Implantable grafts and methods for producing collagenous scaffolds

Abstract

Disclosed are decellularised animal tendons, methods for their preparation and grafts comprising decellularised animal tendons. The animal tendons may be macropod tendons. The method for the preparation of the decellularised animal tendons is adapted to control or limit the swelling of the animal tendon during decellularisation. For example, the method may include an acidic wash step and a basic wash step, wherein the osmolality of the wash solutions may be hyperosmolar relative to the animal tendon. The decellularised tendons and grafts may be suitable for use in the treatment, repair and / or reconstruction of a damaged tendon in a subject in need thereof.

Description

FIELD [1] The present disclosure relates to implantable grafts, in particular xenografts formed from animal tendons. The present disclosure also relates to methods for producing graft scaffolds, in particular utilising macropod source tissue. BACKGROUND [2] There are a limited number of non-synthetic, medical devices available for the repair of ligaments, tendons, and soft tissue in joints. Of these, most lack the correct balance of biomechanical and biochemical properties to allow for stable mobility and function during the healing of the repair much less full reconstruction of the joint. For example, a number of commercially available, implantable, collagenous scaffolds used in soft tissue repair surgeries do not provide full mechanical strength to support the joint. [3] Current methods for the repair of soft tissue in joints such as rotator cuff, ankle ligament, or anterior cruciate ligament typically involve synthetic grafts, allografts and autografts. Failure rates in ligament and tendon implants in humans intra-articular and extra-articular environments are different. Intra-articular recovery, such as for the anterior cruciate ligament (ACL) in the knee, have higher failure rates post-surgery. [4] An autograft utilises graft material harvested from the patient, thereby managing the risk of rejection of the graft as the material is completely biocompatible. Autografts however are associated with longer patient recovery time due to the second (harvest) surgery site. In addition, it is not always the case that the harvested material will have optimal properties for the graft site (for example, the correct width to accommodate the bone tunnel) or can be harvested from all donor sites increasing the risk of post operative failure and future revision. Autograft failure rates of 1015% in adults and 15-20% in teenagers have been reported (Cordasco et al., 2019). Further, there have been reports from revision surgery where a previously implanted autograft had not undergone remodelling. [5] Allografts utilise a graft harvested from a cadaver or living donor. These grafts have the benefit of being available on request and removing the need for a patient harvesting site. However, the age or quality of an allograft may be unknown and the presence of foreign DNA from the donor increases the risk of rejection and / or infection. Allografts are typically expensive and have a high failure rate (up to 25%). Some studies report allografts to negatively impact proprioception (Cruz et al., 2020). Allografts are stored frozen and as such needs to be prepared ahead of planned surgery, they are not available on demand during surgery. [6] Synthetic grafts have found favour for some ligament reconstructions as they provide many of the benefits of an allograft but can be stored and used on demand, for example in a situation where an autograft surgery does not provide the required quality of material. The inert nature of synthetic grafts advantageously removes the risk of reaction to donor DNA. However, synthetic graft have no regenerative activity and can never "remodel" as all collagen grafts are expected to do over time. In addition, there is always the chance of inflammation and rejection of the synthetic material due to foreign body reaction by lymphocytes. Failure of the graft can cause significant post failure complications (Jia, XY et al., 2017; Tiefenboeck et al., 2015). Synthetic grafts have failure rates as high as 33% (Tulloch et al., 2019). [7] Xenografts comprise graft material harvested from a donor animal, typically from bovine and porcine animals, and provide many of the benefits of an allograft and of a synthetic graft. Xenografts can be available packaged to be used at need, mitigate the need for autograft harvesting, and the quality of the material can be managed in manufacture. Xenografts are decellularised and sterilised to manage rejection caused by cellular material, genomic material and pathogens from the external donor tissue. However, decellularised material from bovine and porcine sources yield grafts of reduced strength and require further chemical modifications to match the strength required to provide biomechanical stability at the implantation site. Bovine grafts can only be obtained from stock in places free of Bovine encephalitis to avoid the risk of transmitting Zoonotic disease. [8] The potential of macropod tendons as suitable candidates for xenografts is disclosed in WO2019 / 014698. However, there are significant challenges in processing animal tissue so as to prepare a safe graft with the necessary strength and resilience for use (e.g., in the repair or reconstruction of human tendons or ligaments). A particular challenge is how to process an animal tendon to remove cellular and DNA material without overly compromising its resilience and strength. [9] Collagenous connective tissue, such as tendons and ligaments, comprises a hierarchical structure of collagen. Collagen fibres comprise bundles of collagen fibrils. Each collagen fibril is composed of staggered, cross-linked collagen molecules (or tropocollagen molecules). Collagenous connective tissue comprises a cellular component and extracellular component. The extracellular component comprises mainly collagen fibrils embedded in proteoglycans (themselves comprising glycosaminoglycan (GAG) side chains), elastin, water and other extracellular materials. The characteristic strength and resilience of connective tissues such as tendons and ligaments is at least in part due to the interplay between these extracellular materials. For example, GAG-containing proteoglycans link adjacent collagen fibrils and may therefore contribute to the transverse strength of connective tissue in a transverse direction (relative to the direction of collagen fibrils).

[10] Processing of animal connective tissue for use as grafts can overly disrupt the relationship between key components in the extracellular materials. This can result in a graft with reduced strength and / or resilience.

[11] Collagenous graft materials such as tendons and ligaments are often cross linked to increase their strength. Exogenously cross linking a collagenous material comprises the direct exposure to the collagenous material to an external agent resulting in structural modifications of the collagen macromolecules. Exogenous cross linking is distinct from endogenous cross linking, which occurs as a natural process in the body and is fundamental to maturation and healing processes.

[12] Examples of exogenous cross linking include the application of an external agent to the collagenous material, where the external agent may include chemical, photochemical, enzymatic or thermal processes. For example, a glutaraldehyde cross linked tendon, optionally irradiated with gamma radiation, may exhibit increased ultimate tensile stress compared to a corresponding natural tendon (although prevailing opinion as to whether cross linking treatments necessarily increase UTS).

[13] However, exogenously cross-linked collagenous materials suffer from several drawbacks. For example, while exogenously cross-linked materials may have an increased strength, they tend to suffer from increased brittleness, increased hardness, high creep rate and / or reduced resilience. This may be due to the disruption of intrafibrillar and interfi bril la r associations and the depletion or denaturation of extracellular components such as proteoglycans and elastin.

[14] It is an object of at least preferred embodiments of the present disclosure to address one or more of the above-mentioned disadvantages and / or to at least provide the public with a useful alternative.

[15] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally to provide a context for discussing features of the present disclosure. Unless specifically stated otherwise, reference to such external documents or sources of information is not to be construed as an admission that such documents or such sources of information, in any jurisdiction, are prior art or form part of the common general knowledge in the art. SUMMARY OF INVENTION

[16] In a first aspect, the present disclosure provides a graft for implantation in a patient, comprising a plurality of decellularised tendons; wherein the graft comprises tendons of two or more different widths / diameters held in close proximity.

[17] In an embodiment the tendons are arranged parallel to each other in their folded forms.

[18] In an embodiment, the graft is a xenograft. The decellularised tendons may comprise macropod tendons. Alternatively, the decellularised tendons may comprise bovine or porcine tendons.

[19] In an embodiment, the tendons are arranged in a non-uniform manner or a manner lacking radial symmetry within a nominal outer cross-sectional diameter. The decellularised tendons may be arranged to generally follow the principles of Soddy's circles and / or a Steiner chain when the scaffold is viewed in cross-section. The decellularised tendons are preferably selected and arranged such that the packing density and tensile strength of the graft is enhanced.

[20] In an embodiment, the decellularised tendons each have a maximum cross-sectional folded diameter of between about 1.5 mm and about 6 mm. In some embodiments the graft is for use in the repair of ACL joints and the decellularised tendons each have a maximum cross-sectional width of between about 3.2 - 4.2 mm or a nominal cross-sectional folded diameter of about 3 to 4mm.

[21] The tendons may be folded in half one or more times. For example, once to create a loop at one end, or twice to create a loop at each end of the graft. The graft may have an overall width of between about 2.5 mm to about 15mm, for example. In some embodiments the graft is for use in the repair of ACL joints and the graft has an overall width of between about 6 mm to about 12mm.

[22] The decellularised tendons are between about 180 mm and about 500 mm in length. The graft scaffold may have a length of between about 6 cm and about 12 cm.

[23] In an embodiment, the graft has a force to failure >1725 Newtons.

[24] In an embodiment, the graft has an osmolality that approximately matches an osmolality of synovial fluid at an implant site, for example, between about 347 and about 461 mOsmol / kg.

[25] In an embodiment, the graft is packaged in a storage solution. The storage solution may have a pH of about 5, for example it may be buffered to pH 5 with sodium acetate. In another embodiment, the storage solution may have a pH that is substantially neutral, for example between 7.0 and 7.2, for example it may be buffered to pH 7 with sodium citrate.

[26] In an embodiment, the storage solution comprises PEG4000 and normal saline.

[27] In another embodiment, the storage solution may have an osmolality which substantially matches that of human synovial fluid. In an embodiment, the graft is packaged in a storage solution having an osmolality of approximately 404 ± 57 mOsmol / kg or mOsmol / kg. In one embodiment the storage solution has an osmolality of between 395 and 400 mOsmol / kg.

[28] In a second aspect, the present disclosure provides a method of manufacturing a collagen scaffold, or a method for preparing a decellularised tendon, from animal tendons, the method comprising carrying out decellularisation steps to remove cellular and DNA material from an animal tendon including one or more wash steps, wherein one or more of the decellularisation and / or wash steps utilise a processing solution selected to regulate the swelling of the tendon.

[29] In an embodiment, during the wash step(s), the tendon is washed in a solution that is free from PBS.

[30] In an embodiment, the method comprises a final wash step that utilizes a processing solution comprising a saline solution having a pH of between about 4.5 and about 5.5.

[31] In an embodiment, one or more of the wash step(s) comprise submersing the tendon in a processing solution comprising about 0.25 to IM Na2SO4, or 24 to 26 % ethanol.

[32] In an embodiment, one or more of the wash step(s) comprise submersing the tendon in a processing solution configured to adjust a pH of the tendon to a pH of between about 4 and about 5.5.

[33] In an embodiment, the method comprises an alkali neutralisation step that comprises soaking the tendon in an organic acid such as acetic acid at a pH of about 4, followed by submersing the tendon in a saline solution. Alternatively, citric acid, malic acid, or another suitable acid may be utilised.

[34] In an embodiment, a size of the tendon after the final wash step does not exceed about 58% of an original cross-sectional width of the tendon before the final wash step, preferably not more than about 5% of the original cross-sectional width.

[35] In an embodiment, the tendon comprises a protective sheath in the form of a paratenon or synovial membrane. The method may include the step of removing the protective sheath from the tendon prior to carrying out the steps to remove cellular and DNA material from the tendon.

[36] The step of removing the protective sheath from the tendon may comprise inducing shrinkage of both the tendon and the sheath, then inducing swelling of the sheath at a faster rate than the tendon before stripping the sheath from the tendon. In an embodiment this may comprise soaking the tendon in a solution in PEG8000, for example, in a solution containing about 8% PEG8000 to induce shrinkage. This is followed by soaking the tendon in a solution of normal saline to induce swelling.

[37] The step of removing the protective sheath from the tendon may comprise inducing swelling only of both the tendon and the sheath, before stripping the sheath from the tendon. This method is suited to tendons that have not been frozen for extended periods, for example, less than two months. In an embodiment, the step of removing the protective sheath from the tendon comprises soaking the tendon 0.9% NaCI.

[38] Preferably after removal of the protective sheath, the tendon is substantially free of damage, for example free from fraying, tearing or visible abrasions or cuts.

