Classifications

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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• • A—HUMAN NECESSITIES
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• • A—HUMAN NECESSITIES
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
  • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0062—General methods for three-dimensional culture
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  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
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  • C12N2533/74—Alginate

Definitions

• the present invention relates to biomaterials and methods for making the same for use in tissue engineering applications such as cell culture, tissue regeneration and wound repair.
• Scaffolds that mimic natural extracellular matrices for use in tissue engineering and the method of preparing and using said scaffolds having fibres and porosity preferably for cell growth are provided by the invention.
• the methods use a facile strategy for creating hierarchical 3D architectures with co-aligned nanofibres and optionally macrochannels, by manipulating ice crystallization in solutions of macromolecules.
• the invention also provides for the use of the scaffolds in promoting cell growth and use as a biomedical implant.
• Biomaterials have been of considerable interest in tissue engineering.
• An ideal biomaterial should provide a biomimetic three-dimensional (3D) environment and support, as well as being able to direct cell behaviour and functions by interaction with cells and mediating the complex multicellular interactions both spatially and temporally.
• 3D three-dimensional
• biomaterials are continuously being developed to mimic the structural features and functions of natural extracellular matrix (ECM).
• ECM extracellular matrix
• Natural ECM exists as a 3D porous architecture of intricate nanofibres with diameters ranging between 50 and 500 nm.
• a main component of the ECM is collagen which has various structural arrangements such as orientation of collagen fibres in different tissues. In a specific tissue, cells are fully responsive to the ECM features to maintain their unique behaviours and functions.
• 2D aligned matrices do not mimic the 3D characteristics of native anisotropic tissues and provide support to cells and tissues in a 3D space. Additionally, a drawback of 2D aligned materials are that they have very small pore sizes and low porosity due to mechanical stretching during the fabrication process.
• aligned fibre-based 3D scaffolds in particular, aligned fibre-based 3D scaffolds with interconnected macropores. Furthermore, it has been challenging to obtain the desired alignment of fibres spatially using currently available technologies (such as tubes with fibre alignment towards the short axis or spheres with fibre alignment towards the centre).
• the main forms of aligned fibre-based structures are two-dimensional membranes and tubes with very thin walls (two dimensional) which consist of aligned nanofibres along the long axis of tubes.
• pre-existing 3D scaffolds having random fibre orientation do not have sufficient interconnectivity and pore size.
• An ideal material for regenerating anisotropic tissue should have a 3D biomimetic architecture with aligned nanofibres and interconnected macropores to direct cell growth, facilitate transport/exchange of nutrients/oxygen/waste and intercellular communications.
• a method for preparing a scaffold comprising the steps of: providing a solution comprising fibre-forming molecules; subjecting the solution to a cooling medium to establish a temperature difference at an interface between the cooling medium and solution; and cooling the solution as a result of the temperature difference to induce solvent crystallisation and alignment of fibres in the solution to create the scaffold.
• the scaffolds of the present invention having aligned fibres can in certain embodiments promote at least one of adhesion, proliferation and differentiation of cells as the scaffold mimics the structure of natural extracellular matrix.
• the present invention further comprises subjecting the scaffold to a solution followed by an additional cooling step to induce solvent crystallisation and channels in the scaffold.
• the channels are substantially co-aligned with the aligned fibres.
• the channels formed in the scaffold can in certain embodiments promote at least one of cell adhesion (capturing) and proliferation.
• Channels formed in the scaffold of the present invention can promote three-dimensional cell growth or cell culture for tissue regeneration.
• the present invention provides a porous biomimetic scaffold comprising a matrix of substantially aligned fibres.
• the present invention provides a porous biomimetic scaffold comprising a three- dimensional matrix of substantially aligned fibres.
• the present invention provides a porous biomimetic scaffold comprising a matrix of fibres.
• the fibres are aligned.
• the fibres are radially aligned fibres, linearly aligned fibres or longitudinally aligned fibres.
• the fibres are unidirectionally aligned.
• the scaffolds of the present invention can be used for cell culture and tissue engineering applications.
• the scaffolds provided by the present invention include a method of treating a mammal suffering from a tissue injury and in need of tissue restoration and/or regeneration, comprising applying to the injury site a scaffold of the present invention.
• the inventors have found that the scaffolds are stable in biological systems in certain circumstances and can therefore be used for cell culture, drug delivery, would healing or the treatment of damaged tissue.
• Figure 1 (a) Scaffolds with radially aligned nanofibres and macrochannels.
• the channel walls are made up of aligned nanofibers along the long axis of the channels as well as pores and particles, (b) Scaffolds with vertically aligned nanofibers and macrochannels.
• FIG. 20 Fabricating 3D silk fibroin (SF) scaffolds (A(F&C) scaffolds) with radially co-aligned nanofibres and macrochannels through a facile freeze-drying technology.
• the hole in 4. A(F&C) scaffolds above) is a top view of the central channel in the A(F&C) scaffolds.
• AFb aligned nanofibrous scaffolds
• AF water-resistant aligned nanofibrous scaffolds without macrochannels
• A(F&C) water-resistant scaffolds with radically co- aligned nanofibres and macrochannels.
• FIG. 21 Figure 3 Hierarchical structure of a 3D scaffold with radially aligned nanofibres and channels (A(F&C)).
• A(F&C) Micro-CT images demonstrating the radially aligned channel structure of the scaffold. Scale bars: 1000 ⁇ .
• FIG. 4 Polypropylene porous microfibrous materials modified with locally aligned silk fibroin (SF) nanofibres of the present invention (the nanofibers in a, b and c used 0.0125%, 0.025% and 0.05% (w/v) of silk fibroin solutions, respectively), a', b' and c' are the magnification of a, b, and c, respectively. Scale bars: 200 ⁇ in a, b and c; 10 ⁇ in a' and b'; 30 ⁇ in c'.
• SF silk fibroin
• FIG. 5 Polypropylene porous microfibrous materials modified with locally aligned alginate nanofibres using 0.025% (w/v) of an alginate solution (a,b,c; a, b and c are at different magnifications) and locally aligned gelatin nanofibres using 0.025% (w/v) of a gelatin solution (d,e,f; d, e and f are at different magnifications) of the present invention.
• Scale bars 100, 10, 1 , 200, 20, and 1 ⁇ in a, b, c, d, e and f, respectively.
• HUVECs Human Umbilical Vein Endothelial Cells
• FIG. 7 Aligned nanofibres and channels in 3D A(F&C) scaffolds facilitate the formation of CD31 -positive vessel-like structures by directing the growth, migration and interaction of adherent HUVECs after 21 days of culture (Fig. 6c illustrates how to read images presented in Fig. 7).
• FIG. 8 Aligned nanofibres and channels of 3D A(F&C) scaffolds facilitate capture of the non-adherent Embryonic Dorsal Root Ganglion Neuron cells (DRG), and direct the 3D growth of DRG neurites.
• DRG non-adherent Embryonic Dorsal Root Ganglion Neuron cells
• Scale bars 100 ⁇ for W and W&F scaffolds; 50 ⁇ for AF scaffolds, (c) Aligned nanofibres and channels direct the 3D growth of DRG neurites in 3D A(F&C) scaffolds. Scale bars: from left to right 75, 25 and 25 ⁇ , respectively.
• FIG. 9 3D A(F&C) scaffolds direct the growth, migration and interaction of both adherent HUVECs, and non-adherent DRGs and DRG neurites by radially aligned channels and nanofibres.
• Adherent HUVECs are mainly guided by aligned nanofibres, and nonadherent DRGs and DRG neurites are mainly directed by aligned channels, (a) HUVECs growing and interacting along the aligned nanofibres on channel walls, (b) HUVECs assemble into CD31 -positive vessel-like structures along the aligned nanofibres on channel walls, (c), (d) and (e) DRGs and DRG neurites growing along the aligned channels, suggesting the 3D growth of DRGs and DRG neurites in A(F&C) scaffolds. All scale bars are 25 ⁇ .
• FIG. 10 (a) Representative SEM images showing aligned nanofibres and nanoparticles in AFb scaffolds. Fast Fourier Transform (FFT) pattern in the inset suggests these nanofibres were well aligned in the radial direction. Scale bars: from left to right 2, 1 and 10 ⁇ , respectively, (b) Directional freezing of aqueous silk fibroin solution in liquid nitrogen allows fabricating 3D silk fibroin nanofibrous scaffolds with various geometries (including cylinders, tubes and particles or spheres), diameters and thicknesses as well as different nanofibre alignments.
• FFT Fast Fourier Transform
• FIG. 11 Effects of freezing temperature on the morphology structure of 3D silk fibroin scaffolds
• (a) SEM images reveal freezing aqueous silk fibroin at -80°C leading to 3D scaffolds (W&Fb) with a hybrid structure with short channels/pores/fibres. Scale bars: from left to right 200, 30 and 100 ⁇ , respectively
• (b) SEM images show freezing aqueous silk fibroin at -20°C producing 3D scaffolds (Wb) with wall-like porous structure. Scale bars: from left to right 200, 20 and 100 ⁇ , respectively.
• FIG. 12 Representative images of A(F&C) scaffolds from SF/gelatin mixture (a); sodium alginate (b). Red arrows indicate the channels in scaffolds with aligned nanofibres on the wall of channels. Scale bars: 20 ⁇ in a and 2 ⁇ in inset 1 , b and inset 2.
• FIG. 13 Micro-CT images of the hybrid structure (containing short channels/pores/nanofibres) of W&F and the wall-like porous structure of W 3D scaffolds. The details in structure can be seen clearly in Figure 14. All scale bars are 1000 ⁇ .
• Figure 14 (a) SEM images of the water-resistant W&F scaffolds after post- treatment. Scale bars: from left to right 100, 20 and 100 ⁇ , respectively, (b) SEM images of the water-resistant W scaffolds after post-treatment. Scale bars: from left to right 100, 20 and 100 ⁇ , respectively.
• FIG. 15 ATR-FTIR spectra of 3D silk fibroin scaffolds, (a) ATR-FTIR spectra of silk fibroin scaffolds from different freezing-temperatures: -20°C (Wb), -80°C (W&Fb) and liquid nitrogen (AFb). (b) ATR-FTIR spectra of post-treated silk fibroin scaffolds. All scaffolds (A(F&C), W&F and W) present peaks at around 1517, 1622 and 1700 cm "1 , suggesting the post-treatment made the structure transition of silk fibroin from random coils to ⁇ -sheets.
