides are already known, testing is not performed.

[0184] In one embodiment, selected fractionated bioactive peptides are added prior to carrying out the reconstituted collagen processing and crosslinked to the reconstituted collagen during processing. An alternative to adding bioactive peptides to the reconstituted collagen is to add such peptides after processing of the reconstituted collagen such that the bioactive peptides are not covalently linked to the collagen but are allowed to be released over time from the reconstituted collagen. In another alternative embodiment, the bioactive peptides are covalently linked to the reconstituted collagen after the collagen has been reconstituted. In another embodiment, the bioactive peptides are added to unreconstituted collagen. In another embodiment, the bioactive peptides can be removed prior to reconstitution such that the collagen does not retain its bioactive properties. In another embodiment, a portion of the collagen is removed to increase the concentration of remaining bioactive sites. This portion is then reconstituted to form the device. Combinations and variations of these steps are also possible.

[0185] In various embodiments, after the collagen product is formed according to the above steps (whether reconstituted or not), it can be formed into one or more layers or added to one or more layers (560). This layering or stratification step (560) allows collagen amplification to be engineered in a layered manner. In other words, selected fractionated bioactive peptides are added to collagen to form a target collagen, which is then formed into one or more layers to facilitate control of the biological process.

[0186] In some embodiments, two or more of the target collagen product layers are stacked together to form a layered collagen product, such as a laminate. See FIG. 6, which illustrates a layered collagen product CP having three collagen layers CL1, CL2, and CL3. In various embodiments, the different target collagen layers CL1, CL2, and CL3 are engineered to have different bioactive characteristics. As a non-limiting example, the different collagen layers can be "programmed" to have different release times, e.g., drugs, proteins, or other bioactive substances can be impregnated or selectively absorbed and released from the biological environment within one or more layers.

[0187] In other embodiments, the target collagen layer is used to coat or contact a substrate or other surface that is not the target collagen layer. See FIG. 7, which illustrates a layered hybrid product CP having a substrate S and a collagen layer CL. The collagen layer CL constitutes or includes the target collagen product formed according to the methods described herein, while the substrate S can be untreated collagen or any other biocompatible material. As a non-limiting example, the aforementioned method can be used to form an amplified collagen layer CL with selected peptides that bind to bone morphogenetic proteins (BMPs) and cause osteoblast proliferation, and the substrate S is the outer surface of an orthopedic implant on which the collagen layer CL is deposited. In a further embodiment, a surface coating (not shown) is formed on the collagen layer CL on the side opposite the implant surface / substrate S. The surface coating is designed to bind a high concentration of alpha2beta1 to the surface in order to recruit osteoblasts from the patient's surrounding tissues. Once recruited to the surface coating, the osteoblasts migrate to the underlying amplified collagen layer CL, where the peptides bind to the BMPs and cause the recruited osteoblasts to proliferate. In various embodiments, the substrate S comprises collagen that has not been modified in accordance with the methods disclosed herein (ie, unmodified collagen).

[0188] In other embodiments of the above methods, the bioactive peptides are removed from the collagen (as opposed to added) such that the resulting collagen product does not possess its bioactive properties. The collagen may or may not be reconstituted.

[0189] Thus, targeted collagen products containing specific collagen fractions / peptides are provided or formed as collagen layers designed for specific medical applications. For example, targeted collagen product layers can be designed in this way to address blood clotting, bone formation / osteogenesis, breast repair or wound healing. Programmed multi-layer collagen products can be created with different specific collagen fractions / peptides in different collagen layers, such that the layers have different bioactive characteristics and functions, as described above.

[0190] It will further be appreciated that a layered target collagen product may be formed from the processes described above, as well as any one or more additional processes known in the art.

