High-strength collagen composition and method of use

Abstract

We provide collagen scaffolds that are fabricated without the use of harmful exogenous treatments or cross-linking strategies, offering advantages in the fields of tissue engineering and regenerative medicine. [Solution] The present invention relates to an artificial collagen scaffold having a thickness of about 0.005 mm to about 3 mm and high strength (e.g., a high modulus of elasticity of about 0.5 MPa to about 200 MPa). This artificial collagen scaffold may be non-collapsible and / or non-expandable. This disclosure also relates to methods for using these collagen scaffolds.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 002,644, filed Mar. 31, 2020, under 35 U.S.C. § 119(e), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to an artificial collagen scaffold having a thickness of about 0.005 mm to about 3 mm and high strength [e.g., a high elastic modulus of about 0.5 MPa to about 200 MPa]. The artificial collagen scaffold can be non - collapsible and / or non - expandable. The present disclosure also relates to methods of using these collagen scaffolds.

Background Art

[0003] The ability to replace, repair or regenerate damaged or malfunctioning tissues in patients represents a major challenge in medicine. An important component of all tissues and organs is the extracellular matrix (ECM), which refers to the non - living material in which living cells are distributed and organized. The ECM not only determines the mechanical properties of tissues, but also provides a physical scaffold that supports cells in three dimensions. Furthermore, the ECM serves as an important regulator of cell behavior, informing cells through essential biochemical and biomechanical signaling. Given the importance of the overall tissue structure and function of the ECM, efforts in tissue engineering and regenerative medicine have focused on the development of materials to reconstruct ECM scaffolds for improved tissue repair, recovery and regeneration outcomes.

[0004] Collagen is the most abundant protein in the ECM and the body and serves as a major determinant of the structural and mechanical properties of tissues. In the body, collagen is produced by cells as a single molecule consisting of three polypeptide chains. Individual collagen molecules, also known as tropocollagen, have a central triple helix domain capped at both ends by more randomly organized telopeptides. In vivo, individual collagen molecules (monomers) undergo hierarchical self-assembly to form a polymeric material (e.g., the insoluble fibrous matrix of the ECM), so collagen is not only a protein but also a polymer. During synthesis and assembly in vivo, these polymeric collagen materials are further stabilized by the formation of intermolecular and intramolecular cross-links, which help impart mechanical strength and control collagen turnover (i.e., the balance between collagen degradation and synthesis). Because of its dual role as a structural and cell signaling element of the ECM, collagen is a preferred biomaterial in both research and clinical settings. Its high availability in the body, conservation across tissues and species, predictable degradability by proteolytic enzymes (e.g., matrix metalloproteinases), and high biocompatibility also make collagen ideally suited for tissue engineering and regenerative medicine applications.

[0005] To date, artificial collagen scaffolds known in the art have typically been made from collagen monomers known as telocollagen and atelocollagen. Telocollagen represents the full-length tropocollagen molecule, which is usually isolated from tissue via acid extraction. Atelocollagen represents a modified example of the natural tropocollagen molecule, where the telopeptide terminus is enzymatically cleaved during the protein isolation and purification process. The drawbacks of these collagen monomers in the preparation of collagen materials are well-established and include significant lot-to-lot variability in purity and polymerization ability, long polymerization times (often exceeding 30 minutes), insufficient stability and mechanical integrity of the resulting polymer material, and rapid proteolysis of the resulting polymer material in vitro and in vivo. The instability of collagen materials formed from telocollagen and atelocollagen has led to the need for additional material treatment strategies to improve the stiffness (modulus of elasticity) and strength of the material, as well as its resistance to proteolysis. These material treatment strategies primarily utilize exogenous crosslinking via physical and chemical means or copolymerization with other materials. While such strategies have successfully improved the mechanical properties and stability of collagen materials in various ways, it is known that they have detrimental effects on collagen's inherent biological signaling capabilities, leading to adverse tissue responses, including inflammation and xenobiotic reactions. Furthermore, the achievable densities of materials prepared from telocollagen and atelocollagen are limited, far lower than the collagen density (concentration) found in connective tissue in vivo. This observation is crucial because the physical characteristics of collagen materials, including scaffold stiffness and collagen density, have been shown to directly influence fundamental cellular behavior, including proliferation, migration, and differentiation processes that occur during tissue repair and regeneration. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a need for high-strength collagen scaffolds that approach the in vivo structure and functionality of natural collagen scaffolds, which can be fabricated without the use of harmful exogenous treatments or crosslinking strategies, and which offer advantages in the fields of tissue engineering and regenerative medicine. Surprisingly, the inventors have developed artificial collagen scaffolds with a thickness of about 0.005 mm to about 3 mm and high strength (e.g., high modulus of elasticity of about 0.5 MPa to about 200 MPa). In one embodiment, these collagen scaffolds may be non-disintegrating and / or non-expandable, and their strength is similar to that of high-strength tissues found in vivo, such as the pericardium, amniotic membrane, and heart valves. Furthermore, the high-strength properties facilitate the handling and application of the material when used clinically in tissue replacement and reconstruction procedures. [Means for solving the problem]

[0007] The artificial collagen scaffolds of this disclosure offer several advantages compared to those known in the art. Firstly, the artificial collagen scaffolds of this disclosure have improved mechanical properties compared to those in the art. In particular, the artificial collagen scaffolds of this disclosure are less prone to fracture and have improved mechanical properties (e.g., high modulus of elasticity of about 0.5 MPa to about 200 MPa) without using exogenous crosslinking and processing strategies known to be detrimental to natural collagen biosignaling and in vivo tissue responses. Furthermore, the artificial collagen scaffolds of this disclosure have improved resistance to degradation, a slow turnover, and do not induce inflammation or foreign body reactions.

[0008] In one embodiment, a non-disintegrating and / or non-expanding artificial collagen scaffold is provided. The collagen scaffold has a thickness of about 0.005 mm to about 3 mm and an elastic modulus of about 0.5 MPa to about 200 MPa.

[0009] Another embodiment provides a method for treating a patient to regenerate, restore, or replace damaged or dysfunctional tissue. The method includes implanting a medical transplant tissue, comprising one of the artificial collagen scaffolds described herein, into the patient.

[0010] Further embodiments are also described by the items listed below. Any of the following embodiments may also be combined with any applicable embodiments described in the Background and Summary section, the Detailed Description of Exemplary Embodiments section, the Examples section, or the Claims section.

[0011] 1. An artificial collagen scaffold that is non-disintegrating and / or non-expandable, having a thickness of about 0.005 mm to about 3 mm and an elastic modulus of about 0.5 MPa to about 200 MPa.

[0012] 2. An artificial collagen scaffold as described in item 1, which does not disintegrate when freeze-dried and rehydrated.

[0013] 3. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 2.0 mm, as described in any one of items 1 or 2.

[0014] 4. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 1.0 mm, as described in any one of items 1 or 2.

[0015] 5. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 0.25 mm, as described in any one of items 1 or 2.

[0016] 6. An artificial collagen scaffold having a thickness of approximately 0.1 mm to approximately 1.0 mm, as described in any one of items 1 or 2.

[0017] 7. An artificial collagen scaffold having a thickness of approximately 0.5 mm to 1.0 mm, as described in any one of items 1 or 2.

[0018] 8. An artificial collagen scaffold having a thickness of approximately 0.15 mm to approximately 0.25 mm, as described in any one of items 1 or 2.

[0019] 9. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 18 MPa to approximately 200 MPa.

[0020] 10. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 20 MPa to approximately 180 MPa.

[0021] 11. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 40 MPa to approximately 120 MPa.

[0022] 12. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 60 MPa to approximately 100 MPa.

[0023] 13. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 80 MPa to approximately 180 MPa.

[0024] 14. An artificial collagen scaffold described in any one of items 1 to 8, having an ultimate tensile strength of approximately 0.5 MPa to approximately 20 MPa.

[0025] 15. An artificial collagen scaffold described in any one of items 1 to 8, having an ultimate tensile strength of approximately 1 MPa to approximately 25 MPa.

[0026] 16. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 0.2 MPa to approximately 20 MPa.

[0027] 17. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 5 MPa to approximately 15 MPa.

[0028] 18. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 2 MPa to approximately 20 MPa.

[0029] 19. An artificial collagen scaffold described in any one of items 1 through 18, having a fracture strain of approximately 5% to 70%.

[0030] 20. An artificial collagen scaffold described in any one of items 1 through 18, having a fracture strain of approximately 10% to 40%.

[0031] 21. An artificial collagen scaffold according to any one of items 1 to 20, having a suture-holding peak load of approximately 2N to approximately 8N.

[0032] 22. An artificial collagen scaffold according to any one of items 1 to 20, having a suture-holding peak load of approximately 0.2 N to approximately 2 N.

[0033] 23. An artificial collagen scaffold according to any one of items 1 to 20, having a suture retention peak load of approximately 0.1 N to approximately 4 N.

[0034] 24. An artificial collagen scaffold according to any one of items 1 to 23, wherein the composition comprises an artificial collagen scaffold, and the composition further comprises a fluid.

[0035] 25. An artificial collagen scaffold as described in item 24, in which the percentage of fluid present is approximately 5% to approximately 99%.

[0036] 26. An artificial collagen scaffold according to any one of item 24 or 25, wherein the composition is dried by freeze-drying, vacuum-pressing, or dehydrating heat treatment, or a combination thereof.

[0037] 27. An artificial collagen scaffold described in any one of items 1 through 26, wherein the collagen is type I collagen.

[0038] 28. An artificial collagen scaffold described in any one of items 1 through 27, wherein the collagen is purified type I collagen.

[0039] 29. An artificial collagen scaffold, which is a medical transplant tissue, as described in any one of items 1 through 28.

[0040] 30. An artificial collagen scaffold as described in any one of items 1 through 29, wherein the artificial collagen scaffold is a medical transplant tissue, and the medical transplant tissue is used for the regeneration, restoration, or replacement of damaged or dysfunctional tissue.

[0041] 31. Artificial collagen scaffolds as described in item 30, which are medical transplant tissues for the regeneration, replacement, or restoration of tissues selected from the pericardium, heart valves, skin, blood vessels, airway tissues, body walls, and tissues reconstructed after tumor removal.

[0042] 32. An artificial collagen scaffold described in any one of items 1 through 31, which is final sterilized or prepared aseptically.