• Figure 16 (a) Compressive modulus of 3D W, W&F and A(F&C) silk fibroin scaffolds, (b) Morphology of scaffolds after mechanical test. Of note, after being compressed in the mechanical test, A(F&C) scaffolds still maintained a good radially aligned morphology and structure, and just some minor collapses are seen on the surface of scaffolds, probably resulting from damage of some channels.
• a method for preparing a scaffold comprising the steps of: providing a solution comprising fibre-forming molecules; subjecting the solution to a cooling medium to establish a temperature difference at an interface between the cooling medium and solution; and cooling the solution as a result of the temperature difference to induce solvent crystallisation and alignment of fibres in the solution to create the scaffold.
• the present inventors have found that controlled cooling of a solution comprising fibre-forming molecules induces solvent crystallization in which fibres can align to create a scaffold.
• the alignment of the fibres can be directionally controlled so that crafted scaffolds may be generated having fibres aligned in a direction in which the solvent crystallization forms.
• the method of the invention can be used to prepare any "scaffold" which as used herein preferably refers to a three-dimensional matrix of fibres which is suitable as a template for a cell carrier for cell culture, tissue repair, tissue engineering or related applications.
• the scaffold is a 3D scaffold comprising channels and pores that enable and facilitate cell culture and flow of biochemical and physicochemical factors within the scaffold which are necessary for cell culture and survival.
• the scaffolds are formed from a solution comprising fibre forming molecules.
• the technique used to prepare a scaffold according to the method of the present invention will depend on the solution, fibre-forming molecule and cooling medium used. It will also be appreciated that the technique used will affect the direction of the alignment of the fibres whether they are longitudinally or radially aligned.
• the solution may be subjected directly or indirectly to a cooling medium to establish a temperature difference at an interface between the solution and cooling medium.
• the solution comprising fibre- forming molecules is contained in a receptacle prior and subjected indirectly to the solution for cooling.
• the receptacle may be immersed in the cooling medium followed by addition of the solution comprising fibre-forming molecules to the receptacle to induce alignment of fibres.
• Any suitable receptacle material can be used in the present invention providing a temperature difference is set up at an interface between the solution and the cooling medium.
• the receptacle material is selected from but not limited to glass, metal, plastic, ceramic or combinations thereof.
• the solution comprising fibre-forming molecules can be subjected to a cooling medium directly.
• the solution comprising fibre-forming molecules can be dripped, sprayed or injected directly into a cooling medium to establish a temperature difference at an interface between the cooling medium and solution to induce solvent crystallization and alignment of fibres in the scaffold.
• the inventors believe that the alignment of fibres is controlled by solvent crystallization which occurs when a temperature difference between the solution and the cooling medium is sufficient for nucleation of crystals to form. For instance, where the solvent is water, ice nucleation will form when the temperature difference is sufficient to cause freezing and ice crystals so formed radiate from an interface between the solution and the cooling medium into the solution.
• the solvent crystals and the direction in which they form are believed to act as templates to control the alignment direction of fibres.
• the temperature difference is imperative for the formation of solvent crystallization and alignment of fibres.
• the temperature difference is determined by the difference in temperature between the solution and the cooling medium.
• the temperature difference is sufficient to promote nucleation of solvent crystals at the interface.
• the temperature difference can be measured relative to the solution. For example, if the solution had a temperature of 20°C and the cooling medium had a temperature of -40°C, the temperature difference would be -60°C relative to the solution.
• the temperature difference is at least -120°C relative to the solution.
• the temperature difference is at least -196°C relative to the solution.
• the temperature difference is in a range of from -20°C to -296°C relative to the solution.
• the temperature difference is in a range of from -80°C to -296°C relative to the solution or -180°C to -296°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -120°C to -296°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -20°C to -196°C relative to the solution or -30°C, -40°C, -50°C -60°C or -70°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -80°C to -196°C relative to the solution or -90°C or -100°C relative to the solution.
• the temperature difference is in a range of from -100°C to - 196°C relative to the solution or -1 10°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -120°C to -196°C relative to the solution or - 130°C, -140°C, -150°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -150°C to -196°C relative to the solution or -160°C relative to the solution. In certain embodiments, the temperature difference is in a range of from -170°C to -196°C relative to the solution or -180°C or -190°C relative to the solution.
• the direction of the alignment of fibres can be controlled by adjusting the direction of the temperature difference (i.e., cooling direction).
• the establishment of the temperature difference between the cooling medium and solution comprising fibre-forming molecules induces aligned fibres from the interface between the solution and cooling medium.
• the establishment of the temperature difference between the cooling medium and solution comprising fibre-forming molecules induces unidirectionally aligned fibres from the interface between the solution and cooling medium.
• unidirectionally aligned fibres refers to the fibres in the scaffold being oriented towards a single direction.
• Non-limiting examples of unidirectionally aligned fibres include either fibres which are roughly parallel to each other (linearly aligned) or run roughly towards a point in space (radially aligned). It is to be understood that not every fibre must be oriented towards a single direction, and some deviation in direction is contemplated.
• the temperature difference is established circumferentially to the solution to induce radially aligned fibres in the scaffold. In certain embodiments, the temperature difference is established along a plane of the interface to induce linearly or longitudinally aligned fibres in the scaffold. Therefore the plane may be parallel or perpendicular to the interface.
• the temperature difference is a relative measure of the temperature range between the cooling medium and solution comprising fibre-forming molecules. It can also be convenient to express the temperature sufficient to induce alignment of fibres in absolute terms. For example, the temperature of the cooling medium to induce nucleation of solvent crystals for alignment of fibres can be expressed.
• the cooling medium is at a temperature less than -196°C.
• the cooling medium is at a temperature of from -80°C to -196°C. In some embodiments, the cooling medium is at a temperature less than -80°C or -90°C, - 100°C. In some embodiments, the cooling medium is at a temperature of from -100°C to - 196°C or -1 10°C to -196 °C. In some embodiments, the cooling medium is at a temperature of from -120°C to -196°C or -130°C to -196°C. In some embodiments, the cooling medium is at a temperature of from -140°C to -196°C or -150°C to -196°C. In some embodiments, the cooling medium is at a temperature of from -160°C to -196°C or -170°C to -196°C, or -180°C to -196°C.
• the rate of cooling of the solution comprising fibre-forming molecules can influence alignment of fibres.
• the solution is cooled at a rate of 0.2°C.s “1 to 260°C.s “1 .
• the solution is cooled at a rate of 5°C.s "1 to 260°C.s ⁇ 1 or 10°C.s “1 to 260°C.s “1 or 15°C.s "1 to 260°C.s "1 .
• the solution is cooled at a rate of 20°C.s “1 to 260°C.s “1 or 25°C.s “1 to 260°C.s “1 , 30°C.s ⁇ 1 to 260°C.s “1 , 35°C.s ⁇ 1 to 260°C.s “1 or 40°C.s “1 to 260°C.s “1 .
• the solution is cooled at a rate of 50°C.s "1 to 260°C.s “1 or 60°C.s “1 to 260°C.s “1 or 70°C.s "1 to 260°C.s “1 .
• the solution is cooled at a rate of 80°C.s “1 to 260°C.s “1 or 90°C.s “1 to 260°C.s “1 , 100°C.s “1 to 260°C.s “1 or 1 10°C.s “1 to 260°C.s “1 . In some embodiments, the solution is cooled at a rate of 120°C.s “1 to 260°C.s “1 or 130°C.s “1 to 260°C.s “1 or 140°C.s “1 to 260°C.s “1 .
• the solution is cooled at a rate of 150°C.s “1 to 260°C.s “1 or 160°C.s “1 to 260°C.s “1 , 170°C.s “1 to 260°C.s “1 , 180°C.s “1 to 260°C.s “1 , 190°C.s “1 to 260°C.s “1 , 200°C.s “1 to 260°C.s “1 , 210°C.s “1 to 260°C.s “1 , 220°C.s “1 to 260°C.s “1 , 230°C.s “1 to 260°C.s “1 , 240°C.s “1 to 260°C.s “1 or 250°C.s “1 to 260°C.s “1 .
• the sample of solution comprising fibre-forming molecules can be gradually immersed into the cooling medium to induce alignment of fibres in the scaffold.
• the solution is subjected by immersion in the cooling medium at a rate of 1 to 15 mm.min “1 .
• the solution is subjected by immersion in the cooling medium at a rate of 3 to 15 mm.min "1 .
• the solution is subjected by immersion in the cooling medium at a rate of 1 to 10 mm.min "1 .
• the solution is subjected by immersion in the cooling medium at a rate of 5 to 10 mm.min "1 .
• the solution is subjected by immersion in the cooling medium at a rate of 5 to 8 mm.min "1 .
• any suitable cooling medium can be used in the method of the present invention to induce alignment of fibres in the scaffold.
• the cooling medium could be a solid, a liquid or a gas depending on the exact nature of the cooling medium.
• the cooling medium could be liquid nitrogen, dry ice, air, liquid ethane, liquid C0 2 and combinations thereof.
• the cooling medium is a freezer.
• the cooling medium is dry ice in combination with at least one of tetrachloroethylene, carbon tetrachloride, 1 ,3-dichlorobenzene, o-xylene, m-toluidine, acetonitrile, pyridine, m-xylene, n-octane, isopropyl ether, acetone, butyl acetate, propyl amine.
• the cooling medium is liquid nitrogen in combination with at least one of ethyl acetate, n-butanol, hexane, acetone, toluene, methanol, ethyl ether, cyclohexane, ethanol, ethyl ether, n-pentane, isopentane.
• the cooling medium is liquid nitrogen.
• Deviation in direction of the alignment of the fibres is contemplated. It can be convenient to express the deviation of the alignment of the fibres relative to the surface normal of the interface between the cooling medium and solution comprising fibre-forming molecules.
• the fibres are aligned between 0° to 30° to a surface normal of the interface. In one embodiment, the fibres are aligned between 0° to 25° to a surface normal of the interface. In one embodiment, the fibres are aligned between 0° to 20° to a surface normal of the interface. In one embodiment, the fibres are aligned between 0° to 15° to a surface normal of the interface. In one embodiment, the fibres are aligned between 0° to 10° to a surface normal of the interface. In one embodiment, the fibres are aligned between 0° to 5° to a surface normal of the interface.
• the formation of solvent crystals can function as a template which provides control of fibre alignment in the scaffold.