[39] In some embodiments, the tendon is sourced from the tail of a macropod. The tendon may be a tail tendon extracted from between the 5th and 12th vertebra (Ca5 to Cal2) of a kangaroo tail, for example. In an embodiment the tendon is sourced from a tail having one or more of: a tail circumference between the 7th and 8th vertebrae that is between about 195 mm or greater, the tail weight of about 2.3 kg or greater, or the tail weight between about 2.3 and about 3.4 kg. The tendon may be sourced from the tail of a kangaroo or macropod weighing 3=40 kg.

[40] In an embodiment, the decellularised tendons each have a maximum cross-sectional folded diameter of between about 1.5 mm and about 6 mm. In some embodiments the graft is for use in the repair of ACL joints and the decellularised tendons each have a maximum cross-sectional width of between of between 3.2-4.2 mm or a nominal cross-sectional folded diameter of about 3 to 4 mm.

[41] In an embodiment, prior to the decellularisation process, the tendon has an ultimate tensile strength of between about 76 MPa and about 115 MPa. In an embodiment, the tendon comprises a plurality of fibrils having a fibrillar diameter of at least 200 nm, at least 250 nm, at least 300 nm, or at least 350 nm.

[42] In an embodiment, the tendon has a mass after decellularisation that does not exceed a mass of the tendon prior to decellularisation by more than about 5%.

[43] In an embodiment, the method comprises storing decellularised macropod tendons in a storage solution having an osmolality and / or osmotic pressure which substantially matches that of human synovial fluid. The storage solution may have an osmolality of about 404 ± 57 mOsmol / kg.

[44] In an embodiment, the storage solution may have a pH of about 5. The storage solution may be buffered to pH 5. A suitable buffer may include sodium acetate.

[45] In another embodiment, the storage solution may have a neutral pH. The storage solution may be buffered to pH 7. A suitable buffer may include saline sodium citrate.

[46] In an embodiment, the storage solution comprises PEG4000 and normal saline. In another embodiment, the storage solution comprises a saline sodium citrate buffer.

[47] The graft may comprise one or more of the features described above in relation to the other aspects described herein.

[48] In a third aspect, the present disclosure provides a graft for implantation in a patient, comprising a decellularised macropod tendon, wherein the graft has an osmolality that substantially matches an osmolality of human synovial fluid.

[49] In an embodiment, the graft comprises a plurality of decellularised macropod tendons.

[50] In an embodiment, the, or each, tendon has a maximum cross-sectional folded diameter of between about 1.5 mm and about 6 mm. For example, the, or each, tendon has a maximum crosssectional width of between about 2.5 mm and about 4.2 mm.

[51] In an embodiment, the decellularised tendon(s) have a length between about 180 mm and about 500 mm.

[52] In an embodiment, the graft comprises a force to failure >1725 Newtons.

[53] In an embodiment, the graft has a length of between about 7 cm and about 13 cm and a width of between about 2.5 mm and about 12 mm.

[54] The graft may comprise one or more of the features described above in relation to the other aspects described herein.

[55] In a fourth aspect, the present disclosure provides a method for preparing a decellularised tendon, the method comprising contacting an animal tendon with: an acidic solution and a basic solution, in separate steps; wherein the acidic solution and basic solution each comprise an osmolality of at least 750 mOsmol / kg. In an embodiment, the pH of the acidic solution is less than 2. In an embodiment, the pH of the basic solution is greater than or equal to 13. In an embodiment, the osmolality of the basic solution may be at least 1000 mOsmol / kg.

[56] The method may further comprise a step of contacting the animal tendon with a neutralising solution wherein the neutralising solution comprises an osmolality of at least 750 mOsmol / kg. The neutralising solution may be performed after contacting the animal tendon with the acidic solution and the basic solution, to at least partially neutralise the pH of the animal tendon. In an embodiment, the pH of the neutralising solution is between 6.9 and 7.1.

[57] In an embodiment, each of the acidic solution, basic solution and neutralising solution comprise aqueous solutions.

[58] In an embodiment, each of the acidic solution, basic solution and neutralising solution are free of surfactant or detergent.

[59] In an embodiment, the animal tendon is obtained from a macropod. For example, the macropod may be selected from a kangaroo, wallaby or wallaroo. The animal tendon may be a tail tendon, or an Achilles tendon.

[60] In an embodiment, the method further comprises incorporating the decellularised tendon into a graft for implantation in a subject. The graft may be configured for the repair or reconstruction of a tendon or ligament condition.

[61] In an embodiment, the animal tendon is not subjected to an active step of exogenous chemical cross-linking, and is not subjected to a step of irradiative cross linking except for collateral cross linking caused by an optional step of irradiative sterilisation.

[62] In a fifth aspect, the present disclosure provides a graft for implantation in a subject, comprising a decellularised macropod tendon comprising a plurality of collagen fibrils, wherein the average cross-sectional width of the collagen fibrils is at least 141 nm. In an embodiment, the tendon is not subjected to an active step of cross-linking. For example, the tendon is not exogenously chemically cross linked and the tendon is not irradiatively cross linked except for collateral cross linking caused by irradiative sterilisation.

[63] In a sixth aspect, the present disclosure provides a graft for implantation in a subject, comprising a decellularised macropod tendon comprising a plurality of collagen fibrils, wherein the, or each, decellularised macropod tendon comprises one or more of the following: a. an ultimate tensile strength of at least 58 MPa; b. an elastic modulus of at least 548 MPa; c. a displacement creep rate of less than 0.05 mm / ln(s); d. creep displacement at end of less than 1.24 mm; e. a normalized strain creep rate of less than 0.001 In(s)1; f. normalized strain at test end of less than 1.007; g. a normalized stress relaxation rate of less than -0.57 In(s)1; h. a normalized stress at end of less than 0.70; I. a GAG content of at least 0.46 micrograms / mg; j. an elastin content of at least 16 micrograms / mg; k. an insoluble collagen content of at least 670 micrograms / mg.

[64] In a seventh aspect, the present disclosure provides a method for treating, repairing or reconstructing a tendon or ligament condition in a subject comprising implanting the graft or decellularised tendon described herein at the site of a damaged tendon or ligament.

[65] In an eighth aspect, the present disclosure provides a use of the graft or decellularised tendon described herein in the manufacture of a medical device for the treatment, repair or reconstruction of a tendon or ligament condition in a subject, wherein the graft or decellularised tendon is configured for implantation at the site of a damaged tendon or ligament.

[66] This present disclosure may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features. Where specific integers are mentioned herein which have known equivalents in the art to which this present disclosure relates, such known equivalents are deemed to be incorporated herein as if individually described.

[67] The term 'comprising' as used in this specification and claims means 'consisting at least in part of'. When interpreting statements in this specification and claims that include the term 'comprising', other features besides those prefaced by this term can also be present. Related terms such as 'comprise' and 'comprised' are to be interpreted in a similar manner.

[68] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range and any range of rational numbers within that range (for example, 1 to 6, 1.5 to 5.5 and 3.1 to 10). Therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed.

[69] As used herein the term '(s)' following a noun means the plural and / or singular form of that noun. As used herein the term 'and / or' means 'and' or 'or', or where the context allows, both. BRIEF DESCRIPTION OF DRAWINGS

[70] The present disclosure will now be described by way of example only and with reference to the accompanying drawings.

[71] Figure 1 is a transmission electron microscopy (TEM) image of collagen fibrils within a kangaroo tail tendon.

[72] Figure 2 is a TEM image of collagen fibrils within a bovine tendon.

[73] Figure 3 is a graph illustrating the distribution of collagen fibrils within kangaroo tail tendon of Figure 1, by fibril diameter.

[74] Figure 4 is a graph illustrating the distribution of collagen fibrils within the bovine tendon of Figure 2, by fibril diameter.

[75] Figure 5 shows a skinned and thawed kangaroo tail, with the vertebrae labelled in preparation for dissection.

[76] Figure 6 shows a step of placing cuts along the kangaroo tail at each vertebrae to facilitate the removal of tendons.

[77] Figure 7 shows the removal of dorsal tendons from a kangaroo tail.

[78] Figure 8 shows the ventral tendons exposed in the kangaroo tail of Figures 5 to 7 and illustrating the cut locations for the tendons.

[79] Figure 9 shows a kangaroo tendon separated from the surrounding paratenon*;

[80] Figure 10 is a graph of swelling characteristics of kangaroo tail tendons compared to bovine or porcine sourced tendons after 1 or 3 hours of soaking in PBS.

[81] Figure 11 is a graph of swelling characteristics of kangaroo tail tendons compared to bovine or porcine sourced tendons after 1 or 3 hours of soaking in 0.15 M NaCI.

[82] Figure 12 is a graph of swelling characteristics of kangaroo tail tendons in various PBS or Na?S04 solutions.

[83] Figure 13 is a graph of swelling characteristics of kangaroo tail tendons in various polyethylene glycol solutions.

[84] Figure 14 is a graph of the changes of length, width, and mass during the paratenon removal process.

[85] Figure 15 is a cross-sectional view of a prior art graft comprising a plurality of parallel tendons of substantially the same tending diameter.

[86] Figure 16 is a cross-sectional view illustrating the distribution of tendons of varying tendon thicknesses, arranged according to Soddy's circles and / or a Steiner chain.

[87] Figure 17 shows one embodiment of a graft scaffold for the reconstruction of ACL injuries.

[88] Figure 18 shows a view through a knee joint showing a ACL reconstruction using the graft scaffold of Figure 17.

[89] Figures 19(1) to 19(111) are experimental histology images at lOx magnification showing remodelled tissue in sheep in the intra-articular space after 6 months of graft implantation, where Figure 19(1) shows results from an autograft (control), Figure 19(11) shows a graft scaffold produced according to a first example method of the present disclosure; and Figure 19(111) shows a graft scaffold produced according to a second example method of the present disclosure.

[90] Figures 20(1) to 20(111) are experimental histology images at lOx magnification showing remodelled tissue in sheep in the Femoral bone tunnel after 6 months of graft implantation, where Figure 20(1) shows results from an autograft (control), Figure 20(1i) shows a graft scaffold produced according to a first example method of the present disclosure; and Figure 20(111) shows a graft scaffold produced according to a second example method of the present disclosure.

[91] Figures 21(1) to 21(111) are experimental histology images at lOx magnification showing remodelled tissue in sheep in the Tibial bone tunnel after 6 months of graft implantation, where Figure 21(1) shows results from an autograft (control), Figure 21(11) shows a graft scaffold produced according to a first example method of the present disclosure; and Figure 21(111) shows a graft scaffold produced according to a second example method of the present disclosure.

[92] Figure 22 is a diagram illustrating one example process for the decellularisation of animal tendons.

[93] Figure 23 is a diagram illustrating a second example process for the decellularisation of animal tendons.

[94] Figure 24 shows electron microscope images of the extracellular matrix of decellularised macropod tendons according to an embodiment of the disclosure. Figure 24(1) shows a longitudinal view of a fibre bundle (scanning electron microscopy, 5,000 x). Figure 24(H) shows a longitudinal view of individual fibres that form the fibre bundle (scanning electron microscopy, 5,000 x). Figure 24(111) shows a cross-sectional view of a single fibre (transmission electron microscopy, 30,000 x). Figure 24(iv) shows a longitudinal view of multiple fibrils (scanning electron microscopy, 65,000 x).

[95] Figure 25 is a graph comparing fibril diameters of unprocessed tendons and decellularised tendons prepared according to an embodiment of the present disclosure.

[96] Figure 26 shows a decellularised kangaroo tail tendon according to an embodiment of the present disclosure.

[97] Figure 27 shows graphs of (i) normalised stress vs time, (ii) force vs time, (iii) stress vs In(time) and (iv) force vs In(time) for an embodiment of the present disclosure.

[98] Figure 28 shows graphs of (i) normalised strain vs time, (ii) creep displacement vs time, (iii) strain vs In(time) and (iv) creep displacement vs In(time) for an embodiment of the present disclosure.

[99] Figure 29 is a graph of stress (MPa) over strain for an embodiment of the present disclosure.

[100] Figure 30 is a graph comparing the tendon cross sectional area (mm2) and tensile strength (N).