[0043] 33. An artificial collagen scaffold as described in item 32, which is final sterilized by a process selected from glutaraldehyde treatment, gamma irradiation, electron beam irradiation, or ethylene oxide treatment.

[0044] 34. An artificial collagen scaffold as described in any one of items 1 through 33, wherein the collagen comprises oligomeric collagen, monomeric collagen, telocollagen or atelocollagen, or a combination thereof.

[0045] 35. An artificial collagen scaffold described in any one of items 1 through 34, compressed into a specified shape.

[0046] 36. An artificial collagen scaffold as described in item 35, which is spherical in shape.

[0047] 37. An artificial collagen scaffold as described in item 35, having a tubular shape.

[0048] 38. An artificial collagen scaffold as described in item 35, which is in the form of a sheet.

[0049] 39. An artificial collagen scaffold described in any one of items 35 to 38, wherein the compression is constrained compression.

[0050] 40. Collagen concentration is approximately 50 to 1000 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0051] 41. Collagen concentration is approximately 50 to 900 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0052] 42. Collagen concentration is approximately 50 to 800 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0053] 43. Collagen concentration is approximately 50 to 700 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0054] 44. Collagen concentration is approximately 50-600 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0055] 45. Collagen concentration is approximately 50 to 500 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0056] 46. The artificial collagen scaffold according to any one of items 1 to 39, wherein the collagen concentration is about 50 to about 400 mg / cm 3 .

[0057] 47. The artificial collagen scaffold according to any one of items 1 to 39, wherein the collagen concentration is about 50 to about 300 mg / cm 3 .

[0058] 48. The artificial collagen scaffold according to any one of items 1 to 39, wherein the collagen concentration is about 50 to about 200 mg / cm 3 .

[0059] 49. The artificial collagen scaffold according to any one of items 1 to 48, which does not induce inflammation or foreign body reaction when transplanted into a patient.

[0060] 50. The artificial collagen scaffold according to any one of items 1 to 49, wherein the collagen is selected from porcine collagen, human collagen and bovine collagen.

[0061] 51. The artificial collagen scaffold according to any one of items 1 to 49, wherein the collagen is synthetic collagen.

[0062] 52. The artificial collagen scaffold according to any one of items 1 to 50, wherein the collagen is native collagen.

[0063] 53. The artificial collagen scaffold according to any one of items 1 to 49, wherein the collagen is recombinant collagen.

[0064] 54. The artificial collagen scaffold according to any one of items 1 to 53, further comprising cells.

[0065] 55. The artificial collagen scaffold according to item 54, wherein the cells are stem cells.

[0066] 56. A method for treating a patient to regenerate, restore or replace damaged or dysfunctional tissue, comprising implanting a medical transplant tissue comprising an artificial collagen scaffold as described in any one of items 1 to 55 into the patient.

[0067] 57. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or dysfunctional pericardium.

[0068] 58. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or malfunctioning heart valve.

[0069] 59. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of damaged or dysfunctional skin.

[0070] 60. The method according to item 59, wherein the valve tissue is aortic valve tissue or pulmonary valve tissue.

[0071] 61. An artificial collagen scaffold described in any one of items 1 to 55 or the method described in any one of items 56 to 60, wherein the artificial collagen scaffold is exogenously cross-linked.

[0072] 62. The artificial collagen scaffold or method described in item 61, wherein the artificial collagen scaffold is cross-linked with glutaraldehyde. [Brief explanation of the drawing]

[0073] [Figure 1]Figure 1A shows a chamber compression apparatus and associated densification / dehydration process. The cylindrical chamber was fabricated from Delrin and fitted with a solid spherical sheet at the bottom. The cylinder was filled with liquid collagen, which polymerizes at 37°C to form a complex of low-density collagen matrix consisting of an interconnected network of fibrous collagen surrounded by interstitial fluid. Figure 1B shows that the solid bottom surface can be removed and replaced with a thin spherical sheet of porous polyethylene foam fixed to a porous bottom surface. The thin spherical sheet of porous polyethylene foam can then be placed on top of the collagen scaffold. In the figure, "Porous Platens" is translated as "porous platens" and "Bottom Surface With Holes" is translated as "bottom surface with holes". Figure 1C shows that by applying a compressive load to the upper polyethylene foam in the direction of the gray arrow, fluid can be discharged from the collagen scaffold through both the upper and lower sides (white arrows) to create a hydrated, densified collagen scaffold. [Figure 2] Figure 2A shows a syringe compression device and associated densification / dehydration process in which a composite collagen scaffold is polymerized in a syringe system on top of a stainless steel mesh disc. After polymerization, a piece of Whatman filter paper and a modified plunger are placed inside the syringe to compress the scaffold. Figure 2B shows compressing the plunger in the direction of the gray arrow to expel fluid from the collagen scaffold in both directions (white arrows) to create a densified collagen scaffold. [Figure 3]The left side (Figure 3A) shows a photograph of a typical prototype collagen scaffold (6.3 cm in diameter) prepared by compression dehydration using formulation 3. The right side (Figure 3B) shows a photograph of a typical prototype collagen scaffold (6.3 cm in diameter) prepared by compression dehydration using formulation 4. [Figure 4A] Figure 4A shows the elastic modulus of the collagen scaffold. In the figure, "Collagen Content" is translated as "Collagen content" and "Elastic Modulus" as "Elastic modulus". [Figure 4B] Figure 4B shows the UTS of the collagen scaffold. In the figure, "Collagen Content" is translated as "Collagen content". [Figure 4C] Figure 4C shows the fracture strain as a function of collagen content. In the figure, "Collagen Content" is translated as "Collagen content" and "Failure Strain" is translated as "Failure strain". [Figure 5] Figure 5 shows an image of a prototype collagen scaffold (6.3 cm in diameter) prepared with a total collagen content of 500 mg, followed by vacuum pressing and dehydration by heat of dehydration (DHT) at 90°C. The collagen scaffold was rehydrated in phosphate-buffered saline. [Figure 6] Figure 6 shows photographs of pre-subcutaneous implantation materials in rats, including collagen scaffolds of Formulas 3 and 4 in the absence and presence of glutaraldehyde (GTA) treatment, as well as glutaraldehyde-treated pericardium (PC GTA). [Figure 7] Figure 7 shows photographs of material explants 60 days after subcutaneous transplantation in rats, including collagen scaffolds of Formulas 3 and 4 in the absence and presence of glutaraldehyde (GTA) treatment, as well as glutaraldehyde-treated pericardium (PC GTA). [Figure 8]The upper (Figure 8A) and lower (Figure 8B) sections summarize the semi-quantitative scores (mean ± SD; n=10) for tissue reaction and tissue integration as determined by macroscopic observation of material explants 60 days after subcutaneous transplantation in rats. Figure 8A shows the results for an uncompressed sample (14 mm thick) prepared from 3.5 mg / ml oligomeric collagen, and Figure 8B shows the results for a collagen sheet (2 mm thick and approximately 24.5 mg / ml) obtained after compression at 6 mm / min. In the figures, "Tissue Reaction Score" is translated as "Tissue Reaction Score," "Formula" as "Formula," "Material Implant" as "Material Implant," and "Tissue Integration Score" as "Tissue Integration Score." [Figure 9] Figure 9 shows low (upper) and high (lower) magnification images of histological sections (hematoxylin eosin staining) of skin explants and related collagen scaffolds (Formula 3) in the absence and presence of glutaraldehyde (GTA) treatment. [Figure 10] Figure 10 shows low (upper) and high (lower) magnification images of histological sections (hematoxylin eosin staining) of skin explants and related collagen scaffolds (Formula 4) in the absence and presence of glutaraldehyde (GTA) treatment. [Figure 11] Figure 11 shows low (upper) and high (lower) magnification images of histological sections (hematoxylin-eosin staining) of skin explants and associated glutaraldehyde-treated pericardium (PC GTA). [Modes for carrying out the invention]

[0074] The materials and medical graft tissues described herein include artificial collagen scaffolds that can be prepared in hydrated or desiccated (dry) form. Both can be used for surgical repair, regeneration, recovery, or reconstruction of damaged or dysfunctional tissues, such as pericardium, skin, airway tissue, body wall, and tissue reconstruction after tumor removal, as well as for the production of advanced regenerative tissue replacements (e.g., heart valves, such as aortic and pulmonary valves, and vascular graft tissues). The collagen scaffolds described herein persist in vivo after transplantation, continuously restoring the tissue without inducing inflammation or foreign body reactions, maintaining their physical integrity, and inducing host tissue integration, cellularization, and site-specific tissue regeneration. In various embodiments, the collagen scaffolds described herein include high-strength thin sheet-like materials or high-strength thin material formats in various shapes, having physical and mechanical properties similar to various naturally occurring high-strength tissues and conventional collagen-based materials.

[0075] When used in reference to tissue, the terms “reestablish,” “regenerate,” “replace,” and “repair” refer, respectively, to the re-establishment of tissue presence in a patient area previously characterized by tissue voids or defects, and the regrowth of tissue in this same area. In some embodiments, the restored and / or regenerated and / or repaired tissue may reflect one or more of the appearance, structure, and function of the original tissue being replaced.

[0076] In one embodiment, the invention described herein relates to an artificial collagen scaffold having a thickness of about 0.005 mm to about 3 mm and high strength (e.g., a high modulus of elasticity of about 0.5 MPa to about 200 MPa). In one embodiment, the artificial collagen scaffold may be non-collapsible and / or non-expandable. In another embodiment, a method for using these collagen scaffolds is provided.

[0077] In various embodiments, the artificial collagen scaffold may have a thickness of approximately 0.005 mm to approximately 3 mm, approximately 0.01 mm to approximately 2.0 mm, approximately 0.01 mm to approximately 1.0 mm, approximately 0.01 mm to approximately 0.25 mm, approximately 0.1 mm to approximately 1.0 mm, approximately 0.5 mm to approximately 1.0 mm, or approximately 0.15 mm to approximately 0.25 mm. In other embodiments, the collagen scaffold may have an elastic modulus of approximately 18 MPa to approximately 200 MPa, approximately 20 MPa to approximately 180 MPa, approximately 40 MPa to approximately 120 MPa, approximately 60 MPa to approximately 100 MPa, or approximately 80 MPa to approximately 180 MPa. In other embodiments, the collagen scaffold may have an ultimate tensile strength of approximately 0.5 MPa to approximately 20 MPa, approximately 1 MPa to approximately 25 MPa, approximately 0.2 MPa to approximately 20 MPa, approximately 5 MPa to approximately 15 MPa, or approximately 2 MPa to approximately 20 MPa. In other exemplary embodiments, the artificial collagen scaffold may have a fracture strain of about 5% to about 70%, or about 10% to about 40%. In other embodiments, the artificial collagen scaffold may have a suture-retaining peak load of about 2N to about 8N, about 0.2N to about 2N, or about 0.1N to about 4N.