• the diameter of the solvent crystals will depend on the solvent used, cooling rate, and cooling medium used. Any suitable diameter of solvent crystal can be used in the method of the present invention to induce alignment of fibres.
• the solvent crystals formed from solvent crystallisation has a diameter from 20 nm to 5 mm, 20 nm to 4 mm, 20 nm to 3 mm, 20 nm to 2 mm or 20 nm to 1 mm.
• the solvent crystals formed from solvent crystallisation has a diameter from 1 nm to 500 ⁇ , 10 nm to 400 ⁇ or 10 nm to 300 ⁇ .
• the solvent crystals formed from solvent crystallisation has a diameter from 10 nm to 200 ⁇ . In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 10 nm to 100 ⁇ . In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 10 nm to up to 90 ⁇ , 80 ⁇ , 70 ⁇ , 60 ⁇ , 50 ⁇ , 40 ⁇ , 30 ⁇ , 20 ⁇ or 10 ⁇ . In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 10 nm to 5 ⁇ . In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 100 ⁇ to 2 mm.
• the solvent crystals formed from solvent crystallisation has a diameter from 10 to 3000 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 10 to 3000 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 20 to 2500 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 20 to 2000 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 50 to 2000 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 50 to 1500 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 50 to 1000 nm. In one embodiment, the solvent crystals formed from solvent crystallisation has a diameter from 50 to 700 nm.
• the duration of the cooling step can affect the diameter of the solvent crystals and the resulting fibre diameters. Any suitable duration can be used provided that it is sufficient to induce alignment of fibres in the scaffold.
• the solution comprising fibre-forming molecules is cooled for less than 10 minutes. In some embodiments, the solution comprising fibre-forming molecules is cooled for less than 20 minutes. In some embodiments, the solution comprising fibre-forming molecules is cooled for less than 30 minutes. In some embodiments, the solution comprising fibre-forming molecules is cooled for less than 1 hour. In some embodiments, the solution comprising fibre-forming molecules is cooled for less than 5 minutes. In some embodiments, the solution comprising fibre-forming molecules is cooled for less than 1 minute.
• the scaffolds prepared by the method of the present invention can retain the solvent crystals formed from solvent crystallisation.
• the solvent crystals can be removed from the scaffold using any suitable technique.
• the scaffold prepared by the method of the present invention can be lyophilized (freeze-dried) to remove the solvent crystals.
• the solvent crystals can be thawed into solution state after cooling and solvent removed under reduced pressure such as in a vacuum or vacuum drying oven.
• the solvent crystals can be removed from the scaffold using a desiccator.
• the scaffold can be water soluble.
• the scaffold can be treated to impart water-resistance.
• the scaffold can be treated using any suitable agent to impart water-resistance.
• the scaffold can be subjected to the group consisting of ethanol, methanol, genipin, glutaraldehyde, 1 - ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride, water or combination thereof.
• ethanol, methanol, genipin, glutaraldehyde, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride or water can be in liquid or vapour phase (for example ethanol solution or ethanol vapour).
• the scaffold is water-resistant.
• the scaffold can be treated to induce cross-linking between the aligned fibres.
• the scaffold can be subjected to glutaraldehyde or electromagnetic radiation to induce cross-linking in the scaffold.
• the scaffold can be subjected to at least one of methanol, ethanol, genipin, 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride, water, plasma radiation or combinations thereof to induce cross-linking in the scaffold.
• methanol, ethanol, genipin, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, calcium chloride or water can be in liquid or vapour phase (for example ethanol solution or ethanol vapour).
• any suitable solvent can be used to dissolve the fibre-forming molecules to form a solution.
• the solvent is water, organic solvent, inorganic nonaqueous solvent and combinations thereof.
• the solution comprising fibre-forming molecules is an aqueous solution.
• the solvent crystals formed from crystallisation are ice crystals.
• Suitable organic solvents can be selected from the group consisting of pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1 ,4-dioxane, chloroform, diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethyl formamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, n-butanol, isopropanol, n-propanol, ethanol, methanol, formic acid, acetic acid, hexafluoroisopropanol, trifluoroacetic acid and combinations thereof.
• Suitable inorganic solvents can be selected from the group consisting of liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl chloride fluoride, phosphoryl chloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, neat sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid and combinations thereof.
• the solution comprising fibre-forming molecules can include a mixture of two or more miscible solvents such as a mixture of water and an aqueous soluble solvent, a mixture of two or more organic solvents, or a mixture of an organic and an aqueous soluble solvent.
• miscible solvents such as a mixture of water and an aqueous soluble solvent, a mixture of two or more organic solvents, or a mixture of an organic and an aqueous soluble solvent.
• the amount of fibre-forming molecules dissolved in the solution can be any suitable amount and a person skilled in the relevant art would appreciate that the amount dissolved can depend on the solubility of the fibre-forming molecule and the solvent used.
• the solution comprising fibre-forming molecules is in an amount of from 0.001 % to 35% w/v. In certain embodiments, the solution comprising fibre-forming molecules is in an amount of from 1 % to 20% w/v. In certain embodiments, the solution comprising fibre-forming molecules is in an amount of from 1 % to 25% w/v. In certain embodiments, the solution comprising fibre-forming molecules is in an amount of from 1 % to 15% w/v. In certain embodiments, the solution comprising fibre-forming molecules is in an amount of from 1 % to 10% w/v. In certain embodiments, the solution comprising fibre-forming molecules is in an amount of from 1 % to 5% w/v.
• the present invention also relates to a porous biomimetic scaffold comprising a three-dimensional matrix of substantially aligned fibres.
• the fibres are unidirectionally aligned.
• the fibres are radially aligned.
• the fibres are linearly or longitudinally aligned.
• the diameter of the fibres in the scaffold of the present invention will depend on the solvent, cooling rate, fibre-forming molecule and cooling medium used. In certain embodiments, the diameter of the fibre is from 20 to 5000 nm, 20 to 4000 nm or 20 to 3000 nm. In certain embodiments, the diameter of the fibre is from 20 to up to 2500 nm, 2000 nm or 1500 nm. In certain embodiments, the diameter of the fibre is from 20 to 1000 nm. In certain embodiments, the diameter of the fibre is from 50 to 600 nm. In certain embodiments, the diameter of the fibre is from 20 to 800 nm. In certain embodiments, the diameter of the fibre is from 100 to 500 nm.
• the diameter of the fibre is from 300 to 800 nm. In certain embodiments, the diameter of the fibre is from 300 to 600 nm. [66] It can also be convenient to describe the fibres in terms of the length of the aligned fibres.
• the aligned fibres have a length of at least 50 nm. In certain embodiments, the aligned fibres have a length of from 50 nm to 50 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 4 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 2 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 500 ⁇ .
• the aligned fibres have a length of from 50 nm to 1000 ⁇ . In certain embodiments, the aligned fibres have a length of from 100 nm to 500 ⁇ . In certain embodiments, the aligned fibres have a length of from 50 nm to 5000 nm. In certain embodiments, the aligned fibres have a length of from 50 nm to 1000 nm. In certain embodiments, the aligned fibres have a length of from 100 nm to 500 nm. In certain embodiments, the aligned fibres have a length of from 50 nm to 500 nm. In certain embodiments, the aligned fibres have a length of from 50 nm to 5 mm.
• the aligned fibres have a length of from 50 nm to 10 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 20 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 30 mm. In certain embodiments, the aligned fibres have a length of from 50 nm to 40 mm.
• the scaffold of the present invention is a three- dimensional matrix of fibres suitable for cell culture, tissue repair, tissue engineering or related applications.
• the scaffolds can have pores of any diameter suitable for cell culture, tissue repair, tissue engineering or related applications.
• the scaffold has pores of diameter from 1 nm to 500 ⁇ or 20 nm to 500 ⁇ .
• the scaffold has pores of diameter from 20 nm to 400 ⁇ .
• the scaffold has pores of diameter from 20 nm to 300 ⁇ .
• the scaffold has pores of diameter from 20 nm to 200 ⁇ .
• the scaffold has pores of diameter from 20 nm to up to 100 ⁇ , 90 ⁇ , 80 ⁇ , 70 ⁇ , 60 ⁇ , 50 ⁇ , 40 ⁇ , 30 ⁇ , 20 ⁇ , 10 ⁇ or 5 ⁇ .
• the scaffold has pores of diameter from 20 to 1500 nm.
• the scaffold has pores of diameter from 50 to 1000 nm.
• the scaffold has pores of diameter from 20 to 800 nm.
• the scaffold has pores of diameter from 50 to 600 nm.
• the scaffold has pores of diameter from 100 to 600 nm.
• the scaffold has pores of diameter from 20 to 600 nm.
• the scaffold has pores of diameter from 20 to 500 nm.
• the scaffold of the present invention can also be conveniently described in terms of porosity.
• the porosity of the scaffold can depend on the fibre-forming molecule and solvent used.
• the scaffold porosity was calculated as the ratio of the void volume to the total sample volume. Accordingly, in certain embodiments, the scaffold has a porosity of from 0.01 % to 95%. In certain embodiments, the scaffold has a porosity of from 20% to 95%, 30% to 95% or 40% to 95%. In certain embodiments, the scaffold has a porosity of from 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90% or 85% to 90%. In certain embodiments, the scaffold has a porosity of from 40% to 80%, 40% to 70%, 40% to 60% or 40% to 50%. In certain embodiments, the scaffold has a porosity of from 60% to 80% or 65% to 75%. In certain embodiments, the scaffold has a porosity of from 30% to 60%, 30% to 50% or 30% to 40%.
• At least 5% w/w of the scaffold comprises aligned fibres, based on the total dry weight of the scaffold.
• at least 10% w/w, 20% w/w, 30% w/w, 40% w/w, 50% w/w or 60% w/w of the scaffold comprises aligned fibres, based on the total dry weight of the scaffold.
• at least 70% w/w of the scaffold comprises aligned fibres, based on the total dry weight of the scaffold.
• At least 80% w/w of the scaffold comprises aligned fibres, based on the total dry weight of the scaffold. In some embodiments, at least 90% w/w of the scaffold comprises aligned fibres, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises of from 50% to 90% w/w of aligned fibres, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises of from 60% to 90% w/w of aligned fibres, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises of from 70% to 90% w/w of aligned fibres, based on the total dry weight of the scaffold. In some embodiments, the scaffold comprises of from 80% to 90% w/w of aligned fibres, based on the total dry weight of the scaffold.
