Tissue scaffold

Abstract

To provide a tissue scaffold.SOLUTION: There is provided a tissue scaffold and a method for making the tissue scaffold. The tissue scaffold comprises elastin and optionally fibrin and / or collagen. The elastin in the scaffold may be cross-linked. The elastin that is cross-linked preferably comprises solubilized elastin and is unfractionated. The inventors report novel and economical, biologically active elastin-based materials and methods for fabrication thereof. This invention concerns formation of a scaffold by cross-linking a composition comprising elastin, such as solubilized elastin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to tissue scaffolds, such as elastin-based tissue scaffolds, and methods for forming such scaffolds.

Background Art

[0002] Elastin is an extracellular structural protein found in connective tissues such as skin, fat, lung, tendon, ligament, artery or cartilage. Its main function is to maintain the shape of the tissue after stretching or contraction and it has load-bearing properties (Banga, 1966; Gray, 1973). In vivo, elastin is formed by the process of elastic fiber formation through the assembly and cross-linking of the protein tropoelastin (encoded by the ELN gene).

[0003] Tropoelastin typically consists of a hydrophobic domain having many Gly, Val, Ala and Pro residues (which are often present in repeats of several amino acids such as Gly-Val-Gly-Val-Pro, Gly-Val-Pro-Gly-Val and Gly-Val-Gly-Val-Ala-Pro), and a hydrophobic domain having many Lys and Ala residues (which are important in cross-linking). The formation of elastin by cross-linking of tropoelastin is promoted by lysyl oxidase.

[0004] Elastin is one of the most stable resident proteins in humans and has a half-life of 74 years. Its excellent structural and biological properties have attracted attention for tissue engineering applications (Daamen et al., 2007). For example, elastin provides elasticity to tissues and organs and is abundant in places where elasticity is most important, such as blood vessels, ligaments, lungs, skin. However, since elastin is a very insoluble protein, it remains a challenge to use it as a biomaterial (Leach et al., 2005).

[0005] To overcome this problem, many existing strategies have been developed for α-elastin, a form of soluble elastin obtained after hydrolysis with oxalic acid. However, this process is expensive and time-consuming, and the overall yield is minimal. As a result, its clinical application as a scaffold is questionable.

[0006] In some studies, it has been reported that insoluble elastin is mixed with other materials, such as collagen (Ryan and O’Brien, 2015). However, the resulting scaffolds have weak mechanical properties and altered biological responses compared to collagen itself.

Summary of the Invention

Means for Solving the Problems

[0007] The present inventors report novel economical and biologically active elastin-based materials and methods for their production.

[0008] The present invention relates to the formation of a scaffold by crosslinking a composition containing elastin, such as solubilized elastin.

[0009] The present invention also relates to a tissue scaffold comprising crosslinked elastin.

Embodiments for Carrying Out the Invention

[0010] According to the present invention, there is provided a method for forming a tissue scaffold, the method comprising crosslinking a composition containing solubilized elastin.

[0011] According to the present invention, there is provided a method comprising crosslinking a composition, wherein the composition comprises elastin contacted with a solubilizing agent capable of solubilizing elastin.

[0012] According to the present invention, there is provided a method comprising: a) contacting elastin with a solubilizing agent capable of solubilizing elastin; and b) crosslinking the elastin composition formed in step a).

[0013] According to the present invention, a tissue scaffold containing cross-linkable solubilized elastin is provided.

[0014] Elastin can be extracted from or derived from natural sources. For example, elastin can be derived from mammalian sources. Mammalian sources can be bovine sources, such as bovine neck ligaments, or human sources. Alternatively, elastin can be recombinant elastin.

[0015] Elastin is a very insoluble protein due to intermolecular cross-links. However, it can be solubilized (Daamen (2007)). Solubilized elastin is also referred to as hydrolyzed elastin or elastin peptides.

[0016] Common methods for solubilizing elastin include treating it with 0.25 M oxalic acid at 100 °C or treating it with 1 M KOH in 80% ethanol. In addition, proteolytic enzymes (including serine-type elastase derived from polymorphonuclear leukocytes and some metalloelastases of monocyte / macrophage origin) that can degrade elastic fibers also result in solubilized elastin. Examples of hydrolyzed forms of elastin are shown in the following table.

Table A

[0017] The elastin peptides obtained after hydrolysis with oxalic acid can be coacervated after suspension in 10 mM sodium acetate containing 10 mM NaCl set to pH 5.5 with acetic acid, and heated and centrifuged at 37 °C. As a result, two fractions (α-elastin (viscous coacervate) and β-elastin (in the supernatant)) are formed.

[0018] The prior art has mainly focused on using insoluble elastin in combination with other components, such as collagen, or on the soluble α-elastin component obtained after hydrolysis with oxalic acid and separation from β-elastin. However, surprisingly, the inventors have found that solubilization of elastin and crosslinking of the products of the solubilization process can form promising and cost-effective tissue scaffolds. For this reason, there is no need to separate or isolate fractions of elastin, such as the separation or isolation of α-elastin and β-elastin. Thus, the crosslinked solubilized elastin can be considered to be crude or unfractionated. Advantageously, the present invention can avoid the time, inconvenience and costs associated with the isolation of the α-elastin fraction. Since the step of separating α-elastin and β-elastin can be avoided, the present invention can also improve the overall yield.

[0019] According to the present invention, there is provided a method for forming a tissue scaffold, the method comprising crosslinking a composition comprising unfractionated solubilized elastin.

[0020] According to the present invention, there is provided a method comprising crosslinking a composition comprising elastin, wherein the elastin is unfractionated and the elastin comprises solubilized elastin. The method may involve contacting the elastin with a solubilizing agent capable of solubilizing at least a portion of the elastin and then crosslinking the resulting composition.

[0021] Accordingly, unfractionated elastin can be crude elastin in which one or more elastin fractions have not been purified, isolated, separated or refined, or can be one or more different forms of elastin resulting from the contact of elastin with a solubilizing agent. For example, one or more soluble elastin fractions need not be isolated, or an insoluble elastin fraction need not be separated from the soluble elastin fraction. As a result, the composition can contain different soluble forms of elastin. The composition can contain non-solubilized elastin. Accordingly, the composition can contain both soluble and insoluble forms of elastin. For example, after contact with the solubilizing agent, the elastin need not be subjected to centrifugation. After contact with the solubilizing agent, the elastin need not be fractionated, purified, isolated, separated or refined.

[0022] Surprisingly, the inventors have recognized that an effective elastin-based tissue scaffold can be formed without the need for fractionation of elastin, and one or more fractions of elastin are isolated and subsequently used to form a scaffold using the isolated fraction. For example, the present invention need not require the isolation and utilization of the α-elastin fraction. Advantageously, the present invention need not require conventional steps for fractionating elastin, such as centrifugation and / or coacervation. Also advantageously, a composition containing both soluble and insoluble elastin can be used. This can provide a greater benefit than known methods. For example, US Patent Application Publication No. 2004 / 0136977 requires the isolation of water-soluble elastin involving centrifugation. Japanese Patent Application Publication 2014183886 requires a series of acid fractionations of insoluble elastin involving centrifugation.

[0023] The present invention does not include, for example, methods involving crosslinking of tropoelastin by lysyl oxidase or products of such methods.