[101] Figures 31(1) to (iii) are images of tendons prepared according to different embodiments of the present disclosure, each showing different amounts of swelling at the end of the basic treatment (30 minutes). Figure 31(1) shows decellularised tendons produced according to the decellularisation protocol from Example 5. Figure 31(11) decellularised tendons produced according to the decellularisation protocol from Example 5, with sodium sulfate reduced by 50%. Figure 31(111) shows decellularised tendons produced according to the decellularisation protocol from Example 5 with sodium sulfate reduced by 75%.

[102] Figures 32(1) and (ii) show transmission electron microscope images of tendon fibre cross section. Figure 32(1) shows decellularised tendons produced according to the decellularisation protocol from Example 5 (magnification: 5000x, scale bar: 1.0 um). Figure 32(11) shows decellularised tendons produced according to the decellularisation protocol from Example 5 with 75% reduction in sodium sulfate reduced by 75%.

[103] Figure 33 shows a graph comparing the water content of unprocessed and decellularised tendons as described in Example 6.

[104] Figure 34 shows the collagen content of decellularised tendons as described in Example 6.

[105] Figure 35 shows the ultimate tensile strength (UTS) in MPa of decellularised tendons as described in Example 6.

[106] Figure 36 shows the Modulus of elasticity in MPa of decellularised tendons as described in Example 6.

[107] Figure 37 shows the thermal melting temperature native tissue and decellularised tendons as described in Example 6. DETAILED DESCRIPTION OF INVENTION

[108] The present application relates to novel collagenous scaffolds and grafts for implantation in animals, particularly for use in humans and methods for their preparation. Grafts produced according to the methods described herein may exhibit superior strength properties and / or a lower susceptibility to shrinking in situ after implantation and thereby may exhibit a lower likelihood of failure.

[109] While the present disclosure may be embodied in many different forms, for the purpose of promoting an understanding of the principles of the present disclosure, the disclosure is described in relation to the drawings and a number of examples, including an exemplary graft for the reconstruction of ACL injuries. [HO] Known soft tissue scaffolds typically comprise bovine or porcine source material. Bovine or porcine sourced tendons may be suitable starting materials for the graft scaffolds described herein. The Applicant has identified that macropod tail tendons, for example, taken from kangaroos, may have application in soft tissue scaffolds including for use in the grafts described herein. Macropod tendons may exhibit advantages over bovine or porcine tendons as kangaroo tendons contain thicker tendon fibrils, which may enable stronger grafts. Porcine, bovine and macropod dense connective tissue, such as tendons and ligaments, share fundamental similarities. For example, all sources of tendon comprise cellular and extracellular material, the extracellular material is primarily composed of type I collagen, and the collagen exhibits the same hierarchical organisation of fibres and fibrils. However, macropod tendons are stronger than bovine and porcine tendons. When comparing ultimate tensile strength (UTS), porcine and bovine tendons have been found to be between 7 and 3 times weaker than macropodine tendons (R. Haverkamp report 2022), as exemplified in Table 1, below. Species UTS (MPa) (Mean ± S.D) Fibril diameter (nm) (Mean ± S.D) Macropine (native*) 80 ±19 293 ±96 Bovine (native) 32 ±6 78 ±43 Porcine (native) 12 ±5 N.D. Table 1: Comparison of ultimate tensile strength (UTS) and fibril diameter of tendons of differing source material (R. Haverkamp report 2022)*Tissue removed from the animal in its native state. [Ill] Figures 1 and 2 show the comparative size of collagen fibrils in Kangaroo tail tendons (Figure 1) compared to fibrils in Bovine Achilles tendon (Figure 2), with the distribution of the fibril sizes quantified in the graphs of Figures 3 and 4, respectively.

[112] The present disclosure relates to a graft comprising a decellularised animal tissue. As exemplified, the animal tissue may comprise a macropod tendon.

[113] The decellularised animal tissue may be prepared from an animal tissue according to a process described herein. For example, the decellularised animal tissue may be prepared by exposing an animal tissue to a series of washing steps. The process may comprise one or more treatment steps in which the tissue is treated with an acidic solution, and one or more steps in which the tissue treated with a basic solution. The tissue may be treated with a pH neutralising agent following the acidic and / or basic treatment steps. Each of the steps may comprise treating the tissue with one or more solutions comprising a higher osmolality relative to the animal tissue. The osmolality of the solutions may be controlled by the presence of one or more solutes that contribute to the osmatic pressure of the solution (for example, sodium sulfate, Na2SO4).

[114] The decellularised animal tissue may be prepared without the use of exogenous cross linking agents or an active step of cross linking. Accordingly, the animal tissue itself is free of exogenous cross-linking agents and their cross-linking reaction products or residues.

[115] The decellularised animal tissue may be prepared without the use of a heating step, such as heating to a temperature over 40°C, or over 35°C, or over 30°C.

[116] The decellularised animal tissue may comprise beneficial levels of extracellular components that are present in the unprocessed animal tissue. For example, the decellularised animal tissue may comprise levels of extracellular components that are relatively near the levels of corresponding unprocessed animal tissue. For example, the decellularised animal tissue comprises beneficial levels collagen, elastin, and GAGs.

[117] The decellularised animal tissue may be substantially devoid of DNA of the source animal. For example, the decellularised animal tendon may be substantially devoid of animal DNA, or substantially devoid of DNA not from the recipient, or end user, of the tissue. For example, the decellularised animal tissue may comprise a DNA content below 50 ng / mg, or below 40 ng / mg, or below 30 ng / mg, or below 20 ng / mg, or below 10 ng / mg.

[118] The animal tissue may be dense connective tissue. The dense connective tissue may be selected from tendons and / or ligaments. Exemplary dense connective tissue described herein comprises tendons.

[119] The decellularised animal tendon may be substantially free of visible frays or splits. It may comprise an iridescent surface. The decellularised animal tendon may further comprise plurality of at least partially circumferential visible striations on the surface having a whiter visual appearance.

[120] The decellularised animal tissue may comprise a plurality of collagen fibrils having an average cross-sectional width of collagen fibrils of at least 100 nm, or at least 150 nm, or at least 200 nm, or at least 220 nm, or at least 250nm, or at least 300nm. In an exemplary embodiment, the average cross-sectional width as at least 141 nm.

[121] The packing density of collagen fibrils in the decellularised animal tendon is relatively near that of the unprocessed animal tendon (see, for example, Figure 32 and Examples 5 and 6).

[122] The decellularised  animal tendon may comprise a GAG content of at least 0.4 micrograms / mg, at least 0.6 micrograms / mg, at least 0.8 micrograms / mg, at least 1.0 micrograms / mg, at least 1.2 micrograms / mg, at least 1.4 micrograms / mg, at least 1.6 micrograms / mg, at least 1.8 micrograms / mg, or at least 2.0 micrograms / mg.

[123] The decellularised animal tendon may comprise an elastin content of at least 10 micrograms / mg, at least 15 micrograms / mg, at least 20 micrograms / mg, at least 25 micrograms / mg, at least 30 micrograms / mg, or at least 32 micrograms / mg.

[124] The decellularised animal tendon may comprise a collagen content of at least 600 micrograms / mg, at least 700 micrograms / mg, at least 800 micrograms / mg, or at least 900 micrograms / mg, or at least 950 micrograms / mg.

[125] The decellularised animal tendon may comprise an ultimate tensile strength of at least 50 MPa, at least 58 MPa, at least 60 MPa, at least 70 MPa, or at least 80 MPa, or at least 90 MPa, or at least 100 MPa. As shown below in Example 5, the mean UTS of decellularised macropod tendons is greater than 90 MPa. Higher UTS values can also be achieved.

[126] The decellularised animal tendon may comprise a modulus of elasticity of at least 500 MPa, at least 600 MPa, at least 700 MPa, at least 800 MPa, or at least 900 MPa. As shown below in Example 5, the elastic modulus of decellularised macropod tendons may be greater than 800 MPa. Higher elastic modulus values can also be achieved.

[127] The decellularised animal tendon exhibits a low creep rate. A creep rate is the rate at which a biological tissue continues to deform or elongate over time when subjected to a constant load or stress. During implantation of a graft for repair or reconstruction of a tendon or ligament, the graft must be tensioned and fixed at a precise length and angle. A low creep rate is beneficial for decellularised tendons in grafts, because it ensures that the graft maintains its length over time, avoiding elongation that could compromise function or alignment. The creep rate may be similar to that of native human tendons and ligaments, such as the native anterior cruciate ligament. Therefore, the decellularised animal tendon may comprise a creep rate of less than 0.5 mm / ln(s), less than 0.3 mm / ln(s), less than 0.1 mm / ln(s), less than 0.07 mm / ln(s), or less than 0.05 mm / ln(s). The decellularised animal tendon may comprise a normalised strain creep rate of less than 0.05 ln(s)-1, less than 0.01 ln(s)-1, less than 0.005 ln(s)-1, or less than 0.001 ln(s)-1. For example, known human tendon grafts (such as an ACL allograft), may have a displacement creep rate of 0.038 mm / ln(s) (+ / - 0.007), and a normalised strain creep rate of 0.025 ln(s)-1 (+ / - 0.006).

[128] The decellularised animal tendon may comprise a total water content that is less than 10% greater than the water content of the unprocessed animal tendon. An increased water content during decellularisation is indicative of penetration of water into the tendon's fibrillar structure, which weakens the strength and / or resilience of the tendon. It is therefore desirable to minimise the percentage increase in water content in decellularised tendons. The decellularised animal tendon may comprise a water content of less than 60%.

[129] A decellularised animal tendon of the present disclosure may be used for treating a tendon or ligament condition in any suitable subject, including a human subject. For example, a graft for implantation in a subject may comprise the decellularised animal tendon. The graft may be for treating a tendon or ligament condition in the subject.

[130] The graft may be used to repair or reconstruct any suitable ligament or tendon. For example, the ligament or tendon may be an anterior cruciate ligament (ACL), tibilias posterior (tendon), extensor pollicis longus (EPL), posterior cruciate ligament (PCL), elbow tendinosis, ulna collateral ligament (Tommy John's), medial collateral ligament (MCL) and lateral collateral ligament (LCL), elbow distil biceps, excision arthroplasty, meniscus repair, rotator cuff (tendon), lateral ankle ligament, forearm flexor (tendon), Superior Capsular reconstruction of the shoulder, Achilles (tendon), forearm extensor, acromioclavicular joint. In some embodiments, the ligament is an anterior cruciate ligament (ACL).

[131] In use, the graft may be implanted in the subject at the site of a damaged ligament or tendon in order to repair the damage. The graft may be implanted by any method for graft implantation known in the art. A method is thus provided for treating a tendon or ligament condition in a subject comprising implanting the graft described herein at the site of a damaged ligament or tendon.

[132] A graft scaffold will now be described with reference to Figures 16 to 17. These figures illustrate a multistrand graft scaffold 21 comprising a plurality of decellularised tendons 23. In this example, the tendons are arranged in parallel. In alternative embodiments, the tendons may be intertwined, for example woven or plaited. The tendons may comprise the decellularised kangaroo tendons described herein or may comprise tendons sourced from another animal or prepared using alternative techniques.

[133] The graft 21 comprises elongated decellularised tendons 23 of two or more widths / diameters. The width of a tendon may be taken to be the longest transverse dimension of the tendon and is equivalent to the diameter of a cylindrical tendon or the major dimension for a tendon that is elliptical or oval in cross-section, for example. This contrasts with prior art grafts 221 that are typically of the form shown in cross section in Figure 15, having a plurality of tendons 223 of all the same size I diameter.

[134] In the present graft 21, each tendon is generally circular in cross-section with a crosssectional diameter. Some tendons may not be perfectly circular along all or some of their length but they can be considered generally circular with a nominal cross-sectional diameter. The graft 21 comprises tendons of two or more different cross-sectional diameters.