• the scaffold can take any suitable shape and can be for example in the shape of spheres, cubes, prisms, fibres, rods, tetrahedrons, tubes, or irregular particles.
• the shape of the scaffold can be controlled by using a receptacle as discussed above and the shape of the receptacle can typically determine the shape of the scaffold ultimately produced.
• a radially aligned fibre scaffold can be prepared by providing a solution of fibre-forming molecules in a cylindrical sample tube.
• the sample tube can be immersed in the cooling medium (such as liquid nitrogen) to establish the temperature difference at an interface between the cooling medium and solution circumferentially to induce formation of radially aligned fibres in the scaffold.
• the cooling medium such as liquid nitrogen
• a linearly or longitudinally aligned fibre scaffold can be typically prepared by providing a solution of fibre-forming molecules in a cylindrical sample tube having a flat base.
• the sample tube can be slowly lowered into the cooling medium (such as liquid nitrogen) from the flat base end to establish the temperature difference at an interface between the cooling medium and solution along the plane substantially parallel to the base to induce formation of linearly or longitudinally aligned fibres in the scaffold.
• the cooling medium such as liquid nitrogen
• the scaffold can be of any suitable size with the size being determined, in part by the desired size of the scaffold ultimately produced or the size of the receptacle, if used.
• the size of the scaffold can be controlled by mechanical treatment such as cutting the scaffold using a blade or laser.
• the scaffold is formed by controlling the cooling of the solution comprising fibre-forming molecules such that as the scaffold is formed, the cooling step is terminated once the desired scaffold size is reached.
• the scaffold of the present invention is typically less than 10 cm in at least one dimension.
• the scaffold has a size of from 20 nm to 10 cm in at least one dimension.
• the scaffold has a size of from 1 mm to 10 cm in at least one dimension.
• the scaffold has a size of from 5 mm to 8 cm in at least one dimension.
• the scaffold has a size of from 5 mm to 5 cm in at least one dimension.
• the scaffold has a size of from 1 mm to 3 cm in at least one dimension.
• the scaffold has a size of from 1 mm to 2 cm in at least one dimension.
• the scaffold has a size of from 1 mm to 1 cm in at least one dimension.
• the scaffold of the present invention has a compressive modulus of 5 to 5000 kPa. In certain embodiments, the scaffold of the present invention has a compressive modulus of 5 kPa to up to 4500 kPa, 4000 kPa, 3500 kPa, 3000 kPa, 2500 kPa, 2000 kPa, 1500 kPa, 1000 kPa, 500 kPa, 400 kPa, 300 kPa or 200 kPa. In certain embodiments, the scaffold of the present invention has a compressive modulus of 20 to 160 kPa. In certain embodiments, the scaffold has a compressive modulus of 20 to 140 kPa.
• the scaffold has a compressive modulus of 20 to 120 kPa. In certain embodiments, the scaffold has a compressive modulus of 40 to 100 kPa. In certain embodiments, the scaffold has a compressive modulus of 60 to 100 kPa. In certain embodiments, the scaffold has a compressive modulus of 70 to 100 kPa. In certain embodiments, the scaffold has a compressive modulus of 80 to 100 kPa. Scaffolds with aligned fibres and channels
• the method of the present invention can further comprise subjecting the scaffold to a solution or solvent followed by an additional cooling step to induce solvent crystallisation and channels in the scaffold.
• the channels are substantially co-aligned with the aligned fibres.
• the channels can be microchannels or macrochannels.
• the additional cooling step can be at any suitable temperature to induce channels in the scaffold.
• the additional cooling step is at a temperature of from -5°C to -196°C.
• the additional cooling step is at a temperature of from -10°C to -196°C.
• the additional cooling step is at a temperature of from -5°C to -80°C.
• the additional cooling step is at a temperature of from -10°C to -80°C.
• the additional cooling step is at a temperature of from -10°C to -60°C.
• the additional cooling step is at a temperature of from -10°C to -40°C.
• the additional cooling step is at a temperature of from -10°C to -30°C. In one embodiment, the additional cooling step is at a temperature of from -10°C to -25°C, -1 1 °C to -25°C, -12°C to -25°C, -13°C to - 25°C, -14°C to -25°C, -15°C to -25°C, -16°C to -25°C, -17°C to -25°C, -18°C to -24°C, -18°C to -23°C, -18°C to -22°C or -19°C to -21 °C.
• the solvent crystals formed during the additional cooling step have a diameter from 20 nm to 4 mm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter from 100 ⁇ to 2 mm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter from 50 nm to 1000 nm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter from 100 ⁇ to 2 mm. In one embodiment, the solvent crystals formed during the additional cooling step have a diameter from 100 ⁇ to 1000 ⁇ . In one embodiment, the solvent crystals formed during the additional cooling step have a diameter from 500 ⁇ to 1000 ⁇ .
• the duration of the additional cooling step can affect the diameter of the solvent crystals and the resulting channel diameters. Any suitable duration can be used provided that it is sufficient to induce channel formation in the scaffold.
• the additional cooling step is performed between 5 minutes to 96 hours. In some embodiments, the additional cooling step is performed between 10 minutes to 60 hours. In some embodiments, the additional cooling step is performed between 1 hour to 96 hours. In some embodiments, the additional cooling step is performed between 1 hour to 60 hours. In some embodiments, the additional cooling step is performed between 12 hours to 50 hours. In some embodiments, the additional cooling step is performed between 24 hours to 48 hours. In some embodiments, the additional cooling step is performed between 36 hours to 50 hours. In some embodiments, the additional cooling step is performed between 48 hours to 60 hours.
• the scaffold further comprises a channel.
• the diameter of the channels can vary depending on the fibre-forming molecule, solvent, duration of the additional cooling step and solvent crystal diameter.
• the channel has a diameter from 20 nm to 2 cm, 20 nm to 1 cm, 20 nm to 500 ⁇ , 20 nm to 400 ⁇ , 20 nm to 300 ⁇ , 20 nm to 200 ⁇ or 20 nm to 100 ⁇ .
• the channel has a diameter from 10 ⁇ to 4 mm, 10 ⁇ to 3 mm, 10 ⁇ to 2 mm or 10 ⁇ to 1 mm.
• the channel has a diameter of from 20 nm to 4 mm.
• the channel has a diameter of from 10 ⁇ to 2 mm. In some embodiments, the channel has a diameter of from 50 ⁇ to 1 mm. In some embodiments, the channel has a diameter of from 100 ⁇ to 1000 ⁇ . In some embodiments, the channel has a diameter of from 100 ⁇ to 800 ⁇ . In some embodiments, the channel has a diameter of from 100 ⁇ to 600 ⁇ . In some embodiments, the channel has a diameter of from 100 ⁇ to 400 ⁇ . In some embodiments, the channel has a diameter of from 20 nm to 2 mm. In some embodiments, the channel has a diameter of from 20 nm to 1 mm. In some embodiments, the channel has a diameter of from 400 ⁇ to 1000 ⁇ . In some embodiments, the channel has a diameter of from 400 ⁇ to 800 ⁇ .
• the present inventors have found that in embodiments where the scaffold comprises aligned fibres and channels in the scaffold, the scaffolds of the present invention had significantly higher cell viability than scaffolds comprising aligned fibres without channels.
• the scaffold comprising aligned fibres and channels showed improved cell capturing and proliferation.
• the aligned fibres and co-aligned channels can direct migration of cells and infiltration of tissues, and thus accelerate the regeneration or function reestablishment of damaged tissues.
• the scaffolds of the present invention can be useful for repair of wounds (radial growth of tissue can assist wound closure) and can assist in repair of cracked bones. Fibre-forming molecules
• the scaffold of the present invention and the method of preparing the same can be prepared using any suitable fibre-forming molecule.
• the fibre- forming molecules are selected from the group consisting of a natural polymer, a synthetic polymer and combinations thereof.
• Natural polymers may include polysaccharides, polypeptides, glycoproteins, and derivatives thereof and copolymers thereof.
• Polysaccharides may include agar, alginates, chitosan, hyaluronan, cellulosic polymers (e.g., cellulose and derivatives thereof as well as cellulose production by-products such as lignin) and starch polymers.
• Polypeptides may include various proteins, such as silk fibroin, lysozyme, collagen, keratin, casein, gelatin and derivatives thereof.
• Derivatives of natural polymers, such as polysaccharides and polypeptides may include various salts, esters, ethers, and graft copolymers. Exemplary salts may be selected from sodium, zinc, iron and calcium salts.
• the natural polymer is selected from the group consisting of at least one of silk fibroin, alginate, bovine serum albumin, collagen, chitosan, gelatin, sericin, hyaluronic acid, starch and derivatives thereof.
• the natural polymer is selected from the group consisting of silk fibroin, alginates, gelatin, silk fibroin/alginate, silk fibroin/bovine serum albumin, silk fibroin/collagen, silk fibroin/chitosan, silk fibroin/gelatin and derivatives thereof.
• Synthetic polymers may include vinyl polymers such as, but not limited to, polyethylene, polypropylene, polyvinyl chloride), polystyrene, polytetrafluoroethylene, poly(a- methylstyrene), poly(acrylic acid), poly(methacrylic acid), poly(isobutylene), poly(acrylonitrile), poly(methyl acrylate), poly(methyl methacrylate), poly(acrylamide), poly(methacrylamide), poly(l -pentene), poly(1 ,3-butadiene), polyvinyl acetate), poly(2-vinyl pyridine), polyvinyl alcohol), polyvinyl pyrrolidone), poly(styrene), poly(styrene sulfonate) poly(vinylidene hexafluoropropylene), 1 ,4-polyisoprene, and 3,4-polychloroprene.
• vinyl polymers such as, but not limited to, polyethylene, poly
• Suitable synthetic polymers may also include non-vinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(I O-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(1 1 -undecanoamide), poly(hexamethylene sebacamide), poly(m-phenylene terephthalate), poly(tetramethylene-m-benzenesulfonamide). Copolymers of any one of the aforementioned may also be used.
• non-vinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(I O-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(1 1 -undecanoamide), poly(hexamethylene sebacamide), poly(m-phenylene terephthalate),
• Synthetic polymers employed in the process of the invention may fall within one of the following polymer classes: polyolefins, polyethers (including all epoxy resins, polyacetals, poly(orthoesters), polyetheretherketones, polyetherimides, poly(alkylene oxides) and poly(arylene oxides)), polyamides (including polyureas), polyamideimides, polyacrylates, polybenzimidazoles, polyesters (e.g.
