WO2025199565A1

WO2025199565A1 - Multi-layered construct and methods of fabricating the same

Info

Publication number: WO2025199565A1
Application number: PCT/AU2024/050292
Authority: WO
Inventors: Andrea Janet O'CONNOR; Geraldine Margaret MITCHELL; Zerina TOMKINS; Hazem Murhaf ALKAZEMI (Ali Kazem); Daniel Edward HEATH
Original assignee: St Vincents Institute of Medical Research; University of Melbourne; Monash University
Current assignee: St Vincents Institute of Medical Research; University of Melbourne; Monash University
Priority date: 2024-03-28
Filing date: 2024-03-28
Publication date: 2025-10-02
Anticipated expiration: 2026-09-28

Abstract

The present disclosure provides a multi-layered construct for tissue-engineered grafts. The multi-layered construct comprises a fibrous layer, a hydrogel layer, and cells. The fibrous layer extends along an axis in a first direction from a first end to a second end. The fibrous layer comprises fibres of a first biocompatible polymer. The fibres are at least partially aligned in the first direction. The hydrogel layer is disposed on the fibrous layer and comprises a second biocompatible polymer. The cells are disposed in the hydrogel layer and are substantially aligned in a second direction. The first direction and the second direction are different. The present disclosure also provides methods of fabricating multi-layered constructs for tissue- engineered grafts, and components thereof.

Description

MULTI-LAYERED CONSTRUCT AND METHODS OF FABRICATING THE SAME

FIELD OF THE INVENTION

[0001] The present invention relates generally to multi-layered constructs for tissue- engineered grafts, including vascularized grafts, and methods of fabricating the same,

BACKGROUND

[0002] Cardiovascular disease is the leading cause of death globally (Timmis, A. et al. European Society of Cardiology: Cardiovascular Disease Statistics 2021. European Heart Journal 2022, 43 (8), 716-799). Arterial diseases are responsible for a significant portion of this healthcare burden (Breuer, T. et al. Tissue Engineering of Vascular Grafts: A Case Report From Bench to Bedside and Back. Arterioscler., Thromb., Vase. Biol. 2023, 43, 399). Bypass surgery is often required for severely occluded vessels (Michaels, A. D. et al. Angioplasty Versus Bypass Surgery for Coronary Artery Disease. Circulation 2002, 106 (23), el87-el90). Currently, synthetic grafts are not suitable for bypass surgery for small vessels (<6 mm), such as the coronary artery, because they will occlude due to thrombosis (Baba, T. et al. Evaluation of Heparin-bonded ePTFE Grafts for Forearm Loop Vascular Access: Comparison Between Gore® PROPATEN Vascular Graft and ACUSEAL Vascular Graft. The Journal of Vascular Access 2022, 23 (3), 430-435). Instead, surgeons use autografts such as the saphenous vein and the internal mammary artery. However, this is a suboptimal strategy because donor vessels are limited in supply, an additional surgical site is required, and some patients cannot provide appropriate donor vessels due to previous surgeries or comorbidities (Hu, K. et al. History, Progress and Future Challenges of Artificial Blood Vessels: A narrative Review. Biomaterials Translational 2022, 3 (1), 81).

[0003] An alternative to such autografts is the use of small-diameter tissue-engineered vascular grafts (TEVGs). However, this has so far met with limited success, including because of the challenges in recapitulating the mechanical properties of native blood vessels. The innermost layer, the tunica intima, consists of a monolayer of axially aligned endothelial cells (ECs), commonly known as an endothelium (Naito, H. et al. Mechanisms of New Bloodvessel Formation and Proliferative Heterogeneity of Endothelial Cells. International immunology 2020, 32 (5), 295-305). The middle layer, the tunica media, consists of multiple layers of circumferentially aligned contractile smooth muscle cells (SMCs) (Tennant, M. et al. Blood Vessel Structure and Function: A Brief Update on Recent Advances. Australian and New Zealand Journal of Surgery 1990, 60 (10), 747-753). The EC and SMC alignments are important for appropriate blood vessel function. For instance, the alignment of ECs is required for their atheroprotective properties and barrier function (Karimi, F. et al. Biomaterials Functionalized with Nanoclusters of Integrin-and syndecan-binding Ligands Improve Cell Adhesion and Mechanosensing Under Shear Flow Conditions. J. Biomed. Mater. Res., Part A 2021, 109 (3), 313-325; Heath, D. E. Promoting Endothelialization of Polymeric Cardiovascular Biomaterials. Macromol. Chem. Phys. 2017, 218 (8), 1600574). Similarly, the circumferential alignment of contractile vascular SMCs is necessary to maintain homeostasis and vascular tone, and vascular smooth muscle cell dysfunction is associated with disease states such as hypertension and atherosclerosis (Chan-Park, M. B. et al. Biomimetic Control of Vascular Smooth Muscle Cell Morphology and Phenotype for Functional Tissue-engineered Small-diameter Blood Vessels. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 2009, 88 (4), 1104-1121).

[0004] Despite the need for biomimetic cellular alignment within TEVGs, recapitulation of the tissue niche in in vitro systems remains elusive, including because many TEVGs are fabricated using soft hydrogels, and these materials lack the adequate mechanical strength, suturability, and burst pressure to be directly connected to the patient’s circulatory system (Chen, C., Y. Xi, and Y. Weng, Recent advances in cellulose-based hydrogels for tissue engineering applications. Polymers, 2022. 14(16): p. 3335). While these deficiencies have been at least partly alleviated by multilayered TEVGs, EC alignment often requires costly equipment such as pulsatile flow bioreactors and lengthy processing times (Li, Y. et al. Engineering Cell Alignment in Vitro. Biotechnology advances 2014, 32 (2), 347-365). Moreover, achieving the circumferential alignment of multiple layers of vascular SMCs remains challenging.

[0005] Additionally, vascularization is a major challenge that can limit the clinical implementation of engineered tissues (Paternoster, J.L. and J. J. Vranckx, State of the art of clinical applications of tissue engineering in 2021. Tissue Engineering Part B : Reviews, 2022. 28(3): p. 592-612; and Morrison, W.A., et al., Creation of a large adipose tissue construct in humans using a tissue-engineering chamber: a step forward in the clinical application of soft tissue engineering. EBioMedicine, 2016. 6: p. 238-245). Specifically, most three-dimensional, engineered tissues exhibit poor survival upon implantation, largely due to oxygen transport limitations. In vivo, most tissues require blood to be supplied via a capillary network since sufficient oxygen levels can only diffuse -200 pm from blood vessels in cell-dense tissues (Folkman, J. and M. Hochberg, Self-regulation of growth in three dimensions. The Journal of experimental medicine, 1973. 138(4): p. 745-753; and Carmeliet, P. and R.K. Jain, Angiogenesis in cancer and other diseases. Nature, 2000. 407(6801): p. 249-257). In contrast, engineered tissues are not rapidly connected to the patient’s vascular supply upon implantation. Even if an engineered tissue is generated to contain a capillary network in vitro, its in vivo survival will depend on the slow process of inosculation (functional unification of donor and host capillary networks). This process takes days to weeks before the network is well connected to and perfused by the recipient’s circulating blood, and the diffusion-based transport of oxygen during this time is not sufficient to maintain viability and function of the engineered tissue (Maida, J., et al., Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling. Biotechnology and bioengineering, 2004. 86(1): p. 9-18; and Clark, E.R. and E.L. Clark, Microscopic observations on the growth of blood capillaries in the living mammal. American journal of anatomy, 1939. 64(2): p. 251- 301).

[0006] Despite advances over the last 30 years to address the vascularization problem, including by the use of microfluidic systems and sacrificial molding, engineered tissues typically cannot be directly connected to the patient’s blood supply upon implantation, resulting in poor survival of the engineered tissue and limiting the clinical success of potentially lifesaving tissue engineering strategies (Wang, Y., et al., Advances in hydrogel -based vascularized tissues for tissue repair and drug screening. Bioactive materials, 2022. 9: p. 198- 220).

[0007] Hence, there remains an urgent need for improved constructs for tissue-engineered grafts, including vascularized grafts.

SUMMARY

[0008] In one aspect, the present invention provides a multi-layered construct for tissue- engineered grafts. The multi-layered construct comprises a fibrous layer, a hydrogel layer, and cells. The fibrous layer extends along an axis in a first direction from a first end to a second end. The fibrous layer comprises fibres of a first biocompatible polymer. The fibres are at least partially aligned in the first direction. The hydrogel layer is disposed on the fibrous layer and comprises a second biocompatible polymer. The cells are disposed in the hydrogel layer and are substantially aligned in a second direction. The first direction and the second direction are different. [0009] In some embodiments, the multi-layered construct further comprises cells disposed on the fibrous layer and the cells disposed on the fibrous layer are substantially aligned in the first direction.

[0010] In some embodiments, the fibrous layer is disposed about and extends along the axis in the first direction, the fibrous layer has an interior surface defining a lumen and an exterior surface, and the hydrogel layer is disposed on the exterior surface of the fibrous layer and about the axis. In such embodiments, the first direction may be further defined as an axial direction, and the second direction may be further defined as a circumferential direction about the axis. In some embodiments, the multi-layered construct further comprises cells disposed on the interior surface of the fibrous layer, and the cells disposed on the interior surface of the fibrous layer are substantially aligned in the first direction.

[0011] In some embodiments, at least about 50% of the cells disposed on the fibrous layer are aligned within ± 10° of the first direction. In some embodiments, the cells disposed on the fibrous layer are endothelial cells.

[0012] In some embodiments, the fibres of the first biocompatible polymer are electrospun fibres.

[0013] In some embodiments, the first biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof (e.g., a copolymer thereof (e.g., a bipolymer, terpolymer, a quaterpolymer, etc.)). In some embodiments, the first biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, silk, collagen or derivatives thereof, elastin or derivatives thereof, or any combination thereof (e.g., a copolymer thereof (e.g., a bipolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the first biocompatible polymer is a polyester. In some embodiments, the polyester is polylactide (PLA), poly(lactic acid-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), poly(3 -hydroxybutyrate) (PHB), poly(3 -hydroxybutyrate-co-3 -hydroxy valerate), poly((3 -hydroxyval erate), poly(3 - hydroxyhexanoate), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4- hydroxyhexanoate), polycaprolactone (PCL), poly(ethylene terephthalate), or any combination thereof (e.g., a copolymer thereof (e.g., a bipolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the first biocompatible polymer is PCL. [0014] In some embodiments, the multi-layered construct has a burst pressure of from about 1,000 mm Hg to about 3,500 mm Hg. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 100 gf to about 300 gf. In some embodiments, the multi-layered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 1.5 MPa to about 4 MPa. In some embodiments, the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 10 MPa.

[0015] In some embodiments, the second biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the second biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, silk, gelatin (Gel), gelatin methacryloyl (GelMA), elastin or derivatives thereof, tropoelastin or derivatives thereof, collagen or derivatives thereof, recombinant silk proteins or derivatives thereof, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.).

[0016] In some embodiments, the second biocompatible polymer is a polyester. In some embodiments, the polyester is polylactide (PLA), poly(lactic acid-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), poly(3 -hydroxybutyrate) (PHB), poly(3 -hydroxybutyrate-co-3 -hydroxy valerate), poly((3 -hydroxyval erate), poly(3 - hydroxyhexanoate), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4- hydroxyhexanoate), polycaprolactone (PCL), poly(ethylene terephthalate), or any combination thereof (e.g., a copolymer thereof (e.g., a bipolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the second biocompatible polymer is a polylactide (PLA) derivative, a poly(lactic acid-co-glycolic acid) (PLGA) derivative, a polyglycolic acid (PGA) derivative, a polyhydroxyalkanoate (PHA) derivative, a poly(3 -hydroxybutyrate) (PHB) derivative, a poly(3 -hydroxybutyrate-co-3 -hydroxyval erate) derivative, a poly((3 -hydroxyval erate) derivative, a poly(3 -hydroxyhexanoate) derivative, a poly(4-hydroxybutyrate) derivative, a poly(4-hydroxyvalerate) derivative, a poly(4-hydroxyhexanoate) derivative, a polycaprolactone (PCL), poly(ethylene terephthalate) derivative, polyacrylic acid (PAA) or derivatives thereof, polyethylene glycol (PEG) or derivatives thereof, polyvinyl alcohol (PVA) or derivatives thereof, polyvinylpyrrolidone (PVP) or derivatives thereof, polyacrylamide or derivatives thereof, hyaluronic acid or derivatives thereof, chitin or derivatives thereof, chitosan or derivatives thereof, alginate or derivatives thereof, gelatin (Gel), gelatin methacryloyl (GelMA), xanthan gum, carrageenan or derivatives thereof, dextran or derivatives thereof, starch or derivatives thereof, cellulose or derivatives thereof, elastin or derivatives thereof, tropoelastin or derivatives thereof, collagen or derivatives thereof, silk or derivatives thereof, recombinant silk proteins or derivatives thereof, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the second biocompatible polymer is GelMA. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 2% w/v to about 6% w/v based on the total volume of the hydrogel layer.

[0017] In some embodiments, the hydrogel layer has a Young's modulus of from about 3 kPa to about 20 kPa. In some embodiments, the first and second biocompatible polymers are biodegradable. In some embodiments, one of the first and second biocompatible polymers is biodegradable and the other of the first and second biocompatible polymers is not biodegradable. In some embodiments, the first biocompatible polymer is biodegradable and the second biocompatible polymer is not biodegradable. In some embodiments, the second biocompatible polymer is biodegradable and the first biocompatible polymer is not biodegradable. In some embodiments, the first and second biocompatible polymers are not biodegradable.

[0018] In some embodiments, at least about 35% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 40% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. [0019] In some embodiments, the fibrous layer further comprises a plurality of pores. In some embodiments, the plurality of pores are distributed at least one of along the axis and about the axis. In some embodiments, the plurality of pores each have a diameter of from about 50 pm to about 1,000 pm.

[0020] In some embodiments, the cells disposed in the hydrogel layer are smooth muscle cells.

[0021] In another aspect, the present invention provides a method of fabricating a fibrous layer of at least partially aligned fibres for tissue-engineered grafts. The method comprises: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a biocompatible polymer; and b) applying a mechanical force to the fibres of the biocompatible polymer to at least partially align the fibres in the first direction.

[0022] In some embodiments, providing the fibrous layer of step a) further comprises electrospinning the biocompatible polymer to form the fibrous layer.

[0023] In some embodiments, the method further comprises: c) disposing cells on the fibrous layer after step b); and d) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

[0024] In some embodiments, applying the mechanical force of step b) comprises directional or bidirectional freezing of a solvent to thereby apply the mechanical force to the fibres of the polymer. In some embodiments, applying the mechanical force of step b) comprises: b-1) disposing the fibrous layer in a chamber; b-2) partially filling the chamber with a solvent; and b-3) freezing the solvent, wherein the solvent expands in the first direction during freezing to thereby apply the mechanical force to the fibres of the polymer.

[0025] In some embodiments, the solvent is water. In some embodiments, the chamber is configured to direct freezing in the first direction.

[0026] In a further aspect, the present invention provides a method of fabricating a hydrogel layer of a multi-layered construct for tissue-engineered grafts. The method comprises: a) providing a fibrous layer extending along an axis in a first direction from a first end to a second end, the fibrous layer comprising fibres that are at least partially aligned in the first direction; b) disposing a mixture on a surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; c) curing the cross-linkable polymer composition to form the hydrogel layer on the surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and d) allowing the cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

[0027] In some embodiments, the fibrous layer is disposed about and extends along the axis in the first direction, the fibrous layer has an interior surface defining a lumen and an exterior surface, and the hydrogel layer is formed on the exterior surface of the fibrous layer and about the axis. In such embodiments, the first direction may be further defined as an axial direction, and the second direction may be further defined as a circumferential direction about the axis.

[0028] In some embodiments, the cross-linkable polymer composition comprises polymers having at least one cross-linkable moiety. In some embodiments, the curing step c) comprises heating the mixture, irradiating the mixture with a radiation source, adding a curing agent to the mixture, or any combination thereof. In some embodiments, the curing step c) comprises irradiating the mixture with a radiation source in the presence of a curing agent. In some embodiments, the radiation source is an ultraviolet (UV) light and the curing agent is a photoinitiator.

[0029] In some embodiments, the method further comprises: e) cooling the mixture prior to step c).

[0030] In some embodiments, step e) comprises cooling the mixture to a temperature of from about -10 °C to about 10 °C.

[0031] In some embodiments, the method further comprises: f) treating the fibrous layer to improve a hydrophilicity of the fibrous layer prior to step b).

[0032] In some embodiments, the treating step f) comprises contacting the fibrous layer with plasma, contacting the fibrous layer with an acidic solution, contacting the fibrous layer with a basic solution, or any combination thereof. In some embodiments, the treating step f) comprises contacting the fibrous layer with a basic solution (e.g., a sodium hydroxide (NaOH) solution).

[0033] In yet another aspect, the present invention provides a method of fabricating a multi-layered construct for tissue-engineered grafts. The method comprises: a) forming a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) applying a mechanical force to the fibres of the first biocompatible polymer to at least partially align the fibres in the first direction; c) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; d) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and e) allowing the second cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

[0034] In one embodiment, the method further comprises: f) disposing cells on the fibrous layer, wherein the cells disposed on the fibrous layer are different from the cells disposed in the hydrogel layer; and g) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

[0035] In one aspect, the present invention provides a method of fabricating a multilayered construct for tissue-engineered vascular grafts. The method comprises: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) forming a plurality of pores in the fibrous layer; c) disposing endothelial cells on the interior surface of the fibrous layer; and d) forming a hydrogel layer on the exterior surface of the fibrous layer.

[0036] In some embodiments, forming the hydrogel layer of step d) comprises: dl) disposing a cross-linkable polymer composition on the exterior surface of the fibrous layer; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface.

[0037] In some embodiments, forming the hydrogel layer of step d) comprises: dl) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and smooth muscle cells; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface, wherein the hydrogel layer comprises a second biocompatible polymer that is cross-linked, and wherein the smooth muscle cells are disposed in the hydrogel layer.