[0024] The method of the present invention may include a step of solubilizing elastin. This may involve contacting the elastin with a solubilizing agent capable of solubilizing at least a part of the elastin. For this reason, the present invention may provide a method including: a) solubilizing elastin to form a composition containing unfractionated solubilized elastin; b) crosslinking the product obtained from step a).

[0025] According to the present invention, there is provided a method including: a) solubilizing elastin to form a composition containing solubilized elastin; b) crosslinking the composition obtained from step a).

[0026] According to the present invention, there is provided a method including: a) contacting elastin with a solubilizing agent to form a composition containing solubilized elastin; b) crosslinking the composition obtained from step a).

[0027] According to the present invention, there is provided a method including crosslinking elastin contacted with a solubilizing agent, wherein the elastin is not fractionated.

[0028] According to the present invention, there is provided a method including crosslinking a composition containing elastin, wherein the elastin is unfractionated and contains solubilized elastin.

[0029] According to the present invention, there is provided a tissue scaffold containing crosslinked unfractionated solubilized elastin.

[0030] According to the present invention, there is provided a tissue scaffold containing crosslinked elastin, wherein the composition containing crosslinked elastin is formed by crosslinking a formulation containing elastin containing solubilized elastin, and the elastin is not fractionated.

[0031] According to the present invention, there is provided a tissue scaffold containing crosslinked elastin, wherein the elastin has been contacted with a solubilizing agent and is not fractionated.

[0032] The scaffold can be prepared from a solution containing 1 to 20% (w / v) elastin, such as 5 to 15% (w / v) elastin, such as about 10% (w / v) elastin.

[0033] Preferably, the elastin is solubilized or is solubilized by contact with an acid, most preferably oxalic acid.

[0034] According to the present invention, there is provided a method for solubilizing elastin, the method comprising contacting the elastin with a solubilizing agent capable of solubilizing at least a portion of the elastin. The solubilizing agent is preferably an acid, more preferably oxalic acid.

[0035] According to the present invention, there is provided a method for solubilizing elastin, the method comprising contacting the elastin with an acid, preferably oxalic acid.

[0036] In a particularly preferred embodiment, the elastin is solubilized at a temperature below 100°C, preferably below or equal to 50°C, more preferably at a temperature of 15 to 30°C, such as at room temperature.

[0037] The acid, preferably oxalic acid, can be less than 1 M, preferably less than 0.75 M, more preferably 0.5 M or less. The acid can be at least 0.25 M. For example, the acid can be 0.2 M to 1 M, such as 0.25 M to 0.75 M.

[0038] The method of solubilizing elastin described herein is in contrast to the established method of solubilizing elastin using oxalic acid. Conventional methods of solubilizing elastin using oxalic acid are carried out at 100 °C (see, for example, Daamen et al., (2007)). However, the inventors have found that effective solubilization for forming the scaffold of the present invention can occur at temperatures below 100 °C. Without being bound by theory, the inventors have hypothesized that treatment with oxalic acid at temperatures below 100 °C can result in the formation of a mixture containing α- and β-elastin. In this case, the method of the present invention may include crosslinking of α- and β-elastin, and the scaffold of the present invention may include crosslinked α- and β-elastin. Advantageously, the inventors have recognized that it is not necessary to separate the solubilized fraction, such as isolating the α-elastin fraction, and that an effective scaffold can be formed using a crude or unfractionated mixture of solubilized elastin.

[0039] Compositions containing solubilized elastin, such as unfractionated solubilized elastin, are recognized to potentially contain some elastin that has not been solubilized. Thus, the composition can contain a mixture of insoluble and soluble elastin.

[0040] Thus, the present invention can provide a method that includes crosslinking a composition containing soluble and insoluble elastin. Thus, the present invention can provide a tissue scaffold containing crosslinked elastin, wherein the elastin contains soluble and insoluble elastin.

[0041] Surprisingly, the inventors have recognized that complete solubilization of the elastin to be crosslinked may not be required to obtain an effective tissue scaffold.

[0042] According to the present invention, there is provided a method that includes crosslinking a composition containing insoluble elastin.

[0043] According to the present invention, there is provided a tissue scaffold containing crosslinked insoluble elastin.

[0044] Solubilization or contact with an acid is preferably carried out for at least 30 seconds, more preferably for at least 1 minute. For example, solubilization or contact with an acid can be carried out for about 1 to 3 minutes. Solubilization or contact with an acid can be carried out for a maximum of 5 minutes. This is in contrast to the conventional treatment of elastin with oxalic acid (which is typically carried out for about 1 hour) (see, for example, US Patent Application Publication No. 2004 / 0136777).

[0045] According to the present invention, there is provided a method comprising contacting elastin with an acid, preferably oxalic acid. Preferably, the contact with the acid is carried out at a temperature below 100°C, most preferably at a temperature below or equal to 50°C, more preferably at a temperature of 15 to 30°C, for example at room temperature or ambient temperature. The method may further comprise crosslinking the resulting product.

[0046] According to the present invention, there may be provided a method of forming a tissue scaffold, the method comprising crosslinking a composition comprising α-elastin and β-elastin.

[0047] According to the present invention, there may be provided a tissue scaffold comprising a crosslinked composition comprising α-elastin and β-elastin.

[0048] Crosslinking can occur using any one of a number of crosslinking agents or crosslinking techniques generally known to those skilled in the art, such as chemical methods, radiation methods, and dehydrothermal methods.

[0049] References herein to "crosslinking" relate to covalent crosslinking. Preferably, crosslinking is achieved non-enzymatically using a chemical crosslinking agent.

[0050] Crosslinking can occur in the presence of a solubilizing agent (such as an acid, for example oxalic acid). Thus, the present invention may provide or use a composition comprising elastin, a solubilizing agent, and a crosslinking agent.

[0051] Examples of suitable chemical crosslinking agents include carbodiimide coupling agents such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC); N-hydroxysuccinimide (NHS), azide coupling agents; diisocyanate crosslinking agents such as hexamethylene diisocyanate; epoxide crosslinking agents such as epichlorohydrin, glycidyl ether and glycidyl amine; and aldehyde crosslinking agents such as formaldehyde, glutaraldehyde and glyoxal.

[0052] The chemical crosslinking agent may comprise N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and / or N-hydroxysuccinimide (NHS).

[0053] The chemical crosslinking agent may comprise an aldehyde crosslinking agent such as formaldehyde, glutaraldehyde and glyoxal. The aldehyde crosslinking agent may have the advantage of providing improved biocompatibility to the extracellular matrix composition. In a preferred embodiment, the aldehyde crosslinking agent is glutaraldehyde. The use of glutaraldehyde as a crosslinking agent may provide the advantage of resulting in an optimal crosslink density more rapidly than other aldehydes and can also achieve a relatively high density of crosslinking. In a preferred example, the chemical crosslinking agent is glutaraldehyde.

[0054] During the crosslinking process, the crosslinking agent may be present in an amount of about 0.2 - 5% (v / v), such as 0.5 - 3% (v / v), preferably 0.5 - 1.5% (v / v), such as 1% (v / v).

[0055] When the crosslinking agent comprises glutaraldehyde or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and / or N-hydroxysuccinimide (NHS), the method of the present invention may further comprise the addition of a toxicity reducing agent (such as lysine or sodium borohydride).

[0056] The step of crosslinking the composition containing solubilized elastin can be carried out at a temperature of 20°C to 50°C, preferably about 37°C. The contact or incubation with the crosslinking agent can typically be carried out for 1 minute to 24 hours (e.g., 4 hours). For example, the crosslinking can be carried out for about 1 hour or at least 1 hour. The crosslinking can be carried out in the presence of CO2, for example at least 2% CO2 (by volume), for example 2 to 10% CO2 (by volume) or about 5% CO2 (by volume).