• polylactic acid PLA
• polyglycolic acid PGA
• poly(lactic-co-glycolic acid) PLGA
• polycarbonates polyurethanes, polyimides, polyamines, polyhydrazides, phenolic resins, polysilanes, polysiloxanes, polycarbodiimides, polyimines (e.g. polyethyleneimine), azo polymers, polysulfides, polysulfones, polyether sulfones, oligomeric silsesquioxane polymers, polydimethylsiloxane polymers and copolymers thereof.
• functionalised synthetic polymers may be used.
• the synthetic polymers may be modified with one or more functional groups.
• functional groups include boronic acid, alkyne or azido functional groups.
• Such functional groups will generally be covalently bound to the polymer.
• the functional groups may allow the polymer to undergo further reaction, or to impart additional properties to the fibres.
• the fibre-forming liquid includes a water-soluble or water- dispersible polymer, or a derivative thereof.
• the fibre-forming liquid is a polymer solution including a water-soluble or water-dispersible polymer, or a derivative thereof, dissolved in an aqueous solvent.
• Exemplary water-soluble or water-dispersible polymers that may be present in a fibre-forming liquid such as a polymer solution may be selected from the group consisting of polypeptides, alginates, chitosan, starch, collagen, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides (including poly(N-alkyl acrylamides) such as poly(N-isopropyl acrylamide), polyvinyl alcohol), polyallylamine, polyethyleneimine, polyvinyl pyrrolidone), poly(lactic acid), poly(ethylene-co-acrylic acid), and copolymers thereof and combinations thereof.
• Derivatives of water-soluble or water- dispersible polymers may include various salts thereof.
• the fibre-forming liquid includes an organic solvent soluble polymer.
• the fibre-forming liquid is a polymer solution including an organic solvent soluble polymer dissolved in an organic solvent.
• Exemplary organic solvent soluble polymers that may be present in a fibre-forming liquid such as a polymer solution include poly(styrene) and polyesters such as poly(lactic acid), poly(glycolic acid), poly(caprolactone) and copolymers thereof, such as poly(lactic-co-glycolic acid).
• the fibre-forming liquid includes hybrid polymer.
• Hybrid polymers may be inorganic/organic hybrid polymers.
• Exemplary hybrid polymers include polysiloxanes, such as poly(dimethylsiloxane) (PDMS).
• the fibre-forming liquid includes at least one polymer selected from the group consisting of polypeptides, alginates, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides, polyesters, polyolefins, boronic acid functionalised polymers, polyvinylalcohol, polyallylamine, polyethyleneimine, poly(vinyl pyrrolidone), poly(lactic acid), polyether sulfone and inorganic polymers.
• polypeptides alginates, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides, polyesters, polyolefins, boronic acid functionalised polymers, polyvinylalcohol, polyallylamine, polyethyleneimine, poly(vinyl pyrrolidone), poly(lactic acid), polyether sulfone and inorganic polymers.
• the fibre-forming liquid includes a mixture of two or more polymers, such as a mixture of a thermoresponsive synthetic polymer (e.g. poly(N-isopropyl acrylamide)) and a natural polymer (e.g. a polypeptide).
• a thermoresponsive synthetic polymer e.g. poly(N-isopropyl acrylamide)
• a natural polymer e.g. a polypeptide.
• the use of polymer blends may be advantageous as it provides avenues for fabricating polymer fibres with a range of physical properties (e.g. thermoresponsive and biocompatible or biodegradable properties).
• the process of the invention can therefore be used to form aligned fibres with tuneable or tailored physical properties by selection of an appropriate blend or mixture of polymers.
• Polymers used in the process of the invention can include homopolymers of any of the foregoing polymers, random copolymers, block copolymers, alternating copolymers, random tripolymers, block tripolymers, alternating tripolymers, derivatives thereof (e.g., salts, graft copolymers, esters, or ethers thereof), and the like.
• the polymer may be capable of being crosslinked in the presence of a multifunctional crosslinking agent.
• Fibre-forming molecules employed in the process may be of any suitable molecular weight and molecular weight is not considered a limiting factor provided the method of the invention can align fibres in the scaffold.
• the number average molecular weight may range from a few hundred Dalton (e.g. 250 Da) to more several thousand Dalton (e.g. more than 10,000 Da), although any molecular weight could be used without departing from the invention.
• the number average molecular weight may be in the range of from about 50 to about 1 x 10 7 .
• the number average molecular weight may be in the range of from about 1 x 10 4 to about 1 x 10 7 .
• the scaffold of the present invention and the method of preparing the same can comprise an additive.
• Any suitable additive can be added to impart functionality to the scaffold such as having desired biological activity, improving solubility of the fibre-forming molecule or promoting formation of fibres and/or channels in the scaffolds.
• the additive is selected from the group consisting of a drug, growth factor, polymer, surfactant, chemical, particle, porogen and combinations thereof.
• the additive can be added in the scaffolds of the present invention in any way known in the art.
• the additive can be added in the scaffold by dissolving or dispersing the additive in the solution comprising fibre-forming molecules.
• the scaffold formed using the method of the present invention would encapsulate the additive during the cooling step.
• the additive can be added in the scaffold during the additional cooling step.
• the additive can be added by subjecting the scaffold to a solution comprising the additive followed by the additional cooling step to induce solvent crystallisation and channels in the scaffold.
• the additive in solution is brought into contact with the scaffold such that a certain amount of the additive in solution is adsorbed, absorbed or dispersed into the pores of the scaffold. Adsorption or absorption of the additive in solution can be added in the scaffold by any suitable technique known in the art such as dialysis.
• the additive can be added in the scaffold by chemical reactions (such as catalysis in the scaffold to introduce the desired additive).
• drug refers a molecule, group of molecules, complex, substance or derivative thereof administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes.
• the drug can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions.
• Other suitable drugs can include anti-viral agents, hormones, antibodies, or therapeutic proteins.
• Other drugs include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to drugs through metabolism or some other mechanism.
• Drugs can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixtures or combinations thereof, including, for example, DNA nanoplexes.
• Drugs include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the invention.
• Examples of drugs include a radiosensitizer, a steroid, a xanthine, a beta-2- agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha-agonist, an alpha-1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifingal agent, a vaccine, a protein, or a nucleic acid.
• a radiosensitizer a
• the drug can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2-agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and nonsteroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetominophen, ibuprofen, keto
• Growth factors as additives suitable in the present invention can stimulate cell growth, proliferation, healing or differentiation.
• the growth factor can be a protein or steroid hormone.
• the growth factors can be bone morphogenetic proteins to stimulate bone cell differentiation.
• fibroblast growth factors and vascular endothelial growth factors can stimulate blood vessel differentiation (angiogenesis).
• Growth factors can be selected from the group consisting of adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic proteins, ciliary neurotrophic factor family (such as ciliary neurotrophic factor, leukemia inhibitory factor, interleukin-6), colony- stimulating factors (such as macrophage colony-stimulating factor, granulocyte colony- stimulating factor and granulocyte macrophage colony-stimulating factor), epidermal growth factor, ephrins (such as ephrin A1 , ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1 , ephrin B2 and ephrin B3), erythropoietin, fibroblast growth factor (such as fibroblast growth factor 1 , fibroblast growth factor 2, fibroblast growth factor 3, fibroblast growth factor 4, fibroblast
• the scaffolds can also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of micro-organisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
• polymers suitable as additives in the present invention can be the polymers as already discussed above in relation to the fibre-forming molecule.
• Surfactants as additives suitable in the present invention can increase the solubility of the fibre-forming molecules. Without wishing to be bound by any one theory, the present inventors believe that the surfactants can reduce self-aggregation of the fibre-forming molecules to increase the solubility of the solution comprising fibre-forming molecules.
• the surfactant is anionic, cationic, zwitterionic or non-ionic.
• the surfactant comprises a functional group selected from the group consisting of sulfate, sulfonate, phosphate, carboxylate, amine, ammonium, alcohol, ether and combination thereof.
• the surfactant is selected from the group consisting of sodium stearate, sodium dodecyl sulfate, cetrimonium bromide, 4-(5-dodecyl) benzenesulfonate, 3- [(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, decyl glucoside, lauryl glucoside, octyl glucoside, triton X-100, nonoxynol-9, glyceryl laurate, polysorbate, dodecyldimethylamine oxide, polysorbate (such as polysorbate 20 and polysorbate 80; sold commercially as Tween 20 and Tween 80), coc
• the scaffold of the present invention can further comprise a central channel.
• the central channel can be directed along an axis of the scaffold such as the longitudinal axis of the scaffold.
• the central channel can be formed using any suitable technique known in the art.
• the central channel can be formed by mechanical treatment such as cutting the scaffold to form the channel using a blade or laser.
• the central channel is formed by controlling the cooling of the solution comprising fibre-forming molecules such that as the scaffold is formed, the cooling step is terminated prior to the scaffold being formed completely resulting in a central channel.
• the central channel can be formed upon cooling the fibre-forming solution using a cylindrical tube as the receptacle having an inner tube or cylinder which will define the geometry of the central channel.
• the central channel can be of any suitable dimension. In certain embodiments, the central channel has a diameter greater than 0.1 mm, 0.4 mm, 0.8 mm, 1 cm or 2 cm. In certain embodiments, the central channel has a diameter of from 0.1 mm to 2 cm. In certain embodiments, the central channel has a diameter of from 0.1 mm to 1 cm. In certain embodiments, the central channel has a diameter of from 0.1 to 4 mm. In certain embodiments, the central channel has a diameter of from 0.2 to 4 mm. In certain embodiments, the central channel has a diameter of from 0.1 to 2 mm. In certain embodiments, the central channel has a diameter of from 0.4 to 2 mm. In certain embodiments, the central channel has a diameter of from 0.4 to 1 mm. In certain embodiments, the central channel has a diameter of from 0.4 to 0.8 mm.
• the scaffolds of the present invention can be suitable to promote cell growth, cell culture and tissue formation in the bulk 3D scaffolds. Accordingly, the cells associated with the scaffolds of the present invention have any desirable cell viability and will be determined based on the desired application. As will be understood by a person skilled in the art, the cells can be cultured on the scaffolds of the present invention using any suitable technique known in the art. Typically, the cells can be cultured on the scaffolds after formation of the scaffold.