BRIEF DESCRIPTION OF THE FIGURES

[0038] The figures below are provided by way of example and are not intended to limit the scope of the claimed invention.

[0039] FIG. 1 is an illustration of an exemplary method of fabricating a multi-layered construct for tissue-engineered grafts. FIG. la is an illustration of an exemplary step of providing a fibrous layer, wherein the step comprises electrospinning a biocompatible polymer to form the fibrous layer. FIG lb is an image of an exemplary fibrous layer having a length of about 6 cm and a luminal diameter of about 2.5 mm, with a scanning electron microscope (SEM) image showing randomly aligned fibres of the fibrous layer. FIG. 1c is an illustration of an exemplary step of applying a mechanical force to fibres of the fibrous layer to at least partially align the fibres in an axial direction, wherein the step comprises freezing a solvent that expands in the axial direction during freezing to thereby apply the mechanical force. FIG. Id is an illustration of an exemplary step of treating the fibrous layer to improve a hydrophilicity of the fibrous layer, wherein the step comprises contacting the fibrous layer with a basic solution. FIG. le is an illustration of an exemplary step of disposing a mixture on a surface of the fibrous layer, wherein the mixture comprises a cross-linkable polymer composition and cells (e.g., smooth muscle cells (SMCs)). FIG. If is an illustration of an exemplary step of curing the cross-linkable polymer composition, wherein the curing step comprises irradiating the mixture with an ultraviolet (UV) light for about 1 min. FIG. 1g is an illustration of an exemplary embodiment of the multi-layered construct for tissue-engineered grafts, with an image and an SEM image of the multi-layered construct also shown.

[0040] FIG. 2a is an illustration of a burst pressure test for a multi-layered construct for tissue-engineered grafts, including an image of the burst pressure test, and a graphical representation of burst pressure for multi-layered constructs wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction (red; "FIA-treated"; right bar), and wherein no mechanical force was applied (gray; "Untreated"; left bar). Burst pressures of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation (ns: no signal difference; n = 12; statistical comparisons were not made with the native blood vessel data because the data and error bars were drawn from the literature using different testing protocols and equipment).

[0041] FIG. 2b is an illustration of a suture retention test for a multi-layered construct for tissue-engineered grafts, including an image of the suture retention test, and a graphical representation of suture retention strength for multi-layered constructs wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction (red; "FIA-treated"; right bar), and wherein no mechanical force was applied (gray; "Untreated"; left bar). Suture retention strength of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation (ns: no signal difference; n = 12; statistical comparisons were not made with the native blood vessel data because the data and error bars were drawn from the literature using different testing protocols and equipment).

[0042] FIG. 2c is an illustration of a circumferential tensile strength test for a multi-layered construct for tissue-engineered grafts, including an image of the circumferential tensile strength test.

[0043] FIG. 2c-i is a graphical representation of circumferential tensile strength for multilayered constructs wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction (red; "FIA-treated"; right bar), and wherein no mechanical force was applied (gray; "Untreated"; left bar). Circumferential tensile strength of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation (ns: no signal difference; n = 12; statistical comparisons were not made with the native blood vessel data because the data and error bars were drawn from the literature using different testing protocols and equipment). [0044] FIG. 2c-ii is a graphical representation of Young's modulus for multi-layered constructs wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction (red; "FIA-treated"; right bar), and wherein no mechanical force was applied (gray; "Untreated"; left bar). Young's modulus of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation (ns: no signal difference; n = 12; statistical comparisons were not made with the native blood vessel data because the data and error bars were drawn from the literature using different testing protocols and equipment).

[0045] FIG. 3 is an illustration of an exemplary step of applying a mechanical force to fibres of a fibrous layer to at least partially align the fibres in an axial direction. FIG. 3a is an electrospun fibrous layer, with an SEM image of the fibers of the electrospun fibrous layer also shown (Scale bar = 50 pm). FIG. 3b is an illustration of an exemplary step of disposing the fibrous layer in a chamber and partially filling the chamber with water. FIG. 3c is an illustration of a freezer for freezing the water, wherein the water is frozen in the freezer at -20 °C for about 20 minutes. FIG. 3d is an illustration of the frozen water expanded axially, thereby applying a mechanical force to fibres of the fibrous layer to at least partially align the fibres in the axial direction. FIG. 3e is an image of the fibrous layer, with an SEM image of the fibres at least partially aligned in the axial direction also shown (Scale bar = 50 pm). FIG. 3f is a graphical representation of a percentage of fibres aligned within ± 10° for a fibrous layer wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction ("FIA-treated"), and wherein no mechanical force was applied ("Untreated") (n = 20; ***, P < 0.001).

[0046] FIG. 4a is a perspective view of an exemplary embodiment of a multi-layered construct for tissue-engineered grafts, and a confocal image of endothelial cells (ECs) forming an endothelium on an interior surface of a fibrous layer.

[0047] FIG. 4b provides confocal images of ECs disposed on an interior surface of a fibrous layer of a multi-layered construct for tissue-engineered grafts where a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in an axial direction ("FIA-treated"), and wherein no mechanical force was applied ("Untreated") (Scale bar = 50 pm).

[0048] FIG. 4c is a graphical representation of a percentage of ECs aligned within ± 10° in an axial direction for a fibrous layer wherein a mechanical force was applied to fibres of the fibrous layer to at least partially align the fibres in the axial direction ("FIA-treated"), and wherein no mechanical force was applied ("Untreated"). Quantitative analysis of EC alignment was achieved using Orientation! plugin in Image! comparing EC alignment on Untreated and FIA-treated fibers. Error bars denote upper and lower extreme values (n = 12; ***, p < 0.001). [0049] FIG. 5a is a perspective view of an exemplary embodiment of a multi-layered construct for tissue-engineered grafts, and a confocal image of an arc of the multi-layered construct showing circumferentially aligned (smooth muscle cells (SMCs) in a hydrogel layer (gelatin methacryloyl (GelMA)) with ECs on an interior surface of a fibrous layer, wherein the interior surface defines a lumen (L indicates the location of the lumen); red: aSMA stain, green: CD31 stain).

[0050] FIG. 5b provides confocal images of SMCs in 2, 3, 5, 7, and 10% w/v GelMA hydrogel layers (L indicates the location of the lumen; scale bar = 50 pm).

[0051] FIG. 5c-i is a graphical illustration of Young's modulus of 2, 3, 5, 7, and 10% w/v GelMA hydrogel layers (n = 8; ***, P < 0.001). Error bars denote upper and lower extreme values.

[0052] FIG. 5c-ii is a graphical representation of a percentage of SMCs aligned within ± 10° in a circumferential direction in 2, 3, 5, 7, and 10% w/v GelMA hydrogel layers (n = 12; **, P < 0.01, ***, P < 0.001). Error bars denote upper and lower extreme values.

[0053] FIG. 6a provides confocal images of SMCs in 3% w/v GelMA hydrogel layer after 2, 4, 8, 12, and 24 h of seeding (L indicates the location of the lumen; scale bar = 50 pm).

[0054] FIG. 6b-i is a graphical representation of a percentage of SMCs aligned within ± 10° in a circumferential direction over 7 days of cell culture. Quantitative analysis of SMC morphology was achieved using Orientation! and Analyze Particle plugins in Image! over 7 days of cell culture (n = 12; *, P < 0.05, **, P < 0.01, ***, P < 0.001). Errors bars denote standard deviations.

[0055] FIG. 6b-ii is a graphical representation of cell aspect ratio for the SMCs of FIG. 6b-I (n = 12; *, P < 0.05, **, P < 0.01, ***, P < 0.001). Errors bars denote standard deviations. [0056] FIG. 7a is in illustration of a multi-layered construct for tissue-engineered grafts wherein a fibrous layer of the multi-layered construct includes a v-shaped incision. The V- shaped incision creates a low fibrous layer coverage area ("Low PCL coverage"), a medium fibrous layer coverage area ("Medium PCL coverage"), and a full fibrous layer coverage area ("Full PCL coverage").

[0057] FIG. 7b provides Confocal images of SMCs in the low, medium, and high fibrous layer coverage areas of FIG. 7a (L indicates the location of the lumen; Scale bar = 50 pm).

[0058] FIG. 7c is a graphical representation of a percentage of SMCs aligned within ± 10° in a circumferential direction in the low, medium, and high fibrous layer coverage areas of FIG. 7a (n = 8; **, P < 0.01, ***, P < 0.001).

[0059] FIG. 8a provides an illustration of a method of fabricating a fibrous layer of a multilayered construct for tissue-engineered grafts, wherein the fibrous layer comprises a plurality of pores. FIG. 8a-i depicts fabrication of a fibrous layer via electrospinning. FIG. 8a-ii is an illustration of an exemplary step of forming a plurality of pores in the fibrous layer. FIG. Sa- iii provides an image and SEM images of a fibrous layer comprising plurality of pores (scale bar: 100 pm).

[0060] FIGS. 8b-i and 8b-ii illustrate an exemplary step of forming a hydrogel layer on an exterior surface of a fibrous layer, wherein the step comprises disposing a cross-linkable polymer composition (with or without cells) on the exterior surface of the fibrous layer and curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface.

[0061] FIGS. 8b-iii and 8b-iv illustrate exemplary steps of disposing ECs on the interior surface of the fibrous layer and allowing the ECs to form capillaries in the hydrogel layer.

[0062] FIG. 8b-v is an image of an exemplary embodiment of a multi-layered construct comprising a fibrous layer, wherein the fibrous layer comprises a plurality of pores.

[0063] FIGS. 8c-i and 8b-ii illustrate an exemplary step of forming a hydrogel layer on exterior surfaces of fibrous layers, wherein the step comprises disposing a cross-linkable polymer composition (with or without cells) on the exterior surfaces of the fibrous layers and curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surfaces of the fibrous layers.

[0064] FIGS. 8c-iii and 8c-iv illustrate exemplary steps of disposing ECs on the interior surfaces of the fibrous layers and allowing the ECs to form capillaries in the hydrogel layer.

[0065] FIG. 8c-v is an image of an exemplary embodiment of a multi-layered construct comprising first and second fibrous layers, wherein the fibrous layers each comprise a plurality of pores.

[0066] FIG. 9a is an illustration of a burst pressure test for a multi-layered construct for tissue-engineered grafts, and a graphical representation of burst pressure for multi-layered constructs wherein the fibrous layer comprises a plurality of pores (gray; "Vascular graft + pores"; left bar) and wherein the fibrous layer is free of pores (red; "Vascular graft + pores"; right bar). Burst pressures of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation (Stekelenburg, M., et al., Dynamic straining combined with fibrin gel cell seeding improves strength of tissue-engineered small-diameter vascular grafts. Tissue Engineering Part A, 2009. 15(5): p. 1081-1089; and L'Heureux, N., et al., Human tissue-engineered blood vessels for adult arterial revascularization. Nature medicine, 2006. 12(3): p. 361-365). Error bars represent the standard deviation (n = 12; *: p <0.05; ns: no significant difference, t-test). No statistical comparison was made between the vascular grafts and the multi-layered constructs since the data from the literature was collected using different methods and equipment. [0067] FIG. 9b is an illustration of a suture retention test for a multi-layered construct for tissue-engineered grafts, and a graphical representation of suture retention strength for multilayered constructs wherein the fibrous layer comprises a plurality of pores (gray; "Vascular graft + pores") and wherein the fibrous layer is free of pores (red; "Vascular graft - pores"). Suture retention strengths of native blood vessels, saphenous vein (light blue) and internal mammary artery (dark blue), are also included in the graphical representation. Error bars represent the standard deviation (n = 12; *: p <0.05; ns: no significant difference, t-test). No statistical comparison was made between the vascular grafts and the multi-layered constructs since the data from the literature was collected using different methods and equipment.

[0068] FIG. 9c an illustration of a circumferential tensile strength test for a multi-layered construct for tissue-engineered grafts, and a graphical representation of circumferential tensile strength for multi-layered constructs wherein the fibrous layer comprises a plurality of pores (gray; "Vascular graft + pores"; left bar) and wherein the fibrous layer is free of pores (red; "Vascular graft - pores"; right bar). Circumferential tensile strength of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation. Error bars represent the standard deviation (n = 12; *: p <0.05; ns: no significant difference, t-test). No statistical comparison was made between the vascular grafts and the multi-layered constructs since the data from the literature was collected using different methods and equipment.

[0069] FIG. 9d is a graphical representation of Young's modulus for multi-layered constructs wherein the fibrous layer comprises a plurality of pores (gray; "Vascular graft + pores"; left bar) and wherein the fibrous layer is free of pores (red; "Vascular graft - pores"; right bar). Young's modulus of native blood vessels, saphenous vein (light blue; left bar) and internal mammary artery (dark blue; right bar), are also included in the graphical representation. Error bars represent the standard deviation (n = 12; *: p <0.05; ns: no significant difference, t-test). No statistical comparison was made between the vascular grafts and the multi-layered constructs since the data from the literature was collected using different methods and equipment.

[0070] FIG. 10a is a perspective view of an exemplary embodiment of a multi-layered construct for tissue-engineered grafts, and a confocal image of ECs forming an endothelium on an interior surface of a fibrous layer after 7 days of culture, red: actin filaments stain (ActinRed™ 555), blue: nuclei stain (DAPI). [0071] FIG. 10b provides confocal images of ECs on the interior surface of the fibrous layer at different time points showing the increase of endothelial coverage and proximity of the cells (scale bar = 50 pm).

[0072] FIG. 10c is a graphical representation of EX coverage over the interior surface of the lumen over time. Error bars represent the standard deviation (n = 12; ***: p < 0.001, oneway ANOVA).

[0073] FIG. I la is a perspective view of an exemplary embodiment of a multi-layered construct for tissue-engineered grafts, wherein a fibrous layer of the multi-layered construct comprises a plurality of pores, and a confocal image showing the migration of ECs from an interior surface of the fibrous layer toward a surrounding hydrogel layer (GelMA) through the fibrous layer.

[0074] FIG. 1 lb provides confocal images of ECs at a fibrous layer/hydrogel layer interface in multi-layered constructs wherein the fibrous layer comprises a plurality of pores ("+ pores") and wherein the fibrous layer is free of pores ("- pores"); the dotted line indicates the edge of the fibrous layer. Red: actin filaments stain (ActinRed™ 555), blue: nuclei stain (DAP I) (Scale bar = 50 pm).

[0075] FIG. 11c is a graphical representation of a cell migration in multi-layered constructs having a fibrous layer comprising a plurality of pores ("+ pores") and free of pores ("- pores"). Error bars represent the standard deviation (n = 12; ***: p < 0.001; ns: no significant difference, Student’s t-test).

[0076] FIG. 12a is a perspective view of an exemplary embodiment of a multi-layered construct for tissue-engineered grafts, wherein a fibrous layer of the multi-layered construct comprises a plurality of pores, and a confocal image showing formation of a vascular network in a surrounding hydrogel layer (GelMA 3% w/v, G indicates the GelMA layer; dashed line indicates the outer edge of the sample, red: actin filaments stain (ActinRed™ 555), blue: nuclei stain (DAPI). Scale bar = 50 pm.

[0077] FIG. 12b provides confocal images showing vascular network formation in hydrogel layers of different GelMA concentrations at different time points (red: actin filaments stain (ActinRed™ 555), blue: nuclei stain (DAPI). Scale bar = 50 pm.

[0078] FIG. 12c provides confocal images showing ECs forming vascular networks in a hydrogel layer comprising 3% w/v GelMA in the presence of SMCs (green: GFP-expressing ECs, red: a-SMA (Alexa Fluor 594), blue: Nuclei (DAPI)). Scale bar = 50 pm.

[0079] FIG. 12d provides graphical representations of total vessel length, average vessel area, total number of junctions, and average vessel diameter for vascular networks within hydrogel layers comprising 3% w/v GelMA. Error bars represent the standard deviation (n = 12; ***: p < 0.001, one-way ANOVA).

[0080] FIG. 13a provides confocal images of a hydrogel layer of an exemplary embodiment of a multi-layered construct comprising first and second fibrous layers, wherein the first and second fibrous layers each comprise a plurality of pores. ECs cover interior surfaces of each of the first and second fibrous layers and vascular networks are formed in the hydrogel layer (GelMA) red: actin filaments stain (ActinRed™ 555), blue: nuclei stain (DAP I). L indicates the lumen position, * indicates the fibrous layer (i.e., PCL layer) position and G indicates the hydrogel layer (i.e., GelMA layer) position.

[0081] FIG. 13b provides lightsheet microscope images of the multi-layered construct of FIG. 13a showing the overall vascular network in the hydrogel layer with magnified images of selected regions showing the migration of cells and the vascular network formed; red: actin filaments stain (ActinRed™ 555). L indicates the lumen position, * indicates the fibrous layer (i.e., PCL layer) position and G indicates the hydrogel layer (i.e., GelMA layer) position, arrows indicate some vascular network formation instances.

[0082] FIG. 14a is a NMR spectrum of unmodified 5% w/v gelatin. Far left (green) highlight: Aromatic amino acid. Middle (red) highlight: Methacrylate vinyl group of the MA. Far right (blue) highlight: Lysine methylene.

[0083] FIG. 14b is a NMR spectrum of 5% w/v GelMA. Far left (green) highlight: Aromatic amino acid. Middle (red) highlight: Methacrylate vinyl group of the MA. Far right (blue) highlight: Lysine methylene.

DETAILED DESCRIPTION

[0084] The present invention provides multi-layered constructs for tissue-engineered grafts, including vascularized grafts, and methods of fabricating the same.

[0085] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0086] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0087] I. DEFINITIONS

[0088] The terms "comprises," "comprising," "including," and "having," are inclusive and therefore specify the presence of features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. [0089] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0090] The terminology used herein is for the purpose of describing particular exemplary configurations only and is not intended to be limiting. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. Additional or alternative steps may be employed.