[0078] In another embodiment, a non-collapsible and / or non-expandable artificial collagen scaffold is provided. The collagen scaffold has a thickness of about 0.005 mm to about 3 mm and an elastic modulus of about 0.5 MPa to about 200 MPa. As used herein, the term “non-collapsible” means that the collagen scaffold maintains its thickness and other geometric properties when transitioning from a dehydrated state to a hydrated state. As used herein, the term “non-expandable” means that the material does not expand or swell when freeze-dried or rehydrated. Thus, in one exemplary embodiment, the collagen scaffold described herein does not disintegrate when the collagen scaffold is freeze-dried and rehydrated. In one embodiment, the collagen scaffold is non-collapsible and / or non-expandable due to its high elastic modulus, which is determined in part by the fibril density of the collagen scaffold, along with its hydrophilic (water-retaining) properties.

[0079] In yet another embodiment, a method is provided for treating a patient to regenerate, restore, or replace damaged or dysfunctional tissue. The method includes implanting a medical transplant tissue comprising one of the artificial collagen scaffolds described herein into the patient. As used herein, “medical transplant tissue” means one of the collagen materials described herein administered to the patient.

[0080] Further embodiments are also described by the items listed below. For all embodiments described herein, any applicable combination of embodiments is intended. Any applicable combination of the embodiments described below is considered to be in accordance with the present invention. Any combination of the embodiments described below with embodiments described in the Background and Summary section, the Detailed Description of Exemplary Embodiments section, the Examples section, or the Claims section is considered to be part of the present invention.

[0081] 1. An artificial collagen scaffold that is non-disintegrating and / or non-expandable, having a thickness of about 0.005 mm to about 3 mm and an elastic modulus of about 0.5 MPa to about 200 MPa.

[0082] 2. An artificial collagen scaffold as described in item 1, which does not disintegrate when freeze-dried and rehydrated.

[0083] 3. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 2.0 mm, as described in any one of items 1 or 2.

[0084] 4. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 1.0 mm, as described in any one of items 1 or 2.

[0085] 5. An artificial collagen scaffold having a thickness of approximately 0.01 mm to approximately 0.25 mm, as described in any one of items 1 or 2.

[0086] 6. An artificial collagen scaffold having a thickness of approximately 0.1 mm to approximately 1.0 mm, as described in any one of items 1 or 2.

[0087] 7. An artificial collagen scaffold having a thickness of approximately 0.5 mm to 1.0 mm, as described in any one of items 1 or 2.

[0088] 8. An artificial collagen scaffold having a thickness of approximately 0.15 mm to approximately 0.25 mm, as described in any one of items 1 or 2.

[0089] 9. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 18 MPa to approximately 200 MPa.

[0090] 10. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 20 MPa to approximately 180 MPa.

[0091] 11. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 40 MPa to approximately 120 MPa.

[0092] 12. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 60 MPa to approximately 100 MPa.

[0093] 13. An artificial collagen scaffold described in any one of items 1 to 8, having an elastic modulus of approximately 80 MPa to approximately 180 MPa.

[0094] 14. An artificial collagen scaffold described in any one of items 1 to 8, having an ultimate tensile strength of approximately 0.5 MPa to approximately 20 MPa.

[0095] 15. An artificial collagen scaffold described in any one of items 1 to 8, having an ultimate tensile strength of approximately 1 MPa to approximately 25 MPa.

[0096] 16. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 0.2 MPa to approximately 20 MPa.

[0097] 17. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 5 MPa to approximately 15 MPa.

[0098] 18. An artificial collagen scaffold described in any one of items 1 to 15, having an ultimate tensile strength of approximately 2 MPa to approximately 20 MPa.

[0099] 19. An artificial collagen scaffold described in any one of items 1 through 18, having a fracture strain of approximately 5% to 70%.

[0100] 20. An artificial collagen scaffold described in any one of items 1 through 18, having a fracture strain of approximately 10% to 40%.

[0101] 21. An artificial collagen scaffold according to any one of items 1 to 20, having a suture-holding peak load of approximately 2N to approximately 8N.

[0102] 22. An artificial collagen scaffold according to any one of items 1 to 20, having a suture-holding peak load of approximately 0.2 N to approximately 2 N.

[0103] 23. An artificial collagen scaffold according to any one of items 1 to 20, having a suture retention peak load of approximately 0.1 N to approximately 4 N.

[0104] 24. An artificial collagen scaffold according to any one of items 1 to 23, wherein the composition comprises an artificial collagen scaffold, and the composition further comprises a fluid.

[0105] 25. An artificial collagen scaffold as described in item 24, in which the percentage of fluid present is approximately 5% to approximately 99%.

[0106] 26. An artificial collagen scaffold according to any one of item 24 or 25, wherein the composition is dried by freeze-drying, vacuum-pressing, or dehydrating heat treatment, or a combination thereof.

[0107] 27. An artificial collagen scaffold described in any one of items 1 through 26, wherein the collagen is type I collagen.

[0108] 28. An artificial collagen scaffold described in any one of items 1 through 27, wherein the collagen is purified type I collagen.

[0109] 29. An artificial collagen scaffold, which is a medical transplant tissue, as described in any one of items 1 through 28.

[0110] 30. An artificial collagen scaffold as described in any one of items 1 through 29, wherein the artificial collagen scaffold is a medical transplant tissue, and the medical transplant tissue is used for the regeneration, restoration, or replacement of damaged or dysfunctional tissue.

[0111] 31. Artificial collagen scaffolds as described in item 30, which are medical transplant tissues for the regeneration, replacement, or restoration of tissues selected from the pericardium, heart valves, skin, blood vessels, airway tissues, body walls, and tissues reconstructed after tumor removal.

[0112] 32. An artificial collagen scaffold described in any one of items 1 through 31, which is final sterilized or prepared aseptically.

[0113] 33. An artificial collagen scaffold as described in item 32, which is final sterilized by a process selected from glutaraldehyde treatment, gamma irradiation, electron beam irradiation, or ethylene oxide treatment.

[0114] 34. An artificial collagen scaffold as described in any one of items 1 through 33, wherein the collagen comprises oligomeric collagen, monomeric collagen, telocollagen or atelocollagen, or a combination thereof.

[0115] 35. An artificial collagen scaffold described in any one of items 1 through 34, compressed into a specified shape.

[0116] 36. An artificial collagen scaffold as described in item 35, which is spherical in shape.

[0117] 37. An artificial collagen scaffold as described in item 35, having a tubular shape.

[0118] 38. An artificial collagen scaffold as described in item 35, which is in the form of a sheet.

[0119] 39. An artificial collagen scaffold described in any one of items 35 to 38, wherein the compression is constrained compression.

[0120] 40. Collagen concentration is approximately 50 to 1000 / cm³ 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0121] 41. Collagen concentration is approximately 50 to 900 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0122] 42. Collagen concentration is approximately 50 to 800 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0123] 43. Collagen concentration is approximately 50 to 700 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0124] 44. Collagen concentration is approximately 50-600 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0125] 45. Collagen concentration is approximately 50 to 500 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0126] 46. ​​Collagen concentration is approximately 50 to 400 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0127] 47. Collagen concentration is approximately 50-300 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0128] 48. Collagen concentration is approximately 50 to 200 mg / cm³. 3 An artificial collagen scaffold as described in any one of items 1 through 39.

[0129] 49. An artificial collagen scaffold described in any one of items 1 through 48 that does not induce inflammation or a foreign body reaction when implanted in a patient.

[0130] 50. An artificial collagen scaffold as described in any one of items 1 to 49, wherein the collagen is selected from porcine collagen, human collagen, and bovine collagen.

[0131] 51. An artificial collagen scaffold described in any one of items 1 through 49, wherein the collagen is synthetic collagen.

[0132] 52. An artificial collagen scaffold described in any one of items 1 through 50, wherein the collagen is undenatured collagen.

[0133] 53. An artificial collagen scaffold as described in any one of items 1 through 49, wherein the collagen is recombinant collagen.

[0134] 54. An artificial collagen scaffold as described in any one of items 1 through 53, further containing cells.

[0135] 55. An artificial collagen scaffold as described in item 54, in which the cells are stem cells.

[0136] 56. A method for treating a patient to regenerate, restore or replace damaged or dysfunctional tissue, comprising implanting a medical transplant tissue comprising an artificial collagen scaffold as described in any one of items 1 to 55 into the patient.

[0137] 57. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or dysfunctional pericardium.

[0138] 58. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or malfunctioning heart valve.

[0139] 59. The method described in item 56, wherein medical transplant tissue is for the regeneration, restoration, or replacement of damaged or dysfunctional skin.

[0140] 60. The method according to item 59, wherein the valve tissue is aortic valve tissue or pulmonary valve tissue.

[0141] 61. An artificial collagen scaffold described in any one of items 1 to 55 or the method described in any one of items 56 to 60, wherein the artificial collagen scaffold is exogenously cross-linked.

[0142] 62. The artificial collagen scaffold or method described in item 61, wherein the artificial collagen scaffold is cross-linked with glutaraldehyde.

[0143] Where otherwise described herein, “artificial collagen scaffold” or “collagen scaffold” may refer to a collagen composition that can be synthesized ex vivo or at the time of implantation into a patient’s body to form a collagen fibril-containing scaffold or other collagen structure or material. In one embodiment, polymerization may occur under controlled conditions, which are not limited to but include pH, phosphate concentration, temperature, buffer composition, ionic strength, and the composition and concentration of extracellular matrix components (e.g., collagen molecules, and, if non-collagen ECM components are included, non-collagen molecules). “Artificial collagen scaffold” is either a non-natural collagen scaffold or another non-natural collagen structure or material.

