Chimeric human and animal collagen materials and methods of making same

Abstract

Chimeric collagen compositions and methods of their formation and use to repair damaged collagen-containing structures such as tendons are provided. The chimeric collagen differs from naturally occurring collagen in that it contains different collagens from two or more sources which form a hybrid collagen structure or tissue in a subject. The chimeric collagen composition can include collagen from two or more individuals of the same species, or collagen from two or more different species, or can contain a naturally occurring mammalian collagen and an engineered collagen. Use of the chimeric collagen compositions and methods can speed healing of damaged collagen-containing structures and promote mechanically stronger tendons and other structures than would result from natural healing.

Description

[0001] TITLE

[0002] Chimeric Human and Animal Collagen Materials and Methods of Making Same

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the priority of U.S. Provisional Application No. 63 / 557,834, filed 26 February 2024 and entitled “Chimeric Human and Animal Collagen Materials and Methods of Making Same’’, the whole of which is hereby incorporated by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED

[0006] RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under Grant Number 1 R21 EY029167-01 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0008] BACKGROUND

[0009] Rotator cuff (RC) tears are a common musculoskeletal disorder causing pain and dysfunction of the upper limb in more than 20% of the population.13 38Surgical repair is the gold standard for major RC injuries27 42and more than 300,000 cuff repairs are performed in the United States each year.61Despite the advances in surgical techniques, an unacceptably high re-tear rate for major tears still represents a challenge in orthopedics.22 54Treatments that can accelerate surgical recovery, strengthen surgical repair, and reduce re-tear rates have been extensively investigated.33

[0010] Tendons predominantly comprise a dense extracellular matrix of parallel arrays of collagen type I fibers decorated with proteoglycans and interspersed sparsely with spindle- shaped fibroblast-like cells (tenocytes).1Type I collagen, the primary load-bearing protein in vertebrates and 70% of the dry weight of tendon, is organized into hierarchical structures in the tendon (reviewed in Thorpe ef a / .).58During development, collagen molecules are exported to the extracellular space where they self-assemble into fibrils, putatively under cellular guidance and in response to biochemical and mechanical stimuli.53The fibrils undergo intermolecular cross-linking18and integrate into fibers, which are further bundled into fascicles that define the tendon substructure.21 25Intermolecular cross-linking, parallel organization, and hierarchical structure determine a tendon’s tensile strength and its ability to bear and transmit forces between muscles and bones.34These forces, in turn, can further strengthen the tendon and the collagenous arrays that comprise it56and increase the enzymatic and thermal stability of the constituent fibrils.6 19 20 48 56 62

[0011] In 2016, Paten et al. demonstrated that mechanical strain is sufficient to drive the organized assembly of type I collagen fibrils in the path of extensional strain through a process known as flow-induced crystallization.44To flow-crystallize collagen, the collagen concentration and the extensional strain rate should fall into particular ranges ( / .e., 15 mg / ml and 0.5 s-1were shown to produce fibrils).44Paten et al. concluded that flow-crystallization of collagen is not only possible in vivo, but that it may constitute a mechanism for the self-healing of tissue whereby physiological strains cause preferential assembly within the sites of tissue damage.44The idea opens the possibility of using exogenous soluble collagen as an autonomous direct protein mechanotherapeutic51to repair damage in tendons.

[0012] Although the sequence of events following a tendon rupture are well known, the molecular mechanisms of repair are only partially understood.25After a tendon injury, fibroblasts play an essential role in synthesizing various components of the extracellular matrix in the early response phase. However, the new tissue is composed predominantly of disorganized type III collagen fibrils, which are thinner and less organized than type I collagen fibrils, giving the healed tendon a scar-like appearance and weaker mechanics.5 17 25 59Only during the later stages of healing, at approximately 4 weeks after injury, does the disorganized type III collagen network begin to be replaced with an organized, stronger type I collagen network.23

[0013] To improve tendon healing and reduce the re-tear rate, cell-based approaches, bioinductive and tissue engineering strategies have gained significant interest.2 3 7 14 15 46Nevertheless, none of these “biologic” attempts have achieved regeneration of a tendon-bone attachment that structurally or functionally matches the native enthesis.32Recently, augmentation of rotator cuff repair with xenocollagen has been studied widely.8 10 26 28 29 31 55However, the animal origin of xenocollagen treatments has raised concerns regarding their antigenicity and immunogenicity.35Since xenocollagen antigenicity is more likely to arise from its terminal telopeptides, given their non-homology across species, collagen-based implants have typically used atelocollagen, where the potentially immunogenic telopeptides have been cleaved off enzymatically. Additionally, atelocollagen is more readily extracted from animal tissues than telocollagen, minimizing cost and maximizing yield. In 2017, Suh et al., determined that atelocollagen formulated into a patch improves the healing of rotator cuff tears in a rabbit model.55However, several clinical studies showed no or only modest changes in terms of clinical and functional outcomes after xenogeneic atelocollagen treatment.8 30 There remains a need for compositions and methods capable of promoting the formation, repair, and strengthening of collagen-containing tissues both in vivo and ex vivo.

[0014] SUMMARY

[0015] An aspect of the present technology is a non-naturally occurring chimeric collagen composition that differs from naturally occurring collagen in that it contains collagen from two or more sources to form a hybrid collagen, also referred to herein as “chimeric” collagen, which does not exist naturally. For example, the non-naturally occurring chimeric collagen composition can include collagen from two or more individuals of the same mammalian species, or collagen from two or more different mammalian species. Alternatively, the non- naturally occurring chimeric collagen composition can contain a naturally occurring mammalian collagen and a non-naturally occurring, engineered mammalian collagen. In preferred embodiments, the non-naturally occurring chimeric collagen compositions of the present technology include telocollagen polypeptides but do not include atelocollagen polypeptides. The atelocollagen used in other technologies results from the enzymatic removal of C-terminal telopeptides from telocollagen polypeptides. However, the telocollagen used in the present technology retains the telopeptides, which play a role in assembly and cross-linking of collagen monomers to form collagen fibrils or networks. Chimeric collagen compositions comprising telocollagen but not comprising atelocollagen can be formed in vivo, in tissues of an animal such as a human or non-human mammal, through endogenous processes after the administration of allogeneic telocollagen to the animal. Chimeric collagen compositions so formed have superior mechanical and / or morphological properties compared to collagen formed by natural unaided healing processes or compared to the animal supplemented with atelocollagen. Surprisingly, such chimeric collagen compositions in an animal body are non-immunogenic.

[0016] An engineered mammalian collagen is a collagen polypeptide that has an amino acid sequence that deviates from a naturally occurring mammalian collagen by one or more amino acid residues as a result of substitution, insertion, deletion, or genetic engineering. Engineered collagen can be designed to have one or more altered or improved properties, such as mechanical properties or properties related to its assembly or stability, or its ability to interact with other Types or sources of collagen or other biological structures. Collagen can be engineered with the use of artificial intelligence, for example. Engineered mammalian collagen can be engineered human collagen. The engineered mammalian collagen can be produced, for example, by a method including CRISPR modification or amplification of a collagen gene of a human cell or a non-human mammalian cell, which then synthesizes the collagen. Engineered mammalian collagen also can be produced in a mammalian cell, a nonmammalian cell, or a plant cell using known methods. A non-naturally occurring chimeric collagen composition can contain collagen from two or more different individual humans, a human collagen and a non-human mammalian collagen, a naturally occurring human collagen and a non-naturally occurring engineered human collagen, collagen from two or more non-human mammals, or a naturally occurring non-human mammalian collagen and a non-naturally occurring engineered mammalian collagen. The non-naturally occurring chimeric collagen composition can exist in a variety of forms, such as a solution containing collagen polypeptides, as a solid mixture, such as a lyophilized product, or it can exist as a mixture of collagen fibrils of different composition, or of same condition, with each fibril containing a mixture of two or more different types of collagen, as described above. The non-naturally occurring chimeric collagen composition can be present in a mammalian body, such as a human body, where it is either placed by a surgical procedure or where it is formed from its constituent molecules.