[0091] The terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections. These elements, components, regions, layers and/or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as "first," "second," and other numerical terms do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example configurations.

[0092] As used herein, when an element is referred to as being "on," "engaged to," "connected to," "attached to," or "coupled to" another element, it may be directly on, engaged, connected, attached, or coupled to the other element, or intervening elements may be present. In contrast, when an element is referred to as being "directly on," "directly engaged to," "directly connected to," "directly attached to," or "directly coupled to" another element, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.). As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0093] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells, and so forth. [0094] As used herein, the term "multi-layered construct" refers to a construct for tissue- engineered grafts, including vascularized grafts.

[0095] As used herein, the term "biocompatible polymer" refers to a polymer that does not cause injury or toxicity to living tissue, or an immunological reaction in living tissue.

[0096] As used herein, the term "biodegradable" refers to a material that has a finite halflife in a biological environment (e.g., within a body and/or living tissue).

[0097] As used herein, the term "electrospinning" refers to a fibre fabrication process that uses electric force to draw charged threads of a polymer (e.g., a biocompatible polymer) solution or polymer melt.

[0098] As used herein, the term "cross-linkable moiety" refers to a chemical moiety that forms physical cross-links (i.e., ionic bonds, hydrogen bonds, etc.) and/or chemical cross-links (i.e., covalent bonds) during curing to form a polymer that is cross-linked.

[0099] As used herein, the term "curing agent" refers to a compound that facilitates formation of a chemical cross-link (i.e., covalent bond) between cross-linkable moieties.

[0100] The term "derivative" in relation to any polymer described herein refers to a modified (e.g., chemically modified (e.g., an inclusion or a removal of one or more functional groups) but structurally related polymer that retains or exhibits improved characteristics (e.g., mechanical characteristics) as compared to the underlying polymer.

[0101] II. MULTI-LAYERED CONSTRUCT

[0102] In one aspect, the present invention provides a multi-layered construct for tissue- engineered grafts. The multi-layered construct includes a fibrous layer, a hydrogel layer, and cells.

[0103] A. Fibrous Layer

[0104] The fibrous layer a fibrous layer extending along an axis in a first direction from a first end to a second end. In some embodiments, the fibrous layer has a substantially flat configuration.

[0105] In other embodiments, the fibrous layer is disposed about and extends along the axis in the first direction. In some embodiments, the fibrous layer has an arcuate configuration or a tubular configuration. When the fibrous layer is disposed about and extends along the axis in the first direction, the fibrous layer has an interior surface defining a lumen and an exterior surface, as shown in FIG. 4a. In such embodiments, the first direction may be further defined as an axial direction.

[0106] The fibrous layers comprises fibres of a first biocompatible polymer. In some embodiments, the fibres are at least partially aligned in the first direction. In some embodiments, at least about 35% of the fibres (e.g., about 35%, about 35.5%, about 36%, about 36.5%, about 37%, about 37.5%, about 38%, about 38.5%, about 39%, about 39.5%, about 40%, about 40.5%, about 41%, about 41.5%, about 42%, about 42.5%, about 43%, about 43.5%, about 44%, about 44.5%, about 45%, about 45.5%, about 46%, about 46.5%, about 47%, about 47.5%, about 48%, about 48.5%, about 49%, about 49.5%, about 50%, about 50.5%, about 51%, about 51.5%, about 52%, about 52.5%, about 53%, about 53.5%, about 54%, about 54.5%, about 55%, about 55.5%, about 56%, about 56.5%, about 57%, about 57.5%, about 58%, about 58.5%, about 59%, about 59.5%, about 60%, and so on) of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 37.5% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 40% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 37.5% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 40% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, at least about 42.5% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In other embodiments, from about 37.5% to about 57.5% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, from about 40% to about 50% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction. In some embodiments, the fibres of the first biocompatible polymer are subjected to a mechanical force to thereby at least partially align the fibres in the first direction.

[0107] The first biocompatible polymer may be any polymer that is compatible with a biological environment. Suitable biocompatible polymers will be familiar to persons skilled in the art, illustrative examples of which include a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Thus, in some embodiments, the first biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). In some embodiments, the first biocompatible polymer is a polyester. In some embodiments, the first biocompatible polymer is a polyether. In some embodiments, the first biocompatible polymer is a polyamide. In some embodiments, the first biocompatible polymer is a polyanhydride. In some embodiments, the first biocompatible polymer is a polyesteramide. In some embodiments, the first biocompatible polymer is a polysaccharide. In some embodiments, the first biocompatible polymer is a polysiloxane. In some embodiments, the first biocompatible polymer is a fluoropolymer. In some embodiments, the first biocompatible polymer is a polycarbonate. In some embodiments, the first biocompatible polymer is an acrylate polymer. In some embodiments, the first biocompatible polymer is a polyether ketone. In some embodiments, the first biocompatible polymer is a polyoxazoline. In some embodiments, the first biocompatible polymer is a polysulfone. In some embodiments, the first biocompatible polymer is a polyurethane. In some embodiments, the first biocompatible polymer is a proteinaceous polymer. In some embodiments, the first biocompatible polymer is silk (e.g., silk worm silk, spider silk, etc.) or a derivative thereof. In some embodiments, the first biocompatible polymer is collagen or a derivative thereof. In some embodiments, the first biocompatible polymer is elastin or a derivative thereof. In some embodiments, the second biocompatible polymer is tropoelastin or a derivative thereof (e.g., methacrylated tropoelastin). In other embodiments, the first biocompatible polymer is a combination of any of the foregoing polymers (e.g., a copolymer (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). In some embodiments, the first biocompatible polymer is a polyester, a fluoropolymer, silk or a derivative thereof, or a combination thereof.

[0108] Suitable polyesters will be familiar to persons skilled in the art and include, by way of non-limiting example, polylactide (PLA), poly(lactic acid-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), poly(3 -hydroxybutyrate) (PHB), poly(3 -hydroxybutyrate-co-3 -hydroxy valerate), poly((3 -hydroxyval erate), poly(3 - hydroxyhexanoate), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4- hydroxyhexanoate), polycaprolactone (PCL), poly(ethylene terephthalate), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the first biocompatible polymer is PLA. In some embodiments, the first biocompatible polymer is PLGA. In some embodiments, the first biocompatible polymer is PGA. In some embodiments, the first biocompatible polymer is PHA. In some embodiments, the first biocompatible polymer is PHB. In some embodiments, the first biocompatible polymer is poly(3-hydroxybutyrate-co-3-hydroxyvalerate). In some embodiments, the first biocompatible polymer is poly((3 -hydroxyval erate). In some embodiments, the first biocompatible polymer is poly(3 -hydroxyhexanoate). In some embodiments, the first biocompatible polymer is poly(4-hydroxybutyrate). In some embodiments, the first biocompatible polymer is poly(4-hydroxyvalerate). In some embodiments, the first biocompatible polymer is poly(4-hydroxyhexanoate). In some embodiments, the first biocompatible polymer is PCL. In some embodiments, the first biocompatible polymer is polyethylene terephthalate). In other embodiments, the first biocompatible polymer is a combination of any of the foregoing polyesters (e.g., a copolymer (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[0109] Suitable polyethers will be familiar to persons skilled in the art and include, by way of non-limiting example, polyethylene glycol (PEG), polypropylene oxide), poly(l,3- trimethylene oxide), poly(l,4-tetram ethylene oxide), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyamides will be familiar to persons skilled in the art and include, by way of non-limiting example, nylon 6, nylon 66, nylon 610, Kevlar (poly(azanediyl-l,4- phenyleneazanediylterephthaloyl)), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyanhydrides will be familiar to persons skilled in the art and include, by way of non-limiting example, poly(adipic anhydride), poly(suberic anhydride), poly(sebacic anhydride), and poly(dodecanedioic anhydride), poly(maleic anhydride), poly(benzoic anhydride), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polysaccharides will be familiar to persons skilled in the art and include, by way of non-limiting example, chitin and derivatives thereof (e.g., deacetylated chitin, etc.), chitosan and derivatives thereof (e.g., chitosan acetate, chitosan maleate, chitosan glyconate, chitosan sorbate, chitosan formate, chitosan salicylate, chitosan propionate, chitosan lactate, chitosan itaconate, chitosan niacinate, chitosan gallate, chitosan glutamate, carboxymethyl chitosan, etc.), cellulose and derivatives thereof (e.g., methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), hydroxypropyl methyl cellulose (HPMC), hydroxyethyl cellulose (HEC), etc.), starch, hyaluronic acid, heparin, and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polysiloxanes will be familiar to persons skilled in the art and include, by way of non-limiting example, polydimethylsiloxane (PDMS). Suitable acrylate polymers will be familiar to persons skilled in the art and include, by way of non-limiting example, alkyl(meth)acrylate polymers, aromatic(meth)acrylate polymers, and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyether ketones will be familiar to persons skilled in the art and include, by way of non-limiting example, polyetherketoneketone (PEKK), polyetheretherketone (PEEK), Polyether ether ketone ketone (PEKK), polyetherketoneetherketoneketone (PEKEKK), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyoxazolines will be familiar to persons skilled in the art and include, by way of non-limiting example, poly(2-ethyl-2-oxazoline) (PEOX), poly(2-methyl-2-oxazoline) (PMeOx), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polysulfones will be familiar to persons skilled in the art and include, by way of non-limiting example, polysulfone (PSU), polyethersulfone (PES/PESU), polyphenylene sulfone (PPSU), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyurethanes will be familiar to persons skilled in the art and include, by way of non-limiting example, Bionate® (a polycarbonate polyurethane (PCU) (DSM Biomedical; Exton, Pennsylvania), Bionate® II (DSM Biomedical; Exton, Pennsylvania), Biospan® (a segmented polyether polyurethane (SPU) (DSM Biomedical; Exton, Pennsylvania), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). Suitable polyesteramides will be familiar to persons skilled in the art and include, by way of nonlimiting example, polyesteramides from s-caprolactam and s-caprolactone, polyesteramides from s-caprolactam, 1,4-butanediol, and adipic acid, and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[0110] Suitable proteinaceous polymers will be familiar to persons skilled in the art and include, by way of non-limiting example, gelatin or derivatives thereof (e.g., gelatin methacryloyl (GelMA)), collagen or derivatives thereof, albumin or derivatives thereof, haemoglobin or derivatives thereof, fibrinogen or derivatives thereof, fibroin or derivatives thereof, fibronectin or derivatives thereof, elastin or derivatives thereof, tropoelastin or derivatives thereof (e.g., methacrylated tropoelastin), keratin or derivatives thereof, laminin or derivatives thereof, casein or derivatives thereof, tenascin or derivatives thereof, vitronectin or derivatives thereof, reticulin or derivates thereof, silk or derivatives thereof, recombinant silk proteins or derivatives thereof, a precursor of any of the foregoing proteinaceous polymers or derivatives thereof, and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[oni] Suitable fluoropolymers will be familiar to persons skilled in the art and include, by way of non-limiting example, polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), tetrafluoroethylene (TFE), perfluoro(propyl vinyl ether) (PFA), polychlorotrifluoroethylene (PCTFE), ethylene-chlorotrifluoroethylene (ECTFE), (ETFE), polyvinylidene fluoride (PVDF), polyvinyfluoride (PVF), poly(fluorene-co-thiophene) (PFT), and any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)). In some embodiments, the first biocompatible polymer is PTFE. In some embodiments, the first biocompatible polymer is FEP. In some embodiments, the first biocompatible polymer is TFE. In some embodiments, the first biocompatible polymer is PFA. In some embodiments, the first biocompatible polymer is PCTFE. In some embodiments, the first biocompatible polymer is ECTFE. In some embodiments, the first biocompatible polymer is ETFE. In some embodiments, the first biocompatible polymer is PVDF. In some embodiments, the first biocompatible polymer is PVF. In some embodiments, the first biocompatible polymer is PFT. In other embodiments, the first biocompatible polymer is a combination of any of the foregoing fluoropolymers (e.g., a copolymer (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[0112] In some embodiments, the first biocompatible polymer includes cross-links (i.e., chemical and/or physical cross-links).

[0113] In some embodiments, the fibres of the first biocompatible polymer are electrospun. In some embodiments, the fibres of the first biocompatible polymer are treated (e.g., with a basic solution (e.g., an NaOH solution) to increase hydrophilicity of the fibres.

[0114] In some embodiments, first biocompatible polymer is biodegradable. In other embodiments, the first biocompatible polymer is not biodegradable.

[0115] In some embodiments, the the fibrous layer further comprises a plurality of pores. In some embodiments, the plurality of pores are distributed at least one of along the axis and about the axis. In some embodiments, the plurality of pores are distributed along the axis. In some embodiments, the plurality of pores are distributed about the axis. In some embodiments, the pores are be distributed along and about the axis. The plurality of pores may be distributed evenly or unevenly along and/or about the axis.

[0116] In some embodiments, the pores have a substantially circular configuration. However, the pores may have any suitable configuration for facilitating migrations of cells from the fibrous layer to the hydrogel layer. In some embodiments, the pores have a substantially square, substantially rectangular, substantially triangular, or an irregular configuration.

[0117] In some embodiments, when the pores have a substantially circular configuration, the plurality of pores each have a diameter of from about 50 pm to about 1,000 pm. In some embodiments, the plurality of pores may each have a diameter of from about 50 pm to about 750 pm. In some embodiments, the plurality of pores each have a diameter of from about 50 pm to about 500 pm. In some embodiments, the plurality of pores each have a diameter of from about 50 pm to about 300 pm. In some embodiments, the plurality of pores each have a diameter of from about 100 pm to about 300 pm. In some embodiments, the plurality of pores each have a diameter of from about 100 pm to about 200 pm. In some embodiments, the plurality of pores each have a diameter of about 150 pm. In some embodiments, the plurality of pores each have a diameter of greater than about 0.05 pm (PAC, 1972, 31, 511,' Manual of Symbols and Terminology for Physicochemical Quantities and Units, Appendix II: Definitions, Terminology and Symbols in Colloid and Surface Chemistry, and PAC, 1976, 46, 71. (Manual of Symbols and Terminology for Physicochemical Quantities and Units - Appendix II. Definitions, Terminology and Symbols in Colloid and Surface Chemistry. Part II: Heterogeneous Catalysts'). It is to be understood that the plurality of pores may comprise one or more pores that are not precisely circular in perimeter, but rather comprise an irregular perimeter. In some embodiments, where each of a plurality of pores comprises an irregular perimeter, the plurality of pores each have an average diameter of from about 50 pm to about 1,000 pm, from about 50 pm to about 750 pm, from about 50 pm to about 500 pm, from about 50 pm to about 300 pm, from about 100 pm to about 300 pm, from about 100 pm to about 200 pm, or about 150 pm. In some embodiments, where each of a plurality of pores comprises an irregular perimeter, the plurality of pores each have an average width of from about 50 pm to about 1,000 pm, from about 50 pm to about 750 pm, from about 50 pm to about 500 pm, from about 50 pm to about 300 pm, from about 100 pm to about 300 pm, from about 100 pm to about 200 pm, or about 150 pm.

[0118] Alternatively, or in addition, the fibrous layer may suitably have a thickness such that one or more mechanical properties of the construct approximates one or more corresponding mechanical properties of the vessel it is intended to replace, such as, but not limited to, suture retention strength, modulus, and ultimate tensile strength. Such thicknesses can be readily determined by persons skilled in the art. In some embodiments, the fibrous layer has a thickness of from about 1 pm to about 10,000 pm. In some embodiments, the fibrous layer has a thickness of from about 1 pm to about 1,000 pm. In some embodiments, the fibrous layer has a thickness of from about 1 pm to about 750 pm. In some embodiments, the fibrous layer has a thickness of from about 1 pm to about 500 pm. In some embodiments, the fibrous layer has a thickness of from about 50 pm to about 500 pm. In some embodiments, the fibrous layer may have a thickness of from about 100 pm to about 300 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, or about 300 pm.

[0119] It is to be understood that the construct or fibrous layer described herein may have any suitable length, and/or width (e.g., lumen diameter and thickness of the fibrous layer), and that such length and/or width will be determined, at least in part, by the intended purpose of the construct. For example, where the construct is intended for a TEVG to replace a damaged or diseased natural blood vessel, the construct or fibrous layer will suitably have a length and/or width that approximates the length and/or width of the vessel it is intended to replace. Such lengths and widths can be readily determined by persons skilled in the art. In some embodiments, the fibrous layer has a length of from about 0.1 cm to about 10 cm (e.g., about 0.1 cm, about 0.5 cm, about 1.0 cm, about 1.5 cm, about 2.0 cm, about 2.5 cm, about 3.0 cm, about 3.5 cm, about 4.0 cm, about 4.5 cm, about 5.0 cm, about 5.5 cm, about 6.0 cm, about 6.5 cm, about 7.0 cm, about 7.5 cm, about 8.0 cm, about 8.5 cm, about 9.0 cm, about 9.5 cm or about 10.0 cm). In an embodiment, the fibrous layer has a length of from about 0.1 cm to about

7.5 cm.

[0120] When the interior surface of the fibrous layer defines a lumen, the lumen has a diameter of from about 0.1 mm to about 10 mm (e.g., about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, about 5.5 mm, about 6.0 mm, about 6.5 mm, about 7.0 mm, about

7.5 mm, about 8.0 mm, about 8.5 mm, about 9.0 mm, about 9.5 mm or about 10.0 mm). In an embodiment, the lumen has a diameter of from about 0.1 mm to about 7.5 mm, preferably from about 0.1 mm to about 5 mm, or more preferably from about 1 mm to about 5 mm (e.g., about

2.5 mm). In an embodiment, the lumen has a diameter of about 2.5 mm.

[0121] B. Hydrogel Layer

[0122] The hydrogel layer is disposed on the fibrous layer. The hydrogel layer may be disposed only partially on, or entirely on, a surface of the fibrous layer. In some embodiments, the hydrogel layer is disposed only partially on a surface of the fibrous layer. In other embodiments, the hydrogel layer is disposed on an entire surface of the fibrous layer.