[0144] In one embodiment, the artificial collagen scaffold of the present disclosure is prepared using a compression technique. As used herein, the term “compressed” refers to a reduction in size or an increase in density when a force is applied to a collagen scaffold composition. For example, compression can be achieved through various methods of applying force, such as, but are not limited to, constrained compression, variable compression, physical compression, centrifugation, ultracentrifugation, evaporation, or suction.

[0145] In one embodiment, the compression is variable compression. As used herein, the term "variable compression" refers to the compression of collagen by applying a nonlinear force.

[0146] In yet another embodiment, the compression is physical compression. As used herein, the term “physical compression” refers to the compression of collagen by applying force by physical means. In embodiments where the compression is physical compression, the physical compression may be carried out in a chamber comprising an adjustable mold and a platen (see, for example, Figure 1). Typically, the collagen may be inserted into the mold and then subjected to compression.

[0147] Furthermore, in various embodiments, physical compression may vary depending on the arrangement of the porous platen within the mold. For example, the mold may be adjustable so that the porous polyethylene is arranged as part of the platen and / or along the walls or bottom of the sample mold. In some embodiments, compression is a physical force from at least one direction. In other embodiments, compression is a physical force from two or more directions. In yet another embodiment, compression is a physical force from three or more directions. In some embodiments, compression is a physical force from four or more directions.

[0148] In other embodiments, compression is centrifugal. In other embodiments, compression is ultracentrifugal. In yet another embodiment, compression is evaporation. In some embodiments, compression is suction. In a particular embodiment, suction is vacuum suction.

[0149] In some embodiments of this disclosure, collagen is solubilized from tissue. For example, collagen can be prepared by utilizing acid-solubilized collagen, as described in, for example, U.S. Patent Application No. 11 / 435,635 (published November 22, 2007, publication number 2007-0269476 A1) and No. 11 / 903,326 (published October 30, 2008, publication number 2008-0268052), each of which is incorporated herein by whole reference, and by utilizing predetermined polymerization conditions controlled to obtain a three-dimensional collagen scaffold with controlled assembly dynamics (e.g., polymerization half-life), molecular composition, and fibril microstructure-mechanical properties. In other embodiments, collagen is polymerizable collagen. In yet another embodiment, collagen is type I collagen. In yet another embodiment, collagen is purified type I collagen.

[0150] In some embodiments, the artificial collagen scaffold is a medical transplant tissue. In other embodiments, the artificial collagen scaffold can be used to produce regenerative tissue replacement. In other embodiments, the artificial collagen scaffold may be used in vitro. For example, the in vitro use of the artificial collagen scaffold of this disclosure may be used for research purposes such as cell tissue culture, drug discovery, and drug toxicity testing.

[0151] In some embodiments, the collagen is non-natural collagen. As used herein, the term “non-natural collagen” refers to collagen isolated from a source tissue. In one embodiment, the non-natural collagen isolated from the source tissue may be undenatured collagen. In one embodiment, the non-natural collagen may be solubilized from the tissue source. In other embodiments, the collagen is synthetic collagen. In yet another embodiment, the collagen is recombinant collagen.

[0152] In one embodiment, non-natural collagen or collagen components may be used to construct the collagen scaffolds described herein, which can be obtained from several sources, including, for example, pig skin, human skin, or bovine skin. Suitable tissues useful as collagen-containing source materials for isolating collagen or collagen components to produce the collagen scaffolds described herein are submucosal tissue of warm-blooded vertebrates or any other extracellular matrix-containing tissue. Suitable methods for preparing submucosal tissue are described in U.S. Patents 4,902,508, 5,281,422, and 5,275,826, which are incorporated herein by reference, respectively. In another embodiment, extracellular matrix-containing tissues other than submucosal tissue may be used to obtain collagen and scaffolds according to the methods described herein. Methods for preparing other extracellular matrix-derived tissues for use in obtaining purified collagen or partially purified extracellular matrix components are known to those skilled in the art. For example, see U.S. Patent No. 5,163,955 (pericardial tissue); No. 5,554,389 (bladder submucosa); No. 6,099,567 (gastric submucosa); No. 6,576,265 (extracellular matrix tissue in general); No. 6,793,939 (hepatic basement membrane tissue); and U.S. Patent Application Publication No. US-2005-0019419-A1 (hepatic basement membrane tissue); and International Publication No. WO2001 / 45765 (extracellular matrix tissue in general), each incorporated herein by reference. In various other embodiments, the collagen-containing raw material can be selected from the group consisting of placental tissue, ovarian tissue, uterine tissue, animal tail tissue, and skin tissue. In some embodiments, the collagen is selected from the group consisting of porcine collagen, bovine collagen, and human collagen. Any suitable extracellular matrix-containing tissue can be used as a collagen-containing source material for isolating purified collagen or partially purified extracellular matrix components.

[0153] Exemplary preparation methods for preparing submucosal tissue as a source of purified collagen or partially purified extracellular matrix components are described in U.S. Patent No. 4,902,508, the disclosure of which is incorporated herein by reference. In one embodiment, for example, a portion of vertebrate intestine harvested from, preferably, pig, sheep, or bovine species (without excluding other species) is subjected to scraping using a longitudinal wiping motion to remove cells, or cell removal is achieved by hypotonic or hypertonic dissolution. In one embodiment, the submucosal tissue may be rinsed and sterilized under hypotonic conditions, for example, with water or saline under hypotonic conditions. In another exemplary embodiment, such a composition can be prepared by mechanically removing the luminal portion and outer muscle layer of the mucosa, and / or dissolving resident cells in a hypotonic or hypertonic washing solution, for example, water or saline. In these embodiments, the submucosal tissue may be stored in a hydrated or dehydrated state before isolation of the purified collagen or partially purified extracellular matrix components. In various embodiments, the submucosa may include any delamination embodiment that includes the submucosa detached from both the muscular layer and at least the luminal portion of the mucosa of a warm-blooded vertebrate.

[0154] In some embodiments, collagen is oligomeric collagen. Unlike conventional monomeric collagen preparations, i.e., telocollagen and atelocollagen, oligomers may represent small aggregates of full-length triple-helical collagen molecules (i.e., tropocollagen) having intact carboxy and amino-terminal telopeptides held together by naturally occurring intermolecular crosslinks. The retention of these molecular features, including the carboxy and amino-terminal telopeptide regions, as well as the associated intermolecular crosslinks, results in this biopolymer and collagen material, which forms with desirable but uncommon properties. More specifically, oligomers retain their fibril-forming (self-assembly) ability, which is inherent to fibrous collagen proteins. The presence of oligomeric collagen can enhance self-assembly potential by increasing the assembly rate and by producing collagen compositions with different fibril microstructures and increased mechanical integrity (e.g., rigidity). In some embodiments, collagen comprises oligomeric collagen. In other embodiments, collagen is essentially derived from oligomeric collagen. In yet another embodiment, collagen consists of oligomeric collagen.

[0155] In some embodiments, the collagen is monomeric collagen. In some embodiments, the collagen is atelocollagen. As used herein, the term "atelocollagen" refers to collagen that has been treated in vitro with pepsin or another suitable protease or agent to eliminate or substantially reduce the telopeptide region containing intermolecular crosslinking sites. In other embodiments, monomeric collagen is telocollagen. As used herein, the term "telocollagen" refers to acid-soluble collagen that retains its telopeptide end.

[0156] In certain embodiments, collagen comprises oligomeric collagen and atelocollagen. In other embodiments, collagen comprises oligomeric collagen, monomeric collagen, and atelocollagen. The amounts of oligomeric collagen, monomeric collagen, and / or atelocollagen may be blended in the collagen scaffold composition to advantageously maximize stiffness, strength, fluid and mass transfer, proteolysis, or compatibility of the artificial collagen scaffold.

[0157] In any of the embodiments described herein, the artificial collagen scaffold may have a predetermined percentage of collagen oligomers. In various embodiments, the predetermined percentage of collagen oligomers may be about 0.5% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 100%. In yet another embodiment, the collagen oligomers are obtained from a collagen-rich collagen-containing source material (e.g., pig skin).

[0158] In any of the embodiments described herein, the artificial collagen scaffold may have an oligomer content quantified by the average polymer molecular weight (AMW). As described herein, adjustment of the AMW may affect the polymerization kinetics, fibril microstructure, molecular properties, and fibril structure of the collagen scaffold, such as interfibril branching, pore size, and mechanical integrity (e.g., scaffold stiffness). In another embodiment, the oligomer content of purified collagen quantified by the average polymer molecular weight is positively correlated with scaffold stiffness.

[0159] In some embodiments, the collagen is thermoreversible collagen. As used herein, “thermoreversible collagen” means collagen that can reversibly transition between solution and matrix phases in response to temperature adjustments between 4°C and 37°C or between any other temperatures that cause a reversible matrix-solution transition.

[0160] In some embodiments, the collagen is reduced collagen. As used herein, “reduced collagen” means collagen that has been reduced in vitro to eliminate or substantially reduce reactive aldehydes. For example, collagen may be reduced in vitro by treating the collagen with a reducing agent (e.g., sodium borohydride).

[0161] In some embodiments, the collagen is oligomer 260 collagen. As used herein, “oligomer 260 collagen” is a collagen preparation prepared by a procedure resulting in the isolation of an oligomer (e.g., from pig skin), the collagen preparation having a prominent band at molecular weight 260, which is not prominent or absent in the corresponding monomer preparation. The presence of the band can be determined by SDS polyacrylamide gel electrophoresis. Oligomer 260 collagen is further described in U.S. Patent Application No. 13 / 192,276 (published February 2, 2012, publication number 2012-0027732 A1), which is incorporated herein by reference.

[0162] In other exemplary embodiments, the artificial collagen scaffolds described herein may be crosslinked using, for example, crosslinking agents such as glutaraldehyde, carbodiimide, aldehyde, lysl-oxidase, N-hydroxysuccinimide ester, imide ester, hydrazide, and maleimide, or combinations thereof. In one embodiment, the crosslinking agent may be added before, during, or after polymerization of collagen in the artificial collagen scaffold.

