Tissue engineered scaffold

Abstract

Disclosed are tissue engineered graft materials useful as scaffolds for blood vessel grafts in various surgical procedures and methods of making such scaffolds.

Description

[0001] TISSUE ENGINEERED SCAFFOLD

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to tissue engineered scaffolds that can be used as blood vessel grafts in various surgical procedures, and to methods of making such scaffolds.

[0004] BACKGROUND TO THE INVENTION

[0005] Arteries are vessels responsible for carrying oxygenated blood from the heart to the venous system and tissues throughout the body. Arteries are tubes composed of layers of collagen (largely type I) and elastin-based tissues, lined with smooth muscles responsible for controlling the movement of the arteries [1]. They act as elastic reservoirs, capable of storing a portion of the stroke volume of blood from the heart during each systole and can then discharge blood volume during times of diastole, allowing for a more even flow of blood throughout the cardiac system. Without the elastic and mechanical ability of arteries, the extreme pressure changes within the vessels due to the heart pumping would be too great for the heart to empty and refill again [2].

[0006] Arterial walls thicken with age and lose elasticity. These changes can increase blood velocity and blood pressure, resulting in increased stress on the heart and a greater chance of developing cardiovascular disorders [3]. Cardiovascular disease includes a range of conditions involving damage to the heart and / or surrounding blood vessels. Coronary artery disease and stroke account for 80% of cardiovascular disease related deaths in males, and 75% in females [4]. In some cases, heart disease can be treated, or managed, using drugs such as aspirin or cholesterol- lowering medications, however often patients require invasive procedures to restore normal blood flow. This can involve angioplasty and stent insertion, or coronary bypass surgery. Coronary bypass surgery requires a graft, created from either a vessel from another part of the patient's body, a synthetic graft or a xenograft [5].

[0007] Unfortunately, there are drawbacks associated with the current use of each of the above-described graft materials in coronary bypass procedures as well as in other medical procedures including arteriovenous graft creation (for haemodialysis), peripheral vascular bypass (PVB), and trauma related injuries.

[0008] Although a number of different graft materials are available for clinical use in the above-described applications, each suffers from disadvantages in application. For example, allograft blood vessels taken from another part of a patient's body require additional surgery and the attendant risk of complications arising from both effects related to the removal of the vessel and the surgical procedure per se. These allograft blood vessels also have poor patency in many instances. Particularly where low-pressure blood vessels (veins) are used in high-pressure applications (arteries). For example, although one autologous graft option, the great saphenous vein, is an improvement for treatment of critical limb ischemia over alternative autologous vein bypass, this blood vessel has only a 54% patency rate after 2 years [6]. Cryopreserved veinous allografts for coronary artery bypassgrafts also have poor patency after a short period [7] Synthetic blood vessels can be more prone to infection than natural blood vessels, carry an increased risk of blood clotting (thrombosis), can be prone to aneurysm, are of limited durability, and are at risk of rejection through immunoincompatibility. Similarly, xenograft blood vessels can carry viruses or other pathogens which leads to increased risk of infection, can be of limited durability, and run the risk of rejection due to immunoincompatibility (potentially leading to organ failure). Heterografts from porcine or bovine material may be rejected on cultural and religious grounds, and many diseases carried by pigs or cattle can be transmitted to humans.

[0009] There is a need for alternative graft materials that may be used to reduce at least some of the disadvantages associated with known heterograft materials currently employed in the medical procedures described above.

[0010] It is an object of the present invention to provide at least one heterograft material that goes at least some way towards reducing at least one of the disadvantages associated with the use of current graft materials employed in coronary bypass procedures and / or arteriovenous graft creation (for haemodialysis) and / or peripheral vascular bypass (PVB) and / or and trauma related injuries and / or other procedures where a subject to be treated may benefit from reduction disadvantages described above and / or that at least provides the public with a useful choice.

[0011] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents 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.

[0012] SUMMARY OF THE INVENTION

[0013] In one aspect the present invention relates to a tissue engineered scaffold comprising a decellularised ovine artery or part thereof.

[0014] In another aspect the invention relates to a method of making a tissue engineered scaffold comprising a decellularised ovine artery or part thereof, the method comprising decellularising and optionally, lyophilizing, an ovine artery or part thereof.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will now be described by way of example only and with reference to the drawings in which:

[0017] Figure 1 - Scanning electron microscopy section of decellularised ovine artery.

[0018] Figure 2 - Scanning electron microscopy section of untreated ovine artery [8].Figure 3 - Inflation tests on decellularised arteries. Both the length and diameter can increase by up to 80% before failure, but this occurs at much higher pressures than will be achieved in the human body. In most cases the diameter initially increases in size faster than the length.