[0123] In some embodiments, the hydrogel layer is disposed directly on the fibrous layer. It will be appreciated that when the hydrogel layer is disposed directly on the fibrous layer, the hydrogel layer may penetrate a surface of the fibrous layer.

[0124] When the fibrous layer is disposed about and extends along the axis, and includes the interior surface and exterior surface, the hydrogel layer is disposed on the exterior surface of the fibrous layer and about the axis. In some embodiments, the hydrogel layer is disposed only partially on the exterior surface of the fibrous layer and about the axis. In other embodiments, the hydrogel layer is disposed on the entire exterior surface of the fibrous layer and about the axis. When the hydrogel layer is disposed directly on the fibrous layer, the hydrogel layer may penetrate a surface (e.g., the exterior surface) of the fibrous layer.

[0125] The hydrogel layer comprises a second biocompatible polymer. The second biocompatible polymer may be any polymer that is compatible with a biological environment. In some embodiments, the first and second biocompatible polymers are different. In some embodiments, the first and second biocompatible polymers are the same.

[0126] In some embodiments, the second biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.). In some embodiments, the second biocompatible polymer is a polyester. In some embodiments, the second biocompatible polymer is a polyester. In some embodiments, the second biocompatible polymer is a polyether. In some embodiments, the second biocompatible polymer is a polyamide. In some embodiments, the second biocompatible polymer is a polyanhydride. In some embodiments, the second biocompatible polymer is a polyesteramide. In some embodiments, the second biocompatible polymer is a polysaccharide. In some embodiments, the second biocompatible polymer is a polysiloxane. In some embodiments, the second biocompatible polymer is a fluoropolymer. In some embodiments, the second biocompatible polymer is a polycarbonate. In some embodiments, the second biocompatible polymer is an acrylate polymer. In some embodiments, the second biocompatible polymer is a polyether ketone. In some embodiments, the second biocompatible polymer is a polyoxazoline. In some embodiments, the second biocompatible polymer is a polysulfone. In some embodiments, the second biocompatible polymer is a polyurethane. In some embodiments, the second biocompatible polymer is a proteinaceous polymer. In some embodiments, the second biocompatible polymer is silk (e.g., silk worm silk, spider silk, etc.). In some embodiments, the second biocompatible polymer is gelatin (Gel). In some embodiments, the second biocompatible polymer is gelatin methacryloyl (GelMA). In some embodiments, the second biocompatible polymer is an acrylate polymer. In some embodiments, the second biocompatible polymer is a polyoxazoline. In some embodiments, the second biocompatible polymer is elastin or a derivative thereof. In some embodiments, the second biocompatible polymer is tropoelastin or a derivative thereof (e.g., methacrylated tropoelastin). In some embodiments, the second biocompatible polymer is collagen or a derivative thereof. In some embodiments, the second biocompatible polymer is a recombinant silk protein or a derivative thereof. In other embodiments, the second biocompatible polymer is a combination of any of the foregoing polymers (e.g., a copolymer (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[0127] Suitable polyesters, polyethers, polyamides, polyesteramides, polyanhydrides, polyesteramides, polysaccharides, polysiloxanes, fluoropolymers, polycarbonates, acrylate polymers, polyoxazolines, acrylate polymers, polyether ketones, polyoxazolines, polysulfones, proteinaceous polymers, and polyurethanes will be familiar to persons skilled in the art, illustrative examples of which are described herein in relation to the first biocompatible polymer.

[0128] In some embodiments, the second biocompatible polymer is a polylactide (PLA) derivative, a poly(lactic acid-co-glycolic acid) (PLGA) derivative, a polyglycolic acid (PGA) derivative, a polyhydroxyalkanoate (PHA) derivative, a poly(3 -hydroxybutyrate) (PHB) derivative, a poly(3-hydroxybutyrate-co-3 -hydroxyvalerate) derivative, a poly((3- hy dr oxy valerate) derivative, a poly(3 -hydroxyhexanoate) derivative, a poly(4- hydroxybutyrate) derivative, a poly(4-hydroxyvalerate) derivative, a poly(4- hydroxyhexanoate) derivative, a polycaprolactone (PCL), poly(ethylene terephthalate) derivative, polyacrylic acid (PAA) or derivatives thereof, polyethylene glycol (PEG) or derivatives thereof, polyvinyl alcohol (PVA) or derivatives thereof, polyvinylpyrrolidone (PVP) or derivatives thereof, polyacrylamide or derivatives thereof, hyaluronic acid or derivatives thereof, chitin or derivatives thereof, chitosan or derivatives thereof, alginate or derivatives thereof, gelatin (Gel), gelatin methacryloyl (GelMA), xanthan gum, carrageenan or derivatives thereof, dextran or derivatives thereof, starch or derivatives thereof, cellulose or derivatives thereof, elastin or derivatives thereof, tropoelastin or derivatives thereof, collagen or derivatives thereof, silk or derivatives thereof, recombinant silk proteins or derivatives thereof, or any combination thereof. In some embodiments, the second biocompatible polymer is polylactide (PLA), poly(lactic acid-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), poly(3 -hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3- hydroxyval erate), poly((3-hydroxyval erate), poly(3 -hydroxyhexanoate), poly(4- hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), polycaprolactone (PCL), poly(ethylene terephthalate), polyacrylic acid (PAA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinyl pyrollidone (PVP), polyacrylamide, hyaluronic acid, chitin or derivatives thereof, chitosan or derivatives thereof, alginate or derivatives thereof, gelatin (Gel), gelatin methacryloyl (GelMA), xanthan gum, carrageenan or derivatives thereof, dextran or derivatives thereof, starch or derivatives thereof, cellulose or derivatives thereof, elastin or derivatives thereof, collagen or derivatives thereof, recombinant silk proteins or derivatives thereof, or any combination thereof (e.g., a copolymer thereof (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.).

[0129] In some embodiments, the second biocompatible polymer is PLA. In some embodiments, the second biocompatible polymer is PLGA. In some embodiments, the second biocompatible polymer is PGA. In some embodiments, the second biocompatible polymer is PHA. In some embodiments, the second biocompatible polymer is PHB. In some embodiments, the second biocompatible polymer is poly(3-hydroxybutyrate-co-3- hy dr oxy valerate). In some embodiments, the second biocompatible polymer is polyphydroxy valerate). In some embodiments, the second biocompatible polymer is poly(3- hydroxyhexanoate). In some embodiments, the second biocompatible polymer is poly(4- hy dr oxybutyrate). In some embodiments, the second biocompatible polymer is poly(4- hy dr oxy valerate). In some embodiments, the second biocompatible polymer is poly(4- hydroxyhexanoate). In some embodiments, the second biocompatible polymer is PCL. In some embodiments, the second biocompatible polymer is poly(ethylene terephthalate).

[0130] In some embodiments, the second biocompatible polymer is a PLA derivative. In some embodiments, the second biocompatible polymer is a PLGA derivative. In some embodiments, the second biocompatible polymer is a PGA derivative. In some embodiments, the second biocompatible polymer is a PHA derivative (e.g., PHA sulfonate). In some embodiments, the second biocompatible polymer is a PHB derivative. In some embodiments, the second biocompatible polymer is a poly(3-hydroxybutyrate-co-3-hydroxyval erate) derivative. In some embodiments, the second biocompatible polymer is a polyphydroxy valerate) derivative. In some embodiments, the second biocompatible polymer is a poly(3 -hydroxyhexanoate) derivative. In some embodiments, the second biocompatible polymer is a poly(4-hydroxybutyrate) derivative. In some embodiments, the second biocompatible polymer is a poly(4-hydroxyval erate) derivative. In some embodiments, the second biocompatible polymer is a poly(4-hydroxyhexanoate) derivative. In some embodiments, the second biocompatible polymer is a PCL derivative. In some embodiments, the second biocompatible polymer is a poly(ethylene terephthalate) derivative. In some embodiments, the second biocompatible polymer is PAA or a derivative thereof. In some embodiments, the second biocompatible polymer is PEG or a derivative thereof. In some embodiments, the second biocompatible polymer is PVA or a derivative thereof. In some embodiments, the second biocompatible polymer is PVP or a derivative thereof. In some embodiments, the second biocompatible polymer is polyacrylamide or a derivative thereof. In some embodiments, the second biocompatible polymer is hyaluronic acid or a derivative thereof. In some embodiments, the second biocompatible polymer is chitin or a derivative thereof (e.g., deacetylated chitin, etc.). In some embodiments, the second biocompatible polymer is chitosan or a derivative thereof (e.g., chitosan acetate, chitosan maleate, chitosan glyconate, chitosan sorbate, chitosan formate, chitosan salicylate, chitosan propionate, chitosan lactate, chitosan itaconate, chitosan niacinate, chitosan gallate, chitosan glutamate, carboxymethyl chitosan, etc.). In some embodiments, the second biocompatible polymer is alginate or a derivative thereof (e.g., alginate methacrylate (ALMA)). In some embodiments, the second biocompatible polymer is Gel. In some embodiments, the second biocompatible polymer is GelMA. In some embodiments, the second biocompatible polymer is xanthan gum. In some embodiments, the second biocompatible polymer is carrageenan or a derivative thereof. In some embodiments, the second biocompatible polymer is dextran or a derivative thereof. In some embodiments, the second biocompatible polymer is starch or a derivative thereof. In some embodiments, the second biocompatible polymer is cellulose or a derivative thereof (e.g., methyl cellulose, nitrocellulose, hydroxypropyl cellulose, carboxymethylcellulose (CMC), hydroxypropyl methyl cellulose (HPMC), hydroxyethyl cellulose (HEC), etc.). In some embodiments, the second biocompatible polymer is elastin or a derivative thereof. In some embodiments, the second biocompatible polymer is tropoelastin or a derivative thereof. In some embodiments, the second biocompatible polymer is collagen or a derivative thereof. In some embodiments, the second biocompatible polymer is silk or a derivative thereof. In some embodiments, the second biocompatible polymer is recombinant silk proteins or derivatives thereof. In other embodiments, the second biocompatible polymer is a combination of any of the foregoing polymers (e.g., a copolymer (e.g., a biopolymer, terpolymer, a quaterpolymer, etc.)).

[0131] In some embodiments, the second biocompatible polymer includes cross-links (i.e., chemical and/or physical cross-links).

[0132] In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 1% w/v to about 15% w/v (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% w/v) based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 1% w/v to about 10% w/v based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 2% w/v to about 6% w/v based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 3% w/v to about 6% w/v based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of from about 3% w/v to about 5% w/v based on the total volume of the hydrogel layer.

[0133] In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of about 3% w/v or about 5% w/v based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of about 3% w/v based on the total volume of the hydrogel layer. In some embodiments, the second biocompatible polymer is present in the hydrogel layer in an amount of about 5% w/v based on the total volume of the hydrogel layer.

[0134] In some embodiments, the hydrogel layer has a Young's modulus of from about 1 kPa to about 180 kPa. In some embodiments, the hydrogel layer has a Young's modulus of from about 1 kPa to about 60 kPa. In some embodiments, the hydrogel layer has a Young's modulus of from about 1 kPa to about 30 kPa. In some embodiments, the hydrogel layer has a Young's modulus of from about 1 kPa to about 20 kPa. In some embodiments, the hydrogel layer has a Young's modulus of from about 3 kPa to about 20 kPa.

[0135] In some embodiments, second biocompatible polymer is biodegradable. In other embodiments, the second biocompatible polymer is not biodegradable.

[0136] In some embodiments, the first and second biocompatible polymers are biodegradable. In other embodiments, the first and second biocompatible polymers are not biodegradable. In some embodiments, one of the first and second biocompatible polymers is biodegradable and the other of the first and second biocompatible polymers is not biodegradable. In some embodiments, the first biocompatible polymer is biodegradable and the second biocompatible polymer is not biodegradable. In some embodiments, the first biocompatible polymer is not biodegradable and the second biocompatible polymer is biodegradable.

[0137] When the first and second biocompatible polymers are biodegradable, one of the first and second biocompatible polymers may biodegrade at a faster rate than the other of the first and second biocompatible polymers. For example, the second biocompatible polymer may biodegrade at a faster rate than the first biocompatible polymer. In this manner, the hydrogel layer may biodegrade while the multi-layered construct retains mechanical properties associated with the fibrous layer.

[0138] In some embodiments, the hydrogel layer has a length of about the length of the fibrous layer, as described herein. In some embodiments, the hydrogel layer has a length of from about 0.1 cm to about 10 cm (e.g., about 0.1 cm, about 0.5 cm, about 1.0 cm, about 1.5 cm, about 2.0 cm, about 2.5 cm, about 3.0 cm, about 3.5 cm, about 4.0 cm, about 4.5 cm, about 5.0 cm, about 5.5 cm, about 6.0 cm, about 6.5 cm, about 7.0 cm, about 7.5 cm, about 8.0 cm, about 8.5 cm, about 9.0 cm, about 9.5 cm or about 10.0 cm). In some embodiments, the hydrogel layer has a length of from about 0.1 cm to about 7.5 cm.

[0139] It is to be understood that the hydrogel layer described herein may have any suitable thickness, and that such thicknesses will be determined, at least in part, by the intended purpose of the construct. For example, where the construct is intended for a TEVG to replace a damaged or diseased natural blood vessel, the hydrogel layer may suitably have a thickness such that the total thickness of the construct approximates the thickness of the vessel it is intended to replace. Alternatively, or in addition, the hydrogel layer may suitably have a thickness that, when combined with the thickness of the fibrous layer, one or more of the mechanical properties of the construct approximates one or more corresponding mechanical properties of the vessel it is intended to replace, such as, but not limited to, suture retention strength, modulus, ultimate tensile strength, and burst pressure. Such thicknesses can be readily determined by persons skilled in the art. In some embodiments, the hydrogel layer has a thickness of from about 1 pm to about 10,000 pm. In some embodiments, the hydrogel layer has a thickness of from about 1 pm to about 2,000 pm. In some embodiments, the hydrogel layer has a thickness of from about 100 pm to about 2,000 pm. In some embodiments, the hydrogel layer has a thickness of from about 100 pm to about 1,500 pm. In some embodiments, the hydrogel layer has a thickness of from about 500 pm to about 1,500 pm. In some embodiments, the hydrogel layer has a thickness of from about 600 pm to about 1,000 pm (e.g., about 800 pm). In some embodiments, the hydrogel layer has a thickness of from about 600 pm to about 900 pm. In some embodiments, the hydrogel layer has a thickness of from about 700 pm to about 900 pm. In some embodiments, the hydrogel layer has a thickness of about 800 pm.

[0140] C. Cells

[0141] The multi-layered construct described herein comprises cells disposed on the fibrous layer and/or in the hydrogel layer. [0142] In some embodiments, cells are disposed on the fibrous layer. It is to be understood that the type of cell or cells to be disposed on the fibrous layer will depend on the intended use of the construct. Suitable cell types will be familiar to persons skilled in the art, illustrative examples of which include stem cells, epithelial cells, and endothelial cells. In some embodiments, the cells disposed on the fibrous layer are endothelial cells.

[0143] When the fibrous layer comprises fibres at least partially aligned in the first direction, the cells disposed on the fibrous layer may be substantially aligned in the first direction (for example, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of the cells are aligned within ± 10° of the first direction). Where the fibrous layer comprises fibres at least partially aligned in the first direction, the fibrous layer is disposed about and extends along the axis in the first direction, and the fibrous layer has an interior surface defining a lumen and an exterior surface, the cells may be disposed on the interior surface of the fibrous layer and substantially aligned in the first direction (for example, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of the cells are aligned within ± 10° of the first direction).

[0144] In some embodiments, at least about 50% of the cells (e.g., about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) disposed on the fibrous layer are aligned within ± 10° of the first direction. In some embodiments, at least about 55% of the cells disposed on the fibrous layer may be aligned within ± 10° of the first direction. In some embodiments, at least about 60% of the cells disposed on the fibrous layer are aligned within ± 10° of the first direction. In some embodiments, at least about 65% of the cells disposed on the fibrous layer are aligned within ± 10° of the first direction. Without wishing to be bound by theory or mode of operation, it is believed that partial alignment of the fibres of the first biocompatible polymer in the first direction facilitates spontaneous alignment of the cells on the fibrous layer in the first direction. [0145] Where the fibrous layer includes a plurality of pores, as described herein, cells disposed on the fibrous layer may migrate through the plurality of pores and into the hydrogel layer. In some embodiments, the migrating cells form a vascular network within the hydrogel layer. In some embodiments, the vascular network will extend beyond the surface of the hydrogel layer.

[0146] In some embodiments, cells are disposed on the hydrogel layer. It is to be understood that the type of cell or cells to be disposed on the hydrogel layer will depend on the intended use of the construct. Suitable cell types will be familiar to persons skilled in the art, illustrative examples of which include stem cells, epithelial cells, smooth muscle cells, and endothelial cells. In some embodiments, the cells disposed on the hydrogel layer are smooth muscle cells (e.g., vascular smooth muscle cells).

[0147] When the fibrous layer includes fibres at least partially aligned in the first direction, the cells disposed in the hydrogel layer may be substantially aligned in a second direction (e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70% of the cells are aligned within ± 10° of the second direction). The second direction is different from the first direction. When the fibrous layer fibrous layer is disposed about and extends along the axis in the first direction, and the hydrogel is disposed on the exterior surface of the fibros layer and about the axis, the second direction may be further defined as the circumferential direction about the axis.

[0148] In some embodiments, at least about 30% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 35% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 40% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 45% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 50% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 55% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 60% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 65% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction. In some embodiments, at least about 70% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction.

[0149] Without wishing to be bound by theory or mode of operation, it is believed that partial alignment of the fibres of the first biocompatible polymer in the first direction facilitates spontaneous alignment of the cells in the hydrogel layer in the second direction.