[0017] Another aspect of the present technology is a method of forming collagen fibrils in a mammalian subject, including a human subject. The method includes administering to the subject a non-naturally occurring chimeric collagen composition as described above, or precursors thereof that form said chimeric collagen composition in the mammalian subject. The method can be used in treatment of a wound or damaged or torn tendon, muscle, or ligament of the mammalian subject, or to aid in strengthening of collagen fibrils in the mammalian subject in any collagen containing tissue. The treatment can, for example, be used to aid wound healing such that a wound is healed more rapidly and / or more completely than it would be healed by performing a method lacking administering of the collagen composition. For example, following such treatment, the failure load or failure stress of the wound area or the repaired tissue is greater than it would be after performing a method lacking administering of said collagen composition. In an embodiment, a collagen-containing tissue or structure (e.g., bone, tendon, ligament, or muscle) of a mammalian body is increased by the method to at least the level of strength in the naturally occurring, undamaged or unwounded tissue or structure, or is increased to a higher level of strength than present in the naturally-occurring, undamaged or unwounded tissue or structure. Strength of a tissue or structure can be associated with, for example, an increase in Young’s modulus of the tissue or structure, and / or an increase in cross-sectional area, stiffness, failure load, and / or failure stress.

[0018] The chimeric collagen compositions and their use as described above can be implemented (i.e., present in, incorporated into, or synthesized within) in any collagen- containing tissue. Examples include tendons, ligaments, bone, cartilage, meniscus, and skin. Any known collagen type can be used in the compositions and methods. Preferred collagen types are fibril forming collagen types I, II, III, V, XI, XXIV, and XXVII collagen and network forming collagen type IV. Also preferred are fibril forming collagen types I, II, III, V, and XI, and combinations thereof. The chimeric collagen compositions and methods of their use can be made or carried out using human, non-human mammalian, and other animal collagen polypeptides, collagen genes and the composition can be introduced into or formed within humans, non-human mammals, and other animals.

[0019] The present technology can be further summarized in the following listing of features.

[0020] 1 . A non-naturally occurring chimeric collagen composition, comprising a mixture of two or more different collagens, wherein the composition comprises telocollagen and is devoid of atelocollagen.

[0021] 2. The non-naturally occurring chimeric collagen composition of feature 1 , wherein at least one of said two or more different collagens is an engineered collagen.

[0022] 3. The non-naturally occurring chimeric collagen composition of feature 1 , wherein said two or more different collagens comprise two or more allogeneic collagens.

[0023] 4. The non-naturally occurring chimeric collagen composition of feature 3, wherein said two or more allogeneic collagens are derived from a same animal species, and wherein said composition is non-immunogenic when present within a body of an individual of said animal species.

[0024] 5. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein said composition comprises one or more fibril forming and / or network forming collagen types.

[0025] 6. The non-naturally occurring chimeric collagen composition of feature 5, comprising one or more fibril forming collagens selected from the group consisting of type I, II, III, V, XI, XXIV, and XXVII collagen.

[0026] 7. The non-naturally occurring chimeric collagen composition of feature 6, wherein the fibril forming collagen comprises type I collagen.

[0027] 8. The non-naturally occurring chimeric collagen composition of feature 5, wherein the network forming collagen comprises type IV collagen.

[0028] 9. The non-naturally occurring chimeric collagen composition of feature 3, wherein said two or more allogeneic collagens are derived from a same mammalian species.

[0029] 10. The non-naturally occurring chimeric collagen composition of feature 9, wherein the mammalian species is human.

[0030] 11 . The non-naturally occurring chimeric collagen composition of feature 2, wherein the composition comprises a naturally occurring human collagen and a non-naturally occurring engineered human collagen.

[0031] 12. The non-naturally occurring chimeric collagen composition of feature 10 or feature

[0032] 11 , wherein at least one of said two or more collagens is a naturally occurring human collagen or an engineered human collagen produced by human cells in culture. 13. The non-naturally occurring chimeric collagen composition of feature 12, wherein said human cells have been engineered to increase collagen synthesis.

[0033] 14. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein the collagen composition comprises an animal or engineered collagen obtained by expression in plants.

[0034] 15. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein said two or more collagens differ from one another in amino acid sequence identity by at least 1%, or by at least 2%, or by at least 3%, or by at least 5%, or by at least 7%, or by at least 10%, or by at least 15%, or by at least 20%, or by at least 25%, or by at least 30%, or by at least 50%, or by substitution, deletion, or insertion of from 1-5 amino acids, or from 2-10 amino acids, or from 5-15 amino acids, or from 10-20 amino acids.

[0035] 16. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein the composition further comprises one or more alpha-hydroxy acids.

[0036] 17. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein the composition comprises at least one human collagen having a same amino acid sequence as a collagen from a human subject in need of collagen supplementation, and wherein the collagen composition further comprises an engineered human collagen that has an amino acid sequence modified from that of the collagen from said human subject.

[0037] 18. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein molecules of said two or more collagens are mixed together into composite collagen fibrils or networks.

[0038] 19. The non-naturally occurring chimeric collagen composition of any of the preceding features, wherein the composition exists in vitro or in vivo.

[0039] 20. The non-naturally occurring chimeric collagen composition of feature 19, wherein the composition exists in a human body or a non-human mammalian body.

[0040] 21 . The non-naturally occurring chimeric collagen composition of feature 20, wherein the composition exists in form of an aqueous solution, cell culture, liquid crystalline form, gel, patch, or a non-naturally occurring tissue.

[0041] 22. The non-naturally occurring chimeric collagen composition of feature 21 , wherein the composition exists in form of a non-naturally occurring tissue derived from a cell culture, tissue culture, bioreactor, transgenic plant, or transgenic animal.

[0042] 23. The non-naturally occurring chimeric collagen composition of feature 21 , wherein the composition exists in form of a non-naturally occurring tissue outside of a mammalian body.

[0043] 24. The non-naturally occurring chimeric collagen composition of feature 20, wherein the composition exists in form of a non-naturally occurring tissue present in a non-human mammalian body. 25. The non-naturally occurring chimeric collagen composition of feature 20, wherein the composition exists in a human body.

[0044] 26. The non-naturally occurring chimeric collagen composition of feature 25, wherein the human body is that of a subject who has undergone collagen supplementation therapy.

[0045] 27. The non-naturally occurring chimeric collagen composition of feature 26, wherein the collagen supplementation therapy is for treatment of a wound or condition comprising damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane.

[0046] 28. A pharmaceutical composition comprising the chimeric collagen composition of any of the preceding features and one or more excipients.

[0047] 29. Use of the non-naturally occurring chimeric collagen composition of any of features 1-27 or the pharmaceutical composition of feature 28 for preparation of a medicament for treatment of a wound or medical condition.

[0048] 30. The use of feature 29, wherein said wound or medical condition comprises damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane.

[0049] 31 . A method of forming collagen fibrils in vitro, the method comprising subjecting the non-naturally occurring chimeric collagen composition of any of features 1-27 to conditions that promote assembly or integration of collagen from said composition into collagen fibrils.

[0050] 32. The method of feature 31 , wherein said conditions comprise the presence of lysyl oxidase.

