Silk biomaterials

Abstract

The present invention relates to, inter alia, various scaffold materials including a scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein.

Description

[0001] SILK BIOMATERIALS

[0002] The present invention relates to scaffold materials comprising silk protein. Such scaffold materials are of use as wound dressings and grafts. The scaffold materials also have use in tissue engineering applications, and in particular as scaffolds for cell growth. Also described are layered wound dressings, layered grafts and layered constructs comprising silk protein and hyaluronic acid.

[0003] BACKGROUND OF INVENTION

[0004] Silk fibroin is a protein present in silk produced by various insects such as the larvae of silk moths (Bombyx mori). Silk moth larvae, also known as silkworms, enclose themselves in cocoons made of raw silk, containing two main proteins, sericin and fibroin. The fibroin is a fibrous protein that forms the core of the raw silk fibres, and sericin forms a gum-like coating on the fibres, allowing them to stick together.

[0005] Silk fibroin is biocompatible, it can be used as a wound coagulant and in various cosmetic applications. Furthermore, it can be dissolved in aqueous solutions and can be easily reconstructed into different material formats, including films, mats, hydrogels, and sponges via fabrication techniques including by spin coating, electrospinning, freeze drying, physical, and chemical crosslinking techniques.

[0006] Electrospinning is a method of producing fibres that uses electric force to draw charged threads of polymer solutions, producing fibres having diameters in the nanometres. Broadly speaking, an electrospinning device comprises a power supply, a solution containing a reservoir (such as a syringe) which is tipped with a blunt needle, a pump, and a collector. An electric field is established between the needle and the collector by applying a particular voltage. The pump causes the solution to flow at a constant rate, and charge accumulates at the surface of the liquid. At the point when the electrostatic repulsion is greater than the surface tension of the liquid, a stream of liquid erupts from the surface. The point of eruption is known as the Taylor cone. Once the Taylor cone forms, the charged liquid jet is ejected towards the collector. As the surface area of the ejected jet is large compared to its volume, the solvent evaporates efficiently, and as the charge density is increased due to the decrease in volume, the jet splits and solid fibres are generated. A whipping motion occurs between the Taylor cone and the collector, resulting in a non-woven fibre mat being deposited on the collector. The reconstructed silk fibroin materials have structural properties that allow them to be used as wound dressings and grafts (such as vascular grafts), and also in tissue engineering applications where the material can act as a scaffold material to support the regeneration of growth of cells.

[0007] EP1408146A1 describes a non-woven fabric comprising silk fibroin and a method of preparing the fabric by dissolving silk fibroin in hexafluoroacetone, and then performing electrospinning. The resulting material is said to be of use as a medical material.

[0008] EP2465472A2 describes the preparation of a silk nanofibre by electrospinning a fibrous spinning solution onto a collector coated in polyethyleneoxide. The silk fibre is produced in a conduit shape, which is used a nerve conduit for treating a nerve injury.

[0009] W02004 / 000915A2 describes an all-aqueous process for the production of silk biomaterials. In the process, at least one biocompatible polymer such as polyethylene oxide is blended with silk protein before being processed, e.g. by electrospinning. The biomaterials produced are said to be of use in forming tissue engineered constructs.

[0010] While various scaffold materials formed using silk protein are known, an object of the present invention is to produce new scaffold materials with enhanced functional properties and potential therapeutic benefits. A further object of the invention is to produce wound dressings and grafts with enhanced functional properties and potential therapeutic benefits.

[0011] SUMMARY OF THE INVENTION

[0012] According to one aspect of the present invention, there is provided a scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein.

[0013] According to a further aspect of the invention, there is provided a scaffold material comprising: i) hydroxyapatite; and ii) electrospun fibres comprising silk protein.

[0014] According to a further aspect of the invention, there is provided a scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibres comprising silk protein.

[0015] According to a further aspect of the invention, there is provided a layered wound dressing or layered graft comprising: i) a scaffold material as defined hereinabove; and ii) a support material.

[0016] According to a further aspect of the invention, there is provided a layered construct for tissue engineering applications, comprising: i) a first layer comprising silk protein; ii) a second layer comprising a hydrogel and silk protein; iii) a third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) a fourth layer comprising polycaprolactone and hydroxyapatite.

[0017] According to a further aspect of the invention, there is provided a tissue engineered construct comprising a scaffold material, a layered wound dressing, a layered graft or a layered construct as described herein, and mammalian cells.

[0018] According to a further aspect of the invention, there is provided a method for preparing a tissue engineered construct comprising the steps of: a) preparing a scaffold material as described herein; b) combining the scaffold material with mammalian cells in a suitable medium.

[0019] According to a further aspect of the invention, there is provided a method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

[0020] According to a further aspect of the invention, there is provided a method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material. According to a further aspect of the invention, there is provided a method for preparing a scaffold material comprising i) hydroxyapatite, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with hydroxyapatite to form a scaffold material.

[0021] According to a further aspect of the invention, there is provided a method for preparing a scaffold material comprising i) hydroxyapatite, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hydroxyapatite, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

[0022] According to a further aspect of the invention, there is provided a method for preparing a scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hyaluronic acid, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

[0023] Embodiments and preferences described below with respect to the scaffold material, tissue engineered construct, layered wound dressing, layered graft and layered construct of the invention apply equally to the methods for preparing the scaffold material, tissue engineered construct, layered wound dressing, layered graft and layered construct described herein.

[0024] FIGURES

[0025] Figure 1 shows a layered construct according to an embodiment of the present invention (“Multiphasic Implant”).

[0026] DETAILED DESCRIPTION

[0027] The present inventors have discovered, inter alia, that a scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein, can be used to form materials of use in tissue engineering. The present inventors have also discovered that a scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibres comprising silk protein, can be used to form materials of use in tissue engineering.

[0028] The present inventors have also discovered that a scaffold material comprising: i) hydroxyapatite; and ii) electrospun fibres comprising silk protein, can be used to form materials of use in tissue engineering, in particular to form scaffold materials with a more solid structure.

[0029] A “scaffold material” in the context of the present invention is a material or substance which is of use in, for example, tissue repair and / or support (including reinforcement). The scaffold material can facilitate or contribute to the formation of new bodily materials such as tissue, cartilage, tendon, nerve and bone. This may be by providing support, and / or by enhancing cell growth, attachment, differentiation, cell distribution etc. Suitably, the scaffold material is in the form of a film, a membrane, a mat, or a mesh.

[0030] Platelet-rich plasma (PRP) is typically produced by centrifugation of whole blood to separate the platelets from the red blood cells. A bone marrow concentrate (BMC) can be obtained by a similar process. Additional centrifugation or separation steps may be used to remove the platelet poor plasma. As the cellular composition of PRP, and hence its therapeutic effect, is strongly dependent on the methodology and device used to prepare the PRP, standardization of production of PRP is essential.