[0019] Figure 4 - Inflation tests on decellularised arteries at just the lower pressure portion of the range tested (a, b) and of sheep arteries that have not been decellularised for comparison (c, d). The decellularised arteries provide greater elasticity than the sheep arteries that have not been decellularised.

[0020] Figure 5 - Structure and arrangement of the collagen fibrils in sheep arteries that have not been decellularised from small angle X-ray scattering measurements, measured through the wall of the artery (at right angles to the external surface), a) orientation angle with inflation pressure; b) orientation index with inflation pressure; c) d-spacing with inflation pressure.

[0021] Figure 6 - Structure and arrangement of the collagen fibrils in decellularised sheep arteries, a) orientation angle with inflation pressure; b) orientation index with inflation pressure; c) d-spacing with inflation pressure

[0022] Figure 7 - Structure and arrangement of the collagen fibrils in sheep arteries that have not been decellularised from small angle X-ray scattering measurements measured through a cross section of the artery, a) orientation angle across the artery wall section; b) orientation index across the artery wall section; c) d-spacing across the artery wall section.

[0023] Figure 8 - Structure and arrangement of the collagen fibrils in decellularised sheep arteries from small angle X-ray scattering measurements measured through a cross section of the artery, a) orientation angle across the artery wall section; b) orientation index across the artery wall section; c) d-spacing across the artery wall section. The decellularised arteries provide a smaller change in orientation index through the wall, providing a more uniform structure than in the arteries that have not been decellularised.

[0024] Figure 9 - Histology of cell colonisation of decellularised sheep arteries, a) Endothelial cells (marked by an arrow) with a complete coverage of the luminal surface of an artery decellularised by the pulsed pressure method. The layer of cells has come free from the luminal surface of the artery as a result of the preparation for histology; b) enlargement of an endothelial cell from a; c) decellularised artery prepared without pulsed pressure and after attempted recellularisation with endothelial cells but with no cells visible.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] Definitions and abbreviations

[0027] As used herein the term "comprising" means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or thoseprefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

[0028] The term "about" as used herein means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be construed as including a deviation of+ / - 5% of the value.

[0029] As contemplated herein, an ovine artery or part thereof refers to any portions of the artery that are sufficient in size, when decellularised, to be used as a heterograft material and / or prepared as heterograft material as described herein.

[0030] The term "substantially change" (and grammatical variations thereof) as used herein with reference to D-spacing refers any measurable or observable increase or reduction in the D-spacing of the collagen fibrils comprised in a decellularised ovine artery as described herein when subjected to uniaxial strain applied in either the longitudinal or circumferential direction relative to any measurable or observable increase or decrease in D-spacing in an appropriate control {e.g., untreated) artery. In preferred embodiments the measurable or detectable increase or decrease is a statistically significant increase or decrease, relative to an appropriate control.

[0031] The term "orientation" means the average direction of the collagen fibrils artery materials. The term "orientation index" means the spread of direction of orientation of collagen fibrils in the artery materials. The orientation index (01) is defined as (90° - OA) / 90° where OA is the angle range, centered at the peak collagen fibril direction, that contains 50% of the collagen fibrils. An 01 of 1 indicates the collagen fibrils are completely parallel to each other, an 01 of 0 indicates the collagen fibrils are completely isotropically oriented.

[0032] As used herein, the term "fresh" with reference to an artery or part thereof refers to an artery that has not been decellularised and includes arteries or parts thereof that have been frozen and thawed. 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 (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0033] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and / or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.Detailed description

[0034] A number of characteristics are required for tissue engineered scaffolds, particularly tissue engineered heterograft materials for use in surgical procedures, including coronary bypass procedures. These characteristics include (but are not necessarily limited to) biocompatibility or lack of cytotoxicity for favourable host tissue response; surface structure and chemistry to promote cell attachment and proliferation; interconnected pores of size to permit ingress of cells and diffusion of nutrients for tissue regeneration; controlled biodegradability to facilitate formation of new tissue and adequate mechanical properties to maintain structure and function after implantation.

[0035] The inventors have found that certain decellu larised ovine arteries may be prepared as tissue engineered scaffolds suitable for use as graft material in various surgical procedures, including coronary bypass procedures.

[0036] Surprisingly, the inventors have identified that decellularised ovine arteries, particularly carotid arteries, provide tissue engineered scaffolds that are able to re-cellularise following colonization by human endothelial cells in vitro. Based on the in vitro recellularization observed by the inventors and described herein, a person of skill in the art reasonably expects that the tissue engineered scaffolds disclosed herein will also re-cellularise in vivo following transplantation into a patient. Further without wishing to be bound by theory, the inventors believe that other decellularised ovine arteries, including mammary arteries, can be prepared according to the methods described herein to provide tissue engineered scaffolds that are able to re-cellularise following colonization by human endothelial cells, both in vitro and in vivo.