[0051] 33. A method of forming collagen fibrils in a human or non-human mammalian subject in need thereof, the method comprising administering to the subject a collagen composition comprising allogeneic telocollagen and not comprising atelocollagen, whereby chimeric collagen is formed in the mammalian subject.

[0052] 34. The method of feature 33, wherein the method aids or results in treatment of a wound or damaged or torn tendon, muscle, or ligament of the mammalian subject, or wherein the method aids or results in strengthening of collagen fibrils or networked collagen in the mammalian subject.

[0053] 35. The method of feature 33 or 34, wherein a collagen-containing tissue or structure is at least partially strengthened to a naturally occurring, wound-free or damage-free state of the tissue or structure, or wherein a collagen-containing tissue or structure is strengthened beyond a state obtained without performing said method and relying only on a natural healing process.

[0054] 36. The method of feature 35, wherein said strengthening comprises an increase in Young’s modulus, stiffness, cross-sectional area, failure load, and / or failure stress of the collagen-containing tissue or structure. 37. The method of feature 36, wherein said strengthening comprises an increase in failure load of the collagen-containing tissue or structure.

[0055] 38. The method of feature 37, wherein the collagen-containing tissue or structure is tendon and the subject is in need of repair of said tendon, and wherein failure load of said tendon is increased to at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the failure load of a similar tendon in an intact, undamaged state.

[0056] 39. The method of feature 37, wherein the collagen-containing tissue or structure is tendon and the subject is in need of repair of said tendon, and wherein failure load of said tendon is increased to more than 100% or more than 150% of the failure load obtained without performing said method and relying only on a natural healing process.

[0057] 40. The method of any of features 33-39, further comprising producing said collagen composition by a method comprising CRISPR modification of a collagen gene of a human cell or a non-human mammalian cell.

[0058] 41 . The method of any of features 33-40, further comprising forming and / or administering said collagen composition as a liquid crystalline collagen composition or a collagen-releasing patch.

[0059] 42. The method of any of features 33-41 , wherein said administering comprises implanting or injecting into the subject said collagen composition or a pharmaceutical composition comprising said collagen composition.

[0060] 43. The method of any of features 33-42, wherein damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane is repaired to any degree.

[0061] 44. The method of any of features 33-43, wherein the subject is a human subject.

[0062] 45. The method of any of features 33-43, wherein the subject is a non-human mammalian subject.

[0063] 46. The method of any of features 33-45, wherein a wound is healed more rapidly and / or more completely than it would be healed by performing a method lacking administering of said chimeric collagen composition.

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Fig. 1 depicts a study design for a rat model of rotator cuff tendon repair.

[0066] Fig. 2A shows Verhoeff staining of the supraspinatus tendon at 2, 4, 6 and 8 mm from the insertion. Fig. 2B shows laser scanning the tendon to extract the tendon cross-sectional area. Fig. 2C shows the results averaged at the cross section 6 mm from the tendon insertion (tendons scanned in triplicate). Fig. 3 shows a mechanical testing set up. Custom testing apparatus and recirculating water bath affixed to the Bose ElectroForce 5500.

[0067] Fig. 4 shows a mechanical testing protocol including preconditioning, stress relaxation and a steady ramp to failure.

[0068] Fig. 5A shows the weight progression of the rats from 30 to 60 days, and Fig. 5B shows the cross-sectional areas for each supraspinatus tendon, 6 mm from the insertion point are shown. Note that the 60-day control animals were heavier than the saline or atelocollagen treated animals. (*) and (**) represent statistical significance at p<0.05 and p<0.01 , respectively.

[0069] Fig. 6A shows the ultimate load at failure, and Fig. 6B shows the failure stress, of the supraspinatus tendons at 30 and 60 days are presented above. The telocollagen and uninjured control are both significantly different from all other samples for failure load at 60 days. For failure stress, they are both significantly different from saline treated tendons. (*) and (**) represent statistical significance at p<0.05 and p<0.01 , respectively.

[0070] Fig. 7A shows the stiffness, and Fig. 7B shows the percent relaxation, of all treatments presented above. It is clear that the stiffness of the uninjured control is quite a bit higher than any of our injured treated animals. The uninjured control also had a lower relaxation percentage than either telocollagen or atelocollagen treated tendons. (*) and (**) represent statistical significance at p<0.05 and p<0.01 , respectively.

[0071] Figs. 8A-8C show histological scoring of nuclear roundness (8A), cell density (8B), and vascularity (8C) for each group (S: saline; AT: atelocollagen; T: telocollagen) across the two time points (D30: day 30; D60: day 60). Subpanels i and ii present histological scores across groups at day 30 and 60, respectively. Subpanels iii, iv, and v present histological scores across the two time points for saline, atelocollagen, and telocollagen, respectively. Data is presented as mean ± standard deviation.

[0072] Figs. 9A-9C show histological scoring of inflammation (9A), fiber structure (9B), and fiber arrangement (9C) for each group (S: saline; AT: atelocollagen; T: telocollagen) across the two time points (D30: day 30; D60: day 60). Subpanels i and ii present histological scores across groups at day 30 and 60, respectively. Subpanels iii, iv, and v present histological scores across the two time points for saline, atelocollagen, and telocollagen, respectively. (*) and (**) represent statistical significance at p<0.05 and p<0.01 , respectively. Data are presented as mean ± standard deviation.

[0073] Fig. 10 shows total histological scores for each group (S: saline; AT: atelocollagen; T: telocollagen) across the two time points (D30: day 30; D60: day 60). Subpanels i and ii present histological scores across groups at day 30 and 60, respectively. Subpanels iii, iv, and v present histological scores across the two time points for saline, atelocollagen, and telocollagen, respectively. (*) represent statistical significance at p<0.05. Data is presented as mean ± standard deviation.

[0074] Fig. 1 1 shows several aspects of using collagen self-assembly under applied mechanical force to enhance wound healing.

[0075] Fig. 12 illustrates a process for promoting wound healing in a subject. The process includes producing collagen in a bioreactor using CRISPR-modified cells, packaging the collagen as a liquid crystalline preparation for surgical implantation, followed by tissue integration of the collagen in the subject. At the right are illustrated two forms of collagen for surgical implantation, an injectable form for early interventions with small defects and a patch form for larger defects.

[0076] Fig. 13 shows different phases in the development of rotator cuff disease and conventional treatment thereof.

[0077] Fig. 14 shows results of treatment with complete collagen (complete collagen = telocollagen; incomplete collagen = atelocollagen) on tendon failure load in a rat supraspinatus avulsion model.

[0078] Fig. 15 shows the time course of treatment of a wound (tendon tear) using a patch containing complete human collagen (CHC = human telocollagen) compared with conventional xenopatch (atelocollagen) therapy.

[0079] Fig. 16 shows aspects of a therapeutic process involving producing collagen in a bioreactor using CRISPR-modified cells, packaging the collagen as a liquid crystalline preparation for surgical implantation, followed by tissue integration of the collagen in the subject.

[0080] Fig. 17 shows histology results of treatment of using CHC in a rat supraspinatus avulsion model. Complete collagen = telocollagen; incomplete collagen = atelocollagen.

[0081] Fig. 18 shows histology results indicating reciprocal infiltration by a collagen patch and cells of a tendon wound in a rat Achilles tendon punch tenotomy model.

[0082] Figs. 19A-19D show histology results indicating reciprocal infiltration by implantation of a telocollagen crystal in a tendon wound in a rat Achilles tendon punch tenotomy model.