[0031] Medical devices for obtaining standardized PRP and BMC have been developed, including devices of use in an automated procedure in a closed circuit. For example, centrifugation tubes produced by Regen Lab and described in W02008 / 023026A2, W02011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391 A1 , WO2021 / 198312A1 and WO2022 / 269035A1 (all incorporated by reference herein in their entirety).

[0032] A process for preparing platelet-rich plasma or bone marrow concentrate typically comprises the following steps: a) collecting a bodily fluid or a cell extract in a centrifugation container; b) centrifuging the centrifugation container of step a) to form an enriched fraction; and c) collecting at least a portion of the enriched fraction.

[0033] The centrifugation container is suitably a centrifugation tube or a centrifugation syringe, in particular a centrifugation tube, such as a blood fractionation tube. Suitable materials and coatings for the centrifugation container, as well as the use of density gradient media such as thixotropic gels, are described in in W02008 / 023026A2, W02011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391 A1 , WO2021 / 198312A1 and WO2022 / 269035A1 (all incorporated by reference herein in their entirety).

[0034] The nature of the enriched fraction will depend on the material being centrifuged. Centrifugation separates the components of a solution based on their density, such that particles with a higher density move towards the bottom of the container (distal end), while those of a lower density move towards the top of the container (proximal end). Thus, the “enriched fraction” can be described as a fraction which, following centrifugation, contains a higher concentration of particular cells than the bodily fluid or cell extract that was centrifuged. When the bodily fluid is whole blood, red blood cells (erythrocytes) move to the bottom of the container (below the density gradient medium if present) and platelets move above the red blood cells, in plasma which contains higher concentrations of platelets (and other cell components) compared with the whole blood that was centrifuged. Thus, when the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma. Put another way, the enriched fraction is plasma which is enriched in platelets. When the bodily fluid is bone marrow, the enriched fraction is bone marrow concentrate. Suitably the bone marrow concentrate is enriched in stem cells e.g. mesenchymal stem cells. Suitably, the bodily fluid (in particular the platelet-rich plasma and / or the bone marrow concentrate) is autologous.

[0035] Centrifugation is performed at a force which is sufficient, over a particular length of time, to form a barrier between the components of the bodily fluid that are desired to be separated e.g. when the bodily fluid is whole blood, this is the force / time which results in the density gradient medium (if present) separating the erythrocytes from the platelets and any other desirable whole blood components. In one embodiment, the centrifugation is performed at a force of between about 700 g and about 2800 g, e.g. at a force of between about 1500 g and about 2800 g, between about 1500 g and about 2500 g, such as between about 1500 g and about 2000 g. Suitably, the centrifugation in step b) is performed for a period of time between about 3 minutes and about 40 minutes, e.g. between about 3 minutes and about 15 minutes.

[0036] When the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma. However, following centrifugation the platelet-rich plasma (enriched fraction) is not uniformly distributed, and the platelets are typically concentrated just above the density gradient medium (if present), with a layer of platelet-poor plasma (PPP) above this layer i.e. at the proximal end of the centrifugation container. It can be beneficial to remove a proportion of this platelet-poor plasma e.g. removing between about half and about one third of the enriched fraction (from the top end of the centrifugation container, i.e. from the end furthest from the density gradient medium (if present), the proximal end), thereby removing some or all of the PPP layer, leading to an enriched fraction which is even more concentrated, or enriched.

[0037] Thus, in one embodiment, step b) comprises the steps of: b-1) centrifuging the centrifugation container (of step a)) to form an enriched fraction; b-2) removing and discarding at least a portion of the enriched fraction (of step b-1)).

[0038] The at least a portion of the enriched fraction is typically removed from the proximal end of the centrifugation container. In this embodiment, step c) is collecting at least a portion of the remaining enriched fraction (i.e. the even more concentrated, or enriched fraction).

[0039] Platelet lysate is a liquid that is obtained by subjecting platelets to freeze / thaw cycles. The freeze / thaw cycle causes the platelets to lyse, releasing large quantities of growth factors and cytokines. Platelet lysate is commercially available, or can be readily prepared in house provided a supply of donor platelets is available.

[0040] The platelet lysate may be in lyophilised (or freeze dried) form. The lyophilization process preserves the capability of the platelets to release bioactive molecules, such as growth factors and cytokines, ensuring their stability and extended shelf life without the need for refrigeration.

[0041] The platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate can be mixed with electrospun fibres comprising silk protein. As such, according to one aspect of the invention, there is provided a method for preparing a scaffold material comprising i) plateletrich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

[0042] As discussed in the background of invention, silk protein can be derived from silk produced by insects such as silkworms and spiders. “Silk protein” as used herein is intended to encompass silk fibroin and related proteins. In one embodiment, the silk protein is silk fibroin. Silk fibroin, specifically, is a high molecular weight copolymer composed of a heavy chain (approximately 370 kDa) and a light chain (approximately 26 kDa) linked by a disulfide bond. The silk protein is preferably obtained from a solution containing a dissolved silkworm silk or spider silk. In one embodiment, the silk protein is obtained from a solution containing dissolved silkworm silk, such as from Bombyx mori. In another embodiment, the silk protein is obtained from a solution containing dissolved spider silk, such as from Nephila clavipes. In another embodiment, the silk protein is obtained from a solution containing genetically modified silk, such as obtained from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants.

[0043] In step a), the silk protein solution can be prepared using methods that are well known in the art (see for example Rockwood et al., Nat Protoc. 2011 Sep 22;6(10):1612-31 ; Wdltje et al., Int J Mol Sci. 2021 Sep 29;22(19):10565; Melke et al., Acta Biomaterialia, Volume 31 , 2016, Pages 1-16; and Bucciarelli et al. Biomater Adv. 2022 Aug;139:212982). When the silk protein is derived from silkworms, the silkworm cocoons are typically boiled in an aqueous solution of soap (e.g. sodium oleate) or sodium carbonate (known as a thermal bath), in order to remove the sericin protein and other fats coving the silk fibroin. This process is known as “degumming”. Degumming can also be achieved by a microwave assisted method, which produces high- purity fibroin, preserving intrinsic properties and ensuring superior bioactivity. Following degumming, the silk protein is washed and dried. Further purification by dialysis and / or filtration may be carried out. An exemplary process for preparing a silk protein solution is set out in General Methods.