[0037] Importantly, sheep do not carry most of the diseases that are associated with heterograft materials obtained from other mammals including pigs, and do not carry many diseases that are of danger to humans at all. Furthermore, the size of the artery material from sheep is more suited to the use as heterograft material in humans than arteries from other mammals, such as bovine material.

[0038] Also disclosed herein, the inventors have determined that the tissue engineered scaffolds comprising decellularised ovine artery as described herein are similar in mechanical strength to human arteries and are therefore of sufficient mechanical strength for use in humans as arterial heterografts.

[0039] Further, as disclosed herein, the inventors have determined that fresh ovine arteries have similar increases in length and diameter with inflation to the decellularised arteries. These results demonstrate that decellularisation process disclosed herein does not mechanically degrade the structure of the decellularised ovine heterograft material to any significant extent.

[0040] Accordingly, in one aspect the present invention relates to a tissue engineered scaffold comprising a decellularised ovine artery or part thereof.

[0041] In one embodiment the tissue engineered scaffold consists essentially of or consists of the decellularised ovine artery or part thereof.In one embodiment the decellularised ovine artery or part thereof is a carotid artery or a mammary artery or part thereof of either.

[0042] In one embodiment the decellularised ovine artery or part thereof is from a sheep or a lamb.

[0043] In one embodiment the decellularised ovine artery or part thereof from a lamb is about 3 mm to about 6 mm in diameter, preferably about 3.5 mm to about 5.5 mm, preferably about 4 mm to about 5 mm, preferably 4.1-5.1 mm diameter.

[0044] In one embodiment the decellularised ovine artery or part thereof from a lamb and has a wall thickness of about 0.10 mm to about 0.4 mm, preferably about 0.14 mm to about 0.37 mm, preferably about 0.17 mm to about 0.35 mm.

[0045] In one embodiment the decellularised ovine artery or part thereof is from a sheep and is about 4 mm to about 8 mm in diameter, preferably about 4.5 mm to about 7.5 mm, about 5 mm to about 7 mm, preferably about 5.4 mm to about 6.5 mm in diameter.

[0046] In one embodiment the decellularised ovine artery or part thereof is from a sheep and has a wall thickness of about 0.1 mm to about 0.35 mm, preferably about 0.15 mm to about 0.3 mm, preferably about 0.2 mm to about 0.28 mm.

[0047] In one embodiment the decellularised ovine artery or part thereof is of sufficient length to be used as a heterograft in a medical procedure carried out or intended to be carried out on a mammal, preferably wherein sufficient length is greater than about 80 mm, 90 mm, 100 mm, 110 mm, 120mm, preferably greater than about 130 mm. In one embodiment the mammal is a human.

[0048] In one embodiment the medical procedure is selected from the group consisting of coronary bypass, arteriovenous graft including for haemodialysis, peripheral vascular bypass (PVB) and procedures that treat trauma related injuries.

[0049] In one embodiment uniaxial strain applied to the decellularised artery or part thereof in the longitudinal or circumferential direction does not substantially change the D-spacing of the collagen fibrils comprised in the decellularised artery or part thereof as compared to no applied strain.

[0050] In one embodiment inflationary strain on the decellularised artery or part thereof does not substantially change the D-spacing of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6c).

[0051] In one embodiment inflationary strain on the decellularised artery or part thereof does not substantially increase the orientation of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6a) compared with an artery or part thereof that has not been decellularised. As shown herein, the orientation increases from an average of about 50° to about 90° in an artery or part thereof that has not been decellularised (Figure 5a).In one embodiment inflationary strain on the decellula rised artery or part thereof does not substantially decrease the orientation index of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6b) compared with an artery or part thereof that has not been decellularised. As shown herein, the orientation index decreases from an average of about 0.3 to 0.15 (Figure 5b) in an artery or part thereof that has not been decellularised.

[0052] In one embodiment the decellularised artery or part thereof extends in length by about 20%, preferably by about 21%-25% under 30 kPa inflation pressure (Figure 4a) compared with an artery or part thereof that has not been decellularised. As shown herein, an artery or part thereof that has not been decellularised does not extend as much (0.15 average) (Figure 4c).

[0053] In one embodiment the decellularised artery or part thereof extends in diameter by a fraction of 0.2-0.5 (25-50%) under 30 kPa inflation pressure (Figure 4b) compared with an artery or part thereof that has not been decellularised (0.02-0.2) (Figure 4d).