[0083] DESCRIPTION

[0084] The present technology provides chimeric collagen compositions and methods fortheir use in treating medical conditions associated with repairing wounds and other defects of collagen-containing tissues and biological structures, such as tendons. The compositions and methods also can be used in vitro for the production of artificial collagen-containing tissues and biological structures that can be used as, or in conjunction with, surgical implants, grafts, scaffolds, and other repair-related structures and devices containing a variety of different types of collagen. The present compositions contain chimeric collagen, either present in a pre-formed hybrid or chimeric state or suitable for the formation of such within an animal, such as a human or other mammal. The chimeric collagen compositions of the present technology also can be found within tissues of an animal, such as a human or non-human mammal, where they have been formed with the aid of natural or engineered biological processes, such as alignment, polymerization, cross-linking, and other covalent or non-covalent modifications or interactions with naturally non-naturally occurring collagen and / or other biomolecules in a subject animal, such as a human or non-human mammal. Preferably, the chimeric collagen compositions eventually take the form of morphologically and mechanically typical collagen fibrils or other naturally occurring collagen-containing structures, such as sheets or basement membranes. By their nature as hybrids between two or more different naturally occurring or engineered collagen molecular species, the chimeric collagen compositions of the present technology do not exist in nature, but are formed only through human action to induce the formation of hybrid collagens. The hybrid or chimeric collagens may contain mixtures of two or more different collagens either at the level of individual collagen fibrils or other macromolecular structures, or within a tissue containing mixtures of different fibril types. Different collagens sourced to prepare a chimeric collagen composition will typically differ in amino acid sequence or post- translational modification from one another. The different collagens can be obtained from two or more different individuals of the same species (i.e., allogeneic collagen), or from two or more different species, or from one or more naturally occurring collagens mixed together with one or more engineered collagens (i.e., collagens having a non-naturally occurring amino acid sequence or post-translational modification). The present inventors have surprisingly found that the combination of two or more different collagens, including one or more collagen molecules not found in the subject, generally does not give rise to inflammation or immune reactivity that is harmful to chimeric collagen structures in the subject in which the collagens are present. Further, chimeric collagen structures can be readily formed in vivo or in vitro, and may have superior mechanical and / or morphological properties compared to naturally formed collagen structures present after wound repair.

[0085] Previous technologies for introducing collagen to promote repair of collagen-containing tissues and structures have focused on the use of atelocollagen, which contains collagen polypeptides whose telopeptides have been removed, such as by enzymatic cleavage. Though telopeptide cleavage may be beneficial in preventing immunogenicity,12it may also be detrimental for tendon repair, strength, and function. During collagen fibril formation, telopeptides form covalent cross-links with adjacent collagen molecules, increasing the speed of collagen fibril self-assembly and providing long-term structural strength, including increased resistance to enzymatic and thermal degradation.4 39 60Atelocollagen molecules lack the required lysine and hydroxylysine moieties that participate in lysyl oxidase-mediated covalent cross-linking, and instead can only associate via hydrogen bonding that naturally occurs between collagen molecules, resulting in a weaker structure.11Without telopeptides, atelocollagen treatments are not capable of creating a stable, long-lasting, and mechanically robust repair. Further, atelocollagen may actually impede healing through competitive inhibition by binding and occluding sites where full, length functional telocollagen should incorporate. Therefore, for a more permanent repair, the present technology prefers embodiments that use telocollagen instead of atelocollagen. However, in using telocollagen, the potential immunogenic response must also be considered.

[0086] Telocollagen monomers can associate with collagen fibrils in a cell-free solution, and can associate with the preexisting extracellular matrix in cell culture.52Furthermore, telocollagen molecules can preferentially bind to and incorporate with overload-damaged collagen fibrils under tension.50Thus, telocollagen can be used to promote collagen fibril formation and repair.

[0087] The present technology utilizes a process in which collagen, preferably in the form of telocollagen, binds to and preferentially incorporates into damaged tissue under tension, where it undergoes fibrillogenesis and cross-links with a tendon, resulting in increased mechanical strength. The present technology can utilize allogeneic human telocollagen, or collagen from a different species than the recipient, or even various forms of engineered collagen, which can produce a better, faster repair than either allogeneic atelocollagen or natural healing. Preferably, the use of allogeneic telocollagen does not cause a significant immune response.

[0088] In experiments described in the examples below, soluble allogeneic telocollagen injections produced better mechanical and histological outcomes in a torn / repaired rotator cuff rodent model than either saline (natural healing) or allogeneic atelocollagen treatments. The allogeneic telocollagen treated supraspinatus tendons exhibited high failure strength and a failure stress that was comparable to uninjured controls. Based on histological evaluation scores, fiber structure and arrangement of telocollagen treatment group were better than saline and atelocollagen groups while achieving an inflammation score at day 60 not significantly different from atelocollagen group. The results demonstrate that allogeneic telocollagen improves healing and does not elicit an immunogenic response. Thus, type I telocollagen can be used as a direct protein mechanotherapeutic that can improve healing quality and speed.

[0089] Soluble allogeneic type I telocollagen delivered as a direct protein therapeutic can accelerate and improve the healing of damaged tendons. The mechanochemical properties of type I collagen direct its incorporation into damaged connective tissue.

[0090] In a rodent model study described in the present examples, seventy-eight shoulders (39 Sprague-Dawley rats) had their supraspinatus tendon transected and repaired (72 shoulders) or left uninjured (6 shoulders). The repaired tissue was treated with an injection of 100 ml of saline, 10.0 mg / ml allogeneic atelocollagen, or 10.0 mg / ml allogeneic telocollagen at 0-, 1-, and 2-weeks post-surgery. The treated tendons were assessed by mechanical testing (failure load, failure stress, stiffness, relaxation, and two viscoelastic constants) and by semiquantitative histological scoring at 30- and 60-days post-surgery. Uninjured controls were mechanically assessed at 60 days. The results showed that allogeneic telocollagen improved the failure strength of the supraspinatus (29.93 ± 4.68 N) relative to saline (20.00 ± 3.50 N; p- value = 0.0003) or atelocollagen (23.21 ± 1.53 N; p-value = 0.0251) treated tendons (150% and 130% improvement respectively). The failure strength of telocollagen treated tendons approached that of uninjured controls (36.85 ± 4.99 N; p-value = 0.0205). Allogenic telocollagen improved the failure stress of the supraspinatus (34.1 1 ± 9.30 MPa) relative to the saline treated tendons (21.18 ± 6.09 MPa; p-value = 0.0245; 160% improvement) and was no different than uninjured controls (33.38 ± 9.88 MPa; p-value = 0.9985) or allogeneic atelocollagen (31.89 ± 6.81 MPa; p-value = 0.9598). The stiffness of uninjured controls was far greater than any of injured / treated tendons (>200% stiffer). Histological scoring showed that the allogeneic telocollagen treated tendons produced better collagen fiber arrangement (1.55 ± 0.17) than saline (2.50 ± 0.29; p-value = 0.0027) or allogeneic atelocollagen (2.23 ± 0.28; p-value = 0.0124) treated tendons and that it did not increase markers of immunogenesis (1 . 10 ± 0.42) relative to either saline (1 .44 ± 0.19; p-value = 0.4815) or allogeneic atelocollagen (0.68 ± 0.41 ; p-value = 0.3016).

[0091] Soluble allogeneic telocollagen injections produced better mechanical and histological outcomes in a torn / repaired rotator cuff rodent model than either saline or allogeneic atelocollagen treatments. The failure strength of allogeneic telocollagen treated supraspinatus tendons closely approached that of control animals while the failure stresses were equivalent. Thus, allogeneic telocollagen can be used as a deliverable direct protein mechanotherapeutic that can improve healing quality and speed.