[0191] Reference is now made to Figure 8, which is a flow chart of another exemplary embodiment of a method (600) according to the present disclosure, as further described below. The method (600) includes procuring collagen (610), digesting and fractionating the collagen to obtain peptides (620), testing each of the collagen fractions / peptides for various biological characteristics (630), and isolating collagen fractions / peptides having particular desirable / desired characteristics (640), and includes some of the same steps of the method (100) shown in Figure 1 and discussed above.

[0192] In some embodiments of the method, testing of the collagen fraction / peptides (630) is performed only once. In other embodiments of the method, such testing may be performed more than once. In still other embodiments, testing is not performed, for example, when the biological characteristics of the collagen fraction / peptides are already known.

[0193] Known collagen mimetic peptides (CMPs), single, double, and triple helical CMPs, can be made synthetically by peptide synthesis. Such CMPs can also be added to the reconstituted collagen matrix in various embodiments.

[0194] Selected collagen fractions / peptides are added to a 3D printable collagen mixture (665), and the collagen mixture is 3D printed (670) to form a “targeted collagen” with amplified desired properties of the isolated fractions / peptides, such as collagen targeted to soft tissue wound healing.

[0195] In one embodiment, the selected fractionated bioactive peptides are added before performing the 3D printing collagen process and crosslinked to the collagen during the printing process. One alternative for adding the bioactive peptides to the printed collagen is to add such peptides after the printing collagen process, thereby allowing the bioactive peptides to be released from the reconstituted collagen over time, rather than being covalently linked to the collagen. In another alternative embodiment, the bioactive peptides are covalently linked to the printed collagen after or while the collagen is printed. In another embodiment, the bioactive peptides can be removed before printing, so that the collagen does not have its bioactive properties. Combinations and variations of these steps are also possible.

[0196] Figure 9 illustrates another embodiment of a method (700) in which bioactive peptides are removed before the collagen is 3D printed, such that the collagen does not retain its bioactive properties. The method includes some of the same steps of the method (200) shown in Figure 2 and discussed above, including sourcing collagen (710), digesting and fractionating the collagen to obtain peptides (720), testing each of the collagen fractions / peptides for various undesirable biological characteristics (735), isolating the collagen fractions / peptides with the undesirable characteristics (740), and removing the collagen fractions / peptides with the undesirable characteristics from the collagen mixture (755). The collagen mixture is then 3D printed (770) to form the target collagen product.

[0197] Figure 10 illustrates another embodiment of a method (800) that includes adding fractionated collagen peptides having desired characteristics / properties to a mixture (850). The collagen mixture is then 3D printed (870) to form a target collagen product. In various embodiments, the method (800) can include additional steps as illustrated in Figure 8 and discussed above in connection with its associated method (600).

[0198] 11 illustrates yet another embodiment of a method (900) that includes removing fractionated collagen peptides having undesirable characteristics / properties from the mixture to form a target collagen product (955). The collagen mixture is then 3D printed (970) to form the target collagen product. In various embodiments, the method (900) can include additional steps as illustrated in FIG. 9 and discussed above in connection with its associated method (700).

[0199] In various embodiments, once determined, bioactive peptides can be added to or removed from collagen by genetic modification. DNA or RNA can be modified through various CRISPR techniques as well as zinc finger nucleases (ZFNs) or transcription activator-like endonucleases (TALENS). Once modified collagen is synthesized in cells, it can be isolated by conventional means. In various embodiments, bioactive peptides are removed (rather than added) from genetic modification.

[0200] Thus, targeted collagen products containing specific collagen fractions / peptides are designed for specific medical applications, for example, targeted collagen products may be so designed to address blood clotting, bone formation / osteogenesis, breast repair, or wound healing.

[0201] In some embodiments, peptides with desirable properties can be isolated or added or subtracted from intact collagen. For example, blood clotting properties can be added or enriched from a targeted collagen product if such a product is intended for use in a hemostatic product. In one embodiment, the collagen is intact.