[0054] In one embodiment the decellularised artery or part thereof increases in diameter by less than about 60%, preferably by less than about 50%, 40%, 30%, 25%, preferably by less than about 20% at 20kPa as compared to no applied strain.

[0055] In one embodiment the decellularised artery or part thereof has a relatively uniform orientation index throughout the thickness of the walls (Figure 7a) compared with an artery or part thereof that has not been decellularised. As shown herein, an artery or part thereof that has not been decellularised has a large change in orientation index (0.3-0.5 to 0.55-0.8) between the inner and outer artery wall (Figure 8a).

[0056] In one embodiment the decellularised artery or part thereof has an ultimate tensile strength of about 1 MPa to about 2 MPa, preferably of about 1.1 MPa to about 1.9 MPa, about 1.2 MPa to about 1.8 MPa, preferably about 1.3 MPa to about 1.7 MPa.

[0057] In one embodiment the decellularised artery or part thereof is a decellularised lamb artery or part thereof having an ultimate tensile strength of about 1.4 MPa to about 2 MPa, preferably of about 1.5 MPa to about 1.9 MPa, about 1.6 MPa to about 1.8 MPa, preferably about 1.7 MPa.

[0058] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having has an ultimate tensile strength of about 1 MPa to about 1.6 MPa, preferably of about 1.1 MPa to about 1.5 MPa, about 1.2 MPa to about 1.4 MPa, preferably about 1.3 MPa.

[0059] In one embodiment the decellularised artery or part thereof has Young's modulus of about 1.2 MPa to about 1.9 MPa, preferably of about 1.3 MPa to about 1.8 MPa, about 1.4 MPa to about 1.7 MPa, preferably about 1.5 MPa to about 1.6 MPa.In one embodiment the decellularised artery or part thereof is a decellularised lamb artery or part thereof having a Young's modulus of about 1.3 MPa to about 1.9 MPa, preferably of about 1.4 MPa to about 1.8 MPa, about 1.5 MPa to about 1.7 MPa, preferably about 1.6 MPa.

[0060] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a Young's modulus of about 1.2 MPa to about 1.8 MPa, preferably of about 1.3 MPa to about 1.7 MPa, about 1.4 MPa to about 1.6 MPa, preferably about 1.5 MPa.

[0061] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a pore size of 5-200 pm, preferably 10-50 pm.

[0062] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a wall thickness of 100-350 pm, preferably 150-250 pm.

[0063] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a diameter of 2-8 mm, preferably 4-6 mm.

[0064] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having an Orientation Index measured through the wall of the artery (at right angles to the external surface) of 0.1-0.6, preferably 0.2-0.5.

[0065] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having an Orientation Index measured through a cross section of the artery or part thereof (at right angles to the external surface) varying from 0.5-0.8 at the outer surface to 0.2-0.5 at the inner surface.

[0066] In another aspect the invention relates to a method of making a tissue engineered scaffold comprising a decellularised ovine artery or part thereof, the method comprising decellularising, and optionally lyophilizing, an ovine artery or part thereof.

[0067] In one embodiment decellularising comprises contacting the ovine artery or part thereof with a first decellularising medium comprising 1% sodium dodecyl sulfate (SDS) in dil-bO.

[0068] In one embodiment contacting comprises immersing, preferably fully immersing, the ovine artery or part thereof in the first decellularising medium.

[0069] In one embodiment contacting with the first decellularising medium is for at least 6h, preferably at least 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, preferably at least 72h. In one embodiment contacting is for about 72h.

[0070] In one embodiment decellularising comprises contacting the ovine artery or part thereof with a second decellularising medium comprising 1% Triton X-100 in dil-bO.

[0071] In one embodiment contacting comprises immersing, preferably fully immersing, the ovine artery or part thereof in the second decellularising medium.In one embodiment contacting with the second decellularising medium is for at least 2h, preferably at least 4h, 6h, 8h, lOh, 12h, 14h, 16h, 18h, 20h, 22h, preferably at least 24h. In one embodiment contacting is for about 24h.

[0072] In one embodiment decellularising is carried out under pulsed pressure. In one embodiment the pulsed pressure comprises a pressure fluctuation between 0 kPa and 10 kPa.

[0073] In one embodiment the pressure fluctuation is at a frequence of 0.2 Hz.

[0074] In one embodiment contacting with the first and second decellularising media is sequential beginning with the first decellularisation medium.

[0075] In one embodiment decellularising further comprises washing the ovine artery or part thereof after contacting with the first and second decellularisation media.

[0076] In one embodiment washing comprises contacting the ovine artery or part thereof with diH20 for at least 15m, preferably at least 30m, 45m, preferably at least lh. In one embodiment washing is for about 1 hour.