[0057] According to the present invention, there is provided a composition comprising elastin, a solubilizing agent for solubilizing elastin, and a crosslinking agent.

[0058] The method of the present invention may include casting a composition containing solubilized elastin. Casting may include applying the composition containing solubilized elastin to a mold of a predetermined shape. Casting can occur before or during crosslinking.

[0059] The method of the present invention preferably includes lyophilization after crosslinking. For example, the composition can be frozen at -80°C, preferably overnight, and then lyophilized for about 48 hours. Preferably, the lyophilization is carried out for at least 24 hours.

[0060] According to the present invention, there is provided a method comprising lyophilizing a composition containing crosslinked elastin. According to the present invention, there is provided a method comprising lyophilizing a composition containing solubilized crosslinked elastin. For example, there is provided a method comprising lyophilizing a composition containing unfractionated solubilized elastin that has been crosslinked.

[0061] According to the present invention, there is provided a method of forming a tissue scaffold, comprising lyophilizing a composition containing crosslinked elastin, wherein the composition containing crosslinked elastin is formed by crosslinking a formulation containing unfractionated elastin containing solubilized elastin.

[0062] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking the solubilized elastin obtained from step a); and c) lyophilizing the product of step b).

[0063] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking the unfractionated solubilized elastin obtained from step a); and c) lyophilizing the product of step b).

[0064] According to the present invention, there is provided a method comprising: a) contacting elastin with a solubilizing agent capable of solubilizing elastin to form a composition comprising solubilized elastin; b) crosslinking the composition produced in step a); and c) lyophilizing the product of step b).

[0065] According to the present invention, there is provided a tissue scaffold comprising lyophilized crosslinked elastin.

[0066] According to the present invention, there is provided a tissue scaffold comprising lyophilized crosslinked solubilized elastin.

[0067] According to the present invention, there is provided a tissue scaffold comprising lyophilized crosslinked unfractionated solubilized elastin.

[0068] According to the present invention, there may be provided a method comprising lyophilizing a composition comprising crosslinked α-elastin and β-elastin.

[0069] According to the present invention, there may be provided a tissue scaffold comprising lyophilized crosslinked α-elastin and β-elastin.

[0070] According to the present invention, there may be provided a tissue scaffold comprising lyophilized crosslinked elastin, wherein the crosslinked elastin is formed by crosslinking a composition comprising soluble elastin and insoluble elastin.

[0071] The method of the present invention may include washing or cleaning to remove agents involved in solubilization and / or crosslinking. The washing is preferably performed after solubilization. Particularly when the crosslinking agent includes an aldehyde crosslinking agent, this may include contact with a reducing agent or washing with a reducing agent. The washing may include ultrasonic cleaning. For example, the scaffold can be washed using water in an ultrasonic cleaner.

[0072] The presence of a reducing agent can stabilize the crosslinking process and result in a scaffold with enhanced biological effectiveness. Furthermore, the presence of a reducing agent may reduce the cytotoxic effects caused by the leaching of non-reducing crosslinking agents from the composition.

[0073] Examples of suitable reducing agents include sodium borohydride or similar agents having carbonyl group reactivity. The reducing agent can typically be added in an amount of 0.1% w / v to 10% w / v (e.g., about 1% w / v).

[0074] The step of washing to remove agents involved in solubilization and / or crosslinking can be performed for at least 5 hours, preferably at least 8 hours. For example, to remove oxalic acid and unbound glutaraldehyde, the scaffold can be washed with a reducing agent (e.g., sodium borohydride) for about 8 hours. Preferably, the scaffold is stirred or shaken while in contact with the reducing agent.

[0075] There may be a further washing step that may involve washing with water, such as distilled water and / or ethanol, after contact with the reducing agent. This can help to remove remaining unbound crosslinking agent or oxalic acid.

[0076] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking a composition comprising the solubilized elastin obtained from step a); c) lyophilizing the product of step b); and d) washing the product of step c).

[0077] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking a composition comprising the unfractionated solubilized elastin obtained from step a); c) lyophilizing the product of step b); and d) washing the product of step c).

[0078] According to the present invention, there is provided a method comprising: a) contacting elastin with a solubilizing agent capable of solubilizing elastin; b) crosslinking the composition obtained from step a); c) lyophilizing the product obtained from step b); and d) washing the product of step c).

[0079] After washing, the scaffold can be sterilized. In some embodiments, sterilization involves washing the scaffold with ethanol and PBS.

[0080] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking the composition obtained from step a); c) lyophilizing the product of step b); d) washing the product of step c); and e) sterilizing the product of step d).

[0081] According to the present invention, there is provided a method comprising: a) solubilizing elastin; b) crosslinking a composition comprising the unfractionated solubilized elastin obtained from step a); c) lyophilizing the product of step b); d) washing the product of step c); and e) sterilizing the product of step d).

[0082] According to the present invention, there is provided a method comprising: a) contacting elastin with a solubilizing agent; b) crosslinking the composition produced by a); c) lyophilizing the product of step b); d) washing the product of step c); and e) sterilizing the product of step d).

[0083] Preferably, the scaffold of the present invention is sterilized.

[0084] According to the present invention, there is provided a tissue scaffold obtained or obtainable by the method according to the present invention.

[0085] In some embodiments, the scaffold of the present invention is not a hydrogel.

[0086] The methods and scaffolds of the present invention are also contemplated to be applicable to elastin derivatives or fragments, such as synthetic elastin sequence-based materials or elastin-like peptides (ELPs). ELPs are biopolymers based on important repetitive elastin sequences. For example, an ELP can have repetitive peptides such as pentapeptides or hexapeptides containing Val, Gly, and / or Pro. ELPs can have the elastic properties of elastin using the pentapeptide repeat VPGXG (where X is any amino acid other than proline, such as Val or Ile) (Zhang et al., (2015), Daamen (2007)).

[0087] According to the present invention, there can be provided a method comprising crosslinking an elastin derivative or fragment.

[0088] According to the present invention, there can be provided a tissue scaffold comprising a crosslinked elastin derivative or fragment.

[0089] According to the present invention, there can be provided a method comprising lyophilizing a crosslinked elastin derivative or fragment.

[0090] According to the present invention, there can be provided a tissue scaffold comprising a lyophilized crosslinked elastin derivative or fragment.

[0091] The scaffold of the present invention can include other extracellular matrix components.

[0092] The scaffold of the present invention can include collagen. As a result, according to the present invention, there is provided a scaffold comprising elastin and collagen.

[0093] The scaffold of the present invention may contain fibrin. As a result, according to the present invention, a scaffold containing elastin and fibrin is provided.

[0094] The scaffold of the present invention may contain collagen and elastin. As a result, according to the present invention, a scaffold containing elastin, collagen and fibrin is provided.

[0095] Preferably, the elastin is solubilized. Preferably, the elastin is unfractionated solubilized elastin. The elastin can be unfractionated. The elastin may contain solubilized elastin. The elastin may contain insoluble elastin.

[0096] The scaffold of the present invention can be formed by mixing a composition containing elastin with a) a composition containing collagen and / or b) a composition containing fibrin.