[0150] In some embodiments, more than one type of cell is present in the hydrogel layer. For example, smooth muscle cells may be disposed in the hydrogel layer and a vascular network of endothelial cells may also be present in the hydrogel layer (e.g., when pores in the fibrous layer allow endothelial cells to migrate from the fibrous layer to the hydrogel layer). [0151] In some embodiments, the multi-layered construct is configured to fit within a body lumen (e.g., an artery, an arteriole, a capillary, a vein, a venule, etc.). [0152] In some embodiments, the multi-layered construct comprises endothelial cells disposed on the fibrous layer and smooth muscle cells disposed in the hydrogel layer. The endothelial cells disposed on the fibrous layer may be substantially aligned in the first direction. The smooth muscle cells may be substantially aligned in the second direction.

[0153] In some embodiments, the multi-layered construct has a burst pressure of at least about 500 mm Hg (e.g., at least about 500 mm Hg, at least about 1000 mm Hg, at least about 1,500 mm Hg, at least about 2,000 mm Hg, at least about 2,500 mm Hg, at least about 3,000 mm Hg, or at least about 3,500 mm Hg). In some embodiments, the multi-layered construct has a burst pressure of from about 500 mm Hg to about 3,500 mm Hg (e.g., about 500 mm Hg, about 1000 mm Hg, about 1,500 mm Hg, about 2,000 mm Hg, about 2,500 mm Hg, about 3,000 mm Hg, about 3,500 mm Hg). In some embodiments, the multi-layered construct has a burst pressure of from about 750 mm Hg to about 3,000 mm Hg. In some embodiments, the multilayered construct has a burst pressure of from about 1,000 mm Hg to about 2,500 mm Hg. In some embodiments, the multi-layered construct has a burst pressure of from about 1,000 mm Hg to about 2,500 mm Hg. In some embodiments, the multi-layered construct has a burst pressure of from about 1,250 mm Hg to about 2,500 mm Hg. In some embodiments, the multilayered construct has a burst pressure of from about 1,500 mm Hg to about 2,500 mm Hg. In some embodiments, the multi-layered construct has a burst pressure of from about 1,500 mm Hg to about 2,000 mm Hg.

[0154] In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 50 gf to about 350 gf. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 100 gf to about 300 gf. In some embodiments, the multilayered construct has a suture retention strength measured according to ISO 7198:2017 of from about 125 gf to about 300 gf. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 150 gf to about 300 gf. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 175 gf to about 300 gf. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 200 gf to about 300 gf. In some embodiments, the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 200 gf to about 250 gf.

[0155] In some embodiments, the multi-layered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 0.5 MPa to about 5.5 MPa. In some embodiments, the multi-layered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 1.0 MPa to about 4.5 MPa. In some embodiments, the multilayered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 1.5 MPa to about 4 MPa. In some embodiments, the multi-layered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 1.5 MPa to about 3.5 MPa. In some embodiments, the multi-layered construct has an ultimate tensile strength measured according to ISO 7198:2017 of from about 2.0 MPa to about 3.0 MPa.

[0156] In some embodiments, the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 10 MPa (e.g., about 0.1 mPa, about 0.5 mPa, about 1.0 mPa, about 2.0 mPa, about 3.0 mPa, about 4.0 mPa, about 5.0 mPa, about 6.0 mPa, about 7.0 mPa, about 8.0 mPa, about 9.0 mPa, about 9.5 mPa, or about 10 mPa). In some embodiments, the multilayered construct has a Young's modulus of from about 0.1 MPa to about 8 MPa. In some embodiments, the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 5 MPa. In some embodiments, the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 3 MPa. In some embodiments, the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 2.5 MPa. In some embodiments, the multilayered construct has a Young's modulus of from about 0.1 MPa to about 2.0 MPa. In some embodiments, the multi-layered construct has a Young's modulus of from about 4.0 MPa to about 8.0 MPa.

[0157] In some embodiments, the fibrous layer has a Young's modulus that is greater than a Young's modulus of the hydrogel layer. In such embodiments, it will be readily appreciated that certain mechanical properties (e.g., suture retention strength, ultimate tensile strength, Young's modulus, etc.) of the multi-layered construct approximate the mechanical properties of the fibrous layer. Accordingly, in such embodiments, the fibrous layer has a suture retention strength, ultimate tensile strength, and/or Young's modulus that is about any value or range set forth herein in relation to the multi-layered construct.

[0158] In one aspect, the present invention provides a multi-layered construct for tissue- engineered grafts. The multi-layered construct comprises a fibrous layer, a hydrogel layer, cells disposed in the hydrogel layer, and cells disposed on an interior surface of the fibrous layer. The fibrous layer is disposed about and extends along an axis in a first direction. The fibrous layer has the interior surface defining a lumen and an exterior surface. The fibrous layer comprises fibres of a first biocompatible polymer. The fibres are at least partially aligned in the first direction. The hydrogel layer is disposed on the fibrous layer and comprises a second biocompatible polymer. The cells disposed in the hydrogel layer are substantially aligned in a second direction. The cells disposed on the interior surface of the fibrous layer are substantially aligned in the first direction. The first direction and the second direction are different.

[0159] In another aspect, the present invention provides a multi-layered construct for tissue-engineered grafts. The multi-layered construct comprises a fibrous layer, a hydrogel layer, and cells disposed on an interior surface of the fibrous layer. The fibrous layer is disposed about and extends along an axis in a first direction. The fibrous layer has the interior surface defining a lumen and an exterior surface. The fibrous layer comprises fibres of a first biocompatible polymer. The fibrous layer comprises a plurality of pores, as described herein. The hydrogel layer is disposed on the fibrous layer and comprises a second biocompatible polymer. The hydrogel layer further comprises a vascular network of cells.

[0160] In a further aspect, the present invention provides a multi-layered construct for tissue-engineered grafts. The multi-layered construct comprises a first fibrous layer, a second fibrous layer, a hydrogel layer, and cells disposed on interior surfaces of the first and second fibrous layer. The first fibrous layer is disposed about and extends along a first axis in a first direction. The first fibrous layer has the interior surface defining a first lumen and an exterior surface. The first fibrous layer comprises fibres of a first biocompatible polymer. The second fibrous layer is disposed about and extends along a second axis in the first direction. The second fibrous layer has the interior surface defining a first lumen and an exterior surface. The second fibrous layer comprises fibres of a second biocompatible polymer. The first and second fibrous layers each comprise a plurality of pores, as described herein. The hydrogel layer is disposed on the first and second fibrous layers and comprises a third biocompatible polymer, which may be any of the first and second biocompatible polymers described herein. The hydrogel layer further comprises a vascular network of cells.

[0161] In yet another aspect, the present invention provides a multi-layered construct for tissue-engineered grafts. The multi-layered construct comprises a fibrous layer, a hydrogel layer, cells disposed in the hydrogel layer, and cells disposed on an interior surface of the fibrous layer. The fibrous layer is disposed about and extends along an axis in a first direction. The fibrous layer has the interior surface defining a lumen and an exterior surface. The fibrous layer comprises fibres of polycaprolactone (PCL). The fibres are at least partially aligned in the first direction. The hydrogel layer is disposed on the exterior surface of the fibrous layer and about the axis and comprises gelatin methacryloyl (GelMA). The cells disposed in the hydrogel layer are substantially aligned in a second direction. The cells disposed on the interior surface of the fibrous layer are substantially aligned in the first direction. The first direction and the second direction are different, the first direction is further defined as an axial direction, and the second direction is further defined as a circumferential direction about the axis. The cells disposed on the interior surface of the fibrous layer are endothelial cells. The cells disposed in the hydrogel layer are smooth muscle cells. In some embodiments, the hydrogel layer is disposed directly on the fibrous layer.

[0162] III. METHOD OF FABRICATING A FIBROUS LAYER

[0163] In another aspect, the present invention provides a method of fabricating a fibrous layer of at least partially aligned fibres for tissue-engineered grafts. The method comprises: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a biocompatible polymer; and b) applying a mechanical force to the fibres of the biocompatible polymer to at least partially align the fibres in the first direction.

[0164] The fibrous layer may be any suitable fibrous layer described herein in relation to the multi-layered constructs. In some embodiments, providing the fibrous layer of step a) further comprises electrospinning the biocompatible polymer to form the fibrous layer. The biocompatible polymer may be any biocompatible polymer described herein in relation to the first and second biocompatible polymers of the multi-layered constructs.

[0165] In some embodiments, the method further comprises: c) disposing cells on the fibrous layer after step b); and d) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

[0166] In some embodiments, when the fibrous layer has an interior surface defining a lumen and an exterior surface, the method comprises: c) disposing cells on the interior surface of the fibrous layer after step b); and d) allowing the cells disposed on the interior surface of the fibrous layer to substantially align in the first direction.

[0167] In some embodiments, applying the mechanical force of step b) comprises directional or bidirectional freezing of a solvent to thereby apply the mechanical force to the fibres of the polymer. In some embodiments, the mechanical force of step b) comprises: b-1) disposing the fibrous layer in a chamber; b-2) partially filling the chamber with a solvent; and b-3) freezing the solvent, wherein the solvent expands in the first direction during freezing to thereby apply the mechanical force to the fibres of the polymer. [0168] In some embodiments, the solvent is water. In some embodiments, freezing the solvent of step b-3) is performed with a freezer (e.g., a -20 °C freezer). In some embodiments, step b-3) takes from about 1 minute to about 1 hour, from about 1 minute to about 45 minutes, from about 1 minute to about 30 minutes, from about 5 minutes to about 30 minutes, or from about 10 minutes to about 30 minutes.

[0169] In some embodiments, the chamber is configured to direct freezing in the first direction. For example, the chamber may be constricted radially with respect to the axis of the fibrous layer, thereby directing freezing in the first direction (e.g., the axial direction).

[0170] IV. METHOD OF FABRICATING A HYDROGEL LAYER

[0171] In another aspect, the present invention provides a method of fabricating a hydrogel layer of a multi-layered construct for tissue-engineered grafts, the method comprising: a) providing a fibrous layer extending along an axis in a first direction from a first end to a second end, the fibrous layer comprising fibres that are at least partially aligned in the first direction; b) disposing a mixture on a surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; c) curing the cross-linkable polymer composition to form the hydrogel layer on the surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and d) allowing the cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

[0172] The fibrous layer may be any suitable fibrous layer described herein in relation to the multi-layered constructs. In some embodiments, providing the fibrous layer of step a) further comprises electrospinning fibres to form the fibrous layer (e.g., electrospinning fibres of a biocompatible polymer).

[0173] In some embodiments, the fibrous layer is disposed about and extends along the axis in the first direction and the fibrous layer has an interior surface defining a lumen and an exterior surface. The hydrogel layer is formed on the exterior surface of the fibrous layer and about the axis. The first direction is further defined as an axial direction and the second direction is further defined as a circumferential direction about the axis. [0174] In some embodiments, the cross-linkable polymer composition of step (b) comprises polymers having at least one cross-linkable moiety. In some embodiments, polymers of the cross-linkable polymer composition comprise a plurality of cross-linkable moieties. In such embodiments, each of the cross-linkable moieties may form physical crosslinks and/or chemical cross-links during curing. The polymers comprising at least one crosslinkable moiety may be any polymer suitable for forming the second biocompatible polymer as described herein in relation to the multi-layered constructs.

[0175] In some embodiments, the curing step c) comprises heating the mixture, irradiating the mixture with a radiation source, adding a curing agent to the mixture, or any combination thereof. In some embodiments, the curing step c) comprises heating the mixture. In some embodiments, the curing step c) comprises irradiating the mixture with a radiation source. In some embodiments, the curing step c) comprises adding a curing agent to the mixture. In some embodiments, the curing step c) comprises irradiating the mixture with a radiation source and adding a curing agent to the mixture.

[0176] In some embodiments, the curing step c) comprises irradiating the mixture with a radiation source in the presence of a curing agent. The radiation source may be any suitable radiation source to initiate curing. In some embodiments, the radiation source is a visible light source or an ultraviolet (UV) light source. In some embodiments, the radiation source is a UV light source.

[0177] In some embodiments, the mixture is irradiated for about 1 second to about 10 minutes. In some embodiments, the mixture is irradiated for about 1 second to about 5 minutes. In some embodiments, the mixture is irradiated for about 1 second to about 3 minutes. In some embodiments, the mixture is irradiated for about 1 minute.

[0178] The curing agent may be any curing agent suitable for initiating curing of the crosslinkable polymer composition. The curing agent may itself react with one or more crosslinkable moieties to form a covalent bond comprising one or more atoms from the curing agent. Alternatively, the curing agent may initiate a reaction between cross-linkable moieties without any atoms from the curing agent being incorporated in the resultant chemical cross-link.

[0179] In some embodiments, the curing agent is a radical initiator. In some embodiments, the curing agent may be a photoinitiator or a thermal initiator. Suitable photoinitiators will be familiar to persons skilled in the art and include, by way of non-limiting example, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, acetophenone, benzophenone, 4,4'- di chlorobenzophenone, methyl benzoylformate, 1 -benzoyl cycloh exanol, 2-methyl-4'- (methylthio)-2-morpholinopropiophenone, 2-isonitrosopropiophenone, anthraquinone, 2- isopropylthioxanthone, diphenyliodonium hexafluorophosphate, bis(4-tert- butylphenyl)iodonium tetrafluoroborate, [4-[(2-hydroxytetradecyl)- oxy]phenyl]phenyliodonium hexafluoroantimonate, triphenyl sulfonium bromide, triphenyl sulphonium nonaflate, 4-nitrobenzenediazonium tetrafluorob orate, 2-(4- methoxystyryl)-4,6-bis(trichloromethyl)-l,3,5-triazine, 2-(9-ox oxanthen-2 -yl)propionic acid l,5-diazabicyclo[4.3.0]-non-5-ene salt, and l-(2-formylbenzoyl)piperidine. In some embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate. Suitable thermal initiators include, by way of non-limiting example, 2,2'-azobis[2-(2- imidazolin-2-yl)-propane] dihydrochloride, cumene hydroperoxide, di-/c/7-butyl peroxide, dicumyl peroxide, benzoyl peroxide, ammonium persulfate, dicyandiamide, cyclohexyl tosyl ate, (4-hydroxyphenyl)-dimethyl sulfonium hexafluorophosphate, diphenyl(methyl)sulfonium tetrafluorob orate, benzyl(4-hydroxyphenyl)-methylsulfonium hexafluoroantimonate, (4-hydroxyphenyl)methyl-(2-methylbenzyl)sulfonium hexafluoroantimonate, and triphenyl sulphonium nonaflate.

[0180] In some embodiments, when the polymers comprising the at least one crosslinkable moiety are proteinaceous polymers, the curing agent may be an enzymatic catalyst (e.g., a transferase, hydrolase, oxidoreductase, etc.).

[0181] When the curing step c) comprises adding a curing agent to the mixture, the curing agent may be added to the mixture prior to step b), during step b), or after step b).

[0182] In some embodiments, the method further comprises: e) cooling the mixture prior to step c).

[0183] In some embodiments, step e) comprises cooling the mixture to a temperature of from about -10 °C to about 10 °C. In such embodiments, the mixture may be cooled for about 1 second to about 10 minutes, for about 1 second to about 5 minutes, for about 1 second to about 2.5 minutes, or from about 1 second to about 1 minute. In some embodiments, the mixture is cooled for about 1 minute.

[0184] In some embodiments, the method further comprises: f) treating the fibrous layer to improve a hydrophilicity of the fibrous layer prior to step b).

[0185] In some embodiments, the treating step f) comprises contacting the fibrous layer with plasma, contacting the fibrous layer with an acidic solution, contacting the fibrous layer with a basic solution, or any combination thereof. In some embodiments, the treating step f) comprises contacting the fibrous layer with plasma. In some embodiments, the treating step f) comprises contacting the fibrous layer with an acidic solution (e.g., a hydrochloric acid (HC1) solution). In some embodiments, the treating step f) comprises contacting the fibrous layer with a basic solution (a sodium hydroxide (NaOH) solution).

[0186] In some embodiments, when step f) comprises contacting the fibrous layer with an acidic or basic solution, the fibrous layer may be contacted with the acidic or basic solution for about 1 hour to about 10 hours, for about 2 hours to about 8 hours, for about 2 hours to about 6 hours, or about 4 hours.

[0187] V. METHOD OF FABRICATING A MULTI-LAYERED CONSTRUCT [0188] In one aspect, the present invention provides a method of fabricating a multilayered construct for tissue-engineered grafts. The method comprises: a) forming a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) applying a mechanical force to the fibres of the first biocompatible polymer to at least partially align the fibres in the first direction; c) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; d) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and e) allowing the second cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

[0189] In some embodiments, the method further comprises: f) disposing cells on the fibrous layer, wherein the cells disposed on the fibrous layer are different from the cells disposed in the hydrogel layer; and g) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

[0190] It will be appreciated that any of steps a) to g) may be further defined in accordance with any of the corresponding steps described herein for the methods of fabricating a fibrous layer and fabricating a hydrogel layer. It will be further appreciated that the method may further comprise any additional steps described herein for the methods of fabricating a fibrous layer and fabricating a hydrogel layer.

[0191] VI. METHOD OF FABRICATING A MULTI-LAYERED CONSTRUCT COMPRISING PORES

[0192] In one aspect, the present invention provides a method of fabricating a multilayered construct for tissue-engineered vascular grafts. The method comprises: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) forming a plurality of pores in the fibrous layer; c) disposing endothelial cells on the interior surface of the fibrous layer; and d) forming a hydrogel layer on the exterior surface of the fibrous layer.

[0193] In some embodiments, forming the hydrogel layer of step d) comprises: dl) disposing a cross-linkable polymer composition on the exterior surface of the fibrous layer; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface.

[0194] In some embodiments, forming the hydrogel layer of step d) comprises: dl) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and smooth muscle cells; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface, wherein the hydrogel layer comprises a second biocompatible polymer that is cross-linked, and wherein the smooth muscle cells are disposed in the hydrogel layer.

[0195] In some embodiments, forming the plurality of pores of step b) comprises machining the plurality of pores into the fibrous layer. In some embodiments, the pores are formed with an apparatus comprising an array of needles suitable for puncturing the fibrous layer.