[0077] In one embodiment washing is repeated at least twice, preferably at least three times, preferably four times.

[0078] In one embodiment washing is carried out under pulsed pressure. In one embodiment the pulsed pressure comprises a pressure fluctuation between 0 kPa and 10 kPa.

[0079] In one embodiment the pressure fluctuation is at a frequence of 0.2 Hz.

[0080] In one embodiment lyophilizing comprises freezing the ovine artery or part thereof at -50 °C after washing.

[0081] In one embodiment the frozen ovine artery or part thereof is freeze dried at 0.2 mbar for 48h with a temperature profile ramping up from -20 °C to +20 °C.

[0082] In one embodiment the decellularised ovine artery or part thereof is a carotid artery or a mammary artery or part thereof of either.

[0083] In one embodiment the decellularised ovine artery or part thereof is from a sheep or a lamb.

[0084] In one embodiment the decellularised ovine artery or part thereof from a lamb is about 3 mm to about 6 mm in diameter, preferably about 3.5 mm to about 5.5 mm, preferably about 4 mm to about 5 mm, preferably 4.1-5.1 mm diameter.

[0085] In one embodiment the decellularised ovine artery or part thereof from a lamb and has a wall thickness of about 0.10 mm to about 0.4 mm, preferably about 0.14 mm to about 0.37 mm, preferably about 0.17 mm to about 0.35 mm.In one embodiment the decellularised ovine artery or part thereof is from a sheep and is about 4 mm to about 8 mm in diameter, preferably about 4.5 mm to about 7.5 mm, about 5 mm to about 7 mm, preferably about 5.4 mm to about 6.5 mm in diameter.

[0086] In one embodiment the decellularised ovine artery or part thereof is from a sheep and has a wall thickness of about 0.1 mm to about 0.35 mm, preferably about 0.15 mm to about 0.3 mm, preferably about 0.2 mm to about 0.28 mm.

[0087] In one embodiment the decellularised ovine artery or part thereof is of sufficient length to be used as a heterograft in a medical procedure carried out or intended to be carried out on a mammal, preferably greater than 80 mm, preferably greater than 100 mm, preferably greater than 130 mm. In one embodiment the mammal is a human.

[0088] In one embodiment the medical procedure is selected from the group consisting of coronary bypass, arteriovenous graft including for haemodialysis, peripheral vascular bypass (PVB) and procedures that treat trauma related injuries.

[0089] In one embodiment uniaxial strain applied to the decellularised artery or part thereof in the longitudinal or circumferential direction does not substantially change the D-spacing of the collagen fibrils comprised in the decellularised artery or part thereof as compared to no applied strain.

[0090] In one embodiment inflationary strain on the decellularised artery or part thereof does not substantially change the D-spacing of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6c).

[0091] In one embodiment inflationary strain on the decellularised artery or part thereof does not substantially increase the orientation of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6a) compared with an artery or part thereof that has not been decellularised. As shown herein, the orientation increases from an average of about 50° to about 90° in an artery or part thereof that has not been decellularised (Figure 5a).

[0092] In one embodiment inflationary strain on the decellularised artery or part thereof does not substantially decrease the orientation index of the collagen fibrils comprised in the decellularised artery or part thereof (Figure 6b) compared with an artery or part thereof that has not been decellularised. As shown herein, the orientation index decreases from an average of about 0.3 to 0.15 (Figure 5b) in an artery or part thereof that has not been decellularised. In one embodiment the decellularised artery or part thereof extends in length by at least 20%, preferably by at least 21%-25% under 30 kPa inflation pressure (Figure 4a) compared with an artery or part thereof that has not been decellularised. As shown herein, an artery or part thereof that has not been decellularised does not extend as much (0.15 average) (Figure 4c).In one embodiment the decellularised artery or part thereof extends in diameter by a fraction of 0.2-0.5 (25-50%) under 30 kPa inflation pressure (Figure 4b) compared with an artery or part thereof that has not been decellularised (0.02-0.2) (Figure 4d).

[0093] In one embodiment the decellularised artery or part thereof has a relatively uniform orientation index throughout the thickness of the walls (Figure 7a) compared with an artery or part thereof that has not been decellularised. As shown herein, an artery or part thereof that has not been decellularised has a large change in orientation index (0.3-0.5 to 0.55-0.8) between the inner and outer artery wall (Figure 8a).

[0094] In one embodiment the decellularised artery or part thereof has an ultimate tensile strength of about 1 MPa to about 2 MPa, preferably of about 1.1 MPa to about 1.9 MPa, about 1.2 MPa to about 1.8 MPa, preferably about 1.3 MPa to about 1.7 MPa.