[0092] The results presented herein demonstrate that a low-dose direct protein therapeutic regimen (3 injections) of allogeneic telocollagen significantly improves tendon strength and structural quality within 60 days when compared to either natural healing (saline control) or atelocollagen treatments in a rat model of a full-thickness supraspinatus tendon tear with surgical repair. All treatment groups formed relatively disorganized tendon within 30 days postsurgery that subsequently strengthened and further organized over time. By day 60, telocollagen-treated tendons could withstand significantly more load than either atelocollagen- treated (130%) or saline-treated tendons (150%) and approached the strength of uninjured control animals. The telocollagen and the uninjured controls had significantly higher failure stress values than the saline treated tendons. However, there was no difference between the failure stress of the telocollagen and atelocollagen treated tendons. This suggests that both treatments generate fibers with equivalent mechanical properties, but that telocollagen produces more of them given the substantially higher failure load. The histological findings support the mechanical results and show that telocollagen treated tendons formed more organized collagen than saline and atelocollagen treatments. The total histological scoring was the lowest (not statistically significant) for telocollagen at 60 days, indicating that using full length collagen from the same species did not elicit a sustained overt inflammatory or immune response. Allogenic telocollagen was no different than a saline injection at any time point, supporting the non-immunogenic hypothesis.

[0093] While the ultimate tensile strength of the telocollagen approached that of the uninjured control (80%), the stiffness of the uninjured control tendons was higher than any of the tendons in the injured treatment group. This suggests that the organization and length of fibrils in uninjured tendons remains superior and is more uniformly tuned to nominal loads relative to transected tendons.

[0094] Lysyl oxidase (LOX)-mediated cross-linking following injury begins affecting the repair tissue between day 30 and 60 in the rodent model. In telocollagen treated tendons, the LOX likely finds more available telocollagen as a substrate which assists in the repair. Atelocollagen, while helpful, cannot become a permanent part of the repair tissue. Saline- treated tendons were much slower to heal and had nearly the same tensile strength on day 30 and day 60, suggesting that the tissue formed between day 30 and day 60 was too immature to accept substantial crosslinking. The present results support a mechanochemical mechanism for tendon healing and show that exogenous telocollagen is “active” during the healing process and that the presence of telopeptides promotes a stable, long-lasting, and mechanically robust repair.

[0095] EXAMPLES

[0096] Example 1 . Rodent Model of Rotator Cuff Tear

[0097] Experiments were conducted to demonstrate the effectiveness of allogeneic telocollagen therapy and compare the healing capabilities, including mechanical strength and histological tissue organization, of exogenous allogeneic telocollagen, allogeneic atelocollagen, and saline injection following a full-thickness rotator cuff tear (and repair) in a rodent model.

[0098] Study design

[0099] The Massachusetts General Hospital Institutional Animal Care and Use Committee approved all the procedures in this study. Furthermore, all experiments were conducted and reported in accordance with the recommendations in the ARRIVE guidelines. A total of 39 male adult Sprague-Dawley rats weighing 300-350 g were purchased from Charles River Laboratories (Wilmington, MA) and were acclimatized for three days before surgery in standard animal facilities at the Massachusetts General Hospital. All animals underwent a bilateral detachment and transosseous repair of the supraspinatus tendon immediately, after which the shoulders were randomly allocated to one of the 3 study groups: saline subacromial injection (group 1 ; n = 24 shoulders), atelocollagen subacromial injection (group 2; n = 24 shoulders), telocollagen subacromial injection (group 3; n - 24 shoulders). Figure 1 shows the study design. Each rat received a different, randomized treatment for each shoulder. Seven and fourteen days after the surgery the animals received a second and third injection following their first group allocation. Postoperatively, animals were allowed free cage activity with standard cage amenities and clinical parameters were evaluated daily. The animals were sacrificed at 30 and 60 days after surgical repair. At the endpoints, bilateral shoulders were harvested and allocated for biomechanical testing (n = 48 shoulders) and histology (n = 24 shoulders). Three animals (n = 6 shoulders) without surgery were used as control for mechanical testing.

[0100] Type I telocollagen preparation

[0101] Rat telocollagen solution (#IKD119261001 , Advanced Biomatrix) was concentrated to 1 1 mg / mL using a 100,000 MWCO spin filter tube (PI88532, Fisher Scientific) at 4°C at 6,000xg (5804R, Eppendorf) under sterile conditions. A 10X Penicillin-Streptomycin (P4333, Millipore Sigma) solution containing 9% sodium chloride (S9888, Millipore Sigma) was created and sterilized using a 0.2 pm syringe filter (229743, CELLTREAT). The concentrated collagen was mixed 9:1 with the antibiotic-saline and adjusted to pH 3 to prevent collagen polymerization.

[0102] Type I atelocollagen preparation

[0103] Rat telocollagen solution was diluted with 0.5 M acetic acid (695092, Millipore Sigma) and mixed with pepsin under sterile conditions to yield a final concentration of 3 mg / mL collagen and 0.3 mg / mL pepsin. This solution was incubated at 4°C on a tube rotator for 3 days to cleave telopeptides, creating atelocollagen. After incubation, the collagen was salted out using an established protocol40 43to remove pepsin from the atelocollagen solution. The collagen was precipitated three times by adjusting the solution to 0.7 M sodium chloride, centrifuging at 3,500xg for 10 min at 4°C and redissolving the pellet in 0.5 M acetic acid. Atelocollagen concentration was measured using the DC Protein Assay (50001 11 , Bio-Rad Laboratories), and verified to be atelocollagen by assessing its polymerization kinetics following the protocol established by Siadat et al.52Briefly, collagen was diluted to 50 pg / mL with sterile phosphate buffered saline and adjusted to pH 7. Next, 100 pL samples were pipetted into a 96 well plate, put into a plate reader spectrophotometer (PowerWave XS, BioTek Instruments), and held at 37°C for 2.5 hours to induce collagen polymerization. Every 5 minutes, the optical density of the sample was measured at 313 nm, and lag time (time for optical density to reach 10%) and plateau time (time for optical density to reach 90%) were compared between atelocollagen and telocollagen samples. Atelocollagen polymerizes more slowly than telocollagen and thus has higher lag and plateau times.49After turbidity testing and confirmation, the atelocollagen was concentrated and mixed with the antibiotic-saline solution following the same protocol used for telocollagen.

[0104] Injection preparation

[0105] The 10x concentrated antibiotic-saline solution was diluted to 1x and adjusted to pH 3 (the same pH as telocollagen and atelocollagen treatment solutions) under sterile conditions to create the control saline treatment. For each shoulder, a 1 mL syringe containing 200 mL of telocollagen, atelocollagen, or saline treatment was prepared and kept at 4°C or on ice until injection.

[0106] Surgical procedure

[0107] The procedure was performed during daytime in the animal facility operative room. The surgical procedure has already been generally described57and was performed by two experienced surgeons who were blinded to the treatment group. A second person assisted by preparing all rats and injections ensuring the blinding of the two surgeons. Briefly, animals received pre-operative doses of Buprenorphine (0.01 mg / kg s.c.) and were anesthetized with Isoflurane inhalation (1-5%). Once anesthetized, the fur of the right and left shoulder region on each animal was shaved, and the skin was prepared with betadine. A 2-4 cm incision was made on the lateral aspect of each shoulder of all the animals. The deltoid muscle was identified, and the acromioclavicular joint was elevated with minimal dissection of the deltoid following the direction of its fibers to expose the rotator cuff tendons. Using a micro Adson forceps, space was developed underthe supraspinatus tendon. A double-needled 5-0 prolene suture was used to fashion a modified Kessler suture. A full-thickness tear of the supraspinatus tendon was made by cutting the tendon from the footprint on the humerus with a No. 15 blade. Two crossing bone tunnels were then made with the 5-0 needle. The first tunnel was done from the anterior extent of the footprint in a posteroinferior direction, and the second tunnel was done from the posterior extent of the footprint in an anteroinferior direction. The two 5-0 needles were then passed through their corresponding tunnels, and the sutures were tightened.