[0097] The composition containing collagen may include a collagen hydrogel. For example, the collagen hydrogel can be formed by standard procedures. Using 80% rat tail type I collagen and 10× minimum essential medium, a collagen hydrogel can be prepared, neutralized using 5M and 1M sodium hydroxide, and 10× DMEM (Dulbecco's modified Eagle's medium) can be added.

[0098] The composition containing fibrin may contain a fibrin gel. The fibrin gel can be formed by standard procedures. Using 2% fibrinogen dissolved in 1 ml of PBS, a fibrin gel can be prepared, and then 1% thrombin can be added together with 0.1M CaCl2.

[0099] According to the present invention, there is provided a method comprising mixing a composition containing elastin (preferably a composition containing unfractionated solubilized elastin) with a composition containing collagen (preferably a collagen hydrogel) and / or a composition containing fibrin (preferably a fibrin gel).

[0100] According to the present invention, there is provided a composition comprising elastin (preferably unfractionated solubilized elastin), collagen and / or fibrin. The composition may comprise a crosslinking agent.

[0101] The composition comprising elastin is preferably mixed with a composition comprising collagen and / or a composition comprising fibrin before the crosslinking step. As a result, the resulting scaffold may comprise crosslinked elastin, crosslinked collagen and / or crosslinked fibrin.

[0102] According to the present invention, there is provided a composition comprising elastin; a solubilizing agent for solubilizing elastin; a crosslinking agent; and fibrin and / or collagen.

[0103] Crosslinking can proceed as already described herein. For example, the crosslinking agent may comprise glutaraldehyde and may be carried out in the presence of CO2. The crosslinking agent may be added to a composition comprising elastin, collagen and / or fibrinogen. Alternatively, the crosslinking agent may be added to a composition comprising elastin, a composition comprising collagen and / or a composition comprising fibrinogen before mixing the compositions. For example, the composition comprising elastin may comprise a crosslinking agent. The concentration of the crosslinking agent in the composition comprising elastin may be at a level (e.g., 3 volume %) such that when the composition comprising elastin is mixed with a composition comprising collagen and / or a composition comprising fibrin, the concentration is at a level (e.g., about 1% v / v) desirable for crosslinking to occur.

[0104] Before crosslinking, the composition may be cast as described herein.

[0105] Once crosslinking has been carried out, the scaffold may be lyophilized and / or washed as described herein.

[0106] The relative amounts of elastin, collagen and / or fibrinogen can be adjusted to impart different structural, mechanical and biodegradation properties to the scaffolds. For example, scaffolds containing a high proportion of elastin can result in a high-density structural network, high elasticity and delayed degradation compared to scaffolds having a low proportion of elastin. An increase in the amount of fibrin can increase mechanical strength and accelerate the biodegradation rate. An increase in the amount of collagen can also accelerate the biodegradation rate. The use of specific combinations of elastin, collagen and / or fibrinogen can also enable enhancement of the angiogenic properties of the scaffolds.

[0107] According to the present invention, there is provided a tissue scaffold of the present invention for use as a medicament.

[0108] According to the present invention, there is provided a method for promoting tissue healing, regeneration or repair, the method comprising applying the tissue scaffold of the present invention to a patient. For example, the scaffold can be used in wound healing or tissue grafts (e.g., skin grafts). The scaffolds of the present invention can be particularly applicable to soft tissue regeneration or repair, such as skin regeneration or vascular tissue regeneration. For example, the scaffolds can be used in adipose, skin, vascular grafts, heart valves or lung tissue engineering.

[0109] According to the present invention, there is provided a tissue scaffold of the present invention for use in promoting tissue healing, regeneration or repair.

[0110] According to the present invention, there is provided the use of a tissue scaffold of the present invention in the manufacture of a medicament for promoting tissue healing, regeneration or repair.

[0111] The present invention can provide a method substantially described herein with reference to the drawings.

[0112] The present invention can provide a tissue scaffold substantially described herein with reference to the drawings.

[0113] According to the present invention, there is provided a scaffold as described herein seeded with cells. The scaffold can be in vitro or ex vivo. The cells can be stem cells, such as human adipose-derived stem cells (hADSC).

[0114] According to the present invention, there is provided a method comprising seeding cells onto the scaffold of the present invention.

[0115] According to the present invention, there can be provided a cell or tissue culture comprising a scaffold as defined herein.

[0116] The scaffold of the present invention can have an average pore size of less than 120 μm. The scaffold of the present invention can have an average pore size of less than 100 μm. The scaffold of the present invention can have an average pore size of 10 μm or more. The scaffold of the present invention can have an average pore size of 20 μm or more. For example, the scaffold of the present invention can have an average pore size of 10-120 μm. The scaffold of the present invention can have an average pore size of 20-100 μm.

[0117] The pore size distribution can be changed by including collagen and / or fibrin in elastin or by changing the relative amounts of the components (see, for example, FIGS. 14 and 15).

[0118] The scaffold of the present invention can have a modal pore size of 80 μm or less. For example, the modal pore size can be 60 μm or less. The modal pore size can be 1 μm or more, for example 10 μm or more or 20 μm or more. For example, the modal pore size can range from 1-80 μm, such as 1-60 μm, 20-60 μm or 1-60 μm.

[0119] In one example, the modal pore size can range from 1-20 μm. In another example, the modal pore size can range from 20-40 μm. In one embodiment, the modal pore size can range from 40-60 μm.

[0120] The characteristics of the scaffold, such as pore diameter and porosity, can be calculated using suitable software that is readily available. For example, ND (“Nearest Distance”) is an ImageJ plugin developed to calculate the mean size and the distance between pores and their nearest neighbors in a porous scaffold (see Haeri et al., (2015)). DiameterJ is another example of an ImageJ plugin that can be used to measure pore parameters. Microscopic images of the scaffold (e.g., SEM images) can be used as input.