[0095] In one embodiment the decellularised artery or part thereof is a decellularised lamb artery or part thereof having an ultimate tensile strength of about 1.4 MPa to about 2 MPa, preferably of about 1.5 MPa to about 1.9 MPa, about 1.6 MPa to about 1.8 MPa, preferably about 1.7 MPa.

[0096] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having has an ultimate tensile strength of about 1 MPa to about 1.6 MPa, preferably of about 1.1 MPa to about 1.5 MPa, about 1.2 MPa to about 1.4 MPa, preferably about 1.3 MPa.

[0097] In one embodiment the decellularised artery or part thereof has Young's modulus of about 1.2 MPa to about 1.9 MPa, preferably of about 1.3 MPa to about 1.8 MPa, about 1.4 MPa to about 1.7 MPa, preferably about 1.5 MPa to about 1.6 MPa.

[0098] In one embodiment the decellularised artery or part thereof is a decellularised lamb artery or part thereof having a Young's modulus of about 1.3 MPa to about 1.9 MPa, preferably of about 1.4 MPa to about 1.8 MPa, about 1.5 MPa to about 1.7 MPa, preferably about 1.6 MPa.

[0099] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a Young's modulus of about 1.2 MPa to about 1.8 MPa, preferably of about 1.3 MPa to about 1.7 MPa, about 1.4 MPa to about 1.6 MPa, preferably about 1.5 MPa.

[0100] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a pore size of 5-200 pm, preferably 10-50 pm.

[0101] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a wall thickness of 100-350 pm, preferably 150-250 pm.

[0102] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having a diameter of 2-8 mm, preferably 4-6 mm.In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having an Orientation Index measured through the wall of the artery (at right angles to the external surface) of 0.1-0.6, preferably 0.2-0.5.

[0103] In one embodiment the decellularised artery or part thereof is a decellularised sheep artery or part thereof having an Orientation Index measured through a cross section of the artery (at right angles to the external surface) varying from 0.5-0.8 at the outer surface to 0.2-0.5 at the inner surface.

[0104] In another aspect the invention relates to a tissue engineered scaffold comprising a decellularised ovine artery or part thereof made according to a method of the invention.

[0105] Further aspects and advantages of this invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.

[0106] EXAMPLES

[0107] Example 1: Decellularisation

[0108] Carotid ovine arteries were used in the experiments, sourced from Ovation New Zealand Ltd (Feilding, New Zealand). The arteries were extracted soon after slaughter and thoroughly washed before being frozen at -60° for storage. During transportation they were kept frozen and were thawed just prior to decellularisation.

[0109] Freshly thawed arteries were connected in series and immersed sequentially in first and second decellularisation media in a container on a shaker table. The decellularisation media was also pumped through the interior (i.e., the lumen) of the arteries. The pumping of the decellularisation media through the arteries was with a pulsed pressure with the pressure fluctuation between 10 kPa and 0 kPa-gauge at a frequency of 0.2 Hz. In this context, the term "gauge" refers to pressure above atmospheric pressure, normally 1 atmosphere. Decellularisation was performed by first immersing the arteries in a decellularisation medium comprising 1% sodium dodecyl sulfate (SDS). In this first step, arteries were immersed under pulsed pressure for about 72 hours. The first decellularisation medium was then replaced with a second decellularisation medium comprising 1% Triton X-100. The arteries were immersed in the second decellularisation medium under pulsed pressure for about 24 hours. The arteries were then washed with four times for 1 hour each in deionised water under pulsed pressure. After decellularisation and washing the arteries were frozen at -50 °C and freeze dried in a Buchi Lyovapor L-300 at 0.2 mbar for 48 hours with a temperature profile ramping up from -20 °C to +20 °C.

[0110] Results

[0111] The decellularised arteries show a more open structure than the fresh arteries, as would be expected by the removal of largely non-structural components from the arteries leaving a structural collagen and elastin matrix (Figures 1 and 2).Example 1: Mechanical testing of rehydrated decellularised ovine arteries Tensile, UTS, Young's modulus

[0112] Tensile, ultimate tensile strength and Young's modulus were measured on at Stable Micro Systems TA texture analyser (Stable Micro Systems, Godaiming, UK). A length of artery was clamped between standard tensile grips (10-18 mm lengths between the grips) and strained at 2 mm s-1until rupture. The width and thickness of each artery was measured with a calliper prior to strain for calculation of the thickness and width normalised properties. The Young's modulus was calculated from the average slope of the stress-strain curve from zero strain to the strain for maximum stress.

[0113] Results

[0114] Tensile tests:

[0115]

[0116] None of these apparent differences between lamb and sheep are statistically significant (i.e. lamb and sheep arteries show the same mechanical behaviour with tensile testing).