[135] The parallel tendons 23 are bundled in contact with each other to form an elongate bundle of n tendons with an outer circle 25 defining a nominal outer diameter of the bundle. The decellularised tendons may be arranged with respect to each other to generally follow the principles of Soddy's circles and / or a Steiner chain when the bundle is viewed in cross-section. This arrangement may be a non-uniform arrangement in which the bundled tendons 23 do not exhibit radial symmetry. Figure 16 illustrates one such arrangement for a bundle of 7 tendons. The crosssection may be generally circular or may be non-circular.

[136] As used herein, the term Soddy's circles is intended to refer to three tangent circles each centred on a point of a triangle, with the circles being pairwise tangent to one another. The term "Steiner chain" is intended to refer to a set of n circles, all of which are tangent to two given nonintersecting circles, one being the outer nominal diameter of the graft.

[137] Tendons having a larger cross-sectional diameter / thickness exhibit higher ultimate tensile strength contributing to a stronger graft product. The graft strength is further enhanced by the progressively smaller tendons packed alongside the larger tendon(s) to increase or maximise the packing density of tendons into the nominal diameter ND of the graft. In such an arrangement, the packing density and thereby the tensile strength of the graft 21 is greater than if the graft were to comprise tendons that were all substantially equal in diameter (such as those in Figure 15) for the same nominal cross-sectional diameter of the scaffold.

[138] The thickness and length of the component tendons 23 will depend on the desired application for the graft and the source of the tendons. Some exemplary embodiments are detailed in Example 9.

[139] The bundle of n tendons may optionally be folded in half one or more times to produce a graft with a loop at least one end, and a number of strands that is a multiple of the number of component tendons n.

[140] In one example, a graft may comprise a bundle of two tendons doubled over to form a four-strand graft or doubled over twice to form an eight-strand graft. In an alternative example, a graft scaffold may comprise three or four tendons doubled over to form a six or eight-strand graft, respectively.

[141] Many other configurations of the multistrand graft scaffold with different combinations of numbers of tendons and numbers of folds are anticipated for various medical product applications. The number of tendons, size and arrangement of the tendons, and the number of folds is selected to provide a graft scaffold of a desired length and thickness.

[142] The animal tissue of the present disclosure may be selected from animal dense connective tissue. The dense connective tissue may be selected from tendons and / or ligaments. Exemplary dense connective tissue described herein comprises tendons.

[143] The dense connective tissue may be obtained from macropods. Macropods are animals belonging to the family Macropodidae. The family includes kangaroos, wallabies, and wallaroos. The family also includes a subfamily Macropodinae, which also includes kangaroos, wallabies, and wallaroos. Animals in this family are adapted for hopping as their primary mode of locomotion. Macropods typically have powerful hind legs and a long muscular tail.

[144] An exemplary macropod tendon described herein includes a tail tendon. However, the tendon may also be another tendon of suitable cross section and length. For example, the tendon may be an Achilles tendon. The macropod may be selected from the group consisting of red kangaroo (Macropus rufus), eastern grey kangaroo (Macropus giganteus), western grey kangaroo (Macropus fuliginosus), black wallaroo (Macropus bernardus), antilopine wallaroo (Macropus antilopinus), common wallaroo (Macropus robustus), agile wallaby (Macropus agilis), black-striped wallaby (Macropus dorsalis), red-necked wallaby (Macropus rufogriseus), swamp wallaby (Wallabia bicolor) and whiptail wallaby (Macropus parry!). The macropod may be selected from the group consisting of red kangaroo (Macropus rufus), eastern grey kangaroo (Macropus giganteus) and western grey kangaroo (Macropus fuliginosus).

[145] Average fibril diameter of native tendons obtained may vary between species, or within the species (for example, depending on the size of the donor animal). Preferred macropod tendons for the technology disclosed herein may have a fibril diameter of at least 200 nm, or at least 250 nm, or at least 300 nm, or at least 350 nm.

[146] To harvest macropod tail tendons, tails are severed from the body of the deceased animal, preferably as close to the back of the animal as possible. The tail is then skinned and the fat pad on the bottom removed. The skinned tail should be otherwise intact, with an undamaged fibrous sheath and should be free from broken surface tendons. The tail is then dissected using semi-aseptic techniques to remove the tendons in a manner that keeps the tendons free from fraying, cuts, and nicks.

[147] The tail may be sourced from any macropod / kangaroo species, provided the tail of the specimen is adequately sized to provide tendons of the required lengths and thicknesses. The selection criteria for the required size of the tail and the points where tendons are harvested from may depend on the end use of the tendons and the required strength and length properties of the tendons.

[148] For example, for the harvest of tendons for use in the ACL grafts described herein, a tail preferably has a tail circumference between the 7th and 8th vertebrae (Ca7 and Ca8) of about 19 cm or more. Typically, suitable tails are provided by kangaroos having a weight that is at least 40 kg. Tails outside of these specifications may have fewer tendons that are suitable for the ACL grafts described herein but may be suitable for other applications.

[149] For the harvest of tendons for use in the ACL grafts for described herein, the tendons are preferably longer than about 20cm. However, for other applications such as reinforcement devices where less strength is required, shorter tendons may be utilised. For reinforcement devices that are used with autografts or allografts, which only need to provide partial biomechanical strength, the tendons may be shorter than about 18 cm.

[150] For the harvest of tendons for use in the grafts described herein, the harvested tendons preferably have a maximum cross-sectional dimension (width) of between about 3.2 mm and about 4.2 mm. This corresponds to a nominal diameter when folded over (a common measurement technique employed by surgeons) of between about 3 mm and about 4 mm - that is the folded over tendon can be passed through an aperture in a graft sizer of between 3 and 4 mm. There is typically some compression of the tendon when folded over. However, for other applications the tendons may be thinner (for example, down to a width of about 1.5 mm) or thicker (up to a width of about 6 mm).

[151] Figures 5 to 8 illustrate one example method for obtaining macropod tail tendons. In this example method, the kangaroo tails 1 are first individually wrapped and stored at between -18°C and -20°C until required. Prior to dissection, the tails are placed in a semi-closed environmental chamber with a constant airflow at about 12°C to facilitate gradual thawing and a change in the internal temperature of the tail from about -20°C to about 12°C. Alternatively, the tails may be placed in a closed container at about 21°C for between about 18 to 20 hours or at 5°C for between about 30 to 36 hours.

[152] After thawing, the tails are inspected for the presence of any desiccated regions. If desiccated regions are identified, a cold 0.9% NaCI (5°C) solution is applied to these until hydration is restored. This typically takes between 15-30 minutes. Hydration can be detected visibly as dehydrated regions change from generally transparent to generally opaque when hydrated.

[153] Prior to harvesting the tendons, the vertebrae are identified. In this example the Ca5 vertebra is first identified. The Ca5 vertebra can be identified manually by way of a thick singular bone bulge That is present on the lateral side of the tail, at the Ca6 end of the Ca5 vertebra. This bulge corresponds to the distal transverse process of the Ca5 vertebrae. This feature is unique because the distal transverse process is larger than Ca6 and higher vertebrae. A knife cut 60 roughly 1 cm deep is made on the dorsal and ventral side of Ca5's lateral bulge 62 as shown in Figure 6.

[154] After identifying the Ca5 vertebra, the vertebrae Ca6 through to Cal3 are identified, as illustrated in Figure 5. These are found by feeling (in the direction of the tail tip) with one's index finger and thumb the troughs along the lateral side of the tail which correspond to the gap between the distal transverse process and proximal transverse process of any two adjacent vertebrae. In this example, which obtains tendons suitably sized for use in the ACL graft described herein, tendons are harvested from tails having at least a Cal3 or higher vertebrae present.

[155] Typically, tendon harvesting begins from the tail's dorsal side by making a cut at the Cal2 vertebra between the proximal and distal transverse process of the vertebra. Alternatively, tendon harvesting may begin from the tail's ventral side. The end of the tendon is clamped and pulled out of the tail as illustrated in Figure 7. After collecting one or more tendons, typically 2 or more, outward from the tail midline at this vertebra, tendon collection continues to the next lower vertebra i.e. Call. The process is repeated at each vertebra (Call, CalO, etc) until the tendon length becomes too short for the intended application. For example, for the ACL grafts described herein, a tendon length of at least 20cm is desirable, which generally requires tendons drawn from above the Ca9 vertebra. In this example, tendons 7 pulled between vertebrae Ca5 and Ca9, Ca5 and CalO, Ca5 and Call, and Ca5 and Cal2 had lengths that ranged from about 20cm to 50cm length. Shorter tendons may still be harvested for other applications.

[156] The tendon collection process is repeated on the ventral side of the tail, ensuring tendon length is 20 cm or above yet again from vertebra Cal2 to Ca9. Figure 8 shows an image of an example tail with the ventral tendons exposed. Ventral tendons have a tendon converging pattern unlike the dorsal side where tendons run in parallel towards the tip side. To remove the ventral tendons, they may be first cut at the convergence locations 3 as indicated in Figure 8, clamped, and removed in the same manner as the dorsal side tendons.

[157] The harvested tendons are rinsed with a saline solution to remove excess blood. The rinsed tendons are suitably stored and chilled. For example, at -20C.

[158] The tendons may be categorised into the location where they were obtained from, for example "dorsal" or "ventral". Tendons that are damaged or are not of the required length may be set aside for alternative applications.

[159] To enable use of animal tissue as an implantable, surgical, resorbable medical product, the tissue must be decellularised to remove unwanted cellular components including DNA and RNA material, producing a decellularised tissue comprising an extracellular matrix (ECM) that retains collagen, bio-inductive and biomechanical components, and the desirable structural and mechanical properties of the tissue.

[160] Decellularisation may be carried out using known methods of decellularisation including freezing and thawing, detergent washes, solvent extractions, alternate hypo-tonic and hyper-tonic washes, and enzymes.

[161] The process of decellularizing connective tissue comprises the penetration of washing fluids into the substructure of the tissue. This can cause the substructural elements (such as collagen fibrils) to separate, resulting in the swelling of the tissue. Without wishing to be bound by theory, processes which cause excessive separation of the substructural elements and swelling of the tissue may negatively affect the regenerative properties, strength and / or resilience of the tissue in use as a graft. The processing steps may therefore be adapted to minimise the swelling of the tissue.

[162] Referring to Figure 9, macropod tail tendons comprise a sheath-like connective tissue or paratenon 11 surrounding the tendon 13 (the paratenon allows the tendon to move with minimal friction). The Applicant has discovered that this paratenon introduces complexities into the processing of the tissue with known decellularisation processes by interfering with the decellularisation process and other downstream processes. The paratenon may also contribute to a higher post-processing DNA content. Therefore, removal of the sheath is desirable to enable use of the tendon in a graft.

[163] The Applicant has found that decellularisation of macropod tail tendons with the paratenon attached prevents effective removal of the sheath thereafter. The decellularisation process causes the paratenon to shrink around the tendon during the decellularisation process, distorting the shape and uniformity of the tendon. That in turn-may cause the paratenon to bind tightly to the tendon inhibiting or preventing removal of the paratenon after decellularisation. Removal of the paratenon after decellularisation was found to further damage the tendon tissue and introduce a greater bioburden.

[164] It is preferable to remove the paratenon before the tissue undergoes decellularisation. The Applicant has developed methods to facilitate removal of the paratenon by inducing a size change of the tendon and / or the paratenon that loosens the interface between the paratenon and the tendon. In one method, this may comprise inducing shrinkage of both the tendon and paratenon, then causing the paratenon to swell from its shrunken state and relative to and at a faster rate than the tendon. This method may be particularly useful when the tendons have been frozen for an extended time period contributing to difficulty in paratenon removal.

[165] There are challenges in relation to the decellularisation of animal dense connective tissue, especially macropod tendon. For example, known methods of decellularisation of dense connective tissue can cause significant swelling of the collagen fibrils and loss of mechanical strength. For example, decellularisation processes can cause collagen fibres to absorb water. This causes the distance between collagen fibrils to expand, which weakens inter-fibrillar associations. In addition, the collagen fibrils (within the collagen fibres) themselves absorb water, which weakens intra-fibrillar associations. See, for example, Figures 11 and 12, which show the swelling of bovine, porcine, and kangaroo tendons after exposure to PBS or saline solutions.