[0121] The total porosity of the scaffold of the present invention can be at least 25%. For example, the total porosity can be at least 40%. In certain embodiments, for example, the following are provided: (Item 1) A method for forming a tissue scaffold, comprising crosslinking a composition comprising elastin, wherein the elastin is unfractionated and comprises solubilized elastin. (Item 2) The method according to item 1, comprising the step of solubilizing elastin. (Item 3) A method for forming a tissue scaffold, comprising crosslinking a composition comprising unfractionated solubilized elastin. (Item 4) The method according to item 3, comprising the step of solubilizing elastin to form a composition comprising unfractionated solubilized elastin. (Item 5) The method according to any one of the preceding items, wherein the elastin is solubilized or is being solubilized by contact with oxalic acid. (Item 6) The method according to any one of the preceding items, wherein the elastin is solubilized or is being solubilized at a temperature below 100°C. (Item 7) The method according to item 6, wherein the step of solubilizing the elastin is performed or is being performed at a temperature below or equal to 50°C. (Item 8) The method according to item 7, wherein the step of solubilizing the elastin is carried out or is being carried out at a temperature of 15 to 30°C. (Item 9) The method according to any one of the preceding items, wherein the composition to be crosslinked contains insoluble elastin. (Item 10) A method for forming a tissue scaffold, comprising crosslinking a composition comprising soluble elastin and insoluble elastin. (Item 11) The method according to any one of the preceding items, wherein the composition to be crosslinked contains collagen and / or fibrin. (Item 12) The method according to item 3 or item 4 or any item dependent on item 3 or item 4, comprising crosslinking a composition comprising unfractionated solubilized elastin and a) collagen and / or b) fibrin. (Item 13) The method according to any one of items 11 to 12, wherein the collagen is in the form of a collagen hydrogel. (Item 14) The method according to any one of items 11 to 13, wherein the fibrin is in the form of a fibrin gel. (Item 15) The method according to any one of the preceding items, wherein the crosslinking comprises chemical crosslinking. (Item 16) The method according to item 15, wherein the crosslinking comprises contacting the composition with an aldehyde crosslinking agent. (Item 17) The method according to item 16, wherein the aldehyde crosslinking agent is glutaraldehyde. (Item 18) The method according to any one of the preceding items, wherein the crosslinking is carried out at a temperature of 25 to 50°C. (Item 19) The method according to any one of the preceding items, wherein the crosslinking is carried out in the presence of CO2. (Item 20) The method according to item 19, wherein the crosslinking is carried out in the presence of 2 to 10% CO2. (Item 21) The method according to any one of the preceding items, wherein the crosslinking is carried out for 1 to 24 hours. (Item 22) The method according to item 3 or item 4 or any item dependent on item 3 or item 4, comprising lyophilizing a composition containing crosslinked unfractionated solubilized elastin. (Item 23) The method according to any one of the preceding items, wherein lyophilization is carried out after solubilization and crosslinking. (Item 24) A method of forming a tissue scaffold, comprising lyophilizing a composition containing crosslinked elastin, and optionally, the composition containing crosslinked elastin is an unfractionated one and is formed by crosslinking a formulation containing elastin containing solubilized elastin. (Item 25) The method according to item 24, comprising lyophilizing a composition containing crosslinked unfractionated solubilized elastin. (Item 26) The method according to any one of the preceding items, comprising washing to remove agents involved in solubilization and / or crosslinking. (Item 27) The method according to item 26, comprising lyophilizing and washing, wherein the washing is carried out after lyophilization. (Item 28) The method according to item 26 or item 27, wherein the step of washing to remove agents involved in solubilization and / or crosslinking comprises washing with a reducing agent. (Item 29) The method according to item 28, wherein the reducing agent comprises sodium borohydride or a similar agent having carbonyl group reactivity. (Item 30) The method according to any one of items 26 to 29, wherein the step of washing to remove agents involved in solubilization and / or crosslinking is carried out for at least 5 hours, preferably at least 8 hours. (Item 31) The method according to any one of the preceding items, comprising sterilizing the scaffold. (Item 32) The method according to item 31, including contacting the scaffold with ethanol. (Item 33) a) A composition containing elastin, a solubilizing agent capable of solubilizing the elastin, and a cross-linking agent, or b) A composition containing elastin and a cross-linking agent, wherein the elastin in the composition is unfractionated and contains solubilized elastin. (Item 34) A composition containing unfractionated solubilized elastin and a cross-linking agent. (Item 35) The composition according to item 33 or item 34, containing collagen and / or fibrin. (Item 36) A tissue scaffold obtained or obtainable by the method disclosed in any one of items 1 to 32. (Item 37) A tissue scaffold containing cross-linked elastin, wherein the cross-linked elastin is formed by cross-linking a composition containing unfractionated elastin containing solubilized elastin. (Item 38) A tissue scaffold containing cross-linked unfractionated solubilized elastin. (Item 39) A tissue scaffold containing cross-linked elastin, wherein the cross-linked elastin is formed by cross-linking a composition containing soluble elastin and insoluble elastin. (Item 40) The tissue scaffold according to item 37, wherein the cross-linked elastin is formed by cross-linking a composition containing insoluble elastin, or the tissue scaffold according to item 38, containing insoluble elastin. (Item 41) The tissue scaffold according to any one of items 37 to 40, containing collagen and / or fibrin. (Item 42) The tissue scaffold according to any one of items 37 to 41, which is lyophilized. (Item 43) The tissue scaffold according to any one of items 37 to 44, which is sterilized. (Item 44) A method for promoting tissue regeneration, tissue healing or tissue repair, the method comprising applying the tissue scaffold according to any one of Items 36 to 43 to a patient in need thereof. (Item 45) The method according to Item 44, which is for promoting soft tissue regeneration or repair. (Item 46) A tissue scaffold according to any one of Items 36 to 43 for use in promoting tissue regeneration, tissue healing or tissue repair. (Item 47) A tissue scaffold for use according to Item 46 for promoting soft tissue repair. (Item 48) A method for solubilizing elastin, the method comprising contacting the elastin with oxalic acid at a temperature of less than 100°C. (Item 49) The method according to Item 48, which comprises contacting the elastin with oxalic acid at a temperature of less than 50°C, preferably at a temperature of 15 to 30°C. (Item 50) The method according to Item 48 or Item 49, wherein the elastin is contacted with the acid for 5 minutes or less, preferably for 1 to 3 minutes. (Item 51) A method for solubilizing elastin, the method comprising contacting the elastin with oxalic acid for 5 minutes or less. (Item 52) The method according to Item 51, wherein the elastin is contacted with oxalic acid at a temperature of less than 100°C. (Item 53) The method according to Item 52, which comprises contacting the elastin with oxalic acid at a temperature of less than 50°C, preferably at a temperature of 15 to 30°C. (Item 54) The method according to any one of Items 48 to 53, wherein the oxalic acid is 0.2M to 1M. (Item 55) A composition obtained or obtainable by the method according to any one of Items 48 to 54. (Item 56) A method comprising crosslinking the composition defined in item 55. (Item 57) A tissue scaffold comprising i) elastin; and ii) collagen and / or fibrin, wherein the elastin is crosslinked. (Item 58) The tissue scaffold according to item 56, wherein the collagen and / or fibrin is crosslinked. (Item 59) The tissue scaffold according to item 57 or item 58, wherein the elastin comprises solubilized elastin. (Item 60) The tissue scaffold according to any one of items 57 to 59, wherein the elastin comprises insoluble elastin. (Item 61) The tissue scaffold according to any one of items 57 to 60, wherein the elastin is unfractionated. (Item 62) A composition comprising i) elastin, ii) a crosslinking agent; and iii) fibrin and / or collagen. (Item 63) The composition according to item 61, wherein the elastin comprises solubilized elastin. (Item 64) The composition according to any one of items 62 to 63, wherein the elastin is unfractionated elastin. (Item 65) The composition according to any one of items 62 to 64, comprising a solubilizing agent for solubilizing elastin. (Item 66) A method comprising crosslinking a composition comprising i) elastin and ii) collagen and / or fibrin. (Item 67) The method according to item 66, wherein the elastin is unfractionated. (Item 68) The method according to item 66 or item 67, wherein the elastin comprises solubilized elastin. (Item 69) The method according to any one of items 66 to 68, wherein the elastin comprises insoluble elastin. (Item 70) The tissue scaffold according to any one of Items 57 to 60 for use in promoting tissue healing, regeneration or repair. (Item 71) A method for promoting tissue regeneration, tissue healing or tissue repair, comprising applying the tissue scaffold according to any one of Items 57 to 61 to a patient in need thereof. (Item 72) A tissue scaffold according to any one of Items 36 to 43 or 57 to 61 seeded with cells. (Item 73) A method comprising seeding cells onto a scaffold, wherein the scaffold is as defined in any one of Items 36 to 43 or 57 to 61. (Item 74) A cell or tissue culture comprising a scaffold, wherein the scaffold is as defined in any one of Items 36 to 43 or 57 to 61.

[0122] Examples of the present invention are described by way of example only with reference to the accompanying drawings.