[0044] The silk protein solution to be used in the electrospinning process of step b) suitably has silk protein (in particular silk fibroin) concentration of between about 5 wt.% and about 30 wt.%, e.g. between about 12 wt.% and about 18 wt.%. The higher the concentration of silk protein in the electrospinning solution, the larger the fibre diameter and the thicker the layer of electrospun fibres that is formed. However, if the concentration of silk protein is too high, the solution will be too viscous to be electrospun. The silk protein solution is typically prepared in water.

[0045] In step b), the electrospinning is carried out by electrospinning the silk protein solution of step a), using any means known in the art. A typical electrospinning device operates to apply a voltage (e.g. between about 5 kV and about 30 kV, such as between about 12 kV and about 18 kV) between the needle (which may also be called a spinneret, or a capillary tube) through which the silk protein solution is pumped, (typically via a syringe), and the collector. The collector is placed a short distance from the needle, typically at a distance of 5-20 cm, e.g. 8- 12 cm. Preferably, a constant volume flow rate is maintained using a syringe pump, to maintain the flow rate while avoiding the solution dripping from the tip of the needle. In one embodiment, the flow rate is between about 0.8 mL / hour to about 1.2 mL / hour. The electric field, solution flow rate, and distance between the needle tip and collector are adjusted so that a stable jet of solution is obtained. The collector can be any material that can function as a counter electrode, such as a wire mesh, a polymeric mesh, a metal foil (such as aluminium foil) or a water bath. Suitably, the collector has a non-stick surface to facilitate easy removal of the electrospun fibres e.g. the collector screen can have a Teflon coating, or is antiadhesive paper. Alternatively, the collector can be a substrate onto which the electrospinning solution is cast to form a layered product e.g. the collector can be a sheet material or a support material (as described below). Suitably, the collector spins e.g. at 100-300 rpm, in order to produce a uniform, highly aligned coating of electrospun fibres which form the scaffold material.

[0046] The electrospun fibres suitably have diameter between about 50 nm and about 1 ,000 nm, such as between about 200 nm and about 500 nm. The electrospun fibres are typically in the form of a film, a membrane, a mat or a mesh. If the electrospinning solution contains only silk fibroin, the resulting mesh following electrospinning will be in solid form. If additional components are included in the electrospinning solution (more details of which are set out below) then the resulting scaffold material may have semi-solid characteristics.

[0047] Once the electrospun fibres have been collected, they can be optionally subjected to one or more further processing steps such as washing (e.g. in distilled water) or stretching the fibres.

[0048] In step c) of the embodiment described above, the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material. Suitably, the scaffold material is a semi-solid scaffold material. In one embodiment, the scaffold material is in the form of a film, a membrane, a mat or a mesh.

[0049] “Semi-solid” in the context of the present invention means that the scaffold material is flexibly solid, and has physical properties that allow it to be sutured, but also have sufficient strength to provide the required mechanical and physiological support to act as a scaffold.

[0050] In an alternative process for forming the scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein, rather than being combined with the already-formed electrospun fibres comprising silk fibroin, the platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate can be included in the silk protein solution of step a) i.e. the platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate can included in the electrospinning solution. A mixture of platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and silk protein, then undergoes the electrospinning process. Thus, there is provided an alternative method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

[0051] In another aspect of the present invention is provided a scaffold material comprising: i) hydroxyapatite; and ii) electrospun fibres comprising silk protein. The present inventors have discovered that scaffold materials comprising electrospun fibres comprising silk protein, when combined with hydroxyapatite, form scaffold materials with a more solid structure.

[0052] Hydroxyapatite is a naturally occurring mineral form of calcium apatite with the formula Ca5(PO4)3(OH), often written Caio(P04)6(OH)2.

[0053] The scaffold material can be prepared using the following method comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with hydroxyapatite to form a scaffold material.

[0054] Steps a) and b) can be carried out as described above. In step c) the electrospun fibres comprising silk protein are combined with hydroxyapatite to form a scaffold material. Suitably, the scaffold material is a solid scaffold material. In one embodiment, the scaffold material is in the form of a film, a membrane, a mat or mesh.

[0055] In an alternative process for forming the scaffold material comprising hydroxyapatite and electrospun fibres comprising silk protein, rather than being combined with the already-formed electrospun fibres comprising silk fibroin, the hydroxyapatite can be included in the silk protein solution of step a) i.e. the hydroxyapatite can included in the electrospinning solution. A mixture of hydroxyapatite and silk protein then undergoes the electrospinning process.

[0056] Thus, there is provided an alternative method for preparing a scaffold material comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hydroxyapatite, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

[0057] In another aspect of the invention is provided a method for preparing a scaffold material comprising i) hyaluronic acid, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hyaluronic acid, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

[0058] Step a) can be carried out as described above. In one embodiment, the weight ratio of hyaluronic acid to silk protein in step b) is between about 1 :2 and about 1 :4 e.g. about 1 :3. In one embodiment, the method further comprises step d) cross-linking the scaffold material e.g. by reacting it with an aldehyde-based crosslinking agent such as glutaraldehyde (e.g. vapour phase). In one embodiment, the method further comprises step d) adding platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to the scaffold material of step c).

[0059] In all of the above methods where electrospun fibres comprising silk protein are combined with another material (e.g. platelet-rich plasma, platelet lysate, bone marrow concentrate, hydroxyapatite, hyaluronic acid or an additional component as described below or a mixture thereof) to form a scaffold material, the added material can be extruded. If desired, the electrospinning apparatus can be combined with a microextrusion apparatus, such as described in WO2019 / 211803A1 (incorporated herein by reference). An example of such a combined device is “Electrospider”, which combines electrospinning, fused deposition modelling and pneumatic hydrogel extrusion to fabricate (or “print”) 3D structures which can have multiple layers.

[0060] The scaffold materials described herein above may contain additional components, which can be included before electrospinning (i.e. they are included in the electrospinning solution containing the silk protein to be electrospun) or they can be added to the electrospun fibres comprising silk protein. Alternatively, a combination of additional components can be added, some of which are included in the electrospinning solution, and others which are added to the scaffold material following electrospinning.

[0061] For scaffold materials not already including hyaluronic acid, in one embodiment, the scaffold material further comprises hyaluronic acid. Hyaluronic acid is a glycosaminoglycan. The biological effects of hyaluronic acid are related to its molecular weight. In one embodiment (referring to all possible materials described hereinabove comprising hyaluronic acid), the hyaluronic acid is low molecular weight hyaluronic acid (LMW-HA), with molecular weight between about 400 kDa and about 1 ,000 kDa, or between about 50 kDa and about 100 kDa. In another embodiment, the hyaluronic acid is middle molecular weight hyaluronic acid, with molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa. In another embodiment, the hyaluronic acid is high molecular weight hyaluronic acid (HMW-HA), with molecular weight between about 800 kDa and about 1200 kDa, or greater than 1 ,800 kDa. Preferably, the hyaluronic acid in the centrifugation container of the invention has molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa.