[166] The various steps in the process of decellularisation can induce several cycles of swelling and shrinking of the tendons. While some swelling may be important to enable washes to penetrate the tissue and cellular debris to be washed out of the tissue, excessive swelling can result in a decrease in collagen integrity and reduction of the physical properties of the tissue.

[167] Most known decellularisation methods include one or more washes in saline or phosphate buffered saline (PBS). However, the applicant has identified that such washes may be deleterious as collagen and collagenous tissues, including tendons, swell when repeatedly or prolongedly exposed to saline or PBS (Bowes and Kenten (1950)). Exposure time also contributes to the rate of swelling.

[168] The Applicant has also found that swelling and damage to the decellularised kangaroo tendon may be at least partially controlled by controlling the pH during the decellularisation and the final pH of the decellularised tendon.

[169] The decellularisation methods described herein seek to control or limit the rate or amount of swelling during the decellularisation process. In the present method, the decellularisation steps and one or more subsequent sanitation steps utilise a processing solution selected to minimise swelling of the tendon. For example, the present method may utilise a processing solution having an osmolality that substantially matches that of the tendons to minimise swelling.

[170] The osmolality of tendons is greater than that of cells and is greater than that of PBS. In one embodiment, the method omits the use of PBS in the decellularisation process. The processing solution of the present method is preferably hyperosmolar compared to PBS. Additionally, phosphate ions in PBS can interact with the positively charged amino acids within the collagen molecules to form non-covalent bonds (salt bridges). Without wishing to be bound by theory, these non-covalent bonds may effectively neutralize the positively charged amino acids and disrupt the existing electrostatic balance within the fibril structure. The electrostatic charges between fibrils lead to an increase in the spacing between the fibrils, thus promoting swelling.

[171] In one embodiment, a method of decellularisation may comprise preconditioning the tendon with sodium sulfate (Na2SO4) prior to and throughout the decellularisation and sanitation steps. Figure 12 illustrates the contrast between Na2SO4and PBS - PBS causes swelling due to its osmolality being lower than that of the tendon. In contrast, the osmolality of IM Na2SO4 is greater than the tendon resulting in shrinkage (no greater than 10%) of the tendon.

[172] Alternatively or additionally, ethanol may be used in the sanitation step to reduce the swelling impact of peracetic acid (PAA). The concentration of ethanol may be selected to compete with the swelling effect of PAA on the tissue being decellularised. The Applicants have identified that less tissue damage may be observed with increasing concentrations of ethanol.

[173] In one embodiment, a final step of the decellularisation process may comprise maintaining the pH between 4.5 and 5.5 during a final saline wash.

[174] Alternatively, prior to the final wash, acetic acid may be used to neutralise the NaOH treatment dropping the pH to about 4. The decellularised tendons are then washed several times with normal saline resulting in the pH of the final wash settling at a pH of about 4.5 to 5.5.

[175] In an alternative embodiment, a final step of the decellularisation process may comprise maintaining the pH between about 4.5 and 7.5.

[176] Alternative solutions are envisaged to achieve a wash solution with a desirable osmolality and pH. It will be understood that it is imperative that the wash solution have an acceptable safety profile in view of the intended application of the decellularised tendons.

[177] In one example, tendons with the paratenon attached are first soaked in Polyethylene glycol (PEG) 8000 for 1 hour to cause the tendons to shrink. PEG 8000 at 8% can compete for water, drawing water out of the tendon and subsequently causing the tendon to shrink (see Figure 13). Typical shrinkage may be about 1%.

[178] The shrunken but intact tendon is then washed in a saline solution. The tendon may undergo multiple washes, most commonly two washes. This soak in normal saline causes swelling of both the tendon and the paratenon from their shrunken states. However, because the density of the tendon tissue and the paratenon tissue differ, the paratenon swells at a faster rate than the tendon, loosening the connection between the tendon and the paratenon sheath.

[179] When the difference in swelling between the tendon and the surrounding paratenon sheath is sufficient, the paratenon may be stripped off the tendon. The paratenon 11 may be removed by hand or using tools.

[180] In an alternative method in tails that are fresh or have not been frozen for an extended time period (such as less than about two months), swelling of both the tendon and paratenon may be induced. These methods enable the paratenon to be more readily removed, for example by sliding the paratenon sheath off the tendon.

[181] In one example the tendon may be soaked / washed in an low concentration NaCI solution. A low concentration of NaCI such as of about 0.9% can cause tendons to swell over time as shown in Figure 11 (Percent swelling of tendons immersed in 0.9 % NaCI for 1 and 3 hours from macropine (SWK1 and SWK3), porcine (SWP1 and SWP3) and bovine (SB1 and S7)). In the present example, the tendon experiences 4% swelling after a first 15 minute wash and a further 1.4% increase in swelling after a second wash).

[182] The second soaking step may be repeated one or more times to ensure the paratenon is fully removed.

[183] During paratenon removal the amount of swelling of the tendon is controlled by limiting the soak time. Preferably the swelling is kept below about 8%, more preferably below about 5%.

[184] Another embodiment of decellularizing animal tissue will now be described, with emphasis on decellularisation of an animal tendon.

[185] Washing causes a certain degree of degradation of the connective tissue. This is observed in the reduction of collagen fibril diameter and / or packing density, reduction in GAG content and reduction in elastin content. The processing steps may therefore be adapted to maximise retention of collagen fibril diameter and / or packing, retention in GAG content and retention in elastin content following processing.

[186] Further, and without wishing to be bound by theory, processes of decellularising tissue using surfactants or detergents may excessively strip extracellular components from the tissue. The excessive loss of such extracellular components from the tissue may negatively affect the regenerative properties, strength and / or resilience of the tissue in use as a graft. The processing steps may therefore be adapted to minimise or eliminate the use of surfactants and / or detergents from certain steps in the processing of the tissue.

[187] In addition, and without wishing to be bound by theory, processes of decellularising tissue, such as connective tissue, using exogenous cross linking agents may disrupt the structural properties of the connective tissue, causing a reduction in resilience of the connective tissue. The processing steps may therefore be adapted to minimise or eliminate the use of exogenous cross-linking agents from certain steps in the processing of the tissue.

[188] The process of decellularizing connective tissue may therefore comprise steps configured to minimise swelling of the connective tissue and to reduce or eliminate the use of surfactants and / or detergents.

[189] In certain examples, the process of decellularizing connective tissue comprises a series of washing steps. The process may comprise one or more steps in which the tissue is contacted with an acidic solution, and one or more steps in which the tissue contacted with a basic solution. The tissue may be contacted with a pH neutralising agent following the series of washing steps. An example of a pH neutralising agent includes citric acid, a salt or a buffer composition thereof.

[190] Each of the processing steps may comprise contacting the tissue with one or more solutions comprising a higher osmolality relative to the animal tissue. The osmolality of the solutions may be controlled by the presence of one or more osmolar compounds that contribute to the osmotic pressure of the solution (for example, polyethylene glycol, PEG; sodium sulfate, Na2SO4). Sodium sulfate is described herein as an exemplary osmolality increasing agent. However, other suitable osmolality increasing agents may be used, including water-soluble salts (including salts comprising sodium, potassium, chloride, bromide, and sulfate) and water-soluble sugars. The use of hyperosmolar washing solutions counteracts, or at least partially counteracts, swelling of animal tissue caused by acidic and basic conditions.

[191] The method described herein produces decellularised animal tissue having beneficial levels of collagen, elastin, and GAGs, as well as beneficial biomechanical and viscoelastic properties. The levels of one or more of these components may be nearer to those of corresponding native human tissue than prior art decellularised animal tissues. Without wishing to be bound by theory, it is believed that the use of hyperosmolar solutions in the treatment stages of the method, in combination with acidic and / or basic conditions, effectively decellularises the animal tissue whilst protecting the structure and beneficial components of the tissue, such as collagen, elastin and GAGs.

[192] Advantageously, the length and thickness of tendons decellularised with the methods herein, having had swelling controlled, remain substantially unchanged and are overall in good condition with a smooth, iridescent surface. This is in contrast to tendons processed with conventional decellularisation methods with uncontrolled swelling, which may show lower strength and signs of damage such as fraying, enlarged or distorted ends, and / or may develop a dull and rough surface.

[193] The process may comprise a preconditioning step, in which animal tissue is contacted with a preconditioning wash comprising an osmolality modulating agent. The preconditioning step may acclimate tissue to a hypertonic solution and at least partially break cell membranes, and / or provide an incremental increase to the osmotic pressure prior to subsequent treatment steps, so as to allow the animal tissue to gradually adjust to osmotic conditions in processing.

[194] The preconditioning wash may be hyperosmolar with respect to the animal tissue. For example, the solution may have an osmolality of greater than 500 mOsmol / kg, greater than 600 mOsmol / kg, greater than 700 mOsmol / kg, greater than 750 mOsmol / kg, greater than 800 mOsmol / kg, greater than 900 mOsmol / kg, or greater than 1,000 mOsmol / kg. The osmolality of the preconditioning wash may be lower than the osmolality of a subsequent wash (e.g., lower than the osmolality of an acidic or basic wash).

[195] The preconditioning wash may comprise an aqueous solution having a substantially neutral pH. For example, the aqueous solution may have a pH of between about 6 and 8.

[196] The animal tissue may be contacted with the preconditioning wash for a period of at least 10 minutes, at least 20 minutes, or at least 30 minutes.

[197] The process may comprise a step comprising treating the animal tissue with an acidic solution. The acidic wash may solubilise the cytoplasmic components, degrade nucleic acids, and / or inactivate viruses and other pathogens. The acidic wash may be performed after the preconditioning step.

[198] The acidic wash may comprise an aqueous solution having a pH below 5. In an example, the pH of the aqueous solution is less than 4, or less than 3, or less than 2. The aqueous solution may comprise a strong acid or a mineral acid, such as hydrochloric acid.

[199] The acidic wash may comprise an aqueous solution comprising an osmolality modulating agent. The aqueous solution may be hyperosmolar with respect to the animal tissue. In an example, the aqueous solution may have an osmolality of greater than 500 mOsmol / kg, greater than 600 mOsmol / kg, greater than 700 mOsmol / kg, greater than 750 mOsmol / kg, greater than 800 mOsmol / kg, greater than 900 mOsmol / kg, or greater than 1,000 mOsmol / kg.

[200] The animal tissue may be contacted with the acidic wash for a period of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours.

[201] Following contacting with the animal tissue with the acidic wash, the animal tissue may be subsequently washed with one or more fresh solutions. The one or more fresh solutions may comprise an acidic pH. However, the pH of successive fresh solutions may be gradually increased.

[202] The process may comprise a step comprising contacting the animal tissue with a basic wash. The basic wash may lyse cells, degrade nucleic acid oligomers, and / or inactivate viruses.

[203] The basic wash may comprise an aqueous solution having a pH above 9. In an example, the pH of the aqueous solution is greater than 11, or greater than 12, or greater than 13. The aqueous solution may comprise a strong base or a mineral base, such as sodium hydroxide or potassium hydroxide.

[204] The basic wash may comprise an aqueous solution comprising an osmolality modulating agent. The aqueous solution may be hyperosmolar with respect to the animal tissue. In an example, the aqueous solution may have an osmolality of greater than 750 mOsmol / kg, greater than 1000 mOsmol / kg, greater than 1500 mOsmol / kg, greater than 2000 mOsmol / kg, greater than 2500 mOsmol / kg, greater than 3000 mOsmol / kg, or greater than 3500 mOsmol / kg. The osmolality of the basic wash may be greater than the osmolality of the acidic wash.

[205] The animal tissue may be contacted with the basic wash for a period of at least 10 minutes, at least 20 minutes, or at least 30 minutes.

[206] Following contacting with the animal tissue with the basic wash, the animal tissue may be subsequently washed with one or more fresh solutions. The one or more fresh solutions may comprise a basic pH. However, the pH of successive fresh solutions may be gradually reduced.