[0196] In some embodiments, the method further comprises: e) allowing endothelial cells to migrate from the interior surface of the fibrous layer to the hydrogel layer, thereby forming a vascular network in the hydrogel layer.

[0197] Without wishing to be bound by theory or mode of operation, it is believed that the pores facilitate migration of endothelial cells to the hydrogel layer to thereby form a vascular network.

[0198] It will be appreciated that any of steps a) to e) may be further defined in accordance with any of the corresponding steps described herein for the methods of fabricating a fibrous layer, fabricating a hydrogel layer, and fabricating a multi-layered construct. It will be further appreciated that the method may further comprise any additional steps described herein for the methods of fabricating a fibrous layer, fabricating a hydrogel layer, and fabricating a multilayered construct.

EXAMPLES

[0199] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

[0200] Abbreviations

[0201] The following abbreviations are used throughout this Examples section. [0202] Example 1: Multi-Layered Construct Promoting Spontaneous Cell Alignment [0203] Materials and Methods

[0204] Synthesis of gelatin methacryloyl (GelMA): GelMA was prepared according to the procedure set forth in Loessner, D. et al. Functionalization, Preparation and Use of Cellladen Gelatin Methacryloyl -based Hydrogels as Modular Tissue Culture Platforms. Nature protocols 2016, 11 (4), 727-746. Water purified with a Direct-Q 3 UV Water Purification System (MerckMillipore, USA) was used throughout Example 1. 20 g of gelatin (Sigma-Aldrich, Germany) was dissolved in 200 mL of phosphate-buffered saline (PBS, Gibco, USA) and heated at 50 °C for 1 h. Then, while stirring, 12 mL of methacrylic anhydride (Sigma-Aldrich, Germany) was added dropwise to the solution, and the resultant mixture was left to react for 1 h at 50 °C. 400 mL of PBS was then added and the solution was centrifuged at 3500 g for 3 min. The resultant supernatant was dialyzed for 7 days at 37 °C against water using 12-14 kDa cutoff dialysis tubing (Merck, Germany). After dialysis, the GelMA solution was lyophilized and stored at -20 °C.

[0205] GelMA was analyzed with ¹ H Nuclear Magnetic Resonance (^XH NMR). A solution of 5% w/v GelMA was prepared in deuterium oxide (Sigma-Aldrich, Germany), and NMR spectra were obtained at 20.5 °C using a 400 MHz JEOL spectrometer (JEOL, Japan). 256 scans were taken with a relaxation delay of 4 s and angle of 45°. Gelatin was analyzed under the same conditions as GelMA. Integrals for lysine methylene and aromatic amino acid were determined from the 'H NMR spectra for both GelMA and Gelatin using the MestReNova NMR plugin (Mestrelab, Spain), and the integral of lysine methylene was normalized against the integral for aromatic amino acid. A ratio of the lysine methylene integrals of GelMA to gelatin was used to calculate the degree of methacrylation (DM), as set forth in the following formula:

A(lysine methylene of GelMA)

DM % = (1 )100.

A(lysine methylene of nonmodified gelatin)

[0206] Electrospinning a fibrous layer: Electrospinning was performed with a rotating 2.4 mm stainless-steel collector rod (ER316L TIG filler wire, Hampdon, Australia) and a translation stage that ensured the nanofibres were evenly distributed during deposition.

[0207] A polycaprolactone (PCL) solution was prepared by dissolving 12% w/v PCL (Mw 80 000 Da, Sigma-Aldrich, Germany) in l,l,l,3,3,3-hexafluoro2-propanol (Sigma-Aldrich, Germany) overnight. The PCL solution was electrospun through an 18-gauge needle at a flow rate of 2 mL/h. The needle was connected to a 12 kV voltage source (Spellman, USA) and the collector rod was linked to a -3 kV voltage source. The collector rod rotated at 150 revolutions per minute rpm) and the translation stage moved at a rate of 3 cm/min to form a fibrous layer PCL tube) about the collector rod.

[0208] Fiber alignment of the Fibrous Laver: The fibrous layer (PCL tube), while still on the collector rod, was transferred into a cylindrical container (length: 7.5 cm; inner diameter: 4.4 mm) aligning the tube axially in the center of the container. The container was then partially filled with 500 pL of water and placed upright in a -20 °C freezer for 30 min. Within this time the water froze, encapsulating the fibrous layer in ice. The collector rod was then removed from the fibrous layer by sliding it out of the ice-filled container. The fibrous layers were allowed to thaw and dry overnight.

[0209] Prior to cell seeding, the fibrous layers were immersed in 70% v/v ethanol for 30 min and then placed under a UV light for 20 min for disinfection. The fibrous layers were then washed with PBS three times and placed in EC growth medium.

[0210] Surface Treatment of the Fibrous Laver: Hydrophilicity of surfaces of the fibrous layers (PCL tubes) were enhanced according to the procedure set forth in Bosworth, L. A. et al. Biocompatibility without Compromising Material Properties: An Optimised NaOH Treatment for Electrospun Polycaprolactone Fibres. J. Nanomater. 2019, 11, 4605092. Fibrous layers were immersed in a solution of 1 M sodium hydroxide (Chem-Supply, Australia) for 4 h, then washed five times with water, and left to soak in water overnight.

[0211] Scanning Electron Microscopy: The electrospun PCL fibres were examined with a scanning electron microscope (10 kV, Hitachi, Japan). Multi-layered construct samples were cut into 5 mm sections using a razor blade, placed in water, and left overnight at a temperature of 37 °C before freezing at -80 °C and freeze-drying. The resulting sections were then analyzed by a cross-sectional view.

[0212] Preparation of GelMA Mixtures: The required concentrations of GelMA (2, 3, 5, 7, or 10% w/v) were dissolved in EC growth medium (EGM-2, Lonza, USA) at 37 °C for Ih in an incubator. Then, 0.06% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Sigma-Aldrich, Germany) was added as a photoinitiator to each mixture, followed by 1.6% w/v gelatin. The resulting mixtures were sterile filtered through a 0.22 pm polyethersulfone (PES) syringe filter (Merck, Germany).

[0213] Mechanical Characterization of Hydrogel Layer: GelMA mixtures with various concentrations of GelMA were heated to 37 °C and slowly syringed into polydimethylsiloxane molds with a diameter and height of 10 and 5 mm, respectively. The molds were then placed in an ice bath for 1 min to physically cross-link the GelMA and gelatin prior to photo-cross- linking. The molds were then exposed to UV light (OmniCure SI 500, 320-500 nm filter, 30 mW/cm²) for 1 min until fully cross-linked and returned to the ice bath for 3 min. The resultant hydrogel layers were removed from the mold, deposited into sealed containers containing PBS, and placed in an oven at 37 °C overnight.

[0214] Prior to compression testing, the diameters of the resultant hydrogel disks were measured by using calipers. The hydrogel disks were then uniaxially compressed on an Instron 5848 Microtester at a constant strain rate of 0.5 mm/min either for 4 mm or until failure. The resultant force was measured with a 50 N load cell and the compressive Young’s modulus was calculated from the linear region (10-20% strain) using the methods and custom python script from Trengove et al. Microbial Transglutaminase Improves ex vivo Adhesion of Gelatin Methacryloyl Hydrogels to Human Cartilage. Frontiers in Medical Technology 2021, 3, 773673.

[0215] Construction of Multi-Layered Construct: With reference to FIG. 1(e), a sterilized polytetrafluoroethylene-coated rod with a diameter of 2.5 mm was inserted into a lumen of the fibrous layer (PCL tube) and then placed inside a transparent mold made of polypropylene with a diameter of 4.4 mm. A Pre-heated GelMA mixture of the desired concentration, with or without SMCs (500 000 SMCs/ml of GelMA mixture), was slowly added to the mold until the fibrous layer was completely submerged. The polytetrafluoroethylene-coated rod stopped the ingress of GelMA into the lumen during this process.

[0216] The mold was then placed in an ice-bath for 1 min to physically cross-link the GelMA mixture prior to photo-cross-linking. The GelMA in the mold was exposed to UV light for 60 s (OmniCure SI 500, 320-500 nm filter, 30 mW/cm²), as shown in FIG. If. After crosslinking, the mold was returned to the ice-bath for 3 min before the multi-layered construct (i.e., hydrogel layer/fibrous layer (GelMA/PCL tube)) was separated from the mold and the rod.

[0217] Mechanical testing of the multi-layered construct: The multi-layered constructs were cut into 5 mm sections and affixed to an Instron 5944 Microtester using stainless-steel holders to measure their circumferential tensile strength (UTS) using a 50 N load cell. The segments were prepared and stretched at a speed of 50 mm/min as per the procedure outlined in ISO 7198:2017.

[0218] A burst pressure test was completed by attaching a 2.5 cm long section of multilayered constructs to a pressure transducer (Lutron PS1005BAR) that was then connected to a pressure meter (Sper scientific, USA). Vaseline (Unilever, USA) was pumped into the multilayered constructs using a syringe pump (Harvard apparatus) at a rate of 1 mL/min until the multi-layered constructs burst. [0219] A suture retention test was carried out in accordance with the ISO 7198:2017 guidelines. The multi-layered constructs were sliced into 1 mm sections, and a suture (Ethicon 5-0, Scotland) was placed 2 mm from the end of the multi-layered constructs, penetrating one side to create half a loop. The multi-layered constructs were secured at their base and the multilayered constructs were pulled at a rate of 50 mm/min using an Instron 5944 Microtester until the wall failed.

[0220] Cell Culture: Primary human umbilical vein ECs (Lonza, Switzerland) were grown in EC growth medium (EGM-2, Lonza, USA) and seeded into the vascular graft at passage 5 using a micropipette. Primary human coronary artery SMCs (Lifeline, USA) were grown in SMC Medium (Lifeline, USA) and encapsulated in GelMA during passage 5. Antibiotic- antimycotic solution (Gibco, USA) was added to each growth medium for a final concentration of 1% v/v. All cells were cultivated in 75 cm² culture flasks (Coming, USA) and the growth medium was replaced every other day until 70% confluence was reached.

[0221] Immunoflouresence Analysis: Once the cultivation period was complete, the multilayered constructs were moved to a 15 mL centrifuge tube. The multi-layered constructs were washed with PBS three times and then immersed in 4% paraformaldehyde (Scharlau, Spain) at 4 °C for 4 h to fix the samples. The multi-layered constructs were then cut using a razor blade into 5 mm sections and put into a 96-well plate for staining. The sections were washed three times with PBS before 0.1% Triton X-100 (Labchem, USA) was added to enhance cell permeability. ActinRed 555 ReadyProbes Reagent (Thermo Fisher, USA) was then added for 90 min to stain the actin filaments. To stain the nuclei, a 1 : 10 000 dilution of 4',6-Diamidino- 2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, Germany) was added after washing the sections three times with PBS. To stain the CD31 and aSMA markers, 1% albumin bovine serum (Sigma-Aldrich, Germany) was added for 1 h to block nonspecific binding of the antibodies. The sections were then immersed in Alexa Fluor 488 anti-CD31 antibody and antialpha smooth muscle actin antibody (abeam, United Kingdom) overnight. On the following day, donkey antirabbit IgG H&L (Alexa Fluor 594) was added for 4 h. Finally, the sections were stored in PBS at 4 °C until they were ready to be imaged. Images were captured using a Nikon A1R+ Confocal Microscope (Nikon, Japan).

[0222] Evaluation of Cellular Alignment: The Orientation! plugin in ImageJ was utilized to assess the alignment of the cells in the multi-layered constructs (Rezakhaniha, R. et al. Experimental Investigation of Collagen Waviness and Orientation in the Arterial Adventitia Using Confocal Laser Scanning Microscopy. Biomechanics and modeling in mechanobiology 2012, 11, 461-473; Piispbki, Z. et al. Transforms and Operators for Directional Bioimage Analysis: A Survey. Focus on Bioimage Informatics 2016, 219, 69-93; and Fonck, E. et al. Effect of Aging on Elastin Functionality in Human Cerebral Arteries. Stroke 2009, 40 (7), 2552-2556.). The results of the Orientation! distribution analysis were exported to an Excel spreadsheet for further analysis. To determine the proportion of pixels aligned within a particular range, the peak of the orientation distribution was identified for each image, and the sum of pixels within ±5, 10, or 20° of the peak was divided by the total sum of pixels. The aspect ratio of the cells was assessed using the Analyze Particles plugin in ImageJ.

[0223] Statistical Analysis: Student t tests (for two data sets) or one-way ANOVA with Tukey’s post hoc test (for more than two data sets) were used for statistical analysis with SPSS 27.0 software, p-values below 0.05 indicated statistical significance.

[0224] Results/Discussion

[0225] Polycaprolactone (PCL) fibres were deposited onto a rotating mandrel to produce via electrospinning to produce fibrous layers (PCL tubes) with a length of 6 cm, an inner diameter of 2.5 mm, and a wall thickness of 200 pm. A hydrogel layer (GelMA hydrogel layer) with a thickness of 800 pm was cast around the fibrous layer to produce a multi-layered construct with a total wall thickness of 1 mm. SEM illustrated uniform attachment of the hydrogel layer to the electrospun fibrous layer and no gaps or delamination between layers were observed (FIG. 1g). Prior to casting of the hydrogel layer, the electrospun fibrous layers underwent an alignment process to partially align the electrospun fibres. Furthermore, the fibrous layer was submerged in a solution of NaOH to increase surface hydrophilicity through partial hydrolysis to facilitate subsequent cell attachment (Bosworth, L. et al. Enhancing Biocompatibility without Compromising Material Properties: An Optimised NaOH Treatment for Electrospun Polycaprolactone Fibres. J. Nanomater. 2019, 11, 4605092).

[0226] The multi-layered construct was handleable and had similar mechanical properties to native blood vessels commonly used in bypass surgery, including saphenous veins and internal mammary arterys (Stekelenburg, M. et al. Dynamic Straining Combined with Fibrin Gel Cell Seeding Improves Strength of Tissue-engineered Small-diameter Vascular Grafts. Tissue Engineering Part A 2009, 15 (5), 1081-1089; and L’Heureux, N. et al. Human Tissue- engineered Blood Vessels for Adult Arterial Revascularization. Nature medicine 2006, 12 (3), 361-365), as shown in FIG. 2. Mechanical testing illustrated that the fiber alignment step did not significantly alter the burst pressure, suture retention strength, ultimate tensile strength, or tensile modulus (Young's modulus) of the multi-layered construct. Matching the mechanical properties of native blood vessels is a preferred design parameter for tissue-engineered vascular grafts, as significantly stiffer properties may result in reocclusion of the vessel arising from abnormal flow patterns. Additionally, the fibrous layer may act as a barrier between SMCs and the lumen of the multi-layered construct to prevent SMCs from proliferating excessively and obstructing the lumen, a condition known as intimal hyperplasia. Without wishing to be bound by theory or mode of application, the fibrous layer and the hydrogel layer are expected to enable diffusion of soluble factors between ECs and SMCs, thereby facilitating EC-SMC crosstalk.

[0227] The biomimetic alignment of ECs and SMCs is considered important for the appropriate function of blood vessels, including TEVGs. The fiber alignment process was developed to cause rapid fiber orientation within the fibrous layer in order to promote axial alignment of the endothelium. It was also observed that intermediate hydrogel stiffness coupled with a stiffer fibrous layer unexpectedly resulted in spontaneous circumferential alignment of SMCs.

[0228] A facile method to rapidly and scalably align electrospun fibres in the axial direction was developed using the unique freezing properties of water. As shown in FIG. 3, electrospun fibres were deposited onto a slowly rotating mandrel, resulting in randomly aligned fibres on an interior surface of the resultant fibrous layer. The fibrous layer on the mandrel was then placed in a chamber that was mostly filled with water, and the water was frozen. Upon freezing, the water expanded. The expansion was constrained in the radial direction due to the rigid chamber, resulting in ice expansion in the axial direction. This axial growth of ice applied mechanical forces that partially aligned the electrospun fibres in the axial direction. Afterward, the fibrous layer was removed from the collector rod by sliding the collector rod out of the ice-filled container. This process resulted in a statistically significant increase in the axial alignment of the fibres of the fibrous layer, quantified by assessing the proportion of fibres oriented within ± 10° of the longitudinal axis of the tube (FIGS. 3e and 3 f).