[0062] A mixture of hyaluronic acids with different molecular weights may also be used. Thus, in one embodiment, the hyaluronic acid is a mixture of hyaluronic acids comprising: a low molecular weight hyaluronic acid (LMW-HA), with molecular weight between about 400 kDa and about 1 ,000 kDa; and / or a middle molecular weight hyaluronic acid, with molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa; and / or a high molecular weight hyaluronic acid (HMW-HA), with molecular weight greater than 1 , 800 kDa.

[0063] In one embodiment, the hyaluronic acid is cross-linked. In another embodiment, the hyaluronic acid is linear. Preferably, the hyaluronic acid is linear. The hyaluronic acid is preferably added as a solution in water, wherein the concentration of hyaluronic acid in the solution (added to the centrifugation container) is between about 1 wt.% and about 5 wt.%, such as between about 1.8 % and about 2.2 wt.%. Alternatively, when the scaffold material also contains platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, then the hyaluronic acid can be included in the centrifugation container which is used to form the platelet-rich plasma / platelet lysate / bone marrow concentrate. Suitably, the centrifugation container comprises between about 1.0 mL and about 5.0 mL of the hyaluronic acid, in particular between about 1 .5 mL and about 3 mL, such as about 2 mL. Suitably, the fill volume of the centrifugation container is 10 mL or 15 mL, in particular 15 mL.

[0064] In one embodiment, the scaffold material further comprises an antifibrinolytic substance. An antifibrinolytic substance is a substance (e.g. a compound) which inhibits fibrinolysis. Antifibrinolytic substances prevent or reduce the activation of plasminogen to form plasmin, thereby preventing or reducing blood clot degradation. Antifibrinolytic substances are typically synthetic analogues of the amino acid lysine. In one embodiment, the antifibrinolytic substance is selected from the group consisting of tranexamic acid, aminocaproic acid and aprotinin, or any combination thereof. Suitably, the antifibrinolytic substance is a small molecule e.g. with molecular weight of 500 Da or less, e.g. 400 Da or less, 300 Da or less, or 200 Da or less. In one embodiment, the antifibrinolytic substance is not an enzyme, a polypeptide or a protein. In one embodiment, the antifibrinolytic substance is tranexamic acid, aminocaproic acid or a mixture thereof. In a preferred embodiment, the antifibrinolytic substance is tranexamic acid. Suitably, the antifibrinolytic substance is 5 wt.% tranexamic acid (suitably in water). Reference to “an” antifibrinolytic substance is intended to encompass “at least one” antifibrinolytic substance, and combinations of antifibrinolytic substances are also envisaged.

[0065] In one embodiment, the scaffold material further comprises a coagulation activator.

[0066] A coagulation activator is an agent, for example a compound or an enzyme, that is able to trigger or activate coagulation of plasma and platelets aggregation, forming a clot, which may have the consistency of a gel. Reference to “a” coagulation activator is intended to encompass “at least one” coagulation activator, and combinations of coagulation activators are also envisaged. Typically, the coagulation activator is a compound or moiety which is a thrombin activator and / or a fibrinogen activator. In one embodiment, the coagulation activator is a compound selected from the group consisting of a calcium salt and thrombin. Suitably the calcium salt is selected from the group consisting of calcium gluconate, calcium carbonate, calcium sulphate, calcium saccharate and calcium chloride; or any combination thereof, and in particular is calcium gluconate.

[0067] In one embodiment, the coagulation activator is a calcium salt (in particular calcium gluconate) in a solution of water at between about 1 wt.% and about 20 w.% e.g. between about 5 wt.% and about 15 wt.% such as about 10 wt.%. The calcium salt may comprise a mixture of calcium salt e.g. a combination of calcium gluconate and calcium saccharate.

[0068] In one embodiment, the scaffold material further comprises vitamin K2. In one embodiment, vitamin K2 is selected from the group consisting of menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone- 12 (MK-12), menaquinone-13 (MK-13), and menaquinone-14 (MK-14), and mixtures thereof. References to “vitamin K2” and “menaquinone” are intended to encompass “at least one” menaquinone, and combinations of menaquinones are also envisaged. In one embodiment, vitamin K2 is selected from the group consisting of MK-6, MK-7 and MK-8, and mixtures thereof. In another embodiment, vitamin K2 is selected from the group consisting of MK-4, MK-5, MK-6 and MK-7, and mixtures thereof. In one embodiment, vitamin K2 is MK-4. In one embodiment, vitamin K2 is MK-7.

[0069] In one embodiment, the scaffold material further comprises one or more therapeutic agents. In one embodiment, the therapeutic agent is selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

[0070] For scaffold materials not already including hydroxyapatite, in one embodiment, the scaffold material further comprises hydroxyapatite.

[0071] Particularly preferred scaffold materials also contain hyaluronic acid. Thus, in a preferred embodiment is provided a scaffold material comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibres comprising silk protein; and iii) hyaluronic acid. In another preferred embodiment is provided a scaffold material comprising: i) hydroxyapatite; ii) electrospun fibres comprising silk protein; and iii) hyaluronic acid. In a further preferred embodiment is provided a scaffold material comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibres comprising silk protein; iii) hyaluronic acid; and iv) hydroxyapatite.

[0072] When hyaluronic acid is included in the scaffold material, suitably the ratio of silk protein to hyaluronic acid (w / w) is between about 50:50 and about 95:5. The scaffold materials of the invention have therapeutic use as, inter alia, wound dressings and grafts (e.g. vascular grafts).

[0073] In a further aspect of the invention is provided a layered wound dressing or layered graft comprising: i) a scaffold material comprising: ia) electrospun fibres comprising silk protein; and ii) hyaluronic acid; and ii) a support material.

[0074] Combining electrospun fibres comprising silk protein with hyaluronic acid leverages the mechanical strength of silk and the hydrophilic, cell-friendly environment provided by hyaluronic acid. The combination supports cell viability due to its biocompatibility and conducive environment for cell growth. Hyaluronic acid's hydrophilic nature aids in maintaining a hydrated environment, which is essential for cell survival. The presence of hyaluronic acid also enhances cell attachment due to its role in cell signalling and interaction with cell surface receptors (e.g. CD44). Silk protein, in particular silk fibroin, provides a stable and supportive structure that cells can adhere to.