[207] The process may comprise one or more steps comprising contacting the animal tissue with a neutralising wash following contact with acidic and / or basic washes. Neutralisation of high pH or low pH conditions in the animal tissue, in particular, may reduce or avoid excessive degradation of the tissue extracellular matrix.

[208] The neutralising wash may comprise a neutralising agent to at least partially neutralise the pH of the animal tissue following acidic and / or basic washes. For example, the neutralising wash may be configured to adjust the pH of the animal tissue to between about 5 and 7. The neutralising wash may comprise a weak acid or a buffer solution configured to adjust the pH. In an example, the neutralising wash comprises citric acid.

[209] The neutralising wash may comprise an aqueous solution comprising an osmolality modulating agent. The aqueous solution may by hyperosmolar with respect to the animal tissue. In an example, the aqueous solution may have an osmolality of greater than 500 mOsmol / kg, greater than 600 mOsmol / kg, greater than 700 mOsmol / kg, greater than 750 mOsmol / kg, greater than 750 mOsmol / kg, greater than 800 mOsmol / kg, greater than 900 mOsmol / kg, or greater than 1,000 mOsmol / kg.

[210] The animal tissue may be further conditioned to remove components of the acid wash and / or basic wash, and / or neutralising wash. The conditioning may comprise contacting the animal tissue with an aqueous conditioning solution comprising a pH of between 5 and 7. The conditioning solution may comprise a buffer composition. In an example, the conditioning comprises contacting the animal tissue with a buffer solution of saline sodium citrate at a pH of about 7. In another example, the conditioning comprises contacting the animal tissue with a buffer solution of sodium acetate at a pH of about 5.

[211] In the decellularisation process described herein, the animal tissue is not subjected to an active step of cross linking, for example a step of cross-linking by irradiation with cross-linking radiation or the addition of a cross-linking chemical agent.

[212] Decellularised tendons may be stored in a hydrated state. Typically, this hydrated state may cause some swelling or shrinkage of hydrated decellularised tissue.

[213] When a partially swollen graft is implanted, there is a risk it may shrink in the bone tunnel when exposed to in-vivo osmolality. Shrinking of an implanted graft could create space around the graft - risking free movement of the graft in the bone tunnel and the potential for bone tunnel wear or widening and subsequent failure. Furthermore, a reduction in the graft diameter (shrinkage) post implantation will reduce the pull-out strength when using an interference screw fixation for an reconstruction such as an ACL reconstruction.

[214] Grafts containing a plurality of swollen tendons may exhibit a lower force to failure than an unswollen graft of the same size due to lower interfibriIlar packing density within the tendons, where force to failure is the maximum tensile force the graft can withstand before it begins to fracture or rupture.

[215] Conversely, grafts that have undergone shrinkage during storage may swell when exposed to in-vivo osmolality. Grafts that swell after implantation will increase in diameter and to a diameter greater than the bone tunnel. An implanted graft that is too big for the patient's anatomy (the bone tunnel in this instance) will cause a mechanical block to motion i.e. arthrofibrosis.

[216] To ensure that the hydrated decellularised tendons do not swell or shrink during storage and, therefore undergo minimal shrinkage or swelling after implantation, the storage solution may be selected to have an osmolality and ionic strength substantially the same as that the tissue will be exposed to in the implanted physiological environment.

[217] A reagent that competes for water may be added to minimize the competition for water between the collagen matrix and the vehicle (Figure 13).

[218] Alternatively, the pH of the storage vehicle may be neutral, near 7. The storage solution may be buffered to pH 7. A suitable buffer may include saline sodium citrate.

[219] The decellularised animal tissue may be packaged in a blister tray with liquid. The blister tray may be designed to contain the decellularised animal tissue and the liquid and prevent damage during transportation. The blister tray seal may be designed to be a sterile barrier. The blister tray may be packaged inside a foil pouch. The foil pouch may be designed to be a sterile barrier. The foil pouch will then be inserted into a paperboard carton for distribution

[220] The decellularised animal tissue may be sterilised following preparation and / or prior to use. Sterilisation methods are known in the art. For example, decellularised animal tissue may be sterilised with radiation (e.g. gamma radiation or electron beam radiation), or chemically sterilised (e.g., with ethylene oxide, hydrogen peroxide or peracetic acid), or thermally sterilised.

[221] In an example, the decellularised animal tissue may be exposed to a minimum of 25kGy for the purposes of sterilisation. Dry ice may be used to surround the product during the gamma radiation sterilisation process to keep the decellularised animal tissue frozen throughout the treatment. The decellularised animal tissue may be packaged inside the blister tray with liquid inside the foil pouch during the process.

[222] Whilst sterilisation techniques of the decellularised animal tissue may cause collateral crosslinking within the tendon, cross-linking is not an intended effect. Accordingly, the animal tissue is not subjected to an active step of cross-linking prior to sterilisation (and sterilisation should not be considered an active step of cross linking). For example, the decellularised animal tendon is not exogenously chemically cross linked and the tendon is not irradiatively cross linked except for collateral cross linking caused by irradiative sterilisation. In addition, the process is free of exogenous chemical cross linking agents (e.g. glutaraldehyde) and the product is accordingly free of exogenous chemical cross linking agents, reaction products and residues. And at least prior to a step of sterilisation, the decellularised animal tissue is free of any exogenous cross-linking agents or exogenous cross-linking reaction products. Example 1

[223] In an example procedure, tendons with the paratenon attached were soaked in 8% PEG 8000 for 1 hour at 5°C. The temperature was maintained by positioning the container with the solution on an ice bath. This caused the in-tact tendons to shrink.

[224] The shrunken tendons were then washed by soaking in 0.9% NaCI for 15 mins at 5°C to loosen the paratenon. The paratenon sheaths were then manually removed by gripping one end of the tendon and sliding the paratenon off the tendon. This was done using the thumb and forefinger of the opposite hand to pinch and drag the paratenon off the tendon.

[225] The paratenon were discarded and the tendon soaked again in fresh 0.9% NaCI for 15 mins. The manual paratenon removal was repeated. For this example, further soaking of the tendon in NaCI beyond two cycles was found to cause greater swelling of the tendon, negating the swelling differential between the two tissues. Example 2

[226] In a further example procedure, tendons with the paratenon attached were obtained from kangaroo tails that have either not been frozen or frozen for less than two months. These 'fresh' tendons were soaked in 0.9% NaCI for 15 mins at 5°C to induce swelling. An initial shrinking step was not required.

[227] The paratenons were then manually removed by gripping one end of the tendon and sliding the paratenon off the tendon. The paratenons were discarded and the tendon soaked again in fresh 0.9% NaCI for 15 mins.

[228] For this example, further manual handling of the tendon beyond two cycles was found to cause damage to the tendons (fraying and splitting). Example 3

[229] In one example decellularisation process illustrated in Figure 22, about 250 g of kangaroo tendons were placed in a 5 litre container with 1 Litre of 1% sodium dodecyl sulfate (SDS) in normal saline. The tendons were allowed to soak at room temperature for 4 hours on a rocker. The SDS soak solution was then decanted, and the tendons washed by soaking in 2 L of normal saline for 30 mins and exchanging with fresh normal saline between each wash for a total of 4 washes.

[230] The washed tendons were then soaked in 1 Litre of a solution of 0.18% peracetic acid (PAA) in 24% ethanol in purified water for 3 hours on a rocker. The PAA / ethanol soak solution was then decanted, and the tendons washed by soaking in 2 Litres of normal saline for 60 mins and exchanging with fresh normal saline between each wash for a total of 4 washes.

[231] This decellularisation method eliminated the use of PBS and used a high concentration of ethanol during the sanitation step to control swelling. Despite this method only providing partial swelling control, the processing results in macropine tendons with fibril diameters that are larger than native bovine tendons.

[232] Table 2: Comparison of fibril diameter (Mean ±S.D) and UTS (Mean) between decellularised macropine tendons from example 3 and untreated Bovine tendons. Species Fibril diameter (nm) UTS (MPa) Macropine (decellularised*) 141 ±62 37 Bovine (native**) 78 ±43 32 * decellularisation method from Example 3 above. * tissue removed from the animal in its native / unprocessed state. Example 4

[233] In another example of the decellularisation process illustrated in Figure 23, about 240 g of kangaroo tendons were placed in a 5 L container and preconditioned in 1.3 L of 0.25 M Na2SO4for 0.5 hours. The solution was decanted and the tendons then soaked in 1.3 L of a solution of 0.1 N hydrochloric acid (HCI) in 0.25 M Na2SO4. The tendons were allowed to soak at room temperature for 4 hours on a rocker.

[234] The HCI soak solution was decanted, and the tendons washed by soaking in 1.3 L of 0.25 M Na2SO4 for 20 mins and exchanging with fresh Na2SO4 between each wash for a total of 4 washes. The tendons were stored overnight (16 hours) at 5 °C in 1.3 L of 0.25 M Na2SO4at a pH between 4 and 3.5. The Na2SO4 solution was decanted and the tendons preconditioned in 1.3 L of 0.5 M Na2SO4 for 0.5 hours at room temperature.

[235] The Na2SO4 solution was decanted and the tendons soaked in 1.3 L of a solution of 0.75 M sodium hydroxide (NaOH) in IM Na2SO4 for 3 hours on a rocker. The NaOH solution was then decanted, and the tendons washed by soaking in 1.3 L of 0.5M Na2SO4 for 40 mins. The Na2SO4 solution was decanted and the residual NaOH in the tendons neutralized by soaking the tendons in 1.3L of 0.05M acetic acid in 0.5M Na2SO4for 2 hours at pH 4.5.

[236] The acetic acid solution was then decanted and the tendons washed by soaking in 1.3 L of 0.25 M Na2SO4 for 30 mins and then washed with 2.6L of normal saline for 40 mins, exchanging with fresh normal saline between each wash for a total of 3 washes. The tendons were soaked overnight (16 hours) on a rocker at room temperature in 2.6 L of normal saline at a pH between 4.68 and 4.98. The normal saline solution was decanted and the tendons washed with 2.6L of normal saline for 2.5 hours twice until the pH reached between 5 to 5.5.

[237] This method produced decellularised tendons with swelling of only about 4% compared to the starting tendon. It produced decellularised tendons having an iridescent appearance, smooth surface, flexibility in bending motion, and devoid of frays or splits.

[238] The composition and UTS of decellularised tendons prepared according this Example are shown in Table 3. Mean ±S.D GAG (ug / mg) 1.26 ±0.47 Elastin (ug / mg) 35.64 ±4.42 Collagen (ug / mg) 841.14 ±45.8 UTS (MPa) 50 ±16 Table 3. Biochemical and biomechanical properties of decellularize tendons prepared according to the method of Example 4. Example 5

[239] In another example of a decellularisation process, illustrated in Figure 23, about 240 g of kangaroo tendons were placed in a 5 L container and preconditioned in 1.3 L of 0.25 M Na?S04 (750 mOSmol / kg) for 0.5 hours. The solution was decanted and the tendons then soaked in 1.3 L of a solution of 0.1 N HCI in 0.25 M Na?S04 (950 mOsmol / kg). The tendons were allowed to soak at room temperature for 4 hours on a rocker.

[240] The HCI soak solution was decanted, and the tendons washed by soaking in 1.3 L of 0.25 M Na2SO4 for 20 mins and exchanging with fresh Na2SO4 between each wash for a total of 4 washes. The tendons were stored overnight (16 hours) at 5C in 1.3 L of 0.25 M Na2SO4 (750 mOsmol / Kg) at a pH between 4 and 3.5. The Na2SO4 solution was decanted and the tendons preconditioned in 1.3 L of 0.5 M Na2SO4 (1500 mOsmol / Kg) for 0.5 hours at room temperature.