• the present invention can provide a method of promoting cell growth comprising capturing and culturing cells within a scaffold of the present invention.
• the cell is selected from a neuronal cell, skin cell, fibroblast, vascular cell, endothelial cell, bone cell, muscle cell, cardiac cell, corneal cell, eardrum cell, cancer cell and combinations thereof.
• the cell is selected from a neuronal cell, fibroblast, endothelial cell, stem cell, progenitor cell and combinations thereof.
• the method of promoting cell growth comprises promoting nerve repair or regeneration wherein the cell is a neuronal cell. In some embodiments, the method of promoting cell growth comprises promoting blood vessel repair or formation wherein the cell is an endothelial cell.
• the present invention can provide use of a scaffold of the present invention in the preparation of a biomedical implant for promoting cell growth comprising capturing and culturing cells. In some embodiments, the use comprises promoting nerve repair or regeneration wherein the cell is a neuronal cell. In some embodiments, the use comprises promoting blood vessel repair or formation wherein the cell is an endothelial cell.
• the scaffolds can be used in any suitable application for cell culture, tissue regeneration or tissue repair.
• the scaffold can be used as a biomedical implant.
• the scaffolds can be used as artificial blood vessels.
• the scaffolds can be used to heal wounds, repair bone damage, treat damaged tissue, drug delivery or in vitro cell culture.
• the scaffold can be used as a substrate for in vitro cell culture by providing a coating or layer of the scaffold on cell culture dishes, plates and flasks.
• the scaffolds can be used for tissue or wound repair as radial fibres can promote wound closure.
• the present invention provides a method of treating a mammal suffering from a tissue injury and in need of tissue restoration and/or regeneration, comprising applying to the injury site a scaffold of the present invention.
• the present invention provides use of a scaffold of the present invention in the preparation of a biomedical implant for the treatment of a tissue injury and tissue restoration and/or regeneration.
• the present invention provides use of a scaffold for treating a mammal suffering from a tissue injury and in need of tissue restoration and/or regeneration, comprising applying to the injury site the scaffold of the present invention.
• the method can be carried out, for example, by implanting the scaffold (i.e. porous biocompatible scaffold that fails to cause an acute reaction when implanted into a patient) or biomedical implant into a mammal and then removing the scaffold or biomedical implant from the mammal (such as a human).
• the scaffold or biomedical implant is implanted in direct contact with (i.e. physically touching over at least a portion of its external surface), or adjacent to (i.e. physically separated from) mature or immature target tissue, for a period of time that is sufficient to allow cells of the target tissue to associate with the scaffold or biomedical implant.
• the scaffold or biomedical implant can be pre-seeded with cells of the target tissue.
• the tissue graft includes the removed scaffold and the associated cells of the target tissue.
• Target tissue is tissue of any type that a graft is generated to replace.
• the target tissue is ligament.
• the target tissue is cartilage; when the patient has a damaged tendon, the target tissue is tendon; and so forth.
• the target tissue is "mature” when it includes cells and other components that are naturally found in fully differentiated tissue (e.g. a recognizable ligament in an adult mammal is a mature target tissue).
• the target tissue is "immature” when it includes cells that have not yet differentiated into, but which will differentiate into, mature cells (e.g., immature target tissue can contain mesenchymal stem cells, bone marrow stromal cells, and precursor or progenitor cells).
• Target tissue is also “immature” when it contains cells that induce immature cells to differentiate into cells of a mature target tissue or when it contains cells that sustain mature cells (these events can occur, for example, when cells secrete growth factors or cytokines that bring about cellular differentiation or sustain mature cells).
• the scaffold or biomedical implant of the present invention can be carried out by implanting a scaffold or biomedical implant comprising the scaffold of the present invention in direct contact with, or adjacent to, target tissue or tissue that includes cells that can produce target tissue (by, for example, the processes described herein - differentiation or through the action of growth factors or cytokines).
• the mammal that has the tissue defect and the mammal from which the tissue graft is obtained can be the same mammal or the same type of mammal (e.g. one human patient can have a tissue defect that is treated with a graft generated in another human).
• the mammal that has the tissue defect and the mammal from which the tissue graft is obtained can be different types of mammals (e.g., a human patient can have a tissue defect that is treated with a graft generated in another primate, a cow, a horse, a sheep, a pig, or a goat).
• the scaffold or biomedical implant can be implanted in a mammal at the site of a tissue defect by any surgical technique.
• the scaffold or biomedical implant can be sutured, pinned, tacked, or stapled into a mammal at the site of a tissue defect.
• the scaffold or biomedical implant is implanted by attaching a first portion of the scaffold or biomedical implant to a first support structure at the site of the tissue defect and attaching a second portion of the scaffold or biomedical implant to a second support structure at the site of the tissue defect, such that the scaffold or biomedical implant connects the first support structure to the second support structure.
• the second support structure can be the femur.
• the first support structure is a first articular surface of a joint (e.g. a shoulder, wrist, elbow, hip, knee or ankle joint)
• the second support structure can be a second articular surface of the same joint (i.e., the shoulder, wrist, elbow, hip, knee, or ankle joint, respectively).
• adjacent to means that the scaffold or biomedical implant is separated from the tissue of the target type, or tissue comprising cells that can produce tissue of the target type or both, if both are present, by a distance of up to 10 mm and preferably less than 5 mm.
• the viabilities of cells associated with the scaffolds or biomedical implants can be measured using any suitable technique known in the art.
• the cell viabilities can be measured using colorimetric assays, for example, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay, XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide) assay, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, WST (Water-soluble Tetrazolium salts) assays or the like.
• the viabilities of the cells may be assessed using microscopy techniques with cell staining to differentiate between live and dead cells.
• the present invention also relates to modified materials such as textile fabrics, bandages or other existing products with different types and compositions of fibres as described herein to produce a composite material such as a biomimetic composite material.
• the present invention provides a composite material comprising a matrix of substantially aligned fibres and at least a base material.
• the composite material is porous. In some embodiments, the composite material is non-porous. It is to be understood that the composite materials are suitable to promote cell growth and/or tissue formation.
• the composite materials of the present invention can be used for disease treatment, wound healing, tissue regeneration, drug delivery and the like.
• properties including feeling, comfort, air-permeability, mechanical properties, antimicrobial (such as antiviral, antibacterial and antialgal) properties, hydrophobicity and hydrophilicity can be tailored.
• the composite materials can be used as bandages or dressings for wound healing, tissue regeneration and treatment of diseases such as diabetes.
• the composite material of the present invention can comprise any suitable amount of fibres.
• the functional aspects of the composite material including cell adhesion, proliferation, growth, differentiation, antimicrobial function, and tissue regeneration can be tailored depending on the amount of aligned fibres and the fibre-forming liquid used.
• the composite materials of the present invention can comprise additives such as drugs or growth factors that may be beneficial for cell adhesion, proliferation, growth, differentiation, tissue regeneration or antimicrobial properties.
• additives can be added into the solution of fibre-forming molecules to provide aligned fibres comprising additives loaded, adsorbed or absorbed in the composite material.
• the base material is immersed in a solution of fibre-forming molecules which is then cooled using the present invention to provide the composite material having aligned fibres.
• the base material can be any suitable material which is suitable as a template to incorporate the aligned fibres of the present invention.
• base materials include bandages, dressings and textile fabrics.
• the base material can be a scaffold prepared by the method of the present invention.
• the base material can be of any suitable material which is porous or non-porous which can incorporate the aligned fibres in the composite material of the present invention.
• the base material can be porous or non-porous.
• the aligned fibres can be formed on a surface of the base material.
• the aligned fibres can be formed within in the pores and/or on the surface of the base material. When aligned fibres form on the surface of the base material, the aligned fibres can form a scaffold if there are sufficient fibre-forming molecules.
• the present invention can provide aligned fibres on or in the base material more uniformly and firmly compared to techniques known to an ordinary person skilled in the art including deposition, dispersion and coating technologies.
• the present invention is facile, efficient and cost-effective for modifying various base materials at a large scale to provide the resulting composite materials.
• the base material is selected from the group consisting of a natural polymer, a synthetic polymer and combinations thereof.
• Natural polymers may include polysaccharides, polypeptides, glycoproteins, and derivatives thereof and copolymers thereof.
• Polysaccharides may include agar, alginates, chitosan, hyaluronan, cellulosic polymers (e.g., cellulose and derivatives thereof as well as cellulose production by-products such as lignin) and starch polymers.
• Polypeptides may include various proteins, such as silk fibroin, silk sericin, lysozyme, collagen, keratin, casein, gelatin and derivatives thereof.
• Derivatives of natural polymers, such as polysaccharides and polypeptides may include various salts, esters, ethers, and graft copolymers. Exemplary salts may be selected from sodium, zinc, iron and calcium salts.
• the natural polymer is selected from the group consisting of at least one of silk fibroin, alginate, bovine serum albumin, collagen, chitosan, gelatin, sericin, hyaluronic acid, starch and derivatives thereof.
• the natural polymer is selected from the group consisting of silk fibroin, alginates, gelatin, silk fibroin/alginate, silk fibroin/bovine serum albumin, silk fibroin/collagen, silk fibroin/chitosan, silk fibroin/gelatin and derivatives thereof.
• Synthetic polymers may include vinyl polymers such as, but not limited to, polyethylene, polypropylene, polyvinyl chloride), polystyrene, polytetrafluoroethylene, poly(a- methylstyrene), poly(acrylic acid), poly(methacrylic acid), poly(isobutylene), poly(acrylonitrile), poly(methyl acrylate), poly(methyl methacrylate), poly(acrylamide), poly(methacrylamide), poly(l -pentene), poly(1 ,3-butadiene), polyvinyl acetate), poly(2-vinyl pyridine), polyvinyl alcohol), polyvinyl pyrrolidone), poly(styrene), poly(styrene sulfonate) poly(vinylidene hexafluoropropylene), 1 ,4-polyisoprene, and 3,4-polychloroprene.
• vinyl polymers such as, but not limited to, polyethylene, poly
• Suitable synthetic polymers may also include non-vinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(I O-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(1 1 -undecanoamide), poly(hexamethylene sebacamide), poly(m-phenylene terephthalate), poly(tetramethylene-m-benzenesulfonamide). Copolymers of any one of the aforementioned may also be used.