Brief Description of the Drawings

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Example

[0124] Example 1 - Elastin Scaffold Manufacturing Method and Materials Insoluble elastin powder was obtained from Sigma (the source of elastin was derived from bovine neck ligaments) (Figure 1A). 100 mg of insoluble elastin powder was mixed with 1 ml of freshly prepared 0.5 M oxalic acid (C2H2O4) at room temperature (Figure 1B).

[0125] To crosslink the protein, 1% glutaraldehyde (GTA) (v / v), a homobifunctional crosslinking agent, was added to the solution (Figure 1C). The solution was cast into the wells of a 24-well plate and incubated at 37 °C, 5% CO2 for 1 hour (Figure 1C).

[0126] The mixture was frozen at -80 °C overnight (Figure 1D) and lyophilized for 48 hours to form a scaffold (Figure 1E).

[0127] The fabricated scaffold was brought to room temperature and washed with 0.1 M glycine buffer at pH = 10.4 (two washes for 15 minutes each) and then with Tris-glycine buffer for 15 minutes. To remove excess oxalic acid and unbound glutaraldehyde, the scaffold was washed on a shaker with 0.1% w / v sodium borohydride (NaBH4) (reducing agent) for approximately 8 hours.

[0128] Subsequently, the scaffold was washed with distilled warm water (60 °C) for 15 minutes and then with distilled water twice for 30 minutes each to remove residual unbound glutaraldehyde from the scaffold.

[0129] For sterilization, the scaffold was washed with 70% ethanol for 15 minutes and then with PBS. Structural Integrity and Stability

[0130] The fabricated crosslinked elastin scaffold was not damaged (Figure 2B). However, the non-crosslinked scaffold was broken down / decomposed (Figure 2A).

[0131] An in vitro scaffold stabilization study was conducted by comparing crosslinked and non-crosslinked scaffolds in PBS at 37 °C and 5% CO2 for 28 days. In PBS, it was found that after 28 days, the non-crosslinked scaffolds (Figure 3A) were disassembled / degraded, while in contrast, the crosslinked scaffolds were found to be intact (Figure 3B). This indicates that this manufacturing method effectively generated an integral scaffold. Biological activity

[0132] To evaluate the effectiveness and biological activity of the scaffolds, adipose-derived stem cells (ADSCs) were cultured under standard culture conditions (i.e., incubation at 37 °C, 5% CO2 in MesenPRO RS™ basal cell culture medium (ThermoFisher, UK) supplemented with 2% MesenPRO RS™ growth supplement (ThermoFisher, UK) and 1% penicillin / streptomycin (Sigma-Aldrich, UK)). 50,000 cells were seeded onto scaffolds with a diameter of 6 mm and cultured for 1, 3, and 7 days. The viability and growth of the cells were studied using a live / dead assay and an alamar blue assay, respectively. The ADSCs were viable, adhered to the scaffolds by day 1, and showed a non-aggregated morphology on days 3 and 7 (Figure 4). In addition, the cells maintained non-aggregated behavior and showed a spindle-shaped morphology structure (Figure 4), which suggests that they retained their stem cell characteristics during the culture period.

[0133] Cell proliferation was quantitatively measured by alamar blue activity, a cell metabolism assay, and the absorbance at 570 nm was measured using a spectrophotometer on days 1, 3, and 7 (n = 3 / time point) (Figure 5). Scanning electron microscopy

[0134] The elastin scaffold was washed with distilled water for 3 minutes using an ultrasonic cleaner to remove salts and then dried in a freeze dryer for 24 hours. The scaffold was mounted on a stub and sputter-coated with carbon under vacuum. All images were acquired using the secondary electron detector of a Philips XL 30 Field Emission SEM operated at 5 kV, and the average working distance was 10 mm.

[0135] The SEM images in FIGS. 6A and 6B show that the elastin scaffold has a uniform structure and is porous. FIG. 6A is at a magnification of 50x and FIG. 2 is at a magnification of 250x. Discussion

[0136] At the priority date of this application, 5 mg of insoluble elastin from bovine neck ligaments was £69.70 (E1625), while 1 mg of soluble α-elastin from the commercial supplier Sigma™ was £272.50 (E6527), so this is a very cost-effective and time-efficient method for manufacturing elastin scaffolds.

[0137] The results of the live / dead assay showed that the cells maintained a spindle-shaped morphology, which is one of the characteristics of ADSCs. By using the elastin scaffold within the scope of the present invention, since ADSCs have contact inhibition behavior (Majd et al., 2011), the inventors were able to maintain contact inhibition behavior until day 7 (FIG. 4). This cell morphology can maintain the ADSC phenotype and pluripotency characteristics without undergoing any differentiation (Zhang and Kilian, 2013). The increase in alamar blue absorbance is an indicator of certain cell proliferation. These results also show that the manufactured scaffold was non-toxic to the cells. Example 2 - Elastin / Colagen / Fibrin Scaffold Manufacturing Method and Materials

[0138] Tube 1: Elastin powder (9.7% w / v) + 0.5 M oxalic acid + 3% glutaraldehyde (w / v).

[0139] Tube 2: Prepared using collagen hydrogel - 80% rat tail collagen type I (v / v) (First Link, Birmingham, UK) and 10% of 10× minimum essential medium (Invitrogen, Paisley, UK), neutralized using 5M and 1M sodium hydroxide (Sigma - Aldrich, Dorset, UK), and 10× DMEM was added. Tube 3: Prepared using fibrin gel - 2% fibrinogen (w / v) dissolved in 1 ml of PBS, and 1% thrombin (w / v) was added together with 0.1M CaCl2 for fibril formation.

[0140] Tubes 1 - 3 were mixed in various ratios, cast, and then incubated at 37°C, 5% CO2 for 1 hour. The final volume after mixing the three tubes was always 1 ml, which was then cast. · For the scaffold of 2:1:1 (collagen / elastin / fibrin), 500 μl of Tube 2 was mixed with 250 μl of Tube 1 and 250 μl of Tube 3 (also referred to as Scaffold 3A herein). · For the scaffold of 2:1:1 (elastin / collagen / fibrin), 500 μl of Tube 1 was mixed with 250 μl of Tube 2 and 250 μl of Tube 3 (also referred to as Scaffold 3B herein). · For the scaffold of 2:1:1 (fibrin / elastin / collagen), 500 μl of Tube 3 was mixed with 250 μl of Tube 1 and 250 μl of Tube 2 (also referred to as Scaffold 3C herein). · For the scaffold of 1:1:1, 333.3 μl of each tube was mixed and cast (also referred to as Scaffold 3D herein).

[0141] The mixture was lyophilized for 48 hours.

[0142] Washing: First, wash with Tris-glycine buffer for 15 minutes. Second, to remove excess unbound glutaraldehyde, wash the scaffolds on a shaker with 0.1% w / v sodium borohydride (NaBH4) (reducing agent) for approximately 8 hours. Biocompatibility

[0143] To evaluate the biocompatibility of each composite scaffold, 50,000 adipose-derived stem cells (ADSCs) per scaffold were seeded and cultured for up to 7 days. The live / dead assay and alamar blue assay were used to study cell survival and proliferation at 1, 3, and 7 days post-seeding, respectively.