[0229] The formation of an endothelium and endothelial cell alignment was assessed on both multi-layered structures that were subjected to the alignment process ("FIA-treated") and that were not subjected to the alignment process ("untreated"). After 3 days of growth, both the FIA-treated and untreated fibrous layers supported a large number of endothelial cells on interior surfaces of the respective fibrous layers (FIG. 4A). ECs on the FIA-treated multilayered constructs formed a confluent endothelium and exhibited a significant increase in alignment, as illustrated in FIG. 4b. The majority of the cells on the FIA-treated surfaces were aligned within a range of ± 10° of a longitudinal axis of the fibrous layer, as shown in FIG. 4C. ECs on both the FIA-treated and untreated fiber surfaces showed strong staining for CD31 at cell-cell junctions indicating the formation of a confluent endothelium. [0230] Numerous studies have explored methods for inducing circumferential alignment of SMCs in vitro (van Kampen, K. A. et al. Fabrication of a Mimetic Vascular Graft Using Melt Spinning with Tailorable Fiber Parameters. Biomaterials Advances 2022, 139, 212972; Itai, S. et al. In Vitro Artery Model with Circumferentially Aligned & Contractible Smooth Muscle by Unfixed Molding & Screwing Fabrication. In 2022 IEEE 35th International Conference on Micro Electro Mechanical Systems Conference (MEMSf IEEE, 2022 pp 275-278). Many of these studies rely on seeding SMC on surfaces with circumferentially aligned microgrooves that guide cellular alignment (Rayatpisheh, S. et al. Combining Cell Sheet Technology and Electrospun Scaffolding for Engineered Tubular, Aligned, and Contractile Blood Vessels. Biomaterials 2014, 35 (9), 2713-2719; and Heath, D. E. et al. Biomaterials Patterned with Discontinuous Microwalls For Vascular Smooth Muscle Cell Culture: Biodegradable Small Diameter Vascular Grafts and Stable Cell Culture Substrates. Journal of Biomaterials science, Polymer edition 2016, 27 (15), 14771494). However, these techniques are typically slow, and developing 3D layers of circumferentially aligned cells remains difficult. Using mesenchymal stromal cells and circumferentially aligned fibres, researchers achieved alignment of smooth muscle cell-like layers along microgrooves (Jungst, T. et al. D. Heterotypic Scaffold Design Orchestrates Primary Cell Organization and Phenotypes in Cocultured Small Diameter Vascular Grafts. Adv. Funct. Mater. 2019, 29 (43), 1905987). However, this technique has a long fabrication time and requires cell differentiation. [0231] Advantageously, the work described herein provides rapid circumferential selfalignment of SMCs in a three-dimensional hydrogel. A hydrogel layer was cast around the fibrous layer. The impact of stiffness of the hydrogel layer on SMC alignment was assessed by cultivating the SMCs in hydrogel layers made from 2%, 3%, 5%, 7%, and 10% w/v GelMA. [0232] Unexpected spontaneous self-alignment of SMCs was observed in samples after 7 days of culture, and a confluent endothelium was present on an interior surface of respective fibrous layers (FIG. 5a). Hydrogel stiffness influenced the degree of SMC alignment. For example, cells in hydrogels of intermediate stiffness (5 and 12 kPa) exhibited robust circumferential alignment in comparison to cells in both softer and stiffer hydrogels, as shown in FIGS. 5b and 5c, while SMCs did not noticeably align in 10% w/v GelMA or 7% w/v GelMA hydrogel layers, which exhibited a stiffness of >40 kPa. Some alignment was observed in the 2% w/v GelMA hydrogel layers, although statistically fewer cells in the 2% w/v GelMA hydrogel layers were aligned in comparison to the 5% /v GelMA hydrogel layers. These results indicate that an intermediate stiffness of GelMA hydrogel layers was optimal for promoting SMC alignment in the multi-layered constructs. The observed variation in SMC alignment may be a consequence of the varying stiffness of the hydrogel layers. Compression testing revealed that there was no significant difference in the Young’s modulus values among the 2%, 3%, and 5% w/v GelMA hydrogel layers. However, there was a significant increase in Young’ s modulus when the GelMA concentration was increased to 7% w/v, and the stiffness of the GelMA hydrogel layers increased even more at 10% w/v GelMA. It is possible that stiffer hydrogel layers acted to confine SMCs, preventing elongation and alignment. An increase of GelMA concentration also provides a denser, less swollen scaffold matrix, which decreases the degradability of the hydrogel layer. Therefore, SMCs would be confined for a longer period, reducing the ability for cell-cell signaling and elongation.

[0233] SMCs were imaged at different time points over 24 h to observe the emergence of alignment in 3% w/v GelMA hydrogel layers. FIG. 6a illustrates that the SMCs had a spherical appearance during the first few hours of culture. Unexpectedly, the cells began to align circumferentially after 4 h. Most cells had achieved circumferential alignment by the 8 h time point. The process was quantified by counting the percentage of aligned cells and measuring the aspect ratio of the cells. Approximately 60% of the cells had aligned at the 8 h mark, and this value stayed relatively constant through the remainder of the 24 h experiment (FIG. 6bi). However, the aspect ratio of the cells continued to increase, indicating that the cells spread more with time (FIG. 6bii). The cells closest to the fibrous layer initially elongate and circumferentially align to form a discrete layer. This behavior continues in striated layers arranged around the tube, with SMCs on the outer edge of the GelMA hydrogel layer aligning last. Without wishing the be bound by theory or mode of application, it is believed that the SMCs in the GelMA hydrogel layer directly interfacing with the fibrous layer (PCL tube) experience the underlying stiffness of the fibrous layer, causing an increase in mechanical cell signaling.

[0234] The impact of the underlying fibrous layer (PCL tube) on SMC alignment was also investigated by creating a V-shaped incision in the fibrous layer prior to GelMA hydrogel layer casting. As shown in FIG. 7a, three regions were created: a GelMA hydrogel region with a small area of the underlying fibrous layer (green), a GelMA hydrogel region with a medium area of the underlying fibrous layer (blue), and a GelMA hydrogel region with a complete underlying fibrous layer (red). After 24 h of culture, the cells in the red region, with a full fibrous layer, exhibited similar alignment to the abovementioned results. However, the degree of SMC alignment decreased as the amount of underlying fibrous layer was reduced (blue and green regions). This was true around the entire circumference of these regions, including portions where some fibrous layer remained. These results suggest that the presence of the fibrous layer unexpectedly plays a role in promoting the circumferential alignment of SMCs.

[0235] Axial alignment of a confluent endothelium and circumferential alignment of vascular SMCs are crucial for the development of tissue-engineered vascular grafts. However, achieving this cellular alignment rapidly, easily, and in a cost-effective manner is still a significant obstacle in tissue engineering. The multi-layered construct replicates the alignment of both ECs and SMCs found in native blood vessels within vascular grafts, while also having the mechanical properties needed for their translation. The aligned fibres of the fibrous layer guided EC alignment. Optimization of the mechanical microenvironment of SMCs in the hydrogel layer surrounding the fibrous layer promoted the spontaneous self-organization of SMCs into a biomimetic circumferential pattern of alignment.

[0236] Example 2: Multi-Layered Construct Promoting Formation of a Vascular Network

[0237] Materials and Methods

[0238] Electrospinning a fibrous layer: Fibrous layers were fabricated as described in Example 1. Briefly, a 12% w/v PCL (Mw 80 000 Da, Sigma- Aldrich, Germany) solution was electrospun onto a rotating collector rod (2.4 mm diameter, ER316L TIG filler wire, Hampdon, Australia) with a 12 kV voltage. Prior to cell seeding, the resultant fibrous layers (PCL tubes) were disinfected with ethanol and UV light, rinsed with phosphate-buffered saline (PBS, Gibco, USA), and incubated in complete EC growth medium (Lonza, USA).

[0239] Synthesis of GelMA: A 10% gelatin type A solution in PBS was heated at 50°C for 1 h, then methacrylic anhydride was slowly added (6% volume ratio) and reacted at 50°C for 1 h. Quenching with PBS (2: 1 volume ratio) and centrifugation separated the GelMA, which was then dialyzed in water for a week and lyophilized for storage. The degree of methacrylation was assessed by 'H NMR, comparing the spectra of GelMA and gelatin dissolved in D2O. Integration of specific proton signals revealed the extent of modification.

[0240] Surface Treatment of the Fibrous Laver: Hydrophilicity of surfaces of the fibrous layers were enhanced according to the procedure set forth in Example 1.

[0241] Scanning Electron Microscopy: Pores in the fibrous layers (PCL tubes) were characterized by SEM at an accelerating voltage of 10 kV (FlexSEM 1000, Hitachi, Japan). To prepare the PCL tubes, the samples were cut into 5 mm segments with a razor blade. The side view of the vascular grafts was examined by SEM. The samples were not sputter coated due to the low vacuum technology of the Hitachi SEM microscope that eliminates the need for sputter coating, enabling direct observation of non-conductive specimen surfaces (High-Tech, H. Scanning Electron Microscope FlexSEM 1000 II).

[0242] Preparation of GelMA Mixtures: GelMA solutions were prepared by dissolving GelMA (3, 7, or 10% w/v) in EC growth medium (EGM-2, Lonza, USA) at 37 °C for 60 min in an incubator. Then, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.06% w/v, Sigma-Aldrich, Germany) and gelatin type A (1.6% w/v) were added to the solution. The solution was then sterilized by passing through a 0.22 pm PES syringe filter (Merck, Germany).

[0243] Fabrication of Multi-layered Constructs: Fabrication of the multilayered constructs is illustrated in FIG. 8b. A sterile polytetrafluoroethylene-coated rod (diameter: 2.5 mm) was inserted into the lumen of the fibrous layer (PCL tube) and placed inside a transparent polypropylene mold (4.4 mm inner diameter for the single vessel tissue, or 10.5 mm inner diameter for the double-vessel tissue). The mold was then filled with the preheated GelMA mixture (with or without SMCs at a density of 500 000 cells/mL) until it was fully submerged. The rod prevented the GelMA mixture entering the lumen. The mold was cooled in an icebath for 1 min to induce physical gelation of the GelMA mixture, followed by UV irradiation for 60 s (OmniCure SI 500, 320-500 nm filter, 30 mW/cm²) to achieve photo-crosslinking. The mold was then immersed in the ice-bath for another 3 min before the multi-layered construct (i.e., hydrogel layer/fibrous layer (GelMA/PCL tube) was separated from the mold and the rod.

[0244] Mechanical testing of the multi-layered construct: To measure the circumferential ultimate tensile strength (UTS) and Young’s modulus of the multi-layered constructs, 5 mm segments were cut and secured to stainless-steel holders on an Instron 5944 Microtester with a 50 N load cell. The segments were stretched at a rate of 50 mm/min according to ISO 7198:2017.

[0245] A burst pressure test was performed by connecting a 2.5 cm segment of the multilayered constructs to a pressure transducer (Lutron, USA) and a pressure meter (Sper scientific, USA). The multi-layered constructs were filled with Vaseline (Unilever, USA) using a syringe pump (Harvard Apparatus, USA) at a flow rate of 1 mL/min until rupture. The maximum pressure exerted during this process was then recorded.

[0246] Suture retention testing was conducted following the ISO 7198:2017 protocol. 1 cm segments of the multi-layered constructs were sutured (Ethicon 5-0, Scotland) 2 mm from the edge, creating a half loop on one side. The multi-layered constructs were secured at their base and the multi-layered constructs were pulled at a rate of 50 mm/min using an Instron 5944 Microtester until the wall failed. Young's modulus was obtained from the linear region of the stress/ strain curve.

[0247] Cell Culture: Human umbilical vein ECs (Lonza, USA) and GFP expressing human umbilical vein ECs (Angio-Proteomie, USA) were cultured in EGM-2 medium (Lonza, USA) and endothelial growth medium ECs (Angio-Proteomie, USA), respectively, with 1% v/v antibiotic-antimycotic solution (Gibco, USA). They were seeded into a multi-layered construct at passage 5 using a micropipette. Human coronary artery SMCs (Lifeline, USA) were cultured in SMC Medium (Lifeline, USA) with 1% v/v antibiotic-antimycotic solution and encapsulated in GelMA during passage 5. Cells were grown in 75 cm² flasks (Coming, USA) and the medium was changed every two days until 70% confluence. ECs were seeded (100 pL, 3 x 10⁶ cells/mL) onto an interior surface of a fibrous layer of the multi-layered constructs. Both ends of the multi-layered constructs were subsequently sealed, and the multi-layered constructs were subjected to gentle rotational (2 rpm) within a custom-designed bioreactor for 4 h. This rotational regime promoted uniform cell attachment throughout an inner luminal area of the multi-layered constructs. Subsequently, the EC-seeded multi-layered constructs were transferred to static culture flasks for further analyses.

[0248] Immunofluorescence Analysis: Multi-layered constructs were transferred to a 15 mL tube after the cultivation period and washed with PBS three times. The multi-layered constructs were fixed in 4% paraformaldehyde (Scharlau, Spain) at 4 °C for 4 h and then sliced into 5 mm segments using a razor blade. The segments were placed in a 96-well plate for staining. The segments were washed with PBS three times and permeabilized with 0.1% Triton X-100 (Labchem, USA). ActinRed 555 ReadyProbes Reagent (Thermo Fisher, USA) was used to stain the actin filaments for 90 min. Nuclei were stained with a 1 : 10,000 dilution of DAPI (Sigma-Aldrich, Germany) after washing the segments with PBS three times. For CD31 and aSMA staining, the segments were blocked with 1% albumin bovine serum (Sigma- Aldrich, Germany) for 1 h and then incubated with Alexa Fluor 488 anti-CD31 antibody and anti-alpha smooth muscle actin antibody (abeam, United Kingdom) overnight. The next day, donkey antirabbit IgG H&L (Alexa Fluor 594) was added for 4 h. The segments were stored in PBS at 4 °C until imaging. Confocal images were acquired using a Nikon A1R+ Confocal Microscope (Nikon, Japan). To minimize light scattering and enhance depth penetration during lightsheet imaging, samples were immersed in a 30% v/v glycerol (Thermo Fisher, USA) solution in distilled water, eliminating the need for separate clearing steps due to the inherent transparency of GelMA. Lightsheet images were obtained with an UltraMicroscope II (Miltenyi Biotec, Germany). [0249] Evaluation of Endothelial Coverage: EC coverage was quantified by confocal microscopy images of randomly selected regions of an interior surface of a fibrous layer of the multi-layered constructs ("inner lumen surface"). The red channel containing the actin filaments staining (ActinRed™ 555) of ECs was extracted from the images and binarized using the threshold function in ImageJ. The black pixels, corresponding to the cells, were measured as a percentage of the total area using the measurement function.

[0250] Evaluation of Cell Migration: Migration of ECs was assessed by confocal microscopy. Samples were randomly selected and imaged at the fibrous layer/hydrogel layer (PCL tube/GelMA) interface of samples with and without pores. Cell nuclei were stained and the number of cells in the GelMA hydrogel layer region was determined using ImageJ software with the cell counter plugin. Cell density was calculated by dividing the cell number by the area of GelMA in each image.

[0251] Evaluation of Vascular Network: Vascular networks were quantified by confocal microscopy at day 7 and at day 14 with and without SMCs. Samples were randomly selected, and images taken of the GelMA hydrogel layer. Actin filaments were stained, and the images were analyzed using AngioTool software (version 0.6a, National Institutes of Health, USA). Total vessel length, total number of junctions, and average vessel area were measured. Average vessel diameter was determined using ImageJ software. Evenly spaced horizontal grid lines (approximately 9 per image) were randomly drawn by ImageJ and the diameter of 20 vessels intersecting with the lines was calculated per image.

[0252] Statistical Analysis: Statistical analysis was performed using Minitab 21.4.0 software. To compare the means of two data sets, Student t tests were applied. To compare the means of more than two data sets, one-way ANOVA with a Tukey’ s post hoc test was used. The level of significance was set at/? < 0.05.

[0253] Results/Discussion

[0254] A method of fabricating multi-layered constructs that promote formation of vascular networks is schematically shown in FIG. 8. The electrospun fibrous layers (PCL tubes) were prepared as previously described in Example 1. The fibrous layers were 6 cm in length, 2.5 mm in inner diameter, and the fibrous layer had a 200 pm wall thickness. The fibrous layer was then machined to include 150 pm diameter pores with 1.5 mm spacing, as depicted in FIG. 8a-ii. This processing resulted in fibrous layers with evenly spaced pores, and SEM imaging showed that the pores pierce the fibrous layers without significant damage to the electrospun scaffold (FIG. 8a-iii). FIG. 8b illustrates that an 800 pm thick hydrogel layer (GelMA hydrogel layer), either with or without SMCs, was cast and cured around the fibrous layers. An interior surface of the fibrous layer was then seeded with ECs to allow formation of an endothelial layer on the interior surface of the fibrous layer and growth of a vascular network through the pores of the fibrous layer. The degree of methacrylation of the GelMA was quantified via 'H NMR spectroscopy by comparing the lysine peaks in the spectra of GelMA and gelatin as described in Example 1. The ¹ H NMR spectra of gelatin and GelMA are shown in FIGS. 14a and 14b. The degree of methacrylation of GelMA was found to be 72%, affording the GelMA sufficient mechanical strength after photo-crosslinking, while retaining the thermoresponsive properties of gelatin (He, J., et al., Gelatin Methacryloyl Hydrogel, from Standardization, Performance, to Biomedical Application. Advanced Healthcare Materials, 2023: p. 2300395). FIG. 8c illustrates that multiple fibrous layers can be used together to create larger constructs and to produce a more extensive vascular network (e.g., capillary network).

[0255] To ensure mechanical stability of the vascularized tissue for clinical applications, burst pressure, suture retention strength, and tensile properties of the multi-layered constructs with and without the pores was measured, as shown in FIGS. 9a-9d. Measured values were plotted next to literature-reported values for native blood vessels (Stekelenburg, M., et al., Dynamic straining combined with fibrin gel cell seeding improves strength of tissue- engineered small-diameter vascular grafts. Tissue Engineering Part A, 2009. 15(5): p. 1081- 1089; and L'Heureux, N., et al., Human tissue-engineered blood vessels for adult arterial revascularization. Nature medicine, 2006. 12(3): p. 361-365), although the values were not compared by statistical tests due to differences in equipment and procedures used for data collection. As shown in FIG. 9a, the burst pressure of the fibrous layers (PCL tubes) decreased a little due to the presence of the pores (p < 0.05), but remained comparable to that of native blood vessels. The pores did not significantly impact the suture retention strength (p = 0.113), ultimate tensile strength (p = 0.335), or Young’s modulus (p = 0.262) of the multi-layered constructs, which were all comparable to those of native blood vessels FIGS. 9b-9d. Therefore, the fabricated multi-layered constructs may represent a suitable scaffold for implanting vascularized tissue in vivo. Moreover, mechanical properties of the multi-layered constructs are more similar to native blood vessels than currently used synthetic grafts, which are significantly stiffer. The stiffness mismatch between native blood vessels and synthetic grafts is believed to be a major contributor to synthetic graft failure by causing abnormal blood flow patterns. [0256] Native blood vessels have a confluent endothelial layer that confers anti- thrombogenic properties and regulates the selective permeability of molecules. The ECs also participate in angiogenesis by migrating from the endothelium into the surrounding tissue in response to hypoxia signals. To evaluate endothelialization of an interior surface of a fibrous layer of the multi-layered constructs after seeding ECs, the cells were fixed and the samples were sectioned and imaged at different time points (days 1, 3, 5, 7, 10, and 14). FIG. 10a shows an image of the cross section of a multi-layered construct after 7 days, and it was observed that the entire circumference of the interior surface was covered by ECs. The confocal images of the endothelium at selected time points (FIG. 10b) revealed proliferation of the ECs after seeding, and a confluent endothelium with complete coverage of the interior surface with cells closely positioned next to one another appears to be established by day 5, as indicated by the CD31 staining. Analysis of the confocal images (FIG. 10c) demonstrated that endothelial coverage was minimal at day 1 (17±8%) but increased significantly at days 3 (40±14%) and 5 (87±5%) and remained at the same value for days 5 to 14 (91±3%). Saturation of the interior surface with ECs also illustrates the formation of a full endothelium. This rapid endothelialization of the interior surface is beneficial for the quick fabrication of vascularized tissue, and it provides a source of ECs that can migrate into the surrounding hydrogel layer to form a vascular (e.g., capillary) network.