[241] The Na2SO4 solution was decanted and the tendons soaked in 1.3 L of a solution of 0.75 M NaOH in 0.625M Na2SO4 (3375 mOsmol / Kg) for 0.5 hours on a rocker. The NaOH solution was then decanted, and the tendons washed by soaking in 1.3 L of 0.5M Na2SO4 (1500 mOsmol / Kg) for 0.5 hours. The Na2SO4 solution was decanted and the residual NaOH in the tendons neutralized by soaking the tendons in 1.3L of 0.02M citric acid in 0.5M Na2SO4 (1520 mOsmol / Kg) for 2 hours at pH 3.55.

[242] The citric acid solution was then decanted and the tendons washed by soaking in 1.3 L of 0.25 M Na2SO4 (750 mOsmol / Kg) for 0.5 hours and then washed with 2.6L of a saline citrate buffer composed of 0.154M NaCI in 0.0154 M sodium citrate at pH 7 (369.6 mOsmol / Kg) for 40 mins, exchanging with fresh buffer between each wash for a total of 3 washes. The tendons were soaked overnight (16 hours) on a rocker at room temperature in 2.6 L of saline citrate buffer at a pH 7. The saline citrate buffer was decanted and the tendons washed with 2.6L of saline citrate buffer for 40 mins twice until the pH reached 7 (369.6 mOsmol / Kg).

[243] This method produced decellularised tendons with swelling of only about 6% compared to the starting animal tendon. Examples of the tendons decellularised according to the method described in Example 5 are shown in Figure 26. As was observed with the decellularised tendons produced according to the method of Example 4, Figure 26 shows a decellularised tendon 23 comprising an iridescent appearance, an absence of frays or splits, a smooth surface, and partially circumferential visible striations on the surface having a whiter visual appearance. The decellularised tendons also comprised flexibility in a bending motion.

[244] Figure 24 shows electron microscope images of the extracellular matrix of the decellularised macropod tendons produced according to the method described in Example 5. Figure 24(1) shows a longitudinal view of a fibre bundle composed of multiple uniaxially aligned fibres (scanning electron microscopy, 5,000 x). Figure 24(H) shows individual fibres that form the fibre bundle (scanning electron microscopy, 5,000 x). Figure 24(111) shows a cross-sectional view of a single fibre (transmission electron microscopy, 30,000 x), in which the circular shapes are cross sections of multiple fibrils. Figure 24(iv) shows a longitudinal view of multiple fibrils (scanning electron microscopy, 65,000 x), showing bands along the length of each fibril. The bands are a characteristic feature of fibrillar collagen attributed to the staggered arrangement of collagen molecules.

[245] The decellularised tendons prepared according to the method described in this example were analysed for biochemical and biomechanical features. Details are shown in Table 4. Ultimate tensile strength (UTS) and modulus of elasticity were determined using a uniaxial tensile testing machine (TA.HDPIusC Texture Analyser with Exponent Software) with a 750 Kg loadcell. The tendons were positioned in a non-looped fashion and held in place using vice action grips of 2D sinusoidal profile with a torque of 10.2 N.m. A ramp to failure rate of 2.5 mm / s was later applied to achieve a near mid-substance rupture without sample slipping. Real time force data was recorded at 50 Hz which was normalized by the tendon cross-sectional area to calculate stress values. The tendon crosssectional area was computed using the equation — by identifying the appropriate hole size in the tendon sizer. These stress values were plotted against time (see Figures 27-29). A peak value was defined as the Ultimate Tensile Strength (UTS) and the slope of the linear region was defined as Modulus of Elasticity (MoE).

[246] For stress-relaxation testing, and with reference to Figures 27-29, the tendons were strained at a ramp speed of 2.0 mm / s to a 2.5% strain and held static for 2 minutes. Figure 27 shows graphs of (i) normalised stress vs time, (ii) force vs time, (iii) stress vs In(time) and (iv) force vs In(time). Figure 28 shows graphs of (i) normalised strain vs time, (ii) creep displacement vs time, (iii) strain vs In(time) and (iv) creep displacement vs In(time). Real time force data were collected against time at 2 Hz during the test which were then converted to stress by dividing with the tendon crosssectional area. The normalized stress at end was computed by dividing the stress values at test end with the stress value at the first instance when the required strain was reached. The normalized stress relaxation rate was computed by taking the slope of the stress-time curve, after applying log transformation of the latter. For creep testing, the tendons were strained at a ramp speed of 2.0 mm / s to 100 N force and held static for 2 minutes. Real time creep displacement values were collected at 1 Hz during the test. The displacement observed at test end was defined as Creep displacement. The displacement creep rate was computed by taking the slope of displacement vs log transformed time axis curve. Normalized strain was calculated as a ratio between strain at test end and strain value at the first instance when required force value is reached. The normalized strain creep rate was calculated by taking the slope of the strain vs log transformed time values. Parameters Mean ± S.D Max GAG (ug / mg) 1.47 ±0.46 2.18 2025297765   27 May 2026 Elastin (ug / mg) 27.7 ±6.97 46.29 Collagen (ug / mg) 870 ±97.7 988 UTS (MPa) 95.5 ±13.9 124.87 Modulus of Elasticity (MPa) 856 ±137 1121.12 Normalized stress at test end ( ) 0.70 ± 0.02 Normalized stress relaxation rate (ln(s)-1) -0.57 ± 0.11 Displacement creep rate (mm / ln(s)) 0.05 ± 0.01 Creep Displacement at end (mm) 1.24 ± 0.23 Normalized strain creep rate (ln(s)-1) 0.001 ± 0.0001 Normalized strain at test end ( ) 1.007 ± 0.001 Table 4 Example 6

[247] To determine the effect of decellularisation protocols described herein on the control of swelling during decellularisation, a side-by-side study was performed  comparing two decellularisation test groups. Group 1 samples were decellularised according to the process described in Example 5 and Group 2 samples were decellularised using the process from Example 5 but with a 75% reduction in the volume of sodium sulfate. That is, Group 2 decellularised tendons were prepared according to the method of Example 5, except that the amount of sodium sulfate was reduced by 75% in each of the acidic and basic steps.

[248] Visual observations of Groups 1 and 2 at the end of the basic treatment step showed differences in the degrees of swelling (Figure 31). Group 1 (shown in Figure 31(i)) showed sections of tendons that increased in diameter size. This was limited to a few tendons. All the tendons retained the same opacity as the native tissue and the tendons remained at the base of the treatment vessel. In contrast, Group 2 (shown in Figure 31(iii)) showed an increase in diameter along the entire length of the tendon for all the tendons. All the tendons were translucent and showed partial buoyancy. A third group with a 50% reduction in the amount of sodium sulfate was also included in the study. The swelling characteristics observed were midway between Groups 1 and 2 (Figure 31(ii)). This indicated a linear relationship between swelling and associated impact of collagen integrity. Group 2 tendons also exhibited fraying and loss of iridescent appearance after decellularisation, and thus exhibited clear visual differences to Group 1 tendons described in Example 5.

[249] Transmission electron microscopy of Group 1 and 2 tendon fibre cross sections (Figure 32) showed that Group 1 tendon fibres (Figure 32(i)) comprised collagen fibrils having a smaller inter-fibrillar distance compared to Group 2 inter-fibrillar distance (Figure 32(ii)). The Group 1 tendon fibres thus have greater fibril packing density (62.22%) compared to Group 2 (22.16%); see Table 5. This suggests that the connective structure binding the collagen fibrils of Group 1 tendons was less disrupted by the swelling control decellularisation process than the reduced swelling control process used for Group 2 tendons.

[250] Water content was determined for unprocessed tendons, Group 1 tendons and Group 2 tendons (Figure 33). Compared to unprocessed tendons (55%), Group 1 (59%) showed a 6% increase in water content whereas tendons in Group 2 (63%) showed a 15% increase in water content. While both Groups 1 and 2 increased in water content during the decellularisation process, the impact of the 2.5-fold increase in difference between Group 1 and Group 2 becomes apparent when the structural stability and mechanical properties are considered.

[251] Comparison of thermal melting temperature of unprocessed tendons, Group 1 and Group 2 (Figure 34) showed a denaturation temperature for Group 1 of about 60.0°C, which is about 0.9 °C lower than unprocessed tendons (60.9°C), whereas a denaturation temperature for Group 2 (58.6°C) was 2.3 °C lower than unprocessed tendons. A decrease in thermal melting or denaturation temperature is an indicator of a loss of protein structure and subsequently stability.

[252] Table 6 gives further details of the ultrastructural features (fibril diameter mean and range, and D-spacing mean and range) of the decellularised tendon obtained from Example 5. The distribution of fibril diameters of the decellularised tendon compared to the unprocessed animal tendon is shown in Figure 32. The frequency distribution of fibril sizes shows bimodality in both native and swelling controlled decellularised tendons. Fibril packing density (%) Fibril diameter (nm) (Mean + / - S.D) Figure D-spacing (Mean + / -S.D) Group-1 62.22 370.44 + / - 111.87 29 (i) 56.34 + / - 6.14 Group-2 22.16 219.78 + / - 95.4 29 (ii) 56.04 + / - 5.98 Table 5

[253] The data thus shows that Group 1 tendons had greater fibril packing density, lower water content and a higher denaturation temperature than Group 2 tendons. This data indicates that Group 2 was less structurally stable than Group 1. Without wishing to be bound by theory, the diminished stability of Group 2 is likely due to damage from increased swelling during the decellularisation process.

[254] The UTS and modulus of elasticity (Young's Modulus) (Figures 35 and 36, respectively) were determined for Group 1 and Group 2. Tendons from Group 2 (UTS: 75 MPa; Modulus: 651 MPa)) showed a 19 MPa drop in UTS and a 205 MPa drop in modulus of elasticity when compared to tendons from Group 1 (UTS: 96 MPa, Modulus: 856 MPa).

[255] The collagen content is similar for both tendons of Group 1 and 2 (Figure 37). Example 7

[256] In one example, decellularised tendons were stored in a storage vehicle comprising 0.5-1% PEG 4000 in normal saline buffered to pH 5 with 10-15mM sodium acetate. The percent swelling of a decellularised tendon stored over a 24-hour period was less than 1%. This compares favourably with the measured swelling of a similar decellularised tendon stored over a 24-hour period in normal saline at pH 7 of 36%. Example 8

[257] In another example, decellularised tendons were stored in a saline sodium citrate buffer comprising 15.4 mM sodium citrate in normal saline at pH7. The percent swelling of a decellularised tendon stored for over a month was less than 1%. Example 9

[258] A graft was prepared from a plurality of folded decellularised tendons prepared according to the decellurisation methods described herein.

[259] Irregular shaped tendons were folded in half and measured via a tendon sizer to produce biological scaffolds of circular cross-section and allow the application of Soddy circle principles. Converting tendons into multiple Soddy circle forms and combining them to form grafts allows the advantage that circles provide in terms of isoperimetric equality i.e. [Perimeter2 = 4nArea], to be exploited. The isoperimetric equality property is combined with the tangency property of Soddy circles to increase the tendon packing density within a graft.

[260] Tendons arranged in a Soddy circles arrangement have a tissue remodelling advantage to each tendon within the graft by decreasing diffusion time (t) It = Characteristicdlffuslonle^ 1 Thus the arrangement supports natural tissue regeneration and integration over time because of the improved ability of each tendon to support host cell infiltration. As circles offer lower characteristic diffusion length in terms of its radii while also maximizing its cross-sectional area via isoperimetric equality property, we decrease (t) and thus enhance graft remodelling (Table 4).

[261] Tendons having a larger cross-sectional area / diameter exhibit higher tensile strength contributing to a stronger graft product. The graft strength is further enhanced by the progressively smaller tendons packed alongside the larger tendon(s) to increase or maximise the packing density of tendons into the nominal diameter ND of the graft. In such an arrangement, the packing density and thereby the tensile strength of the graft 21 is greater than if the graft were to comprise tendons that were all substantially equal in diameter (such as those in Figure 15) for the same nominal crosssectional diameter of the scaffold.