[0144] As an example, the results for the three-component scaffolds showed that the ADSCs survived, adhered to the scaffolds (Figure 7), and proliferated up to day 7 (Figure 8). Similar results were observed for the two-component scaffolds (i.e., scaffolds containing elastin and collagen or elastin and fibrin). Microstructure

[0145] SEM was used to study the microstructure of each scaffold. The results for the three-component scaffolds (Figure 9) showed that each scaffold combination had a unique ultrastructural fibril network and pore size. Similar observations were made for the two-component scaffolds. This structural change can alter the behavior and differentiation of ADSCs as well as the biomechanical properties of the scaffolds (see Ghasemi-Mobarakeh et al. (2015)). Example 3 - Water Contact Angle (WCA)

[0146] By developing an experimental setup, the wettability of the elastin scaffolds was investigated. 30 μL of distilled water droplets were dispensed onto each scaffold, and several images were taken at time intervals of 0 - 5 seconds. The 0-second time was considered the initial contact time with the liquid medium (water). The WCA was calculated using the Young's equation, and the angle was measured from the tangent of the water-scaffold boundary to the perimeter of the water droplet (Fu et al (2014)). The calculated WCA is an evidence of the water-material interaction.

[0147] The calculated WCA of elastin at the 0 - second time point was 102 ± 7.75°, which decreased to 73.88 ± 5.90° at the 4 - second time point. Over time, the WCA continued to decrease over time and reached 0° at the 9 - second time point, which indicates complete wettability of the elastin scaffold (Figure 10).

[0148] However, by combining elastin with other natural polymers, such as fibrin and collagen, in different ratios, the WCAs of 3A (from 68.18 ± 3.38° at the 0 - second time point to 0° at the 3 - second time point) and 3C (from 67.46 ± 4.51° at the 0 - second time point to 0° at the 4 - second time point) changed and showed complete wettability by 4 seconds. Interestingly, the WCAs of 3B (from 112.34 ± 5.37° at the 0 - second time point to 99.32 ± 14.55° at the 10 - second time point) and 3D (from 120.18 ± 5.36° at the 0 - second time point to 113.23 ± 8.93 at the 10 - second time point) (Figure 11) did not show complete wettability even at the 10 - second time point, so they are hydrophobic. Since any material with > 90° is considered hydrophobic, the scaffolds of elastin, 3A, and 3C showed hydrophilic properties, high cohesion to water, and achieved complete wettability by 9 seconds, while scaffolds 3B and 3D showed hydrophilic properties but low cohesion to water. Example 4 - Acceleration of Trypsin Degradation

[0149] To measure the stability of the scaffolds, an accelerated degradation profile was performed by using 1× trypsin. The initial weight of the scaffolds was measured using an XS205 Mettler Toledo® digital scale. The scaffolds were placed in 24 - well plates containing 1× trypsin and incubated at 37 °C and 5% CO2. At each time point, the scaffolds were washed with distilled water, lyophilized, and weighed.

[0150] As a parameter of degradation, the net weight change was measured. The results of in vitro accelerated degradation showed that the elastin scaffolds started to degrade on the first day (136.06 ± 11.90 mg). By the fifth day, the weight had decreased by 25%, and this trend continued. By the 42nd day, the scaffolds had degraded by 70% (Figure 12).

[0151] For 3A, 3C, and 3D, the degradation profiles of the elastin-based copolymers were the same. By day 7, nearly 40% of the scaffolds had degraded, and this pattern continued until day 42 when nearly 70% of the scaffolds had degraded. However, 3B with 50% elastin was the most stable scaffold, with a degradation of 55% by day 42 (Figure 13). This indicates that elastin-based scaffolds with different degradation patterns can be used for various tissue engineering applications according to the regeneration characteristics of each tissue type. Example 5 - Structural Properties

[0152] To measure the pore size range and porosity, all SEM images were quantitatively analyzed using ImageJ bundled with 64-bit Java (registered trademark) 1.6.0 (NIH, USA). The threshold function was used to visualize all the pores of the scaffolds. In addition, the aspect ratio, which is a particle analysis function, was used.

[0153] The calculated pore size ratio of the scaffolds ranged from 0 to 120+μm, with 28% of the pores in the range of 0 to 19μm, 48% of the pores in the range of 20 to 79μm, and the remaining 24% in the range of 80 to 120+μm (Figure 14), and the total porosity of the scaffolds was 48%.

[0154] When elastin was combined with other polymers, in 3A, 70% of the pores were within 0 to 59, and the remaining 50% were in the range of 60 to 120+μm. In 3B, most of the pores (65%) were in the range of 20 to 59μm, while in 3C, the pore pattern was uniform, and 55% of the pores were in the range of 20 to 59μm. However, in 3D, 75% of the pores were in the range of 0 to 59μm (Figure 15).

[0155] Pores and porosity play important roles in angiogenesis and nutrient diffusion. The results suggest that elastin-based scaffolds can be used for various tissue engineering applications. Example 6 - Mechanical Properties

[0156] The elastin scaffolds were tested to failure using a biaxial biotester (CellScale Biomaterials Testing, Canada). This system includes two high-performance actuators with a temperature-controlled media bath to prevent scaffold drying during testing of the cell-seeded scaffolds. A time-synchronized high-resolution CCD camera was used for acquisition and processing of test results to analyze the real-time stress distribution.

[0157] The wet mechanical properties of the elastin scaffolds on day 0 were 154 ± 1 mN, and after seeding with hADSC cells for 28 days, the strength of the scaffolds increased significantly (p < 0.0001) to 185.5 ± 1.5 mN (Figure 16). This demonstrates that the cells seeded on the scaffolds increased the mechanical integrity of the scaffolds through tissue remodeling mechanisms.

[0158] The calculated breaking strength for 3A was 74.33 ± 3.78 mN, for 3B was 119.33 ± 33.12 mN, for 3C was 103.34 ± 20.23 mN, and for 3D was 71.68 ± 4.72 mN. This demonstrates that after adding another copolymer, the mechanical properties of the elastin decreased. This is thought to be due to the non-fibrillar arrangement of the polymer (Lake et al., (2012)). Example 7 - Angiogenesis

[0159] Pathogen-free fertilized eggs were obtained from a commercial supplier and incubated at 38 °C and 40 - 45% humidity for 3 days. On embryonic day 3 (ED3), an ex ovo glass bowl setup was constructed to grow the embryo cultures, which were maintained at 37.5 °C, 3% CO2, and an average humidity in the range of 80 - 85% (3). At ED9, the elastin scaffolds were placed on the developing chorioallantoic membrane (CAM) to allow vascular invasion, and at ED12, the embryos were euthanized according to home office guidelines, the scaffolds were excised, fixed with 4% glutaraldehyde, and analyzed.

[0160] The total calculated vascular area of ED10 was 4.78±2.12%, and in ED11, this vascular area increased to 6.01±3.34%. Although this increase was not statistically significant, two large blood vessels with many capillary networks developed. This trend continued in ED12, and the calculated vascular area was 8.34±2.67% (Figure 18).