• non-vinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(I O-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(1 1 -undecanoamide), poly(hexamethylene sebacamide), poly(m-phenylene terephthalate),
• Synthetic polymers employed in the process of the invention may fall within one of the following polymer classes: polyolefins, polyethers (including all epoxy resins, polyacetals, poly(orthoesters), polyetheretherketones, polyetherimides, poly(alkylene oxides) and poly(arylene oxides)), polyamides (including polyureas), polyamideimides, polyacrylates, polybenzimidazoles, polyesters (e.g.
• polylactic acid PLA
• polyglycolic acid PGA
• poly(lactic-co-glycolic acid) PLGA
• poly(lactide-co-£-caprolactone) PLCL
• polycarbonates polyurethanes, polyimides, polyamines, polyhydrazides, phenolic resins, polysilanes, polysiloxanes, polycarbodiimides, polyimines (e.g. polyethyleneimine), azo polymers, polysulfides, polysulfones, polyether sulfones, oligomeric silsesquioxane polymers, polydimethylsiloxane polymers and copolymers thereof.
• functionalised synthetic polymers may be used.
• the synthetic polymers may be modified with one or more functional groups.
• functional groups include Arg-Gly-Asp (RGD) peptides, boronic acid, alkyne, amino, carboxyl or azido functional groups.
• RGD Arg-Gly-Asp
• Such functional groups will generally be covalently bound to the polymer.
• the functional groups may allow the polymer to undergo further reaction, or to impart additional properties to the fibres.
• the base material includes a water-soluble or water- dispersible polymer, or a derivative thereof.
• the base material comprises a water-soluble or water-dispersible polymer, or a derivative thereof.
• Exemplary water-soluble or water-dispersible polymers include polypeptides, alginates, chitosan, starch, collagen, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides (including poly(N- alkyl acrylamides) such as poly(N-isopropyl acrylamide), polyvinyl alcohol), polyallylamine, polyethyleneimine, polyvinyl pyrrolidone), poly(lactic acid), poly(ethylene-co-acrylic acid), polyesters (e.g.
• PLA polylactic acid
• PGA polyglycolic acid
• PLGA poly(lactic-co-glycolic acid)
• PLCL poly(lactide-co-£-caprolactone)
• PHA polylactic acid
• PGA polyglycolic acid
• PLGA poly(lactic-co-glycolic acid)
• PLCL poly(lactide-co-£-caprolactone)
• PCC polycarbonates
• Purethanes polypropylene
• the base material includes organic solvent soluble polymers selected from the group consisting of poly(styrene) and polyesters such as poly(lactic acid), poly(glycolic acid), poly(caprolactone) and copolymers thereof, such as poly(lactic-co-glycolic acid).
• the base material includes a hybrid polymer.
• Hybrid polymers may be inorganic/organic hybrid polymers.
• Exemplary hybrid polymers include polysiloxanes, such as poly(dimethylsiloxane) (PDMS).
• the base material includes at least one polymer selected from the group consisting of polypeptides, alginates, gelatin, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polypropylene, polyacrylamides, polyesters, polyolefins, boronic acid functionalised polymers, polyvinylalcohol, polyallylamine, polyethyleneimine, polyvinyl pyrrolidone), poly(lactic acid), polyether sulfone and inorganic polymers.
• polypeptides alginates, gelatin, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polypropylene, polyacrylamides, polyesters, polyolefins, boronic acid functionalised polymers, polyvinylalcohol, polyallylamine, polyethyleneimine, polyvinyl pyrrolidone), poly(lactic acid), polyether sulfone and inorganic
• the base material includes a mixture of two or more polymers, such as a mixture of a thermoresponsive synthetic polymer (e.g. poly(N-isopropyl acrylamide)) and a natural polymer (e.g. a polypeptide).
• a thermoresponsive synthetic polymer e.g. poly(N-isopropyl acrylamide)
• a natural polymer e.g. a polypeptide
• Silk cocoons were boiled 4 times (20 min/time) in an aqueous 0.5% (w/v) Na 2 C0 3 solution to remove sericin protein.
• the degummed silk fibres were rinsed with ultrapure water thoroughly to remove the residual of serein. Following drying, they were dissolved in a mixture of CaCI 2 , H 2 0 and CH 3 CH 2 OH (in a molar ratio of 1 :8:2) at 65 °C to get a clear solution. Subsequently, the resulting solution was dialysed against ultrapure water (18.2 mQ-cm) using cellulose dialysis tubes (molecular weight cut-off: 14 kDa; Sigma Aldrich, Australia) at ambient temperature for 4 days.
• regenerated SF sponge was obtained by lyophilizing the centrifuged solution using a freeze dryer (FreeZone 2.5 Liter Benchtop Freeze Dryer; Labconco, Kansas City, MO, USA). SF solution (2%) was obtained by dissolving 2 g of regenerated SF sponge in 100 mL ultrapure water for further use.
• SF solution in a glass tube was directly immersed into liquid nitrogen.
• Target scaffolds were produced by freeze-drying the frozen samples using a freeze dryer. The fabrication scheme is shown in Fig. 2.
• scaffolds were also formed in freezers at -20 °C and -80 °C, respectively, rather than by instant freezing with liquid nitrogen.
• SF solution in the glass tube was frozen at -20 °C for 53 h.
• W&Fb scaffolds from -80 °C SF solution in the glass tube was frozen at -80 °C for 53 h.
• Wb and W&Fb scaffolds are respectively obtained.
• Wb and W&Fb scaffolds above were further processed with the same procedures for obtaining A(F&C) scaffolds, i.e., the scaffolds were post-treated by immersing in ethanol at ambient temperature for 12 h. After removing ethanol and thoroughly rinsing with ultrapure water, the scaffolds in the ultrapure water were frozen at -20 °C for 72 h. Following freeze- drying, W and W&F scaffolds were obtained, respectively.
• SF/gelatin (Sigma-Aldrich, Australia) solution (2%) was obtained by dissolving 2 g of regenerated SF/gelatin mixture (in a weight ratio of 95:5) in 100 mL ultrapure water for further use. Then the SF/gelatin composite A(F&C) scaffolds were fabricated by the same protocol for producing SF A(F&C) scaffolds above.
• Sodium alginate (Sigma-Aldrich, Australia) solution (0.3% w/v) was fabricated by dissolving 0.3 g of sodium alginate in 100 mL ultrapure water at 50 °C under stirring.
• the sodium alginate A(F&C) scaffolds were prepared by the same protocol for fabricating SF A(F&C) scaffolds.
• a solution of fibre-forming molecules and a base material for example polypropylene porous microfibrous material in a container; or a base material with an absorbed solution of fibre-forming molecules (such as silk fibroin solution, alginate solution, gelatin solution, or combination thereof) were directly immersed into liquid nitrogen or slowly lowered into liquid nitrogen to induce a temperature difference.
• the composite material was produced by freeze-drying the frozen samples using a freeze dryer.
• the resulting composite scaffolds can be post-treated by immersing in a suitable cross-linker (such as an ethanol solution) or in a vapour environment of cross-linker (such as 75% ethanol vapour).
• the resulting composite material was obtained by drying at room temperature or thoroughly rinsing with ultrapure water and then freeze- drying. Representative micrographs are shown in Figures 4 and 5.
• Cylindrical scaffolds with a diameter of 10 mm and height of 4 mm were measured at a crossing-head speed of 5 mm/min (six samples were measured for each group). Compressive stress and strain were graphed, and the compressive modulus was calculated as the slope of the initial linear section of the stress-strain curve.
• the architecture of silk scaffolds was imaged using Micro X-ray Computed Tomography (micro-CT) by an Xradia ⁇ micro XCT200 (Carl Zeiss X-ray Microscopy, Inc., USA). An X-ray tube with a voltage of 40 kV and a peak power of 10 W was used.
• 361 equiangular projections (exposure time: 8 seconds/projection) over 180 degrees were taken for one complete tomographic reconstruction.
• Phase retrieval tomography with 3D reconstruction algorithm was introduced to obtain clear projections and a final 3D visualization.
• the size of reconstructed 3D images was 512x512x512 voxels with a 4.3 ⁇ voxel size along each side. Scaffold cell capturing, growth and in vitro vascularisation of Human Umbilical Vein Endothelial Cells in 3D SF scaffolds.
• HUVECs Human Umbilical Vein Endothelial Cell (HUVEC; Life Technologies, Australia) Culture and Scaffold Seeding: HUVECs were cultured in Medium 200 with Low Serum Growth Supplement (LSGS; Life Technologies, Australia). Scaffolds (diameter around 10 mm and thickness around 3 mm) were placed in 24-well plates (Greiner Bio-One) after sterilization in an environment of 75% ethanol vapour. HUVECs suspended in cell medium were evenly seeded onto scaffolds at a corresponding density (1 ⁇ 10 5 /well, 1 .5 ⁇ 10 5 /well and 2 x 10 5 /well for in vitro cell adhesion, proliferation and vascularization study, respectively). Cell-seeded scaffolds were maintained in vitro under standard culture conditions (37 °C, 5% C0 2 ) with medium change every 2-3 days.
• the composites were then incubated in Image-iT® FX Signal Enhancer Ready ProbesTM reagent (Life Technologies, Australia) for 30 min and rinsed with PBS. Subsequently, the composites were incubated with Alexa Fluor® 568 Phalloidin (1 :100; Life Technologies, Australia) for 1 hour. After rinsing in PBS, the composites were incubated in DAPI (Life Technologies, Australia) in the dark for 10 min. As-treated samples were assessed using the confocal fluorescence microscope.
• cell-scaffold composites were rinsed with PBS, and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. Following rinsing with PBS, the composites were permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich, Australia) for 10 min, followed by rinsing with PBS. The composites were then incubated in Image-iT® FX Signal Enhancer Ready ProbesTM reagent (Life Technologies, Australia) for 30 min.
• the composites were incubated for 10 min with 10% Normal Goat Serum blocking solution (Life Technologies, Australia) to block non-specific binding and then rinsed with PBS. Subsequently, the composites were incubated with CD31 Monoclonal Antibody (1 :50; Life Technologies, Australia) overnight at 4 °C. Following rinsing with PBS, the composites were incubated with Goat-anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1 :200; Life Technologies, Australia) for 1 hour. The scaffolds were rinsed again with PBS and incubated in DAPI (Life Technologies, Australia) in dark for 10 min. As-treated samples were assessed using the confocal fluorescence microscope.