[0075] In one embodiment, the scaffold material further comprises platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate (described in detail above). In one embodiment, the scaffold material further comprises hyaluronic acid (as described in detail above). In one embodiment, the scaffold material further comprises an antifibrinolytic substance, such as tranexamic acid (described in detail above). In one embodiment, the scaffold material further comprises a coagulation activator, such as calcium gluconate (as described in detail above). In one embodiment, the scaffold material further comprises vitamin K2 (as described in detail above). In one embodiment, the scaffold material further comprises hydroxyapatite (as described in detail above).

[0076] The support material is any support material that can typically be used in a wound dressing or graft. In one embodiment, the support material comprises a synthetic polymer such as poly(glycolic acid), poly(lactic acid), polyethylene glycol (PEG), low density polyethylene (LDPE), polycaprolactone (PCL) or a naturally occurring polymer such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran or albumen. The scaffold material of the invention can be attached to the support material using conventional techniques that are well known to the skilled person including by bonding (e.g. using an adhesive) or by laminating. In a preferred embodiment, the support material comprises a plastic material, such as low density polyethylene (LDPE) or polycaprolactone (PCL).

[0077] The scaffold materials of the invention also have use in tissue engineering applications. In one embodiment, the scaffold material of the invention can be combined with cells to form a tissue engineering construct for use in regenerating or replacing damaged or degenerated tissue or organs.

[0078] A tissue engineered construct can be prepared by various methods. In one embodiment, a tissue engineered construction comprises i) a scaffold material as described herein; and ii) cells e.g. mammalian cells. The particular type of cells will depend on the intended application of the construct, but suitable mammalian cells included cells selected from the group consisting of hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal- hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smooth muscle cells, skeletal muscle cells, oscular cells, integumentary cells, bone marrow cells, keratinocytes, pluripotent cells and stem cells and combinations thereof.

[0079] In one embodiment is provided a method for preparing a tissue engineered construct comprising the steps of: a) preparing a scaffold material as described herein; and b) combining the scaffold material with cells (e.g. mammalian cells) in a suitable medium.

[0080] The suitable medium is a culture medium and will depend on the type of cells being cultured, and can be easily prepared by the skilled person.

[0081] In a further aspect of the invention is provided a layered construct for tissue engineering applications, comprising: i) a first layer comprising silk protein; ii) a second layer comprising a hydrogel and silk protein; iii) a third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) a fourth layer comprising polycaprolactone and hydroxyapatite. A layered construct according to an embodiment of the invention is shown in Figure 1 (“Multiphasic Implant”). This innovative construct is specifically designed for the treatment of cartilage damage caused by degeneration or trauma, including applications in sports medicine. The construct mimics the structure and function of native osteochondral tissue by utilizing a layered, multiphasic approach. It is fabricated through GMP-compliant 3D printing and electrospinning techniques to ensure clinical-grade quality.

[0082] Suitably, the first layer comprising silk protein has a thickness between about 40 pm and about 60 pm, e.g. about 50 pm. Suitably, the first layer comprising silk protein is a cover layer. Suitably, the silk protein in the first layer is composed of electrospun fibres of silk protein. In one embodiment, the electrospun fibres of silk protein are prepared by electrospinning silk fibroin solution at a concentration between about 5% (w / v) and 15% (w / v) e.g. about 7% (w / v); under a voltage of 12-18 kV e.g. about 15 kV; at a flow rate of between about 0.8 mL / hour to about 1.2 mL / hour, e.g. at 0.8 mL / hour; and a collector distance of 5-20 cm, e.g. about 10 cm. The first layer comprising silk protein (in particular silk fibroin, preferences described hereinabove apply) and provides a protective barrier for underlying layers. The first layer also provides structural integrity to the layered construct and prevents degradation during early implantation. Finally, it offers excellent biocompatibility, promoting cellular attachment.

[0083] In one embodiment, the hydrogel of the second layer comprises or is hyaluronic acid and / or gelatin. Suitably, the hyaluronic acid is a mixture of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid (e.g. in a weight ratio of about 1 :1). In one embodiment, the silk protein in the second layer is composed of electrospun fibres of silk protein. In one embodiment, the electrospun fibres of silk protein are prepared by electrospinning silk fibroin solution at a concentration between about 5% (w / v) and 15% (w / v) e.g. about 7% (w / v); under a voltage of 12-18 kV e.g. about 15 kV; at a flow rate of between about 0.8 mL / hour to about 1.2 mL / hour; e.g. at 0.8 mL / hour, and a collector distance of 5-20 cm, e.g. about 10 cm. The second layer can be formed by combining the hyaluronic acid and / or gelatin with the electrospun silk protein, or alternatively the hyaluronic acid and / or gelatin can be combined with the silk protein solution prior to electrospinning, according to the methods described hereinabove. Thus, in one embodiment, the second layer is composed of electrospun fibres of hydrogel (e.g. hyaluronic acid) and silk protein. In one embodiment, the electrospun fibres of hyaluronic acid and silk protein are prepared by electrospinning a hyaluronic acid and silk fibroin solution at a concentration between about 5% (w / v) and 15% (w / v) e.g. about 7% (w / v); under a voltage of 12-18 kV e.g. about 15 kV; at a flow rate of between about 0.8 mL / hour to about 1.2 mL / hour; e.g. at 0.8 mL / hour, and a collector distance of 5-20 cm, e.g. about 10 cm. In one embodiment, the second layer has thickness of 1-2 mm. In one embodiment, the second layer further comprises platelet-rich plasma, platelet lysate and / or bone marrow concentrate, and in particular further comprises platelet lysate. The hydrogel-based second layer mimics cartilage middle zone, enabling chondrogenesis. It facilitates controlled delivery of growth factors for tissue regeneration and enhances water retention, contributing to the construct’s biomechanical properties.

[0084] In one embodiment, the third layer comprises polycaprolactone (PCL) and hydroxyapatite, and is a layer formed by electrospinning. In one embodiment, the third layer comprises hyaluronic acid and hydroxyapatite, and is a layer formed by electrospinning. Suitably, the third layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 80:20. In one embodiment, the third layer has thickness of 90 pm to 110 pm. The third layer mimics the calcified layer of the osteochondral tissue, providing a transition interface between soft cartilage and the underlying bone. The third layer combines mechanical strength with bioactivity to ensure integration with subchondral bone.

[0085] In one embodiment, the fourth layer comprising polycaprolactone and hydroxyapatite is formed using fused deposition modelling (FDM). FDM is a type of 3D printing in which a thermoplastic polymer is heated past its melting point and then fed through a nozzle at a precise rate. The molten polymer is deposited on a build plate at a lower temperature, causing it to rapidly cool and solidify. In one embodiment, the fourth layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 70:30. In one embodiment, the fourth layer has thickness of 450 pm to 550 pm, e.g. about 500 pm. The fourth layer mimics the subchondral bone layer and provides load-bearing functionality and support, facilitating long-term integration with native bone tissue.