[0163] The concentration of collagen present in the various embodiments of the artificial collagen scaffolds of this disclosure may vary. In some embodiments, the collagen concentration is approximately 50 to approximately 1000 mg / cm³. 3 , about 50~900mg / cm 3 , about 50~800mg / cm 3 , about 50~700mg / cm 3 , about 50~600mg / cm 3 , about 50~500mg / cm 3 , about 50~400mg / cm 3 , about 50~300mg / cm 3 , about 50~200mg / cm 3 , about 100~about 1000mg / cm 3 , about 100~900mg / cm 3 , about 100~about 800mg / cm 3 , about 100~700mg / cm 3 , about 100~about 600mg / cm 3 , about 100~500mg / cm 3 , about 100~400mg / cm 3 , about 100~300mg / cm 3 , about 100~200mg / cm 3 , about 200~about 1000mg / cm 3 , about 200~900mg / cm 3 , about 200~about 800mg / cm 3 , about 200~700mg / cm 3 , about 200~600mg / cm 3 , about 200~500mg / cm 3 , about 200~400mg / cm 3 , about 200~300mg / cm 3 , about 300~about 1000mg / cm 3 , about 300~900mg / cm 3 , about 300~800mg / cm 3 , about 300~700mg / cm 3 , about 300~600mg / cm 3 , about 300~500mg / cm 3 , about 300~400mg / cm 3, about 400~1000mg / cm 3 , about 400~900mg / cm 3 , about 400~800mg / cm 3 , about 400~700mg / cm 3 , about 400~600mg / cm 3 , about 400~500mg / cm 3 , about 500~about 1000mg / cm 3 , about 500~900mg / cm 3 , about 500~800mg / cm 3 , about 500~700mg / cm 3 , about 500~about 600mg / cm 3 , about 50~2000mg / cm 3 , or approximately 50 to 1500 mg / cm³ 3 It is present in the artificial collagen scaffold at this concentration.

[0164] In other embodiments, collagen may be present in the starting composition used to polymerize collagen to produce a collagen scaffold in concentrations of about 1 mg / ml to about 50 mg / ml, about 1 mg / ml to about 40 mg / ml, about 1 mg / ml to about 30 mg / ml, about 1 mg / ml to about 20 mg / ml, about 1 mg / ml to about 15 mg / ml, about 1 mg / ml to about 12 mg / ml, about 1 mg / ml to about 10 mg / ml, about 1 mg / ml to about 9 mg / ml, about 1 mg / ml to about 8 mg / ml, about 1 mg / ml to about 7 mg / ml, about 1 mg / ml to about 6 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 4 mg / ml, or about 1 mg / ml to about 3 mg / ml.

[0165] In some embodiments, the collagen scaffold further comprises a polymer. As used herein, the term “polymer” refers to a molecule consisting of individual chemical parts, which may be the same or different, but preferably the same and bonded together. As used herein, the term “polymer” refers to individual chemical parts bonded together at their ends to form a linear molecule, as well as individual chemical parts bonded together in the form of a branched (e.g., “multi-armed” or “star-shaped”) structure. In other embodiments, the collagen scaffold further comprises a copolymer. As used herein, the term “copolymer” refers to a polymer derived from two or more monomer species, including copolymers that can be obtained by copolymerizing two monomer species, copolymers that can be obtained from three monomer species (“ternary polymers”), copolymers that can be obtained from four monomer species (“quaternary polymers”), and so on.

[0166] In various embodiments of this disclosure, the collagen scaffold as described herein may be polymerized under controlled conditions to obtain specific physical properties. For example, the collagen scaffold may have a desired collagen fibril density, pore size (fibril-fibril branching), modulus, tensile strain, tensile stress, linear modulus (modulus), compressive modulus, ultimate tensile strength, fracture strain, suture-holding peak load, loss modulus, fibril area fraction, fibril volume fraction, collagen concentration, cell seeding density, shear storage modulus [G' or elastic (solid-like) behavior], and phase angle delta [δ or measurement of fluid (viscous) to solid (elastic) behavior; δ is 0 for Hooke solid]. o and 90 about Newtonian fluids o It may have [equal to].

[0167] As used herein, “modulus of elasticity” may be the modulus of elasticity or linear modulus of elasticity (defined by the slope of the linear region of the stress-strain curve obtained using conventional mechanical testing protocols; i.e., stiffness), the compressive modulus of elasticity, the loss modulus of elasticity, or the shear storage modulus (e.g., the storage modulus of elasticity). These terms are well known to those skilled in the art.

[0168] As used herein, “fibril volume fraction” (i.e., fibril density) is defined as a percentage of the total area occupied by fibrils in three dimensions.

[0169] As used herein, tensile or compressive stress "σ" is a force per unit area, and is expressed by the formula:

[0170]

number

[0171] A force (P) produces a stress perpendicular to the cross-section of the material (for example, if the stress tends to lengthen the material, it is called a tensile stress, and if the stress tends to shorten the material, it is called a compressive stress).

[0172] As used herein, “strain” refers to mechanical strain, which is the deformation of a material as a result of mechanical stress. Strain is usually defined as the ratio of displacement to a reference length. As used herein, “tensile strain” is the elongation of a material subjected to tension.

[0173] In any embodiment described herein, the fibril volume fraction of the collagen scaffold may be about 1% to about 60%. In various embodiments, the collagen scaffold may contain fibrils having a fibril volume fraction (i.e., density) of a particular characteristic, for example, about 2% to about 90%, about 2% to about 80%, about 2% to about 70%, about 2% to about 60%, about 2% to about 50%, about 2% to about 40%, about 5% to about 60%, about 15% to about 60%, about 2% to about 30%, about 5% to about 30%, about 15% to about 30%, about 20% to about 30%, about 5% to about 90%, about 15% to about 90%, about 2% to about 80%, about 5% to about 80%, about 15% to about 80%, or about 20% to about 80%.

[0174] In any of the exemplary embodiments described herein, the collagen scaffold is, but is not limited to, approximately 18 MPa to approximately 200 MPa, approximately 20 MPa to approximately 200 MPa, approximately 20 MPa to approximately 180 MPa, approximately 20 MPa to approximately 170 MPa, approximately 20 MPa to approximately 160 MPa, approximately 20 MPa to approximately 150 MPa, approximately 20 MPa to approximately 140 MPa, approximately 20 MPa to approximately 130 MPa, approximately 20 MPa to approximately 120 MPa, and approximately 20 MPa. It may contain fibrils having specific characteristics, including elastic moduli (e.g., compression modulus, loss modulus, modulus, or storage modulus) of approximately 110 MPa, 20 MPa to 100 MPa, 20 MPa to 90 MPa, 20 MPa to 80 MPa, 20 MPa to 70 MPa, 20 MPa to 60 MPa, 20 MPa to 50 MPa, 20 MPa to 40 MPa, 20 MPa to 30 MPa, or 20 MPa to 25 MPa. In other embodiments, the collagen scaffold is used in pressures of approximately 0.5 MPa to 200 MPa, approximately 0.5 MPa to 190 MPa, approximately 0.5 MPa to 180 MPa, approximately 0.5 MPa to 170 MPa, approximately 0.5 MPa to 160 MPa, approximately 0.5 MPa to 150 MPa, approximately 0.5 MPa to 140 MPa, approximately 0.5 MPa to 130 MPa, approximately 0.5 MPa to 120 MPa, approximately 0.5 MPa to 110 MPa, and approximately 0.5 MPa to 1 It can have elastic moduli of 0.5 MPa, approximately 0.5 MPa to approximately 100 MPa, approximately 0.5 MPa to approximately 90 MPa, approximately 0.5 MPa to approximately 80 MPa, approximately 0.5 MPa to approximately 70 MPa, approximately 0.5 MPa to approximately 60 MPa, approximately 0.5 MPa to approximately 50 MPa, approximately 0.5 MPa to approximately 40 MPa, approximately 0.5 MPa to approximately 30 MPa, approximately 0.5 MPa to approximately 20 MPa, approximately 0.5 MPa to approximately 10 MPa, approximately 0.5 MPa to approximately 5 MPa, and approximately 0.5 MPa to approximately 1 MPa.

[0175] In any of the embodiments described herein, the collagen composition may contain fibrils having certain characteristics including phase angle deltas (δ) of about 0° to about 12°, about 0° to about 5°, about 1° to about 5°, about 4° to about 12°, about 5° to about 7°, about 8° to about 10°, and about 5° to about 10°.

[0176] In various embodiments, the artificial collagen scaffold may have a thickness of approximately 0.005 to 3 mm, approximately 0.005 to 2 mm, approximately 0.005 to 1 mm, approximately 0.005 to 0.5 mm, approximately 0.01 mm to 3.0 mm, approximately 0.01 mm to 2.0 mm, approximately 0.01 mm to 1.0 mm, approximately 0.01 mm to 0.5 mm, approximately 0.01 mm to 0.25 mm, approximately 0.1 mm to 1.0 mm, approximately 0.5 mm to 1.0 mm, approximately 0.15 mm to 0.25 mm, approximately 0.02 mm to 0.2 mm, approximately 0.02 mm to 0.15 mm, or approximately 0.02 mm to 0.1 mm.

[0177] In other embodiments, the collagen scaffold may have an ultimate tensile strength of approximately 0.5 MPa to approximately 20 MPa, approximately 1 MPa to approximately 25 MPa, approximately 0.2 MPa to approximately 20 MPa, approximately 5 MPa to approximately 15 MPa, approximately 2 MPa to approximately 20 MPa, approximately 1 MPa to approximately 20, approximately 1 MPa to approximately 15 MPa, approximately 1 MPa to approximately 10 MPa, approximately 1 MPa to approximately 5 MPa, approximately 1 MPa to approximately 4 MPa, approximately 1 MPa to approximately 3 MPa, approximately 1 MPa to approximately 2 MPa, approximately 0.5 MPa to approximately 25 MPa, approximately 0.5 MPa to approximately 20, approximately 0.5 MPa to approximately 15 MPa, approximately 0.5 MPa to approximately 10 MPa, approximately 0.5 MPa to approximately 5 MPa, approximately 0.5 MPa to approximately 4 MPa, approximately 0.5 MPa to approximately 3 MPa, approximately 0.5 MPa to approximately 2 MPa, or approximately 0.5 MPa to approximately 1 MPa.

[0178] In other exemplary embodiments, the artificial collagen scaffold may have fracture strain of about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or about 10% to about 20%.