• DRGs were cultured in Primary Neuron Basal Medium (PNBM; Lonza, USA) supplemented with PNGMTM SingleQuotsTM (Lonza, USA) and 150 ng/ml of Nerve Growth Factor (NGF; Sigma-Aldrich, Australia).
• PNBM Primary Neuron Basal Medium
• NGF Nerve Growth Factor
• the scaffolds were rinsed with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, Australia) for 30 min at ambient temperature. Following rinsing with PBS, the composites were permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich, Australia) for 30 min and then rinsed with PBS again. Subsequently, the scaffolds were incubated in 10% Normal Goat Serum blocking solution (Life Technologies, Australia) for 10 min to block non-specific binding, followed by rinsing with PBS.
• the scaffolds were incubated with Anti-Neurofilament-200 antibody from rabbit (1 :50; Sigma-Aldrich, Australia) overnight at 4 °C. After rinsing with PBS, the scaffolds were incubated with Goat-anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1 :200; Life Technologies, Australia) for 1 hour. Finally, the treated samples were analysed using the confocal fluorescence microscope.
• the nanofibrous scaffold was put in water and frozen again but at a higher temperature of -20°C. This relatively higher temperature led to the formation of larger ice crystals which were directed by radially aligned nanofibres to grow along the direction of fibres. The formation of the crystals reduces the free space for nanofibres, which pushes and squeezes the nanofibres to around the crystals. After removing these crystals with freeze-drying, macrochannels with nanofibrous walls are created in the aligned 3D nanofibrous scaffolds.
• the present inventive 3D scaffolds with co-aligned nanofibres and macrochannels can capture more cells that are both adherent and non-adherent. More interestingly, the scaffolds not only significantly promote cell proliferation, but also direct Human Umbilical Vein Endothelial Cells (HUVECs) to assemble into vessel-like structures and the 3D growth of Embryonic Dorsal Root Ganglion Neurons (DRGs) and neurites.
• UUVECs Human Umbilical Vein Endothelial Cells
• Example 2 Formation of 3D architectures with co-aligned nanofibres and macrochannels.
• SF nanofibres presented a smooth morphology and were well aligned radially (see Fig. 10a). This method is facile and allows the fabrication of samples with varied geometries (even including tubes and particles), diameters and thicknesses (see Fig. 10b).
• the alignment direction of scaffold nanofibres can be controlled by directionally freezing SF solution in liquid nitrogen (see Fig. 10b).
• vertically aligned nanofibres can be fabricated by slowly lowering the SF solution-containing tube into liquid nitrogen. By directly dropping SF solution into liquid nitrogen, particles with radially aligned nanofibres were obtained.
• dripping or spraying the solution comprising fibre-forming molecules (silk fibroin) into liquid nitrogen produced particles or spheres with radially aligned nanofibres similar to that of Fig. 10b.
• SF solutions contained in the same glass tubes were frozen in freezers at -80°C and -20°C, respectively, followed by the removal of ice crystals using freeze-drying.
• SF scaffolds from -80°C freezing have a hybrid structure with random short channel-like structures, pores and nanofibres, but these structures are not interconnected (see Fig.
• each radially aligned channel (diameter, 100-1000 ⁇ ) connected the surface and centre of scaffold.
• the channel walls are composed of SF nanoparticles and nanofibres (diameter, 50-600 nm) aligned along the direction of channels (indicated by large yellow arrows). Zooming in on a representative channel wall, many pores (diameter, 50-1000 nm) were seen which appeared to align in the orientation of nanofibres.
• Example 3 Secondary structure and mechanical characteristics of scaffolds.
• [171 ] Secondary structures of the scaffolds were investigated to understand the effect of preparation method on structural change of silk fibroin. It is known that the conformation change of SF can be indicated by the shift of characteristic absorption peaks (1600-1500 cm “1 for amide II and 1700-1600 cm “1 for amide I) in ATR-FTIR spectra. All three scaffolds before post-treatment with ethanol showed one main characteristic peak at around 1644 cm "1 suggesting random coils (see Fig. 15a).
• the Wb and W&Fb scaffolds showed another main characteristic peak at 1517 cm “1 (indicating dominant ⁇ -sheet structure), whereas the AFb scaffold showed another main characteristic peak at 1533 cm “1 (indicating dominant random coil structure), suggesting the low temperature treatment with liquid nitrogen could be beneficial for the formation of random coils (see Fig. 15a).
• all three scaffolds presented main characteristic peaks at around 1700, 1622 and 1517 cm “1 , suggesting the treated scaffolds mainly consisted of ⁇ -sheet structure (see Fig. 15b).
• FIG. 16a Compressive modulus of scaffolds was demonstrated in Fig. 16a.
• 3D A(F&C) nanofibrous scaffolds have a compressive modulus of around 80 kPa, which is lower than those of the wall-like W and W&F scaffolds (around 100 and 140 kPa, respectively). This could be due to their large channel-based structure with nanofibres.
• A(F&C) scaffolds still maintained a good radially aligned morphology and structure, with just some minor collapses seen on their surface, probably due to damage of some channels (see Fig. 16b).
• Example 4 Co-aligned channels and nanofibres enhance cell capture, and direct growth, behaviour and function of adherent Human Umbilical Vein Endothelial Cells (HUVECs) in 3D SF scaffolds.
• HUVECs Human Umbilical Vein Endothelial Cells
• AF scaffolds after post treatment in ethanol were used as cell culture substrates.
• AF scaffolds had the same radially aligned nanofibous structure as AFb scaffolds shown in Fig. 2 and Fig. 10a, but they did not have channels as presented in the A(F&C) scaffolds.
• A(F&C) scaffolds demonstrated significantly higher cell viability than AF scaffolds at all time points, demonstrating the advantages of channels in cell capturing and proliferation.
• W and W&F scaffolds also showed higher cell viability in comparison with AF scaffolds. This is probably due to the fact that W, W&F and A(F&C) scaffolds provide more space for cell adhesion and proliferation due to their larger pores or channels.
• AF scaffolds Although cells were also observed in AF scaffolds (Fig. 6d), it was difficult to find them during scanning under confocal microscopy due to the small number of cells in the inner (internal) region of the scaffold. Cells in AF scaffolds were not well aligned and elongated in the direction of nanofibres, exhibiting relatively flat and polygonous morphology. This is probably due to the fact that the loosely aligned nanofibres provide cells with many surrounding signals from different directions. Cells on the walls of A(F&C) scaffolds were elongated and aligned along with nanofibres well.
• HUVECs are a classic endothelial cell model for studying vascularization.
• A(F&C) scaffolds can promote the proliferation of HUVECs. It is believed that the cell migration and elongation induced by aligned channels and nanofibres should enhance the intercellular interaction to facilitate formation of vessel-like structures.
• the present inventors cultured HUVECs up to 21 days to observe the vascularization behaviours of cells in the scaffolds (Fig. 7, Fig 6c illustrates how to read the images).
• Example 5 Co-aligned channels and nanofibres enhance the capturing of scaffolds for non-adherent Embryonic Dorsal Root Ganglion Neurons (DRGs) and induce 3D growth of neurites in the scaffolds.
• DRGs Dorsal Root Ganglion Neurons
• Fig. 8a illustrates the areas of scaffolds that were scanned and the corresponding images. Affluent neurites were aligned in the direction of nanofibres on the surface of AF scaffolds, but they were not observed in the inner portion of the scaffolds.
• Fig. 8c illustrates the scanned areas of A(F&C) scaffolds and the corresponding images. DRGs can be clearly seen, and a significant amount of long neurites had grown through the channel (the channels, channel walls and neurites were indicated by white arrows, respectively). Interestingly, zooming in on the channel revealed that all DRGs and neurites were mainly growing along the channels, suggesting a 3D growth mode of neurites. This was totally different from the 2D growth of DRGs and neurites along the aligned nanofibres on the surface of AF scaffolds (Fig. 8b). From the last image in Fig. 8c, neurites in bundles were observed clearly, which is very important for the formation of nerve tissues. These observations demonstrated that the aligned channels and nanofibres can not only promote the adhesion and proliferation of both adherent and non-adherent cells, but also direct them to grow, migrate and interact in the 3D space similar to the nature ECM.
• radially aligned channels (diameter, 100-1000 ⁇ ) towards the centre of scaffolds provided enough space for the migration and 3D growth of cells, as shown in Fig. 6d, Fig. 7a,b and Fig. 8c.
• a common issue in tissue engineering is the necrosis of cells or tissues in the 3D scaffolds due to insufficient supply of oxygen and nutrients.
• the channels with porous walls (diameter of pores: 50-1000 nm) in the A(F&C) scaffolds are very important for the transport of oxygen, nutrients and wastes.
• the large central channel (diameter, 0.4-2 mm) of the scaffold should also facilitate nutrient exchange and waste disposal.
• nanofibres (diameter, 50-600 nm) on channel walls played an important role in cell capturing, proliferation and directing cells to migrate and grow along the alignment direction (Fig. 6a,b,d, Fig. 7a,b and Fig. 8a). Furthermore, nanofibres and nanoparticles are good carriers for the delivery of growth factors or drugs. As shown in Fig. 7a and Fig. 8c, the channels still showed good morphology and structure after 21 days of cell culture, indicating the stability of scaffolds.
• A(F&C) scaffolds were developed as a model platform for proof-of-concept that the creation of ECM-mimicking 3D structure plays an important role in insight into cell behaviours and functions in vitro. Based on this platform, the present inventors found that adherent HUVECs preferred to grow along the materials in 3D scaffolds. Therefore, they were mainly directed by the aligned nanofibres on the wall of A(F&C) scaffolds (Fig. 9a,b). In contrast, non-adherent DRGs and neurites preferred to grow along the 3D space. As shown in Fig. 9c,d,e, the neurites mainly grew along the channels.
• the present inventors have developed a facile freeze-drying strategy for creating biomimic 3D scaffolds with aligned nanofibres and macrochannels.
• the 3D scaffolds showed significantly higher cell capturing and proliferation-promoting capability than widely-used wall-like 3D scaffolds and 3D aligned nanofibrous scaffolds without channels for both adherent HUVECs and nonadherent DRGs.
• aligned nanofibres and channels not only direct the growth, migration, and interaction of HUVECs to assemble into blood vessel-like structures in the scaffolds in vitro, but also direct the neurite growth of DRGs in the 3D space.

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