[0117] Inflation

[0118] Inflation measurements were performed on lengths of artery tied off at one end and inflated with air from the other end. The arteries were kept fully hydrated throughout the test, by partially filling them with water. A pressure gauge was positioned close to a T-junction near the end of the artery. The air pressure was increased in steps and the pressure and artery dimensions recorded by a camera that recorded the artery, the pressure gauge and a scale ruler in each image. The inflation pressure was increased until each artery developed a leak.

[0119] Results

[0120] Inflation tests:

[0121] The blood pressure of humans is typically less than 150 mmHg (a systolic pressure of 120 mmHg and below is considered normal healthy blood pressure). Therefore, the inventors considered the changes to the arteries at pressures up to 150 mmHg (but measured to pressures over 1800 mmHg).

[0122] For decellularised ovine arteries made as described herein, the following was observed (Figure 3): There is a 20-60% increase in artery diameter at 20 kPa (150 mmHg).

[0123] There is a 20-25% increase in artery length at 20 kPa (150 mmHg).

[0124] The burst pressure of the decellularised arteries is greater than 100 kPa (50% of samples burst at over 200 kPa).There are no systematic differences between the inflation measurements for sheep and lamb arteries. The inventors also observed that fresh arteries have similar increases in length and diameter with inflation to the decellularised arteries suggesting that the decellularisation process does not mechanically degrade the structure to any significant extent (Figure 4).

[0125] Example 2: Cell colonization of decellularised tissue engineered scaffold

[0126] Decellularised arteries were cut into approximately 15-20 mm long sections using sterile techniques and hydrated for 30 minutes in phosphate buffered saline before being transferred to a six or ten well plate. Human large vessel endothelial cells (HUVECs C0035C) were purchased from ThermoFisher Scientific and cultured in Human Large Vessel Endothelial Cell Basal Medium Phenol Red Free (M200PRF500; ThermoFisher) supplemented with Large Vessel Endothelial Supplement (LVES, A1460801; ThermoFisher) and 1% Antibiotic / Antimycotic (Anti / Anti, 15240062, ThermoFisher) at 37 °C and 5 % CO2. Cryopreservation of the endothelial cells was achieved by mixing the basal medium with 10 % FBS (10099141, ThermoFisher) and 5 % DMSO (ATCC4X; In Vitro) after which the new cell stocks were slowly brought down to -80 °C before transferring to liquid nitrogen storage.

[0127] Cell passaging was completed by rinsing confluent cells with sterile PBS before detaching the endothelial cells using Trypsin-EDTA 0.5 % Phenol Red Free (15400054, ThermoFisher) then resuspended in basal medium with 10 % FBS before centrifuging at 1500 RPM for five minutes at room temperature.

[0128] Cells were seeded onto the arteries by adding the concentrated cells to fresh supplemented media in the hydrated arteries in six-well plates before incubating at 37 °C and 5 % CO2. Cell media was changed every 48 hours to ensure maximum growth. Histology of the decellularised arteries was carried out by the Massey School of Veterinary Sciences Histology Services, using formalin fixatives with haematoxylin and eosin (H&E) staining (Figure 1).

[0129] Results

[0130] Cell colonisation:

[0131] When the arteries were decellularised (including washing) with the pressure-pulsed pumped method described herein a complete coverage of the luminal surface with human endothelial cells was obtained (Figure 9a, b). When arteries were decellularised by the pumped method without pulsing, endothelial cell coverage was not obtained (Figure 9c).

[0132] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0133] Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

[0134] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0135] The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0136] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and / or enforceability of such patent documents.

[0137] The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

[0138] All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0139] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0140] INDUSTRIAL APPLICATION

[0141] The ovine heterografts described herein have utility and application in various surgical procedures, including, but not limited to coronary bypass surgery. Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto.REFERENCES

[0142] [1] M.K. Pugsley, R. Tabrizchi, J of Pharmacol Tox Met, 44 (2000) 333-340.

[0143] [2] IE. Wagenseil, R.P. Mecham, Physiol. Rev., 89 (2009) 957-989.

[0144] [3] H.Y. Lee, B.H. Oh, Circulation journal : official journal of the Japanese Circulation Society, 74 (2010) 2257-2262.

[0145] [4] S. Mendis, Puska, P., Norrving, B., Global Atlas on Cardiovascular Disease Prevention and Control, World Health Organisation in collaboration with the World Heart Federation and the World Stroke Organisation., World Health Organization, Geneva, Switzerland, 2011.