[0086] The scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs described herein have therapeutic utility. Thus, in one embodiment is provided the scaffold material, layered wound dressing, layered graft, layered construct as described herein, or the tissue engineered construct as described herein, for use in therapy. The scaffold material is a medical scaffold material. In one embodiment, the scaffold material is in the form of a wound dressing or a graft.

[0087] The scaffold material, tissue engineered construct, layered wound dressing, layered graft, and layered constructs of the invention can provide mechanical support at the site of injury, and can also provide physiological support by facilitating the augmentation of native tissue repair. In one embodiment is provided a scaffold material, a tissue engineered construct, a layered wound dressing, a layered graft, or a layered construct as described herein, for use in wound healing, wound sealing, tissue repair, tissue regeneration, cartilage repair, cartilage regeneration, tendon repair, tendon regeneration, nerve repair, nerve regeneration, bone repair and / or bone regeneration. Tissue includes muscle, skin and organs.

[0088] Specific examples of wound healing and tissue repair include the treatment of gynaecological conditions (e.g. pelvic organ prolapse), bowel injuries, bladder injuries, vascular injuries, other non-healing wounds, burns, and diabetic foot.

[0089] In one embodiment is provided a scaffold material, a tissue engineered construct, a layered wound dressing, a layered graft, or a layered construct as described herein, for use in tissue augmentation.

[0090] In one embodiment is provided a scaffold material, a tissue engineered construct, a layered wound dressing, a layered graft, or a layered construct as described herein, for use as a delivery vehicle for a therapeutic agent.

[0091] Scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention comprising hydroxyapatite are particularly suited for use in bone regeneration.

[0092] In one embodiment, is provided the scaffold material, tissue engineered construct, layered wound dressing, layered graft, or layered construct as described herein, for use in treating or preventing a joint disorder or condition. Suitably, the joint disorder or condition is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia and rheumatica. Arthritis includes osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, cervical spondylitis, psoriatic arthritis, enteropathic arthritis, oligoarthritis, polyarthritis and secondary arthritis.

[0093] The scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention can be in any suitable form, including in the form of a film, a membrane, a mat, or a mesh. The scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention can be used, inter alia, as wound dressings, grafts such as vascular grafts and scaffolds for cell growth. Scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention have excellent biocompatibility, and in certain embodiments contain pores or interstices which allow bodily fluids to be exchanged within their structure. Scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention are suitably biodegradable and are hence resorbed following application (e.g. implantation).

[0094] The scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention can be layered onto conventional materials (e.g. wound dressing materials) to provide enhanced mechanical strength. In one embodiment, the scaffold material of the invention is layered onto a suitable sheet material e.g. a backing layer. In one embodiment, the scaffold material, tissue engineered construct, layered wound dressing, layered graft, or layered construct of the invention is layered onto a sheet comprising a viscose polyester matrix. In one embodiment, the scaffold material, tissue engineered construct, layered wound dressing, layered graft, or layered construct of the invention is layered onto a sheet comprising a synthetic polymer such as poly(glycolic acid), poly(lactic acid), polyethylene glycol (PEG), low density polyethylene (LDPE), polycaprolactone (PCL) or a naturally occurring polymer such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran or albumen. The scaffold material, tissue engineered construct, layered wound dressing, layered graft, or layered construct of the invention can be attached to the sheet material using conventional techniques that are well known to the skilled person including by bonding (e.g. using an adhesive) or by laminating.

[0095] In an alternative process of the invention, a composition comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein can be prepared by forming the platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate using a centrifugation container which already contains a silk protein solution. The silk protein solution can be prepared as described above. In this embodiment, the bodily fluid or cell extract is collected in a centrifugation container already containing the silk protein solution. The centrifugation container is then centrifuged to form an enriched fraction and a silk protein solution. This solution can then be subjected to an electrospinning process, as described herein, to form a scaffold material of the invention.

[0096] In one embodiment, is provided a centrifugation container comprising a silk protein (details of which are as described above). The centrifugation container can further comprise hyaluronic acid (details of which are described above) and / or an antifibrinolytic substance (details of which are described above) and / or a coagulation activator (details of which are described above) and / or vitamin K2 and / or one or more therapeutic agents (details of which are described above) and / or hydroxyapatite. When the centrifugation container comprises hyaluronic acid, trehalose can also be included in the container as a stabilizer for the hyaluronic acid. The centrifugation container can further comprise a density gradient medium (details of which are described above).

[0097] In one embodiment is provided a centrifugation container comprising a silk protein, a density gradient medium, and hyaluronic acid.

[0098] The use of electrospinning to form scaffold materials of the invention provides a number of benefits. Firstly, it enables the creation of a standardized scaffold material, which can be produced in bulk and subsequently cut to the desired dimensions, making it adaptable for various clinical applications. Secondly, the technique also allows the fabrication of custom- tailored scaffold materials, which can be precisely designed and manufactured to match the specific pathology and anatomical requirements of individual patients. As such, the methods of the invention have dual capabilities for both standardized mass production and personalized fabrication.

[0099] In one embodiment, the scaffold material of the invention has a swelling ratio of at least 300%. The swelling ratio is defined at the difference between the initial weight of the material in its dry state and the weight of the material in its swollen state, divided by the initial weight.

[0100] Scaffold materials, tissue engineered constructs, layered wound dressings, layered grafts, and layered constructs of the invention are expected to have, in certain embodiments, one or more of the following merits / advantages: suitable porosity for nutrient and / or cell exchange; suitable structure to support cell adhesion and / or elongation; facilitate controlled delivery of bioactive agents such as growth factors; enhance cell growth and hence enhance healing; biocompatibility; anti-apoptotic effects.

[0101] Further specific advantages of the layered construct of the present invention:

[0102] Biomimetic design:

[0103] Each layer replicates the architecture and function of natural osteochondral tissue zones, from the articular cartilage surface to the subchondral bone.

[0104] Controlled Growth Factor Release: The hydrogel layer incorporates platelet lysate proteins, enabling sustained release of growth factors to enhance tissue regeneration.

[0105] Fabrication Techniques:

[0106] Electrospinning: Produces the nanoscale fibrous architecture in the electrospun layer for enhanced cell adhesion and mechanical strength.

[0107] 3D Printing: Ensures precise and scalable production of the FDM layer and overall construct. GMP Compliance:

[0108] All processes are carried out under GMP conditions, ensuring regulatory and clinical applicability.

[0109] Scalability and Customization:

[0110] The construct can be tailored to patient-specific needs using advanced 3D printing technologies.