[0202] In various embodiments, the amplifying peptide can be covalently, ionically, hydrogen bonded, or linked to the collagen via hydrophobic associations and / or released from the collagen over time, hi another embodiment, the amplifying peptide can be formulated with a biodegradable carrier for release over time.

[0203] In other embodiments, amplifying peptides are added to or removed from the reconstituted collagen to affect the pharmacokinetics or release properties of the bioactive protein. Collagen can also be genetically modified to affect the release of the bioactive protein or peptide. Examples of bioactive proteins that can be added include BMP, PDGF, BDNF, EGF, VEGF, NGF, TNF, etc.

[0204] In some embodiments, the target collagen with amplified peptides can be produced by genetically engineered prokaryotes and eukaryotes, which are further isolated through processing.

[0205] Of course, it is recognized that multiple desirable properties can be engineered into or out of the target collagen. For example, in skin wound healing, collagen amplification via bioactive peptides can be added for clotting, epithelial growth, and faster absorption. Clotting tests can identify peptides that induce this process, epithelial cell assays can identify bioactive peptides of cell activity, and peptides targeted by the enzyme collagenase can be added to increase collagen absorption.

[0206] It will further be appreciated that the target collagen product may be formed from the processes described above, as well as any one or more additional processes known in the art.

[0207] In general, any combination of the disclosed features, components, and methods described herein is possible. The method steps can be carried out in any order that is physically possible.

[0208] All cited references are incorporated herein by reference.

[0209] Although embodiments have been disclosed, the invention is not limited thereto.

Claims

1. A method for forming a target collagen product having an amplified desired property, the method comprising the step of adding a peptide exhibiting the desired property to collagen to form the target collagen product.

2. 10. The method of claim 1, further comprising the step of digesting collagen to obtain peptides therefrom that exhibit desirable properties.

3. 10. The method of claim 1, further comprising the step of sourcing collagen for the digesting step, wherein the sourcing step comprises obtaining collagen from the group consisting of eukaryotic organisms, genetically modified prokaryotes, and genetically modified plant cells.

4. The method of claim 2 further comprising testing the peptide for a desirable property.

5. 5. The method of claim 4, further comprising the step of isolating peptides that exhibit desirable properties.

6. 10. The method of claim 1, wherein the step of adding comprises adding a peptide exhibiting desirable properties to the mixture to reconstitute the collagen into a target collagen product.

7. 7. The method of claim 6, wherein the step of adding reconstituted collagen is carried out to control the release profile of peptides exhibiting desirable properties.

8. 7. The method of claim 6, wherein the step of adding is carried out such that the reconstituted collagen is crosslinked to peptides that exhibit desirable properties.

9. 10. The method of claim 1, wherein the step of adding comprises adding a peptide exhibiting a desired property to the intact collagen.

10. 2. The method of claim 1, wherein the desirable properties of the peptide have uses selected from the group consisting of wound healing, bone generation, blood clotting, organ scaffolding, dura repair, nerve repair, ocular repair, epithelial repair, and cardiovascular repair, breast repair, and endothelial repair.

11. A method for forming a target collagen product lacking an undesirable property, the method comprising removing peptides exhibiting the undesirable property from collagen to form the target collagen product.

12. 12. The method of claim 11, further comprising the step of digesting collagen to obtain peptides therefrom that exhibit undesirable properties.

13. The method of claim 12 further comprising testing the peptide for undesirable properties.

14. 14. The method of claim 13, further comprising the step of isolating peptides that exhibit undesirable properties.

15. 12. The method of claim 11, wherein the removing step comprises removing peptides exhibiting undesirable properties from the mixture and reconstituting the collagen into the target collagen product.

16. 12. The method of claim 11, wherein the removing step comprises removing peptides exhibiting undesirable properties to reconstitute the remaining collagen containing the desired properties, thereby enriching the desired properties.

17. 12. The method of claim 11, wherein the removing step comprises removing peptides that exhibit undesirable properties from the intact collagen.

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