[262] The number of tendons, size and arrangement of the tendons, and the number of folds is selected to provide a graft scaffold of a desired dimensions and strength. Table 6 gives example graft diameters consisting of tendons each having a nominal diameter between about 3 mm and about 4 mm in its single folded form. Table 7 gives examples of grafts comprising tendons of two or more different widths. The tendons were held parallel to each other and in close proximity. No. of tendons No. of folds No. of strands Length (cm) Graft diameter (mm) 1 2 4 10 5.0 - 6.0 3 1 6 10 6.0 - 7.0 4 1 8 10 6.5 - 8.0 Table 6: Relationship between graft diameters and the number of tendons / strands used to make the graft assembly No. of tendons No. of strands Graft size (mm) 3.0 mm 3.5 mm 4.0 mm 4.5 mm 5.0 mm - 4 - - 1 10 8 - 4 - 1 - 10 8 - - 4 - 1 10 9 - - 4 - 1 10 9 - - 4 1 - 10 9.5 - - 4 1 - 10 9.5 - - 4 - 1 10 9.5 1 8 - - - 18 9.5 - 8 1 - - 18 10 Table 7: Relationship between folded tendons of different sizes and final graft sizes after implementing Soddy circles to improve packing efficiency. Clinical uses / applications

[263] The engineered scaffold described herein has application in medical products for the repair or reconstruction of injuries of the following dense connective tissues: anterior cruciate ligament (ACL), tibilias posterior (tendon), extensor pollicis longus (EPL), posterior cruciate ligament (PCL), elbow tendinosis, ulna collateral ligament (Tommy John's), medial collateral ligament (MCL) and lateral collateral ligament (LCL), elbow distil biceps, excision arthroplasty, meniscus repair, rotator cuff (tendon), lateral ankle ligament, forearm flexor (tendon), Superior Capsular reconstruction of the shoulder, Achilles (tendon), forearm extensor, acromioclavicular joint. It will be understood that this list is not exhaustive and other applications are possible.

[264] In addition, the engineered scaffold described herein has application in medical products for the repair or reconstruction of injuries of the following areas or specialized connective tissues: bony voids, meniscus and articular cartilage.

[265] In addition, the engineered scaffold described herein has application in medical products for the repair or reconstruction of injuries of the following soft tissues: intestinal, urogenital, abdominal, vascular, and neuronal soft tissue.

[266] Figures 17 and 18 show one example embodiment graft 21 for use in an ACL reconstruction for illustrative purposes. However, grafts of similar forms may be used in other applications. This embodiment comprises 2 parallel tendons bundled and folded in half one or more times to form a graft having 4 or more strands such that the graft scaffold 21 has a total length of between about 70mm and about 130mm and / or a diameter between about 6 mm and about 12 mm.

[267] In one embodiment, the graft 21 is configured to have a first end of the graft 21 operatively attached to a first fixation device 24 for anchoring the device to bone; and a second end operatively attached to a second fixation device 25 for anchoring the device to bone. The fixation devices may be any suitable fixation device, for example a screw or button, as commonly used in the art.

[268] Referring to Figure 17, the first end of the graft 21 may comprise a fixation feature for anchoring the graft in situ. In the embodiment shown, the fold of the tendons produces a first end with a looped portion 29. This looped portion 29 may be utilised to attach a fixation component. In this example, the suture 31 is tied to the loop portion of the device to form a continuous loop, suspensory fixation device that can then be attached to any suitable fixation device.

[269] One or more securing members in the form of a collar, sleeve, thread 27 or other elongate member may extend around the tendons 23 to hold them in tight arrangement. In the case of a thread or elongate member, the member may be wrapped around the tendons in a generally annular manner and one or more discrete points or alternatively may be wrapped in a helical-like manner along a portion of or a full length of the graft or bundled tendons.

[270] The decellularised tendons 23 may comprise macropod tendons, for example kangaroo tendons, or may comprise tendons sourced from another animal species. In some embodiments the tendons may be decellularised according to the method described above, but in other embodiments, the tendons may be decellularised according to alternative processes utilizing the swelling control protocol.

[271] Kangaroo tendons, particularly when prepared as described herein are likely to enable a graft with superior strength properties. The required strength of the graft will differ according to the application. For ACL construction in an adult, the graft scaffold may have a tensile force to failure that is greater than or equal to about 1725 Newtons, where failure is taken to be the onset of rupture or fracture.

[272] The graft scaffold 21 comprises extracellular matrix (ECM) consisting of proteins, glycoproteins, and peptides, and capable of integrating into the patient's body. After implantation, the ECM provides a scaffold for the ingrowth of cells and undergoes controlled degradation and the release of bioactive peptides (i.e. cytokines or chemokines) that influence physiological processes such as cell migration, cell recruitment, angiogenesis, and other immunological responses.

[273] In some embodiments, the decellularised tendons 23 and thereby the graft has an osmolality that matches an osmolality of synovial fluid at an implant site. For example, the tendons may be prepared using one of the methods described above.

[274] The graft scaffold may be provided to a surgeon in a hydrated form or in a dry form. For supply in a hydrated form, the graft scaffold is package in a vehicle that ensures biochemical and physical stability of the graft, preventing degradation and swelling. The vehicle may also be selected to have an osmolality matching that of the synovial environment, as described above.

[275] The graft 21 may be sterilised prior to packaging or in the packaging. For example, by way of gamma irradiation or other suitable sterilisation method. Example 10

[276] A pilot animal study was performed using an established sheep model of ACL (or CCL in sheep) reconstruction following the known animal study methods described in Edwards et. al., 2021 and Kondo et. al., 2011. (Edwards_2021, Kondo_2011) Twelve 50-70 kg adult sheep were enrolled in this study. Two sets of graft scaffolds were prepared from decellularised kangaroo tail tendons using two different decellularisation / sanitation processes (example 3 and example 4). Decellularised tendons were packaged individually in vacuum sealed bags and sterilised via gamma irradiation. All three groups underwent a cranial cruciate ligament (CCL) surgery in the stifle (knee), receiving one of three different graft treatments:

[277] Group 1 - reconstruction using autograft (lateral extensor tendons)

[278] Group 2 - reconstruction using decellularised kangaroo tail tendons produced by example 3

[279] Group 3 - reconstruction using decellularised kangaroo tail tendons produced by example 4.

[280] Grafts were assembled in-situ using three decellularised kangaroo tail tendons with lengths of 20 cm and diameters ranging from 3 to 4 mm. The three tendons were then fold in half so that they formed a loop with both ends of the tendons meeting at one end thus producing a six stranded, 10 cm long graft with a graft diameter size of 7 mm. Grafts were implanted and the animals euthanised 3 and 6 months after the reconstruction surgery.

[281] Referring to Figures 19(1) to (iii), histology results of graft in the intra-articular space, showed evidence of cell infiltration, ligamentisation, and neovascularisation of the grafts in all three groups within 6 months of implantation. In Figures 20(11) to (iii), and 21(11) to (iii) there was evidence within and around the xenografts (Group 2 and 3) of cell differentiation and ordered cell structures represented by, blood vessel formation, and new bone formation. These results echoed those of the autograft, Group 1 (Figure 20 (i) and 21 (i)) demonstrating that the graft remodels into new, host tissue. REFERENCES Cordasco FA, Black SR, Price M, et al. Return to sport and reoperation rates in patients under the age of 20 after primary anterior cruciate ligament reconstruction: risk profile comparing 3 patient groups pre-dicated upon skeletal age; Am J Sports Med. 2019;47:628-639. Cruz Jr, A. L, Beck, J. J., Ellington, M. D., Mayer, S. W., Pennock, A. T., Stinson, Z. S., ... & Ellis Jr, H. B. (2020). Failure rates of autograft and allograft ACL reconstruction in patients 19 years of age and younger: a systematic review and meta-anaiysis. JBJS Open Access, 5(4), e20. Jia, ZY., Zhang, C., Cao, Sq. et al. Comparison of artificial graft versus autograft in anterior cruciate ligament reconstruction: a meta-analysis. BMC Musculoskelet Disord 18, 309 (2017). https: / / doi.org / 10.1186 / sl2891-017-1672-4 Tiefenboeck, T. M., Thurmaier, E., Tiefenboeck, M. M., Ostermann, R. C., Joestl, J., Winnisch, M., ... 8i Hofbauer, M. (2015). Clinical and functional outcome after anterior cruciate ligament reconstruction using the LARS™ system at a minimum follow-up of 10 years. The Knee, 22(6), 565568. Tulloch, S. J., Devitt, B. M., Porter, T., Hartwig, T., Klemm, H., Hookway, S., & Norsworthy, C. J. (2019). Primary ACL reconstruction using the LARS device is associated with a high failure rate at minimum of 6-year foilow-up. Knee Surgery, Sports Traumatology, Arthroscopy, 27, 3626-3632. Haverkamp, R. (2022). Report on Kangaroo, Bovine and Porcine Tendon Structure and Strength Bowes, J. H., & Kenten, R. H. (1950). The swelling of collagen in alkaline solutions. 1. Swelling in solutions of sodium hydroxide. Biochemical Journal, 46(1), 1. Edwards, J. H., Jones, G. L., Herbert, A., Fisher, J., & Ingham, E. (2021). Integration and functional performance of a decelluiarised porcine superflexor tendon graft in an ovine model of anterior cruciate ligament reconstruction. Biomaterials, 279, 121204. Kondo, E., Yasuda, K., Katsura, T., Hayashi, R., Kotani, Y., & Tohyama, H. (2012). Biomechanical and histological evaluations of the doubled semitendinosus tendon autograft after anterior cruciate ligament reconstruction in sheep. The American journal of sports medicine, 40(2), 315-324.

Claims

1. A graft for implantation in a subject, comprising a decellularised macropod tendoncomprising a plurality of collagen fibrils, wherein the average cross-sectional width of the collagen fibrils is at least 141 nm, and wherein the tendon is not exogenously chemically cross linked and the tendon is not irradiatively cross linked except for collateral cross linking caused by irradiative sterilisation.

2. The graft of claim 1, comprising a plurality of decellularised macropod tendons.

3. The graft of claim 1 or 2 , wherein the, or each, tendon comprises an ultimate tensile strengthof at least 58 MPa.

4. The graft of any one of the preceding claims, wherein the, or each, tendon comprises anelastic modulus of at least 548 MPa.

5. The graft of any one of the preceding claims, wherein the, or each, tendon comprises adisplacement creep rate of less than 0.5 mm / ln(s), or less than 0.3 mm / ln(s), or less than 0.1 mm / ln(s), or less than 0.07 mm / ln(s).

6. The graft of claim 5, wherein the, or each, tendon comprises a displacement creep rate ofless than 0.06 mm / ln(s).

7. The graft of any one of the preceding claims, wherein the, or each, tendon comprises creepdisplacement at end of less than 1.47 mm.

8. The graft of any one of the preceding claims, wherein the, or each, tendon comprises anormalized strain creep rate of less than 0.05 ln(s)-1, or less than 0.01 ln(s)-1, or less than 0.005 ln(s)-1.

9. The graft of claim 8, wherein the, or each, tendon comprises a normalized strain creep rateof less than 0.0011 ln(s)-1.

10. The graft of any one of the preceding claims, wherein the, or each, tendon comprisesnormalized strain at test end of less than 1.008.

11. The graft of any one of the preceding claims, wherein the, or each, tendon comprises anormalized stress relaxation rate of less than -0.46 ln(s)-1.

12. The graft of any one of the preceding claims, wherein the, or each, tendon comprises anormalized stress at end of less than 0.70.

13. The graft of any one of the preceding claims, wherein the, or each, tendon comprises a GAGcontent of at least 0.46 micrograms / mg.2025297765   27 May 202614.    The graft of any one of the preceding claims, wherein the, or each, tendon comprises anelastin content of at least 16 micrograms / mg.

15. The graft of any one of the preceding claims, wherein the, or each, tendon comprises aninsoluble collagen content of at least 670 micrograms / mg.

16. The graft of any one of the preceding claims, wherein the, or each, tendon is a decellularisedkangaroo tendon.

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