[0108] Rats were then randomized into one of the three experimental groups. In group 1 , 12 rats (n = 24 shoulders) received a subacromial injection of 100 ml of pharmaceutical grade saline with a 25 G needle. In group 2, 12 rats (n = 24 shoulders) received a subacromial injection of 100 ml of 10 mg / ml allo-atelocollagen with a 25 G needle. Finally, in group 3, 12 rats (n = 24 shoulders) received a subacromial injection of 100 ml of 10 mg / ml allo-telocollagen with a 25 G needle. The wounds were closed with surgical clips to facilitate re-opening the sites for the injections at 1 and 2 weeks.

[0109] Multiple injections

[0110] Seven days and then fourteen days after the first surgery, the animals received preoperative doses of Buprenorphine 30 minutes before induction of anesthesia. All animals were anesthetized via isoflurane inhalation. Once anesthetized, the animals had the fur of their right and left shoulder region shaved and the skin prepared with chlorhexidine and Betadine. A smaller incision (2 cm) was made on the lateral aspect of each shoulder of all the animals following the previous one. The subacromial space and the repaired supraspinatus tendon were exposed. Injections were done with a 25 G needle following the randomization plan in the subacromial space. All injections were performed by 1 person who was blinded to the treatment groups. The wounds were then closed with surgical clips.

[0111] Rats were euthanized 30 and 60 days after the surgical treatment. Each shoulder was designated for mechanical testing or histological analysis from every animal. After euthanasia, the humerus, the supraspinatus tendon, and the tendon-to-bone attachment were carefully collected.

[0112] Mechanical testing

[0113] After collecting the humerus and supraspinatus tendon, excess tissue on the bone and tendon-to-bone attachment was carefully removed. On some samples, a scar formed at the tendon-to-bone attachment, and this scar tissue was not removed. Using the blunt side of a #1 1 blade scalpel, the muscle was carefully scraped from the supraspinatus tendon to reveal a bright white, clean tendon. In each experimental group, 8 shoulders were dissected and mechanically tested. Additionally, six shoulders of uninjured control 655 g rats were tested. After dissection, the shoulders were frozen and were thawed before mechanical testing.

[0114] Mechanical testing was performed by investigators masked to the treatment of the tissue and following the procedure established by Soslowsky’s group.24 47Figure 2 shows the marking and area measurement procedure. Verhoeffs stain was used to create thin perpendicular lines along the tendon, at 2, 4, 6 and 8 mm away from the humeral head.16Tendon cross-sectional area was measured at the 6 mm line (to avoid measurements near the humeral head where the scar tissue was present) using a 670 nm laser (optoNCDT, MicroEpsilon)45and an nanomotion stage (A3200, Aerotech, Inc.). Next, the humerus was potted in polymethyl methacrylate, the tendon was inserted into the grip approximately at the 6 mm line (gauge length was measured in situ however), and sandpaper was used to prevent slipping. The sample was then loaded into the mechanical testing machine (ElectroForce 5500, TA Instruments) and kept in a 37°C, phosphate buffered saline bath during testing (Figure 3).

[0115] The mechanical testing protocol adapted from Gimbel et al.24is shown in Figure 4. Briefly, the tendon was subject to the following mechanical perturbations: 1) Preconditioning (0.5-1 N at 1 % / sec; 10 cycles); 2) A 300 second hold; 3) A viscoelastic stress relaxation test (delta step to 5% strain at 5% / s with a 600 second hold and return to baseline at 5% / s); 4) A 60 second hold; and 5) A steady (quasistatic) ramp to failure (0.3% / s). From the mechanical data collected, we extracted the failure load, failure stress, stiffness, and percent relaxation. The viscoelastic parameters: ii and T2, were calculated by fitting the stress-time data to the following equation:

[0116] Histological analysis

[0117] Once muscle-tendon-bone units were dissected and removed from the rat, they were fixed in 10% buffered formalin for 3 days and were then decalcified for 24 hours. After that, the specimens were cut to obtain three coronal sections: anterior, middle, and posterior. Sections were embedded in paraffin, sectioned at 5 mm with a microtome, and subsequently stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome. All the sections were then scanned to digital images. The histological evaluators agreed on the most appropriate location to score based on tendon attachment and humeral head landmarks. X by X window masks were then consistently applied to the sections at the agreed location. The six parameters: (1) fiber structure ( / .e., fiber length and continuity), (2) fiber arrangement ( / .e., fiber compactness and parallelism), (3) rounding of the nuclei, (4) inflammation, (5) increased vascularity, and (6) cell density — were evaluated according to a semiquantitative histology scoring system. These variables were quantified using the Maffulli’s and Chen’s scoring system9 36 37two independent researchers, masked to the sample type, scored the samples using a 0-3 scale, with 0 being normal and 3 being maximally abnormal. Hence, a perfectly normal tendon would score 0 and a maximally abnormal tendon would score 18. A third independent researcher, masked to the sample type, was brought in to score any samples where the scores diverged.

[0118] Statistical analysis

[0119] For each tendon and histological parameter assessed, the researchers’ scores for the anterior, middle, and posterior sections were averaged to obtain a final score. After scoring, two tendons were excluded from statistical analysis because they had scores greater than 3 standard deviations from the mean and visually appeared very different from the other tendons in their treatment category. Nine mechanically tested tendons were excluded from statistical analysis for extensive lingering failure due to uneven gripping (n = 5), PMMA resin covering insertion (n = 1), accidental rapid pull to breaking (n = 1), and greater than 2 standard deviations from mean (n = 2).

[0120] For each treatment type and parameter assessed, the mean and standard deviation was calculated, and the Shapiro- Wilk normality test was performed. T-tests (2 tailed, unpaired, variance determined with F-test) were performed in Excel to compare results at 30 days vs 60 days for each treatment and parameter. ANOVA1 followed by a Tukey’s comparison of means test was performed in GraphPad Prism (version 9.5.0) to compare treatments for each time point and parameter.

[0121] Example 2. Mechanical Testing.

[0122] The weights of the animals at the time of sacrifice and the cross-sectional areas at 6 mm from the insertion points are shown in Figure 5. The weight of the control animals at 60 days (655.5 ± 53.7 g) was significantly higher than the atelocollagen (579.0 ± 38.3 g; p-value = 0.0180) or saline (575.3 ± 36.6 g; p-value = 0.0073) treated animals. The control animals were also heavier than the telocollagen treated animals (597.9 ± 35.5 g; p-value = 0.0821), and while the difference was appreciable (~9%), it was not significant (Figure 5A). With regard to cross-sectional area, the saline treated tendons were the only ones that increased in area with time (0.69 ± 0.21 mm2at 30 days to 0.99 ± 0.21 mm2at 60 days; p-value = 0.2798), but the atelocollagen (0.76 ± 0.23 mm2at 30 days to 0.76 ± 0.16 mm2at 60 days; p-value = >0.9999) and telocollagen (0.89 ± 0.20 mm2at 30 days to 0.91 ± 0.21 mm2at 60 days; p-value = >0.9999) treated tendons did not change and were not different from each other for either time point (p-value = 0.5897 and 0.6393 for 30 and 60 days respectively; Figure 5B). However, the trend shows that the telocollagen-treated animals may have slightly thicker tendons than atelocollagen-treated animals. In Figure 6, the failure loads and failure stresses are shown for all conditions. Notably, there was a significant improvement failure load for the telocollagen (29.9 ± 4.7 N) relative to saline (20.0 ± 3.5 N; p-value = 0.0003) and atelocollagen (23.2 ± 1.5 N; p-value = 0.0251) treated tendons at 60 days. However, the failure load for telocollagen treated tendons approached, but did not reach that of the uninjured controls (36.9 ± 5.0 N; p-value - 0.0205) at 60 days. The failure stress for telocollagen (34.1 ± 9.3 MPa) at 60 days was significantly improved relative to saline (21 .2 ± 6.1 N; p-value = 0.0245) but was not different from atelocollagen (31.9 ± 6.8 N; p-value = 0.9598) treated tendons or uninjured controls (33.4 ± 9.9 N; p-value = 0.9985). Atelocollagen had similar failure stress to telocollagen treated tendons, possibly due to its generally smaller cross-sectional area.