[0179] In other embodiments, the artificial collagen scaffold is approximately 2N to 8N, approximately 0.2N to 2N, or approximately 0.1N to 4N, approximately 2N to 7N, approximately 2N to 6N, approximately 2N to 5N, approximately 2N to 4N, approximately 2N to 3N, approximately 0.1N to 8N, approximately 0.1N to 7N, approximately 0.1N to 6N, approximately 0.1N to 5N, approximately 0.1N to 4 The suture retention peak load may be N, approximately 0.1N to 3N, approximately 0.1N to 2N, approximately 0.1N to 1N, approximately 0.2N to 8N, approximately 0.2N to 7N, approximately 0.2N to 6N, approximately 0.2N to 5N, approximately 0.2N to 4N, approximately 0.2N to 3N, approximately 0.2N to 2N, approximately 0.2N to 1N, or approximately 0.2N to 0.5N.

[0180] In all embodiments described herein, “about ~” and “~about” include the numbers referred to at both ends of the range. For example, “about 20% to about 80%” includes 20% and 80%, and “about 20 MPa to about 200 MPa” includes 20 and 200 MPa, and so on. Where used herein, “about” referring to a number including integers, fractions and percentages generally refers to a range of numbers (e.g., + / - 5% to 10% of the stated value) that a person skilled in the art would consider equivalent to the listed value (e.g., having the same function or result).

[0181] In any of the exemplary embodiments described herein, the qualitative and quantitative microstructural features of the collagen scaffold can be determined by cryostage scanning electron microscopy, transmission electron microscopy, confocal microscopy, or second-harmonic generation multiphoton microscopy, etc. In another embodiment, tensile, compressive, and viscoelastic properties can be determined by flow measurements or tensile tests. All of these methods are either publicly known in the art or are described in U.S. Patent Application No. 11 / 435,635 (published November 22, 2007, publication number 2007-0269476 A1), U.S. Patent Application No. 11 / 914,606 (published January 8, 2009, publication number 2009-0011021 A1), U.S. Patent Application No. 12 / 300,951 (published July 9, 2009, publication number 2009-0175922 A1), U.S. Patent Application No. 13 / 192,276 (published February 2, 2012, publication number 2012-0027732 A1), and U.S. Patent Application No. 13 / 383,796 (published 2012-0115222 Further details are provided in or incorporated herein by reference in A1 (published May 10, 2012), Roeder et al., J. Biomech. Eng., vol. 124, pp. 214-222 (2002); Pizzo et al., J. Appl. Physiol., vol. 98, pp. 1-13 (2004); Fulzele et al., Eur. J. Pharm. Sci., vol. 20, pp. 53-61 (2003); Griffey et al., J. Biomed. Mater. Res., vol. 58, pp. 10-15 (2001); Hunt et al., Am. J. Surg., vol. 114, pp. 302-307 (1967); and Schilling et al., Surgery, vol. It is described in 46, pp. 702-710 (1959).

[0182] In various embodiments, the collagen scaffold composition further comprises cells. Any cell type within the knowledge of those skilled in the art can be used with the collagen scaffold composition of this disclosure. In some embodiments, the cells are stem cells. As used herein, “stem cells” refers to undifferentiated cells of embryo, fetus, or adult origin that are capable of self-renewal or self-regeneration and can develop into various differentiated cell types (i.e., differentiation potential). As used herein, the term encompasses oligopotent cells (cells that can differentiate into a few cell types, e.g., lymphoid or myeloid) and unipotent cells (cells that can differentiate into only one cell type), unless otherwise specified. In some embodiments, hematopoietic stem cells may be isolated, for example, from bone marrow, circulating blood, or umbilical cord blood, by methods well known to those skilled in the art. Cell markers can be used to select and purify hematopoietic stem cells. For example, preferred markers are Lin-, Sca1+ and c-Kit+ mouse or Lin-, CD34+ and c-Kit+ human hematopoietic stem cell markers. In one embodiment, cell markers may be used alone or in combination to select and purify desired cell types for use in the compositions and methods described herein. In one embodiment, the collagen scaffold composition may be seeded with autologous cells isolated from the patient being treated. In an alternative embodiment, the cells may be heterogeneous or homogeneous in nature.

[0183] In any of the embodiments described herein, cells are added to the collagen scaffold composition in a manner of about 1 × 10 6 ~Approx. 1×10 8 Cell density of 10 cells / ml, or approximately 1 × 10⁶ 3 ~Approx. 2×10 6 The cells are seeded at a density of 5 × 10⁶ cells / ml. In one embodiment, the cells are 5 × 10⁶ 4 Cells are seeded at a density of less than 10 cells / ml. In another embodiment, cells are seeded at a density of 1 × 10⁶ 4 Cells are seeded at a density of less than 1 cell / ml. In another embodiment, the cells are approximately 1 × 10⁶ 2 ~Approx. 5×10 6 , about 0.3×10 4 ~Approx. 60×10 4cells / ml, and approximately 0.5 × 10⁶ 4 ~Approx. 50×10 4 The cells are seeded at a density selected from a range of 10 cells / ml. The cells are maintained, proliferated, or differentiated according to the methods described herein or methods well known to those skilled in the art of cell culture.

[0184] In any of the various embodiments described herein, the artificial collagen scaffold composition of the present invention may be combined with hematopoietic stem cell culture or nutrients, including minerals, amino acids, sugars, peptides, proteins, vitamins (e.g., ascorbic acid), or glycoproteins, that promote the culture of other cell types, such as laminin and fibronectin, hyaluronic acid, or platelet-derived growth factor or transforming growth factor beta, and glucocorticoids such as dexamethasone. In other exemplary embodiments, fibrillation inhibitors, such as glycerol, glucose, or polyhydroxylated compounds, may be added before or during polymerization. According to one embodiment, cells may be added to the collagen scaffold and other extracellular matrix components as the final step before or after the polymerization of collagen. In other exemplary embodiments, crosslinking agents, such as carbodiimide, aldehydes, lysyl oxidase, N-hydroxysuccinimide esters, imide esters, hydrazides, and maleimides, may be added before, during, or after the polymerization of collagen. In yet another embodiment, the artificial collagen scaffold composition may include components such as a buffer (e.g., phosphate-buffered saline), hydrochloric acid (e.g., 0.01 N), and glucose. In one embodiment, glucose may be added if cells are present. In another embodiment, non-collagenous components of the ECM that are normally present in the natural collagen matrix are absent.

[0185] In certain embodiments, the collagen scaffold composition further comprises a fluid. Some of the fluid is removed from the collagen scaffold composition in response to compression, but a certain amount of fluid is retained in the compressed collagen scaffold composition. In some embodiments, the percentage of fluid present is about 25% to about 99%, about 5% to about 99%, about 5% to about 95%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, or about 5% to about 30%, about 10% to about 99%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or about 10% to about 20%. In some embodiments, the percentage of fluid present is approximately 20% to 99%, 30% to 99%, 40% to 99%, 45% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, or 80% to 99%. In some embodiments, the percentage of fluid present is approximately 50% to 80%. In some embodiments, the percentage of fluid present is approximately 60% to 70%.

[0186] In various embodiments, the collagen scaffold composition is freeze-dried. As used herein, the term “freeze-dried” typically refers to the removal of water from the composition by freeze-drying under vacuum. However, drying can be carried out by any method known to those skilled in the art, and the method is not limited to freeze-drying under vacuum. Typically, the freeze-dried collagen scaffold composition is freeze-dried to dryness, and in one embodiment, the moisture content of the freeze-dried collagen scaffold composition is below a detectable level. In another embodiment, the collagen scaffold may be dried by freeze-drying the composition, vacuum-pressing the composition, or by dehydration heat treatment, or a combination thereof.

[0187] Another embodiment provided herein offers a method for treating a patient to regenerate, restore, or replace damaged or dysfunctional tissue. The method involves implanting a medical transplant tissue, which includes one of the artificial collagen scaffolds described herein, into the patient. In various embodiments, the medical transplant tissue may be for the regeneration, replacement, or restoration of tissue selected from the pericardium, heart valves (heart value), skin, blood vessels, airway tissue, body wall, and tissue reconstructed after tumor removal. In another embodiment, the valve tissue may be aortic or pulmonary valve tissue. In one embodiment, the medical transplant tissue (i.e., collagen scaffold) does not induce inflammation or a foreign body reaction when implanted in the patient.

[0188] In one embodiment, the artificial collagen scaffold is sterilized or prepared aseptically before implantation in the patient. In various embodiments, the sterilization method may be a process selected from glutaraldehyde treatment, gamma irradiation, electron beam irradiation, peracetic acid sterilization, formaldehyde tanning at an acidic pH, propylene oxide treatment, ethylene oxide treatment, or gas plasma sterilization. Sterilization techniques that do not adversely affect the structure of the collagen can be used.

[0189] Another embodiment provided herein provides a method for producing an artificial collagen scaffold by compression, for example, constrained compression. Any embodiment of the artificial collagen scaffold described herein may be produced by the production method. In some embodiments, the production method includes a step of polymerizing collagen before compressing the collagen scaffold composition into a predetermined shape. In certain embodiments, the production method includes a step of modifying the physical properties of the collagen scaffold before compressing the collagen scaffold composition into a predetermined shape. As used herein, the term “modify” refers to the modification of the collagen scaffold under controlled conditions to obtain desired physical properties. For example, before compression, the collagen scaffold has the following physical properties of a desired value or amount: fibril density, pore size (fibril-fibril branching), modulus of elasticity, thickness, tensile strain, tensile stress, linear modulus of elasticity, ultimate tensile stress, fracture strain, suture-holding peak load, compressive modulus of elasticity, loss modulus of elasticity, fibril area fraction, fibril volume fraction, collagen concentration, cell seeding density, shear storage modulus [G' or elastic (solid-like) behavior], and phase angle delta [δ or measurement of fluid (viscous) to solid (elastic) behavior; δ is 0 for Hooke solids]. o and 90 about Newtonian fluids o Under control conditions, it is modified to produce one or more of the following:

[0190] By adjusting the physical properties of the collagen scaffold composition before compression, high levels of interfibril association can be induced in the pre-compression collagen scaffold. This process allows for control of key mechanical properties before the final collagen scaffold is formed, and these controlled mechanical properties are retained after compression of the final collagen scaffold. Therefore, the design features of the collagen scaffold can be optimized for the purpose of predictively inducing desired cellular mechanisms in the collagen scaffold.