[0111] ABBREVIATIONS

[0112] BMC bone marrow concentrate

[0113] CBD cannabidiol

[0114] DMA dynamic mechanical analysis

[0115] DM EM Dulbecco’s Modified Eagle Medium

[0116] DMMB dimethylmethylene blue

[0117] DMF dimethylformamide

[0118] ELISA enzyme-linked immunosorbent assay

[0119] EtO ethylene oxide

[0120] FDM fused deposition modelling

[0121] GAG glycosaminoglycan

[0122] GF gingival fibroblasts

[0123] GMP good manufacturing practice

[0124] HA hyaluronic acid

[0125] HMW-HA high molecular weight hyaluronic acid

[0126] HLIVECs human umbilical vein endothelial cells

[0127] LDPE low density polyethylene

[0128] LMW-HA low molecular weight hyaluronic acid

[0129] MK menaquinone

[0130] MPV mean platelet volume

[0131] MSC mesenchymal stem cells

[0132] NSAID non-steroidal anti-inflammatory drug

[0133] PBS phosphate buffered saline PCL polycaprolactone

[0134] PDGF platelet-derived growth factor

[0135] PDL periodontal ligament cells

[0136] PEG polyethylene glycol

[0137] PL platelet lysate

[0138] PPP platelet-poor plasma

[0139] PRP platelet-rich plasma rpm revolutions per minute

[0140] SDS sodium dodecyl sulfate

[0141] SEM scanning electron microscopy

[0142] SF silk fibroin

[0143] THC tetrahydrocannabinol

[0144] VF vaginal fibroblasts

[0145] EXAMPLES

[0146] General Methods

[0147] Cell preparation - fibroblasts

[0148] Gingival tissues were collected from adult patients interdental papillae during tooth extractions. After collection, tissues were immediately stored in sterile saline solution for one to four hours before processing. Gingival tissues were washed 10 times in phosphate-buffered saline (PBS) to dilute the oral bacterial flora of the gingival tissue. Following the PBS wash, the tissues were cut into small pieces of between 1 and 2 mm2using a No. 10 surgical blade. Explants were then plated over 100 mm tissue culture dishes and incubated for 48 h, undisturbed, at 37 °C in a humidified incubator with 5% CO2. After 48 h, the medium was replaced. Cells were cultivated in complete growth medium (Dulbecco's Modified Eagle's medium [DMEM]; Life Technologies, Paisley, UK) supplemented with 10% FBS, 1% HEPES 1 M buffer solution (Life Technologies), 1 % nonessential amino acid mixture 100* (Life Technologies), 1% L-glutamine 100x (Life Technologies), 1% penicillin / streptomycin 100* (Life Technologies), 1% sodium pyruvate 100* (Life Technologies). Fibroblasts from other tissues can be cultivated using a similar process.

[0149] Silk fibroin solution preparation

[0150] A degumming process is first carried out by cutting 5 g of Bombyx mori cocoons into small pieces. The pieces are then boiled for 30 minutes in 2 L of 0.02 M carbonated carbonate, before being removed and squeezed to remove excess water from the silk. The silk pieces are then rinsed three times for 20 min with 1 L of deionized water, before being squeezed to remove excess water. The pieces of degummed silk fibroin are dried overnight in a fume hood, and can be stored indefinitely at room temperature.

[0151] A silk fibroin solution is prepared by dissolving the degummed silk fibroin in 9.3 M lithium bromide at a 20 w / v% solution, for 4 hours at 60 °C until a clear and transparent solution if formed. The solution can be desalted by dialysis, e.g. 12 mL in 1 L of DI water (renew 6 times water content), followed by centrifuging three times at 9,000 rpm (approximately 12,700 g) at 4 °C for 20 minutes. 5 g of silk cocoons generally yield 25 ml of 7 to 8 w / v% silk solution.

[0152] Example 1a - Preparation of platelet-rich plasma

[0153] A blood sample was obtained from a healthy donor into a centrifugation tube (CuteCell™ PRP; Regen Lab SA). The collected blood was centrifuged for 5 min in a standard laboratory centrifuge at 1500 g at room temperature. Subsequently, the red and white blood cells accumulated at the bottom of the tube under the separator gel, whereas the plasma and platelets remained above the gel layer. Plasma containing platelets was homogenized by inverting the tube five times. The resulting 6 mL of platelet-rich plasma was transferred in a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) until use.

[0154] Example 1 b - Preparation of platelet-rich plasma containing hyaluronic acid

[0155] A blood sample was obtained from a healthy donor into a centrifugation tube containing hyaluronic acid (CellularMatrix™ PRP; Regen Lab SA). The collected blood was centrifuged for 5 min in a standard laboratory centrifuge at 1500 g at room temperature. Subsequently, the red and white blood cells accumulated at the bottom of the tube under the separator gel, whereas the plasma and platelets remained above the gel layer. Plasma containing platelets was homogenized by inverting the tube five times. The resulting 6 mL of platelet-rich plasma and hyaluronic acid was transferred in a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) until use.

[0156] Example 2 - Preparation of scaffold material in the form of a membrane comprising electrospun fibres comprising silk protein and hyaluronic acid

[0157] A solution of 0.5% w / v hyaluronic acid in formic acid was prepared by dissolving hyaluronic acid in formic acid at 55 °C for 3 hours. The solution was cooled down and then 7.5% (w / v) of silk fibroin was added, and the resulting mixture stirred for 30 minutes. The solution was then ready to be electrospun, with previously optimised parameters, using antiadhesive paper as collector. The silk fibroin / hyaluronic acid nanofibers were electrospun and collected directly on the substrate, with a deposition time of about 25 minutes. The resulting membrane was cut using a laser cutting machine, and the cut samples were sterilized using EtO.

[0158] Example 3 - Preparation of scaffold material comprising electrospun fibres comprising silk protein and hyaluronic acid layered onto a non-woven viscose / polyester substrate

[0159] A solution of 0.5% w / v hyaluronic acid in formic acid was prepared by dissolving hyaluronic acid in formic acid at 55 °C for 3 hours. The solution was cooled down and then 7.5% (w / v) of silk fibroin was added, and the resulting mixture stirred for 30 minutes. The solution was then ready to be electrospun, with previously optimised parameters, using a sheet of viscose / polyester substrate as collector. The silk fibroin / hyaluronic acid nanofibers were electrospun and collected directly on the substrate, with a deposition time of about 12 minutes. The resulting sheet was cut using a laser cutting machine, and the cut samples were sterilized using EtO.