[0123] Figure 7A indicates that the stiffness of an uninjured tendon (61 .7 ± 9.6 N / mm) is far higher than any of the injured, treated tendons at 60 days (25.1 ± 9.9, 26.0 ± 12.6, and 21.7 ± 4.6 N / mm for saline, atelocollagen, and telocollagen respectively; p-value = <0.0001 for all three treatments). None of the treated tendons were statistically different in terms of stiffness from one another at either time point. With regard to relaxation (Figure 7B), the uninjured tendon had a lower percent relaxation value (35.8 ± 3.1) than either the telocollagen (48.3 ± 7.5; p-value = 0.0013) or the atelocollagen treated tendons (44.9 ± 3.6; p-value = 0.0261) at 60 days. None of the injured tendons displayed relaxation differences at any time point. The time constants, TI and T2, showed no differences among the groups (data not shown).

[0124] Example 3. Histological Assessment.

[0125] The sections were examined at low power (8x) to consistently select a tendinous region immediately adjacent to the tendon-to-bone interface. The histological evaluation scores of the tendon were then obtained by the masked observers at higher power. Figure 8, Figure 9, and Figure 10 show the results with significant differences indicated. Tenocyte nuclei were moderately rounded without any definite differences between the three groups (Figure 8A). At day 60 a general shift to a flattened and spindle shaped organization of the tenocyte nuclei was observed. At 30 days all the groups showed hypercellularity (Figure 8B) and hypervascularity (Figure 8C) at the tendon to bone interface. Cell density and vascularity decreased at day 60, however the scores were not significantly different.

[0126] A substantial portion of the supraspinatus tendon was covered with inflammatory cells. At 30 days, the histology scores of the three groups in the assessment of the inflammation was similar (Figure 9A). At 60 days inflammatory cellular infiltration was lower in all groups. Atelocollagen had the lowest inflammation score (0.68 ± 0.41) which was significantly different from the saline group (1.44 ± 0.20; p-value = 0.0166). Most notably, inflammation score was significantly reduced for telocollagen from 2.06 ± 0.45 at day 30 to 1.10 ± 0.42 at day 60 (p- value = 0.0286) and was not significantly different from atelocollagen group at day 60.

[0127] Collagen fibers were fragmented, poorly organized, and arranged in a moderately wavy pattern. At 30 days, the histology scores of the three groups in the assessment of the fiber structure and arrangement were similar (Figure 9B). At 60 days, the telocollagen group had the lowest fiber structure score (1 .16 ± 0.24) and was significantly different from the saline treated tendons (2.22 ± 0.42; p-value = 0.011 1). Furthermore, the fiber arrangement of the telocollagen group appeared denser than that of the control groups and with a more compact and parallel collagen orientation. The fiber arrangement scores were significantly lower for the telocollagen (1 .55 ± 0.17) group than for the atelocollagen (2.23 ± 0.28; p-value = 0.0419) and saline (2.50 ± 0.28; p-value = 0.0099) groups (Figure 9C).

[0128] The total scores (sum of scores of all 6 categories) were similar for the three groups at 30 days (Figure 10). At 60 days, the histological total scores showed improvement both for the telocollagen and atelocollagen-treated tendons compared to their scores at 30 days (telocollagen at 30 days: 12.21 ± 2.02 vs telocollagen at 60 days: 8.82 ± 1.54; atelocollagen at 30 days: 12.6 ± 1 .35 vs atelocollagen at 60 days: 9.85 ± 1 .31). Most notably, the total score for telocollagen at day 60 was significantly lower than the saline group (1 1 .50 ± 0.44; p-value = 0.0195).

[0129] Figure 1 1 shows in vitro evidence for the mechanism of action of the invention (from the prior art). The four lines of evidence are: 1) Force driven healing, Illustrated by the direct physical production of new collagen structures using mechanical tension and flow-induced crystallization; 2) Self-cleaning, Illustrated by the retention of only loaded fibrils when collagenase is applied to mechanically strained collagen material; 3) Injury targeting, Illustrated by the homing of labelled collagen to damaged, loaded collagenous fibers and fibrils; and 4) Enhanced cell healing, Illustrated by a pulse chase experiment showing that labelled exogenous collagen molecules are incorporated into new tissue constructs produced by cells.

[0130] Figure 12 shows a typical workflow for the use of the present technology comprising the production of collagen via cell engineering methods, the packaging of the collagen into a protected delivery material and the injection or placement of the material into a patient proximal to a connective tissue injury.

[0131] Figure 13 indicates the progressive nature of tendon degeneration (e.g rotator cuff disease). More than half of all patients diagnosed with rotator cuff disease are not eligible for surgery and are given palliative treatment as there are no available curative approaches.

[0132] Figure 14 shows the results of a study in which Sprague-Dawley rats were treated with exogenous type I telocollagen (complete collagen) with the intent to create a chimeric collagen repair. The results show that three injections of telocollagen extracted from a different animal (same species) did not elicit an immunogenic response (i.e. was no different than injecting saline) and resulted in increased strength relative to controls (saline and type I atelocollagen (incomplete collagen))

[0133] Figure 15 compares the time course of current xenopatch treatment with the expected time course of treatment with exogenous type I collagen that is designed to create a chimeric collagen repair. Because the xenopatch needs to be removed and replaced with the patients own collagen, it will take longer to heal.

[0134] Figure 16 shows a possible process for producing, packaging and delivering the collagen to an injury where it will form a chimeric material within the animal or patient.

[0135] Figure 17 shows the total histological score and histological images (H&E and Masson’s T richrome) after 60 days of treatment of a rat supraspinatus tendon with exogenous collagen. The histology shows little immune response and repaired tissue that is likely chimeric. Figure 18 integration of exogenous type I collagen following implantation in a rat Achilles’ tendon. The collagen, which was implanted in the form of a liquid crystal was integrating with the host tissue forming a new chimeric material

[0136] Figures 19A-19D show donor type I telocollagen collagen implanted in the form of a liquid crystal into a recipient rat Achilles tendon. The images suggest that a chimeric material has likely formed at the interface of the donor implanted liquid crystal and the recipient tissue.

[0137] As used herein, "consisting essentially of" allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a listing of components of a composition or elements of a device, constitutes disclosure of alternative embodiments in which “comprising” is replaced with "consisting essentially of" or "consisting of".

[0138] While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

[0139] The publication “Soluble allogeneic telocollagen as a direct protein therapeutic: results of serial injections in a rodent rotator cuff tear model”, by L.S. Oh, et al., J. Shoulder and Elbow Surg. (2024) doi.org / 10.1016 / j.jse.2024.08.018, is hereby incorporated by reference in its entirety.