[0257] FIG. I la shows collective migration of ECs from the interior surface of the fibrous layer (PCL tube) into the hydrogel layer (GelMA hydrogel layer). This migration commenced shortly after seeding the ECs on the interior surface and increased with time as the endothelial coverage reached confluence (-90%) and the ECs had limited space for proliferation. FIG. 11b shows that the presence of the pores was required for significant migration of ECs from the interior surface of the fibrous layer into the hydrogel layer. Without pores, few ECs are seen in the hydrogel layer, even after 7 days of culture. In contrast, many ECs are observed in the hydrogel layer in multi-layered constructs that were micro-machined with 150 pm diameter pores. FIG. 11c provides a quantification of the number of cells within the hydrogel layer at various timepoints post-seeding with ECs, and the quantitative data supports the qualitative observations from the confocal images. Specifically, multi-layered constructs free of pores have few ECs in the hydrogel layer, likely due to the dense PCL fiber network hindering passage of the cells. In contrast, a statistically significant increase (p < 0.001) in the number of cells was observed for multi-layered constructs comprising a plurality of pores, and the number of cells in the hydrogel layer increased over time during culture. These data demonstrate that the pores are important for the migration of a large number of ECs from the interior surface of the fibrous layer into the hydrogel layer. This migration behavior is crucial for formation of a vascular network in the hydrogel layer and its connection with the fibrous layer to support delivery of oxygen and nutrients to the hydrogel layer.

[0258] FIG. 12a provides an illustration of the formation of a vascular network in the hydrogel layer through EC migration from the interior surface of the fibrous layer. The density of a biodegradable hydrogel network regulates the ability of cells to migrate through the hydrogel layer, and the stiffness of the hydrogel layer can impact the mechanobiology of embedded cells. For this reason, the ability of hydrogel layers with varying matrix densities to allow EC migration and vascular network formation was assessed. Specifically, EC migration and vascular network formation was assessed in 3, 7, and 10% w/v GelMA hydrogel layers. The Young’s modulus of the hydrogel layers increased with the concentration, as shown in Example 1 : 4.3±0.9 kPa for 3% w/v, 43.7±5 kPa for 7% w/v, and 139±16 kPa for 10% w/v. Interestingly, different migration and vascular network formation behavior was observed as a function of GelMA concentration, as shown in FIG. 12b. Specifically, few ECs were found within the hydrogel layers having a concentration 7 and 10% w/v GelMA at 7 days. Additionally, minimal to no vascular network formation was observed. However, a large number of ECs were found at the outer surface of the hydrogels. These results indicate that ECs migrated from the fibrous layer to the edge of the hydrogel layer and formed a monolayer on an external surface of the hydrogel layer. Similar results were observed after 14 days, though some apparent vascular network formation was observed near the outer edge of the 7% w/v GelMA hydrogel layer at the end point of the experiment. The difference in the vascularization behavior of ECs in the 7% and 10% w/v GelMA hydrogel layers may be attributable to the difference in polymer density, noting the invasion of ECs after 14 days of culture in 7% w/v GelMA hydrogel layers, but not in 10% w/v GelMA hydrogel layers. In contrast, ECs in 3% w/v GelMA hydrogel layers formed a vascular network after 7 days of culture and network density was increased at day 14. Accordingly, pores in the fibrous layer appear to facilitate formation of a hydrogel permeated with a vascular network.

[0259] To function as an engineered free tissue flap, it is generally desirable that other cells beyond ECs must be able to cohabitate in the hydrogel layer of the multi-layered construct without impeding the formation of the vascular network. To assess the effect of co-culturing, EC network development was assessed in hydrogel layers that were pre-seeded with SMCs. FIG. 12c illustrates that GFP-expressing ECs in 3% w/v GelMA hydrogel layers formed a vascular network within the SMC-laden hydrogel layer after 14 days of culture. The confocal images were analyzed to quantify total vessel length, total number of junctions, average vessel area, and average vessel diameter. Quantitative analysis of the vascular networks (no SMCs) showed that both the total vessel length and total number of junctions increased significantly from day 7 to 14 (p < 0.001), but no significant difference was observed between the samples with and without SMCs at day 14 (p = 0.325), as shown in FIG. 12d. The analysis also demonstrated that the average vessel area was significantly larger between day 7 (0.03±0.01 mm²) and day 14 in the presence of the SMCs (0.07±0.01 mm²), but not without SMCs (0. l±0.07 mm²). The average vessel diameter was similar between day 7 (6.3±1.1 pm) and 14 without SMCs (7.9±1.2 pm) but increased significantly in the presence of SMCs (20±2.8 pm). This increase in vessel diameter may reflect the maturity of the vascular network in the presence of SMCs. These results demonstrate that ECs can form vascular networks in hydrogel layers in the presence of co-cultured cells, and that the presence of SMCs may impact vessel maturation.

[0260] The size of the multi-layered construct was increased by fabricating a multi-layered construct containing two fibrous layers, seeded with ECs as described for constructs containing a single fibrous layer. The double-fibrous layer construct was thicker, at 10 mm diameter, as compared to the 4 mm diameter described above. Based on the observation that ECs migrated from the interior surface of the fibrous layer (PCL tube) to the surrounding GelMA hydrogel layer and formed a vascular network, it was anticipated that ECs would migrate from the lumens of both fibrous layers and form a more extensive network in the GelMA hydrogel layer, especially in the region between the fibrous layers. FIG. 13a shows confocal images of the multi-layered constructs after 14 days of culture. Endothelialized interior surfaces were observed, indicating that the endothelialization was not adversely affected by the increase in the size of the multi-layered construct. Additionally, ECs formed a dense vascular network in the GelMA hydrogel layer, with a higher density network observed in the regions between the fibrous layers. Using light-sheet microscopy, images of the whole structure were obtained. FIG. 13b shows various patterns of EC network formation, including migration from the fibrous layers, and network formation from the fibrous layer side and from the GelMA hydrogel layer side, as indicated by the arrows. These results suggest that vascular network formation was not impaired by an increase in size of the multi-layered construct. The larger multi-layered construct was 1 cm in diameter, contained an embedded and three-dimensional vascular network, and included endothelialized fibrous layers that have the potential be sutured to native blood vessels, representing a significant advancement toward creating large and vascularized engineered tissues that can survive once transplanted in vivo. The multi-layered construct, including two central, medium sized fibrous layers, may potentially be anastomosed to both arterial and venous circulations at an in vivo implant site.

EQUIVALENTS AND SCOPE

[0261] The invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0262] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A multi-layered construct for tissue-engineered grafts, the construct comprising:

(i) a fibrous layer extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibres are at least partially aligned in the first direction;

(ii) a hydrogel layer disposed on the fibrous layer and comprising a second biocompatible polymer; and

(iii) cells disposed in the hydrogel layer, wherein the cells are substantially aligned in a second direction, and wherein the first direction and the second direction are different.

2. The multi-layered construct of claim 1, further comprising cells disposed on the fibrous layer, wherein the cells disposed on the fibrous layer are substantially aligned in the first direction.

3. The multi-layered construct of claim 1, wherein the fibrous layer is disposed about and extends along the axis in the first direction, the fibrous layer having an interior surface defining a lumen and an exterior surface; and the hydrogel layer is disposed on the exterior surface of the fibrous layer and about the axis; wherein the first direction is further defined as an axial direction, and wherein the second direction is further defined as a circumferential direction about the axis.

4. The multi-layered construct of claim 3, further comprising cells disposed on the interior surface of the fibrous layer, wherein the cells disposed on the interior surface of the fibrous layer are substantially aligned in the first direction.

5. The multi-layered construct of claim 2 or claim 4, wherein at least about 50% of the cells disposed on the fibrous layer are aligned within ± 10° of the first direction.

6. The multi-layered construct of claim 2, claim 4, or claim 5, wherein the cells disposed on the fibrous layer are endothelial cells.

7. The multi-layered construct of any one of claims 1 to 6, wherein the fibres of the first biocompatible polymer are electrospun fibres.

8. The multi-layered construct of any one of claims 1 to 7, wherein the first biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof.

9. The multi-layered construct of claim 8, wherein the first biocompatible polymer is a polyester, and wherein the polyester is polylactide (PLA), poly(lactic acid-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), poly(3 -hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly((3-hydroxyval erate), poly(3- hydroxyhexanoate), poly(4-hydroxybutyrate), poly(4-hydroxy valerate), poly(4- hydroxyhexanoate), polycaprolactone (PCL), poly(ethylene terephthalate), or any combination thereof.

10. The multi-layered construct of any one of claims 1 to 9, wherein the multi-layered construct has a burst pressure of from about 1,000 mm Hg to about 3,500 mm Hg.

11. The multi-layered construct of any one of claims 1 to 10, wherein the multi-layered construct has a suture retention strength measured according to ISO 7198:2017 of from about 100 gf to about 300 gf.

12. The multi-layered construct of any one of claims 1 to 11, wherein the multi-layered construct an ultimate tensile strength measured according to ISO 7198:2017 of from about 1.5 MPa to about 4 MPa.

13. The multi-layered construct of any one of claims 1 to 12, wherein the multi-layered construct has a Young's modulus of from about 0.1 MPa to about 10 MPa.

14. The multi-layered construct of any one of claims 1 to 13, wherein the second biocompatible polymer is a polyester, a polyether, a polyamide, a polyanhydride, a polyesteramide, a polysaccharide, a polysiloxane, a fluoropolymer, a polycarbonate, an acrylate polymer, a polyether ketone, a polyoxazoline, a polysulfone, a polyurethane, a proteinaceous polymer, or any combination thereof.

15. The multi-layered construct of any one of claims 1 to 13, wherein the second biocompatible polymer is a polylactide (PLA) derivative, a poly(lactic acid-co-glycolic acid) (PLGA) derivative, a polyglycolic acid (PGA) derivative, a polyhydroxyalkanoate (PHA) derivative, a poly(3 -hydroxybutyrate) (PHB) derivative, a poly(3-hydroxybutyrate-co-3- hy dr oxy valerate) derivative, a poly((3 -hydroxy valerate) derivative, a poly(3- hydroxyhexanoate) derivative, a poly(4-hydroxybutyrate) derivative, a polyphydroxy valerate) derivative, a poly(4-hydroxyhexanoate) derivative, a polycaprolactone (PCL), poly(ethylene terephthalate) derivative, polyacrylic acid (PAA) or derivatives thereof, polyethylene glycol (PEG) or derivatives thereof, polyvinyl alcohol (PVA) or derivatives thereof, polyvinylpyrrolidone (PVP) or derivatives thereof, polyacrylamide or derivatives thereof, hyaluronic acid or derivatives thereof, chitin or derivatives thereof, chitosan or derivatives thereof, alginate or derivatives thereof, gelatin (Gel), gelatin methacryloyl (GelMA), xanthan gum, carrageenan or derivatives thereof, dextran or derivatives thereof, starch or derivatives thereof, cellulose or derivatives thereof, elastin or derivatives thereof, tropoelastin or derivatives thereof, collagen or derivatives thereof, silk or derivatives thereof, recombinant silk proteins or derivatives thereof, or any combination thereof.

16. The multi-layered construct of any one of claims 1 to 15, wherein the second biocompatible polymer is present in the hydrogel layer in an amount of from about 2% w/v to about 6% w/v based on the total volume of the hydrogel layer.

17. The multi-layered construct of any one of claims 1 to 16, wherein the hydrogel layer has a Young's modulus of from about 3 kPa to about 20 kPa.

18. The multi-layered construct of any one of claims 1 to 17, wherein the first and second biocompatible polymers are biodegradable.

19. The multi-layered construct of any one of claims 1 to 18, wherein at least about 35% of the fibres of the first biocompatible polymer are aligned within ± 10° of the first direction.

20. The multi-layered construct of any one of claims 1 to 19, wherein at least about 40% of the cells disposed in the hydrogel layer are aligned within ± 10° of the second direction.

21. The multi-layered structure of any one of claims 1 to 20, wherein the fibrous layer further comprises a plurality of pores.

22. The multi-layered construct of claim 21, wherein the plurality of pores are distributed at least one of: along the axis; and about the axis.

23. The multi-layered construct of claim 21 or claim 22, wherein the plurality of pores each have a diameter of from about 50 pm to about 1,000 pm.

24. The multi-layered construct of any one of claims 1 to 23, wherein the cells disposed in the hydrogel layer are smooth muscle cells.

25. A method of fabricating a fibrous layer of at least partially aligned fibres for tissue- engineered grafts, the method comprising: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a biocompatible polymer; and b) applying a mechanical force to the fibres of the biocompatible polymer to at least partially align the fibres in the first direction.

26. The method of claim 25, wherein providing the fibrous layer of step a) further comprises electrospinning the biocompatible polymer to form the fibrous layer.

27. The method of claim 25 or claim 26, further comprising: c) disposing cells on the fibrous layer after step b); and d) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

28. The method of any one of claims 25 to 27, wherein applying the mechanical force of step b) comprises directional or bidirectional freezing of a solvent to thereby apply the mechanical force to the fibres of the polymer.

29. The method of claim 28, wherein applying the mechanical force of step b) comprises: b-1) disposing the fibrous layer in a chamber; b-2) partially filling the chamber with a solvent; and b-3) freezing the solvent, wherein the solvent expands in the first direction during freezing to thereby apply the mechanical force to the fibres of the polymer.

30. The method of claim 29, wherein the solvent is water.

31. The method of claim 29 or claim 30, wherein the chamber is configured to direct freezing in the first direction.

32. A method of fabricating a hydrogel layer of a multi-layered construct for tissue- engineered grafts, the method comprising: a) providing a fibrous layer extending along an axis in a first direction from a first end to a second end, the fibrous layer comprising fibres that are at least partially aligned in the first direction; b) disposing a mixture on a surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; c) curing the cross-linkable polymer composition to form the hydrogel layer on the surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and d) allowing the cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

33. The method of claim 32, wherein the fibrous layer is disposed about and extends along the axis in the first direction, the fibrous layer having an interior surface defining a lumen and an exterior surface; the hydrogel layer is formed on the exterior surface of the fibrous layer and about the axis; and wherein the first direction is further defined as an axial direction; and wherein the second direction is further defined as a circumferential direction about the axis.

34. The method of claim 32 or claim 33, wherein the cross-linkable polymer composition comprises polymers having at least one cross-linkable moiety.

35. The method of any one of claims 32 to 34, wherein the curing step c) comprises heating the mixture, irradiating the mixture with a radiation source, adding a curing agent to the mixture, or any combination thereof.

36. The method of claim 35, wherein the curing step c) comprises irradiating the mixture with a radiation source in the presence of a curing agent.

37. The method of claim 36, wherein the radiation source is an ultraviolet (UV) light, and wherein the curing agent is a photoinitiator.

38. The method of any one of claims 32 to 37, further comprising: e) cooling the mixture prior to step c).

39. The method of claim 38, wherein step e) comprises cooling the mixture to a temperature of from about -10 °C to about 10 °C.

40. The method of any one of claims 32 to 39, further comprising: f) treating the fibrous layer to improve a hydrophilicity of the fibrous layer prior to step b).

41. The method of claim 40, wherein the treating step f) comprises contacting the fibrous layer with plasma, contacting the fibrous layer with an acidic solution, contacting the fibrous layer with a basic solution, or any combination thereof.

42. A method of fabricating a multi-layered construct for tissue-engineered grafts, the method comprising: a) forming a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) applying a mechanical force to the fibres of the first biocompatible polymer to at least partially align the fibres in the first direction; c) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and cells; d) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface of the fibrous layer, wherein the hydrogel layer comprises a biocompatible polymer that is cross-linked, and wherein the cells are disposed in the hydrogel layer; and e) allowing the second cells disposed in the hydrogel layer to substantially align in a second direction, wherein the first direction and the second direction are different.

43. The method according to claim 42, further comprising f) disposing cells on the fibrous layer, wherein the cells disposed on the fibrous layer are different from the cells disposed in the hydrogel layer; and g) allowing the cells disposed on the fibrous layer to substantially align in the first direction.

44. A method of fabricating a multi-layered construct for tissue-engineered vascular grafts, the method comprising: a) providing a fibrous layer disposed about and extending along an axis in a first direction from a first end to a second end, wherein the fibrous layer comprises fibres of a first biocompatible polymer, and wherein the fibrous layer has an interior surface defining a lumen and an exterior surface; b) forming a plurality of pores in the fibrous layer; c) disposing endothelial cells on the interior surface of the fibrous layer; and d) forming a hydrogel layer on the exterior surface of the fibrous layer.

45. The method of claim 44, wherein forming the hydrogel layer of step d) comprises: dl) disposing a cross-linkable polymer composition on the exterior surface of the fibrous layer; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface.

46. The method of claim 44, wherein forming the hydrogel layer of step d) comprises: dl) disposing a mixture on the exterior surface of the fibrous layer, wherein the mixture comprises, a cross-linkable polymer composition, and smooth muscle cells; and d2) curing the cross-linkable polymer composition to form the hydrogel layer on the exterior surface, wherein the hydrogel layer comprises a second biocompatible polymer that is cross-linked, and wherein the smooth muscle cells are disposed in the hydrogel layer.