[0191] The collagen scaffolds described herein can be compressed into several different predetermined shapes. In some embodiments, the predetermined shape is a tube. In other embodiments, the predetermined shape is a sheet. In yet another embodiment, the predetermined shape is a sphere. In some embodiments, the predetermined shape is a slab. In other embodiments, the predetermined shape is a cylinder. In yet another embodiment, the predetermined shape is a cone.

[0192] In another embodiment, the methods, uses, and collagen scaffolds and collagen scaffold compositions described herein include the following examples. These examples further illustrate additional features of various embodiments of the invention described herein. However, it should be understood that these examples are illustrative and should not be construed as limiting other embodiments of the invention described herein. Furthermore, it should be recognized that other variations of the examples are included in various embodiments of the invention described herein. [Examples]

[0193] [Example 1] Formation of hydrated collagen scaffolds by compression dehydration Chamber compression system: Type I oligomeric collagen was isolated and purified from porcine dermis. Animal hides were obtained from market-weight castrated pigs in closed groups located in the United States, proven to be free from infectious or contact-transmitted diseases. Acid-solubilized collagen oligomers were sterile filtered and quality-controlled based on purity, molecular composition, and polymerization parameters. High-density collagen scaffolds were produced sterile using a specially designed confinement compression procedure with cylindrical chambers fabricated from Delrin (Figure 1). The bottoms of these chambers were removable to accommodate two shapes: 1) a solid bottom for containing liquid collagen before and during polymerization (Figure 1A), and 2) a porous bottom with holes to facilitate controlled fluid removal from the bottom during compression (Figures 1B and C). To produce the collagen scaffolds, liquid collagen was neutralized at specific concentrations and volumes and added to sterile cylindrical chambers (either 6.3 cm or 3.4 cm in diameter) (Figure 1A). These chambers were sealed and incubated at 37°C to induce collagen polymerization, resulting in the formation of a complex collagen scaffold containing a network of fibrous collagen surrounded by interstitial fluid. After collagen polymerization, sterile porous polyethylene foam was placed on the top and bottom surfaces of the complex collagen scaffold, replacing the solid bottom surface of the chamber with the porous bottom surface (Figure 1B). The collagen was then compressed to the desired thickness at a strain rate of 0.05% per second, and fluid removal occurred from both the top and bottom surfaces (Figure 1C). After compression, the hydrated collagen scaffold was aseptically removed from the chamber and stored in a sealed, airtight sterile container before testing.

[0194] Syringe compression system: Bidirectional fluid removal during compression dewatering was also achieved using a modified syringe system (Figure 2). The syringe (6cc Covidien Monoject, 1.2cm diameter) was modified by removing the plunger and placing a stainless steel mesh disc (100x100 openings per 1"x1" unit, 0.006" opening; McMaster-Carr, Douglasville, GA) directly in front of the tip inside the syringe. The syringe's rubber plunger was modified by cutting out the central region and attaching another disc of stainless steel mesh to the resulting hole. A notch was also created at the end of the rubber plunger to facilitate fluid flow and prevent air pressure buildup when inserting the plunger into the syringe. After capping the syringe tip, the neutralized collagen solution was poured through the stainless steel mesh. The viscous collagen solution was added to the body of the syringe at the top (due to the surface tension between the viscous collagen solution and the small grids of the mesh, the fluid could not flow through the mesh). The syringe was sealed and incubated at 37°C to allow the collagen to polymerize. After polymerization, the modified plunger was placed inside the syringe along with a piece of Whatman filter paper to provide a buffer between the stainless steel mesh and the composite collagen scaffold (to prevent mesh grip marks from being left on the scaffold during compression). The syringe was then placed on a syringe pump (Model NE-1600, New Era Pump Systems, Farmingdale, NY) to compress to the desired thickness at a strain rate of 0.05% per second.

[0195] [Example 2] Quantification of collagen scaffold thickness The thickness and uniformity of the collagen scaffold were determined using a Mitutoyo 547-526S high-precision thickness gauge (Aurora, IL; + / - 5 μm accuracy). At least five measurements (n≧5) were taken along the material sheet, including the central and edge regions, and the relevant mean and standard deviation were determined. [Example 3]

[0196] Uniaxial tensile test Uniaxial tensile tests were performed in ambient air on dogbone-shaped material samples with gauge lengths and widths of 4 mm and 3 mm, respectively (n≧3). The average duration of the mechanical tests from setup to completion was less than 10 seconds, and no dehydration of the samples was observed. All samples were tested under uniaxial tension to fracture at a strain rate of 40% per second (1.6 mm / min) using a servo-electrical materials testing system (TestResources, Shakopee, MN) fitted with a 25N load cell at a sampling rate of 32 Hz. The modulus of elasticity (ET) was calculated from the linear region of the stress-strain curve. The ultimate stress (σU) represents the peak stress experienced by the sample, and the fracture strain (εf) is the strain at which the material underwent total failure.

[0197] [Example 3] Suture retention test The suture retention test was performed on 2 × 1 cm rectangular samples in ambient air. To ensure high reproducibility in placing the sutures on these samples, a suture guide was used to place the sutures along the long axis center of the sample at a bite distance of 2 mm. After passing a single suture (5-0 Nylon, monofilament) through the sample, the guide was used to assist in securing the suture 2.5 cm from the end of the sample with two double square knots. After tying the suture in place, the sample was placed on a tensile testing machine (the same one used for the tensile test) by winding the suture around a hook held in the top grip of the machine and securing the bottom end of the sample to the bottom grip. The suture was pulled at a speed of 10 mm / min until fracture, and the maximum load in units of Newtons (N) was recorded as the suture retention strength.

[0198] [Example 4] Hydrated collagen scaffold A representative image of a prototype hydrated collagen scaffold prepared using a specially designed chamber compression system is shown in Figure 3. Table 1 provides an overview of various scaffold formulations and their associated properties.

[0199] Table 1. Overview of the properties of various collagen scaffolds prepared by compression dehydration. [Table 1]

[0200] [Example 5] Relationship between collagen concentration and collagen scaffold design To identify predictive relationships for the design of custom-designed collagen scaffolds, scaffolds were fabricated over a wide range of collagen concentrations (approximately 100 mg / mL to 700 mg / mL) using either a chamber or syringe compression dehydration method. Mechanical properties of the scaffolds, including modulus of elasticity, ultimate tensile strength (UTS), and fracture strain, were measured and plotted as a function of collagen content (Figure 4).

[0201] [Example 6] Collagen scaffolds produced by compression dehydration To produce dry collagen scaffolds, hydrated, high-density scaffolds were dehydrated and dried using freeze-drying or vacuum pressing. Prior to freeze-drying or vacuum pressing, all scaffolds were thoroughly rinsed in water. For freeze-drying, the collagen scaffolds were secured to a frame, gripping the ends to prevent curling. The scaffolds were then flash-frozen with liquid nitrogen and freeze-dried overnight. For vacuum pressing, the scaffolds were placed between two sheets of porous polyethylene foam and compressed under vacuum until dry. In some cases, the dried material was further subjected to dehydration heat (DHT) treatment, a process in which the material is heated under vacuum to further remove water molecules and create intermolecular crosslinks. For DHT treatment, the dried scaffolds were placed in a vacuum oven for 24 hours at a specific vacuum level and temperature. The vacuum was set to 50 mTorr and the temperature to 60, 90, or 120°C. Figure 5 shows an example of a dried collagen scaffold after rehydration in phosphate-buffered saline. Table 2 summarizes the properties of various collagen scaffold formulations prepared by compression dehydration and drying. In general, treatment with DHT improved the mechanical integrity of the scaffold (i.e., elastic modulus, UTS) and the scaffold's ability to maintain its shape (i.e., thickness) after rehydration. This was particularly noticeable for scaffolds with high total collagen content (>500 mg;>500 mg / mL).

[0202] Table 2. Summary of the properties of various collagen scaffolds prepared by compression dehydration followed by freeze-drying or drying via vacuum pressing. The thickness values ​​for the dried scaffolds prepared by vacuum pressing and freeze-drying were 377±25 μm and 604±33 μm, respectively. [Table 2]

[0203] [Example 7] Evaluation of biocompatibility and tissue response after subcutaneous transplantation of collagen scaffolds in rats. Preclinical evaluation of biocompatibility and tissue response of materials: The biocompatibility and tissue response of the materials were evaluated using an established rat subcutaneous transplantation model. This study included adult male Sprague Dawley (Envigo, Indianapolis, Indiana) rats, and all materials were evaluated in 10 replicates. All animals were housed under standard conditions (e.g., 25°C temperature, 12-hour light / dark cycle) and given a standard rat solid pellet diet and free access to water. At the time of the procedure, the animals weighed between 272g and 308g. After induction of anesthesia, the animals' backs were shaved, and they were thoroughly washed from the rump to the shoulders with a surgical scrub and dried. Four transverse incisions, approximately 2 cm in length, were made on both sides of the back parallel to the midline. The fascia was randomly cut to form small pockets directly lateral to the incisions. The test specimens (circular, 8 mm in diameter) were subcutaneously transplanted directly beneath the cutaneous truncai muscle, and the incisions were closed with non-absorbable sutures. Sutures were removed 10–14 days after surgery. All animals were observed at least three times a week, weighed weekly, and evaluated for both their physiological and mental state. 60±2 days later, the animals were euthanized, shaved, and their dorsal sides were photographed. Subcutaneous tissue of the back was then exposed, photographed, and radiographed. Each graft site and associated normal tissue were then collected, photographed, and divided in half for follow-up histopathological and calcium analysis. Explant tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin, eosin, and von Kossa. Blinded macroscopic examination of all graft sites and associated materials was performed immediately after surgical dissection / exposure and after preparation for histopathological analysis. Specimens were graded based on the level of tissue reaction and tissue integration, as summarized below. Histopathological analysis of explants was performed by blinded pathologists.