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Claims

CLAIMS1 . A non-naturally occurring chimeric collagen composition, comprising a mixture of two or more different collagens, wherein the composition comprises telocollagen and is devoid of atelocollagen.

2. The non-naturally occurring chimeric collagen composition of claim 1 , wherein at least one of said two or more different collagens is an engineered collagen.

3. The non-naturally occurring chimeric collagen composition of claim 1 , wherein said two or more different collagens comprise two or more allogeneic collagens.

4. The non-naturally occurring chimeric collagen composition of claim 3, wherein said two or more allogeneic collagens are derived from a same animal species, and wherein said composition is non-immunogenic when present within a body of an individual of said animal species.

5. The non-naturally occurring chimeric collagen composition of claim 1 , wherein said composition comprises one or more fibril forming and / or network forming collagen types.

6. The non-naturally occurring chimeric collagen composition of claim 5, comprising one or more fibril forming collagens selected from the group consisting of type I, II, III, V, XI, XXIV, and XXVII collagen.

7. The non-naturally occurring chimeric collagen composition of claim 6, wherein the fibril forming collagen comprises type I collagen.

8. The non-naturally occurring chimeric collagen composition of claim 5, wherein the network forming collagen comprises type IV collagen.

9. The non-naturally occurring chimeric collagen composition of claim 3, wherein said two or more allogeneic collagens are derived from a same mammalian species.

10. The non-naturally occurring chimeric collagen composition of claim 9, wherein the mammalian species is human.11 . The non-naturally occurring chimeric collagen composition of claim 2, wherein the composition comprises a naturally occurring human collagen and a non-naturally occurring engineered human collagen.

12. The non-naturally occurring chimeric collagen composition of claim 10, wherein at least one of said two or more collagens is a naturally occurring human collagen or an engineered human collagen produced by human cells in culture.

13. The non-naturally occurring chimeric collagen composition of claim 12, wherein said human cells have been engineered to increase collagen synthesis.

14. The non-naturally occurring chimeric collagen composition of claim 1 , wherein the collagen composition comprises an animal or engineered collagen obtained by expression in plants.

15. The non-naturally occurring chimeric collagen composition of claim 1 , wherein said two or more collagens differ from one another in amino acid sequence identity by at least 1%, or by at least 2%, or by at least 3%, or by at least 5%, or by at least 7%, or by at least 10%, or by at least 15%, or by at least 20%, or by at least 25%, or by at least 30%, or by at least 50%, or by substitution, deletion, or insertion of from 1-5 amino acids, or from 2-10 amino acids, or from 5-15 amino acids, or from 10-20 amino acids.

16. The non-naturally occurring chimeric collagen composition of claim 1 , wherein the composition further comprises one or more alpha-hydroxy acids.

17. The non-naturally occurring chimeric collagen composition of claim 1 , wherein the composition comprises at least one human collagen having a same amino acid sequence as a collagen from a human subject in need of collagen supplementation, and wherein the collagen composition further comprises an engineered human collagen that has an amino acid sequence modified from that of the collagen from said human subject.

18. The non-naturally occurring chimeric collagen composition of claim 1 , wherein molecules of said two or more collagens are mixed together into composite collagen fibrils or networks.

19. The non-naturally occurring chimeric collagen composition of claim 1 , wherein the composition exists in vitro or in vivo.

20. The non-naturally occurring chimeric collagen composition of claim 19, wherein the composition exists in a human body or a non-human mammalian body.21 . The non-naturally occurring chimeric collagen composition of claim 20, wherein the composition exists in form of an aqueous solution, cell culture, liquid crystalline form, gel, patch, or a non-naturally occurring tissue.

22. The non-naturally occurring chimeric collagen composition of claim 21 , wherein the composition exists in form of a non-naturally occurring tissue derived from a cell culture, tissue culture, bioreactor, transgenic plant, or transgenic animal.

23. The non-naturally occurring chimeric collagen composition of claim 21 , wherein the composition exists in form of a non-naturally occurring tissue outside of a mammalian body.

24. The non-naturally occurring chimeric collagen composition of claim 20, wherein the composition exists in form of a non-naturally occurring tissue present in a non-human mammalian body.

25. The non-naturally occurring chimeric collagen composition of claim 20, wherein the composition exists in a human body.

26. The non-naturally occurring chimeric collagen composition of claim 25, wherein the human body is that of a subject who has undergone collagen supplementation therapy.

27. The non-naturally occurring chimeric collagen composition of claim 26, wherein the collagen supplementation therapy is for treatment of a wound or condition comprising damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane.

28. A pharmaceutical composition comprising the chimeric collagen composition of claim 1 and one or more excipients.

29. Use of the non-naturally occurring chimeric collagen composition of claim 1 for preparation of a medicament for treatment of a wound or medical condition.

30. The use of claim 29, wherein said wound or medical condition comprises damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane.31 . A method of forming collagen fibrils in vitro, the method comprising subjecting the non-naturally occurring chimeric collagen composition of claim 1 to conditions that promote assembly or integration of collagen from said composition into collagen fibrils.

32. The method of claim 31 , wherein said conditions comprise the presence of lysyl oxidase.

33. A method of forming collagen fibrils in a human or non-human mammalian subject in need thereof, the method comprising administering to the subject a collagen composition comprising allogeneic telocollagen and not comprising atelocollagen, whereby chimeric collagen is formed in the mammalian subject.

34. The method of claim 33, wherein the method aids or results in treatment of a wound or damaged or torn tendon, muscle, or ligament of the mammalian subject, or wherein the method aids or results in strengthening of collagen fibrils or networked collagen in the mammalian subject.

35. The method of claim 33, wherein a collagen-containing tissue or structure is at least partially strengthened to a naturally occurring, wound-free or damage-free state of the tissue or structure, or wherein a collagen-containing tissue or structure is strengthened beyond a state obtained without performing said method and relying only on a natural healing process.

36. The method of claim 35, wherein said strengthening comprises an increase in Young’s modulus, stiffness, cross-sectional area, failure load, and / or failure stress of the collagen-containing tissue or structure.

37. The method of claim 36, wherein said strengthening comprises an increase in failure load of the collagen-containing tissue or structure.

38. The method of claim 37, wherein the collagen-containing tissue or structure is tendon and the subject is in need of repair of said tendon, and wherein failure load of said tendon is increased to at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the failure load of a similar tendon in an intact, undamaged state.

39. The method of claim 37, wherein the collagen-containing tissue or structure is tendon and the subject is in need of repair of said tendon, and wherein failure load of said tendon is increased to more than 100% or more than 150% of the failure load obtained without performing said method and relying only on a natural healing process.

40. The method of claim 33, further comprising producing said collagen composition by a method comprising CRISPR modification of a collagen gene of a human cell or a non-human mammalian cell.41 . The method of claim 33, further comprising forming and / or administering said collagen composition as a liquid crystalline collagen composition or a collagen-releasing patch.

42. The method of claim 33, wherein said administering comprises implanting or injecting into the subject said collagen composition or a pharmaceutical composition comprising said collagen composition.

43. The method of claim 33, wherein damage to skin, bone, a ligament, a tendon, a meniscus, a vertebral disc, cartilage, a muscle, a blood vessel, an intestine, or a basement membrane is repaired to any degree.

44. The method of claim 33, wherein the subject is a human subject.

45. The method of claim 33, wherein the subject is a non-human mammalian subject.

46. The method of claim 33, wherein a wound is healed more rapidly and / or more completely than it would be healed by performing a method lacking administering of said chimeric collagen composition.

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