[0161] When elastin was combined with fibrin and collagen in different ratios, by day 12, the total vascular area percentage increased. The calculated % vascular area was 12.97±0.61% for 3A, 11.33±1.52% for 3B, 14.41±0.67% for 3C, and 16.52±0.57 for 3D (Figure 19). Therefore, it seems beneficial to use combinations of polymers to enhance the angiogenic properties of elastin. The CAM assay functioned as an ex vivo bioreactor to understand vascular invasion into elastin scaffolds. Considering that the scaffolds had a pore distribution in the range of 0μm to 120+μm and acted as angiogenic promoters for vascular invasion. Example 8 - Cell Differentiation

[0162] To understand the differentiation pathway of human adipose-derived stem cells (hADSCs) on an elastin scaffold, a total of 5×10 3 per 1 mm 5 passage 4 hADSCs were seeded onto the scaffold. On days 1, 7, and 14, RNA was isolated by using the TRIzol (Invitrogen, Paisley, UK) method, and the total RNA yield was quantified by using a spectrophotometer (Spectronic Camspec Ltd, Garforth, UK). cDNA synthesis was performed using the Precision nanoscript 2 reverse transcription kit (Primer Design, Southampton, UK), and quantitative PCR was carried out using custom-designed synthetic primers (Table 1) (Primer Design, Southampton, UK).

Table 1

[0163] The gene expressions of adipogenic markers (CEBPA and PPARG), osteogenic marker (RUNX2), myogenic marker (MYOD1), chondrogenic marker (SOX9), and MSC markers (OCT4 and REX1), which are mesenchymal lineage-specific differentiation markers, were studied in hADSCs.

[0164] Differentiation profiles of hADSCs on an elastin scaffold. OCT4, CEBPA, PPARG, and MYOD1 showed the same tendency of significant upregulation by 0.03 - 0.04 units at days 7 and 14 compared to day 1 (p < 0.0001). However, there was no significant upregulation at day 14 compared to day 7. RUNX2 showed no tendency. SOX9 showed negligible expression (<0.027) at all three time points the same as all other scaffolds reported above, but it showed significant upregulation at day 14 (0.025, p < 0.05) compared to day 1 (0.027). REX1 showed the first downregulation at day 7 (0.036 - 0.028, p < 0.0001), followed by a tendency of significant upregulation at day 14 (0.031, p < 0.0001) (Figure 20).

[0165] In 3A, Oct-4 showed significant downregulation from day 1 (0.031) at days 7 and 14 (0.028, p < 0.0001). Rex-1 was significantly downregulated at days 7 (0.026, p < 0.0001) and 14 (0.029, p < 0.0001) compared to day 1 (0.031). However, the expression on day 14 was significantly higher than that on day 7 (p < 0.0001), while MyoD-1 remained constant at 0.032. CEBP showed slight upregulation on day 7 (p < 0.05) and was significantly downregulated to 0.025 on day 14 (p < 0.0001). In 3B, Oct-4, RUX-2, and CEBP showed significant downregulation at days 7 and 14 (p < 0.0001), and there was no significant difference in expression between days 7 and 14. In 3C, Oct-4 showed a steady and significant downregulation from 0.030 on day 1 to 0.029 on day 14 (p < 0.001). Rex-1 and RUNX-2 were significantly downregulated from 0.032 on day 1 to 0.030 and 0.029 respectively on day 7 (p < 0.0001). In 3D, Oct-4, CEBP, PPAR-γ, and MyoD-1 showed the same trend of significant downregulation by 0.04 - 0.06 units at days 7 and 14 compared to day 1 (p < 0.0001) Example 9 - Two - component elastin - based scaffolds ·Elastin / collagen - 1:1 ratio ·Elastin / fibrin - 1:1 ratio

[0166] As shown in Example 2, elastin, collagen, and fibrin were prepared. ·Swelling ratio

[0167] Figure 22 shows the difference in swelling ratio between the elastin / collagen scaffold and the elastin / fibrin scaffold. The swelling ratio is an indicator of the interaction between the solvent and the polymer. It indicates the affinity and entropy exchange between the two phases. The higher the cross - link density inside the polymer, the lower the swelling property, and vice versa. The following formula [Number] (where M d is the dry weight of the scaffold, and M w is the wet weight of the scaffold) was used to measure the swelling ratio (SR) of elastin and its composites from the dry and wet masses. The wet mass of the scaffold was measured by immersing it in 2 ml of distilled water. The dry and wet masses were measured using a digital scale (XS205 Mettler Toledo (registered trademark)), and the SR was calculated using Equation (1). · Degradation

[0168] Figure 23 shows the difference in the degradation profiles between the elastin / collagen scaffold and the elastin / fibrin scaffold. The experimental protocol was the same as that described in Example 4. · Microstructure

[0169] Figure 24 shows the microstructure of the elastin / collagen scaffold (A) and the elastin / fibrin scaffold (B) using SEM. · Pore size distribution

[0170] Figure 25 shows the pore size distribution of the elastin / collagen scaffold and the elastin / fibrin scaffold. · Biological activity

[0171] Figure 26 shows the results of the live / dead assay of the elastin / collagen scaffold and the elastin / fibrin scaffold. The experimental protocol was the same as that described in Example 1. · Angiogenesis

[0172] Figure 27 shows the vascular area of the elastin / collagen scaffold and the elastin / fibrin scaffold on day 12. The experimental protocol was the same as that described in Example 7. References [Chemistry]

Chem.

Claims

1. A tissue scaffold, comprising: i) elastin or an elastin derivative, wherein the elastin derivative comprises a repetitive pentapeptide or hexapeptide containing Val, Gly, and Pro; ii) collagen; and iii) fibrin, wherein the elastin or elastin derivative is crosslinked, and a) the tissue scaffold is lyophilized; and / or, b) the elastin is unfractionated and contains solubilized elastin; and / or, c) the elastin contains soluble elastin and insoluble elastin, Tissue scaffold.

2. The tissue scaffold according to claim 1, wherein the collagen and / or fibrin is crosslinked.

3. The tissue scaffold according to claim 1 or claim 2, wherein the elastin contains soluble elastin and insoluble elastin.

4. The tissue scaffold according to any one of claims 1 to 3, wherein the elastin is unfractionated and contains solubilized elastin.

5. The tissue scaffold according to any one of claims 1 to 4, which is lyophilized.

6. The tissue scaffold according to any one of claims 1 to 5, wherein the elastin derivative is an elastin-like peptide (ELP), and the ELP contains a pentapeptide repeat of the sequence VPGXG, where X is any amino acid other than proline.

7. A method for forming the tissue scaffold according to any one of claims 1 to 6, comprising: i) elastin or an elastin derivative, wherein the elastin derivative comprises a repetitive pentapeptide or hexapeptide containing Val, Gly, and Pro; ii) collagen; and, iii) fibrin comprising crosslinking a composition comprising, wherein, a) the composition is lyophilized after crosslinking; and / or, b) the elastin is undifferentiated and comprises solubilized elastin; and / or, c) the elastin comprises soluble elastin and insoluble elastin, method.

8. The method according to claim 7, wherein the elastin is undifferentiated.

9. The method according to claim 7 or 8, wherein the elastin comprises soluble elastin and insoluble elastin.

10. The method according to any one of claims 7 to 9, wherein the elastin derivative is an elastin-like peptide (ELP), wherein the ELP comprises a pentapeptide repeat of the sequence VPGXG, wherein X is any amino acid other than proline.

11. A tissue scaffold obtained by the method according to any one of claims 7 to 10.

12. The tissue scaffold according to any one of claims 1 to 6 for use in promoting tissue healing, regeneration or repair.

13. The tissue scaffold according to any one of claims 1 to 6 seeded with cells.

14. A method comprising seeding cells onto a scaffold, wherein the scaffold is as defined in any one of claims 1 to 6.

15. A cell culture or tissue culture comprising a scaffold, wherein the scaffold is as defined in any one of claims 1 to 6.

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