[0204] Tissue reaction (evaluated immediately after surgical dissection / exposure) No orders 1 = very little 2 = minor 3=moderate 4 = Widespread

[0205] Tissue integration (evaluated during sample preparation for calcium analysis) No orders 1 = very little 2 = minor 3=moderate 4 = Widespread

[0206] result: The biocompatibility and tissue response of collagen scaffolds prepared with or without glutaraldehyde treatment, particularly formulations 3 and 4 (as originally described in Example 1, Table 1), were evaluated in an established 60-day rat subcutaneous graft model using glutaraldehyde-treated pericardium as a reference material. Representative pre-graft photographs of each group are shown in Figure 6, and Table 3 provides an overview of the material and mechanical properties. Glutaraldehyde treatment of the collagen scaffolds resulted in i) increased elastic modulus and UTS, ii) a slight increase in suture retention, and iii) a decrease in fracture strain. The collagen scaffolds of formulations 3 and 4 appeared white, while the glutaraldehyde-treated materials were brown in various shades. Representative images of skin exgrafts containing the relevant graft materials 60 ± 2 days after transplantation are shown in Figure 7. Figure 8 summarizes the results of macroscopic observation and grading of differences in the degree of tissue response (i.e., fibrous tissue associated with transplantation) and tissue integration (i.e., adhesion between the material transplant and the surrounding host tissue). In general, all materials except the untreated formulations 3 and 4 showed varying levels of foreign body response, along with significant fibrous tissue overgrowth. Histopathological analysis revealed that the scaffolds of formulations 3 and 4 appeared to be homogeneous materials with linearly oriented fibers similar to those of undenatured collagen. For these materials, the inflammatory pattern was generally mild, and the fibrotic response was generally mild, with a smooth transition from the transplanted tissue to the surrounding tissue (Figures 9 and 10). The inflammatory response to the glutaraldehyde-treated collagen scaffolds was also mild (Figures 9 and 10); however, a moderate maturing fibrotic response was typically observed around the transplanted tissue. Consistent with other published studies, glutaraldehyde-treated pericardium appeared as a coarse fibrous material with linearly oriented fibers and showed a moderate inflammatory response consistent with the periphery (Figure 11). Lymphocytes and other inflammatory cells were often observed to infiltrate between fibrils, and a moderate fibrotic response was consistently present. No detectable calcifications related to either material were present.

[0207] Table 3. Summary of the properties of collagen scaffolds and reference materials evaluated for biocompatibility and tissue response in established rat subcutaneous transplant models. [Table 3]

[0208] Collagen polymerization The composition of the artificial collagen scaffold for polymerization may include type I oligomeric collagen in 0.01 N hydrochloric acid, which is neutralized by mixing it with the 10× buffer presented below in a 10:1 ratio. Glucose may be included if cells are present. The combination of the collagen solution in dilute acid with this 10× buffer induces a fibril formation reaction. This composition can be added to a chamber compression system as described herein and incubated at 37°C to induce collagen polymerization.

[0209] (10 × phosphate-buffered saline) 1.37M NaCl 0.027M KCl 0.081M Na2HPO4 0.015M KH2PO4 0.1N NaOH 55.5 mM glucose

Claims

1. A non-disintegrating and / or non-expandable artificial collagen scaffold having a thickness of about 0.005 mm to about 3 mm and an elastic modulus of about 0.5 MPa to about 200 MPa.

2. The artificial collagen scaffold according to claim 1, which does not disintegrate when freeze-dried and rehydrated.

3. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.01 mm to approximately 2.0 mm.

4. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.01 mm to approximately 1.0 mm.

5. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.01 mm to approximately 0.25 mm.

6. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.1 mm to approximately 1.0 mm.

7. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.5 mm to approximately 1.0 mm.

8. An artificial collagen scaffold according to any one of claims 1 to 2, having a thickness of approximately 0.15 mm to approximately 0.25 mm.

9. An artificial collagen scaffold according to any one of claims 1 to 8, having an elastic modulus of approximately 18 MPa to approximately 200 MPa.

10. An artificial collagen scaffold according to any one of claims 1 to 8, having an elastic modulus of approximately 20 MPa to approximately 180 MPa.

11. An artificial collagen scaffold according to any one of claims 1 to 8, having an elastic modulus of approximately 40 MPa to approximately 120 MPa.

12. An artificial collagen scaffold according to any one of claims 1 to 8, having an elastic modulus of approximately 60 MPa to approximately 100 MPa.

13. An artificial collagen scaffold according to any one of claims 1 to 8, having an elastic modulus of approximately 80 MPa to approximately 180 MPa.

14. An artificial collagen scaffold according to any one of claims 1 to 8, having an ultimate tensile strength of approximately 0.5 MPa to approximately 20 MPa.

15. An artificial collagen scaffold according to any one of claims 1 to 8, having an ultimate tensile strength of approximately 1 MPa to approximately 25 MPa.

16. An artificial collagen scaffold according to any one of claims 1 to 15, having an ultimate tensile strength of approximately 0.2 MPa to approximately 20 MPa.

17. An artificial collagen scaffold according to any one of claims 1 to 15, having an ultimate tensile strength of approximately 5 MPa to approximately 15 MPa.

18. An artificial collagen scaffold according to any one of claims 1 to 15, having an ultimate tensile strength of approximately 2 MPa to approximately 20 MPa.

19. An artificial collagen scaffold according to any one of claims 1 to 18, having a fracture strain of approximately 5% to approximately 70%.

20. An artificial collagen scaffold according to any one of claims 1 to 18, having a fracture strain of approximately 10% to approximately 40%.

21. An artificial collagen scaffold according to any one of claims 1 to 20, having a suture-holding peak load of approximately 2 N to approximately 8 N.

22. An artificial collagen scaffold according to any one of claims 1 to 20, having a suture-holding peak load of approximately 0.2 N to approximately 2 N.

23. An artificial collagen scaffold according to any one of claims 1 to 20, having a suture-holding peak load of approximately 0.1 N to approximately 4 N.

24. An artificial collagen scaffold according to any one of claims 1 to 23, wherein the composition comprises an artificial collagen scaffold, and the composition further comprises a fluid.

25. The artificial collagen scaffold according to claim 24, wherein the percentage of fluid present is approximately 5% to approximately 99%.

26. The artificial collagen scaffold according to any one of claims 24 or 25, wherein the composition is dried by freeze-drying, vacuum-pressing, dehydration heat treatment, or a combination thereof.

27. An artificial collagen scaffold according to any one of claims 1 to 26, wherein the collagen is type I collagen.

28. An artificial collagen scaffold according to any one of claims 1 to 27, wherein the collagen is purified type I collagen.

29. An artificial collagen scaffold according to any one of claims 1 to 28, which is a medical transplant tissue.

30. The artificial collagen scaffold according to any one of claims 1 to 29, wherein the artificial collagen scaffold is a medical transplant tissue, and the medical transplant tissue is used for the regeneration, restoration, or replacement of damaged or dysfunctional tissue.

31. An artificial collagen scaffold according to claim 30, which is a medical transplant tissue for the regeneration, replacement, or restoration of tissue selected from the pericardium, heart valves, skin, blood vessels, airway tissue, body wall, and tissue reconstructed after tumor removal.

32. An artificial collagen scaffold according to any one of claims 1 to 31, which is final sterilized or prepared aseptically.

33. The artificial collagen scaffold according to claim 32, which is ultimately sterilized by a process selected from treatment with glutaraldehyde, gamma irradiation, electron beam irradiation, or ethylene oxide treatment.

34. An artificial collagen scaffold according to any one of claims 1 to 33, wherein the collagen comprises oligomeric collagen, monomeric collagen, telocollagen or atelocollagen, or a combination thereof.

35. An artificial collagen scaffold according to any one of claims 1 to 34, compressed into a predetermined shape.

36. The artificial collagen scaffold according to claim 35, wherein the shape is spherical.

37. The artificial collagen scaffold according to claim 35, wherein the shape is tubular.

38. The artificial collagen scaffold according to claim 35, wherein the shape is that of a sheet.

39. An artificial collagen scaffold according to any one of claims 35 to 38, wherein the compression is constrained compression.

40. Collagen concentration: approximately 50 to 1000 mg / cm³ ３ The artificial collagen scaffold according to any one of claims 1 to 39.

41. Collagen concentration is approximately 50 to 900 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

42. Collagen concentration is approximately 50 to 800 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

43. Collagen concentration is approximately 50 to 700 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

44. Collagen concentration is approximately 50 to 600 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

45. Collagen concentration is approximately 50 to 500 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

46. Collagen concentration is approximately 50 to 400 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

47. Collagen concentration is approximately 50 to 300 mg / cm³. ３ The artificial collagen scaffold according to any one of claims 1 to 39.

48. The collagen concentration is from about 50 to about 200 mg / cm ３ The artificial collagen scaffold according to any one of claims 1 to 39, which is such.

49. An artificial collagen scaffold according to any one of claims 1 to 48, which does not induce inflammation or a foreign body reaction when implanted in a patient.

50. An artificial collagen scaffold according to any one of claims 1 to 49, wherein the collagen is selected from porcine collagen, human collagen, and bovine collagen.

51. An artificial collagen scaffold according to any one of claims 1 to 49, wherein the collagen is synthetic collagen.

52. An artificial collagen scaffold according to any one of claims 1 to 50, wherein the collagen is undenatured collagen.

53. An artificial collagen scaffold according to any one of claims 1 to 49, wherein the collagen is recombinant collagen.

54. An artificial collagen scaffold according to any one of claims 1 to 53, further comprising cells.

55. The artificial collagen scaffold according to claim 54, wherein the cells are stem cells.

56. A method for treating a patient to replace, restore or regenerate damaged or dysfunctional tissue, comprising implanting a medical transplant tissue comprising an artificial collagen scaffold according to any one of claims 1 to 55 into the patient.

57. The method according to claim 56, wherein the medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or dysfunctional pericardium.

58. The method according to claim 56, wherein the medical transplant tissue is for the regeneration, restoration, or replacement of a damaged or malfunctioning heart valve.

59. The method according to claim 56, wherein the medical transplant tissue is for the regeneration, restoration, or replacement of damaged or dysfunctional skin.

60. The method according to claim 59, wherein the valve tissue is aortic valve tissue or pulmonary valve tissue.

61. An artificial collagen scaffold according to any one of claims 1 to 55, wherein the artificial collagen scaffold is exogenously crosslinked, or the method according to any one of claims 56 to 60.

62. The artificial collagen scaffold according to claim 61, wherein the artificial collagen scaffold is crosslinked with glutaraldehyde.

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