[0146] [5] P.W. Serruys , F. Unger , J.E. Sousa , A. Jatene , H.J.R.M. Bonnier , J.P.A.M. Schonberger , N. Buller , R. Bonser , M.J.B. van den Brand , L.A. van Herwerden , M.-A.M. Morel , B.A. van Hout New Engl J Med, 344 (2001) 1117-1124.

[0147] [6] Arvela, E.; Venermo, M.; Soderstrom, M.; Alback, A.; Lepantalo, M. Ann Vase Surg 26 (2012) 396-403.

[0148] [7] Fang, S.; Ellman, D. G.; Andersen, D. C. CellslO (2021), 10, 713.

[0149] [8] H.C. Wells, K.H. Sizeland, N. Kirby, A. Hawley, S. Mudie, C.W. Cunningham, R.G. Haverkamp, Measured collagen fibril response to arterial inflation using SAXS, Int J Biol Macromol 137 (2019) 1020-1029.

Claims

WHAT WE CLAIM:

1. A tissue engineered scaffold comprising a decellularised ovine artery or part thereof.

2. The tissue engineered scaffold of claim 1 wherein the decellularised ovine artery or part thereof is from a sheep or lamb.

3. The tissue engineered scaffold of claim 1 or claim 2 wherein the decellularised ovine artery or part thereof is of sufficient length to be used in a medical procedure carried out or intended to be carried out on a mammal, preferably wherein sufficient length is greater than about 80 mm, 90 mm, 100 mm, 110 mm, 120mm, preferably greater than about 130 mm, preferably wherein the mammal is a human.

4. The tissue engineered scaffold of claim 3 wherein the medical procedure is selected from the group consisting of coronary bypass, arteriovenous graft including for haemodialysis, peripheral vascular bypass (PVB) and procedures that treat trauma related injuries.

5. The tissue engineered scaffold of any one of claims 1 to 4 wherein uniaxial strain applied to the decellularised artery or part thereof in the longitudinal or circumferential direction does not substantially change the D-spacing of the collagen fibrils comprised in the decellularised artery as compared to no applied strain.

6. The tissue engineered scaffold of any one of claims 1 to 5 wherein inflationary strain on the decellularised artery or part thereof does not substantially increase the orientation of the collagen fibrils comprised in the decellularised artery or part thereof compared with an artery or part thereof that has not been decellularised.

7. The tissue engineered scaffold of any one of claims 1 to 6 wherein inflationary strain on the decellularised artery or part thereof does not substantially decrease the orientation index of the collagen fibrils comprised in the decellularised artery or part thereof compared with an artery or part thereof that has not been decellularised.

8. A method of making a tissue engineered scaffold comprising a decellularised ovine artery or part thereof, the method comprising decellularising, and optionally lyophilizing, an ovine artery or part thereof.

9. The method of claim 8 wherein decellularising comprises contacting the ovine artery or part thereof with a first decellularising medium comprising 1% sodium dodecyl sulfate (SDS).

10. The method of claim 8 or claim 9 wherein decellularising comprises contacting with the first decellularising medium for at least 6h, preferably at least 12h, 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, preferably at least 72h, preferably for about 72h.

11. The method of any one of claims 8 to 10 wherein decellularizing comprises contacting the ovine artery or part thereof with a second decellularising medium comprising 1% Triton X- 100.

12. The method of claim 11 wherein decellularising comprises contacting with the second decellularising medium is for at least 2h, preferably at least 4h, 6h, 8h, lOh, 12h, 14h, 16h, 18h, 20h, 22h, preferably at least 24h, preferably for about 24h.

13. The method of any one of claims 8 to 12 wherein decellularising is carried out under pulsed pressure, preferably wherein the pulsed pressure comprises a pressure fluctuation between 0 kPa and 10 kPa.

14. The method of claim 13 wherein the pressure fluctuation is at a frequence of 0.2 Hz.

15. The method of any one of claims 8 to 14 wherein decellularising further comprises washing the ovine artery or part thereof after contacting with the first and second decellularisation media.

16. The method of claim 15 wherein washing comprises contacting the ovine artery or part thereof with diH20 for at least 15m, preferably at least 30m, 45m, preferably at least lh, preferably for about 1 hour.

17. The method of claim 15 or 16 wherein washing is repeated at least twice, preferably at least three times, preferably four times.

18. The method of any one of claims 15 to 17 wherein washing is carried out under pulsed pressure, preferably wherein the pulsed pressure comprises a pressure fluctuation between 0 kPa and 10 kPa.

19. The method of claim 18 wherein pressure fluctuation is at a frequence of 0.2 Hz.

20. The method of any one of claims 8 to 19 wherein the decellularised ovine artery or part thereof is a carotid artery or a mammary artery or part thereof of either.

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