[0160] Example 4 - Preparation of scaffold material comprising electrospun fibres comprising silk protein and hyaluronic acid

[0161] 5 g of degummed silk fibroin is dissolved in 9.3 M lithium bromide at 60 °C for 4 hours. The solution is dialyzed against distilled water for 48 hours and adjusted to 6% (w / v). Hyaluronic acid is dissolved in PBS to form a 2% (w / v) solution, prepared separately for LMWHA (50- 100 kDa) and HMW HA (800-1200 kDa). The silk fibroin and HA solutions are mixed at a 3:1 ratio under gentle stirring to form a homogeneous blend. The SF-HA mixture is electrospun under the following parameters: Voltage: 15 kV.

[0162] Flow rate: 1 mL / hour.

[0163] Collector distance: 10 cm.

[0164] Nanofibers are deposited onto anti-adhesive paper and dried at room temperature. The resulting matrix is crosslinked with vapor-phase glutaraldehyde for 12 hours to stabilize its structure. The resulting scaffold material is expected to exhibit favorable mechanical properties, swelling capacity, and uniform fiber morphology, making it a promising scaffold for tissue engineering applications.

[0165] Example 5 - Use of silk-HA scaffold material in cellular assays

[0166] The electrospun HA-Silk matrix prepared according to Example 4 (1 cm2samples) is immersed in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum for 1 hour to reduce hydrophobicity. The matrix is expected to absorb the medium and settle at the bottom of the dish. The bioactive HA-Silk layer is then oriented upwards in the culture dish to maximize exposure to seeded cells. A total of 100,000 cells suspended in 500 pL of culture medium are added as a drop on the surface of the matrix. The cells are allowed to adhere for 1 -2 hours before adding the experimental media, as follows:

[0167] Experimental Conditions:

[0168] Control (Medium-Only): DM EM + 10% FBS.

[0169] PRP Condition: DMEM supplemented with platelet-rich plasma (10% PRP).

[0170] PRP-HA Condition: DMEM supplemented with platelet-rich plasma and hyaluronic acid (10% PRP-HA).

[0171] The use of the HA-silk matrix for biological essays is then evaluated. The Live / Dead Kit (Invitrogen L3224) is used to evaluate cell viability at Days 7 and 15. Imaging is performed using a system to observe cell proliferation and mortality.

[0172] Morphology Analysis:

[0173] Cell Types Tested: Gingival fibroblasts (GF), periodontal ligament cells (PDL), and endothelial cells (HLIVECs). Observations are recorded using phase-contrast microscopy.

[0174] The bioactive HA-Silk matrix is expected to demonstrate biocompatibility, supporting cell adhesion, proliferation, and viability under various experimental conditions. The addition of PRP-HA is expected to enhance cellular responses, highlighting its potential for tissue engineering applications.

[0175] Example 6 - Cell Adhesion and Elongation Assay

[0176] The ability of cells to adhere and elongate on the HA-Silk matrix and assess structural alignment along the fibers is assessed. Electrospun HA-Silk matrices are prepared and rehydrated as described in Example 5. The HA-Silk side of the matrix is oriented upwards to optimize interaction with seeded cells.

[0177] Cell Types:

[0178] Tested Primary Cell Lines:

[0179] Gingival fibroblasts (GF).

[0180] Periodontal ligament cells (PDL).

[0181] Endothelial cells (HUVECs).

[0182] Experimental Conditions:

[0183] Control: DMEM supplemented with 10% FBS.

[0184] PRP Condition: DMEM supplemented with 10% platelet-rich plasma (PRP).

[0185] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1 % w / v).

[0186] Assay Protocol:

[0187] Cells are cultured on the HA-Silk matrix for 7 and 15 days. Adhesion and elongation are assessed using phase-contrast microscopy, focusing on cell alignment along the fibers. The HA-Silk matrix is expected to support strong cell adhesion and promote structural alignment along the fibers. The PRP-HA condition is expected to enhance elongation and network formation, highlighting the matrix’s potential for applications in tissue engineering.

[0188] Example 7 - Hyaluronic acid layer stability

[0189] The stability of the HA layer in the HA-Silk matrix under different culture conditions (control, PRP, and PRP-HA) is assessed over time. Electrospun HA-Silk matrices are prepared and rehydrated as described in Example 5. The HA layer is oriented upwards during all assays. Experimental Conditions:

[0190] Control: DMEM supplemented with 10% FBS.

[0191] PRP Condition: DMEM supplemented with 10% platelet-rich plasma (PRP).

[0192] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1 % w / v).

[0193] Assay Protocol:

[0194] Matrices are cultured in each condition for up to 15 days. Observations are recorded on Days 7 and 15, focusing on: Macroscopic matrix integrity; and Microscopic layer stability via phasecontrast microscopy. The stability of the HA layer is expected to be enhanced in PRP-HA conditions, demonstrating the importance of combining PRP with hyaluronic acid. This feature is expected to support the HA-Silk matrix’s use as a robust scaffold for prolonged tissue engineering applications.

[0195] Example 8 - Synergistic effects of PRP-HA

[0196] The combined effects of platelet-rich plasma (PRP) and hyaluronic acid (HA) on cell proliferation, network formation, and bioactivity when cultured on the HA-Silk matrix are evaluated. Electrospun HA-Silk matrices are prepared and rehydrated as described in Example 5. The HA-Silk layer is oriented upwards during all assays.

[0197] Cell Types:

[0198] Tested Primary Cell Lines:

[0199] Gingival fibroblasts (GF).

[0200] Periodontal ligament cells (PDL).

[0201] Endothelial cells (HUVECs).

[0202] Experimental Conditions:

[0203] Control: DMEM supplemented with 10% FBS.

[0204] PRP Condition: DMEM supplemented with 10% platelet-rich plasma (PRP).

[0205] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1 % w / v).

[0206] Assay Protocol:

[0207] Cells are cultured on the HA-Silk matrix for up to 15 days. Cell proliferation and network formation are assessed using high-throughput imaging and phase-contrast microscopy. It is

[0208] T1 expected that the synergistic effects of PRP and HA will significantly enhance the bioactivity of the HA-Silk matrix, promoting robust cell proliferation and network formation. This is expected to underscore the matrix’s potential for applications in tissue engineering and regenerative medicine.

[0209] Example 9 - Platelet Lysate Optimization for Enhanced Bioactivity

[0210] The impact of different platelet lysate (PL) concentrations on the bioactivity of the HA-Silk matrix, focusing on cell adhesion, proliferation, and elongation is assessed. Electrospun HA- Silk matrices are prepared and rehydrated as described in Example 5.

[0211] Platelet Lysate Concentrations:

[0212] Experimental groups included matrices cultured in media supplemented with:

[0213] 0.25% (v / v) PL.

[0214] 0.5% (v / v) PL.

[0215] 1.0% (v / v) PL.

[0216] Control: DMEM supplemented with 10% FBS (no PL).

[0217] Cell Types:

[0218] Tested with primary human cell lines:

[0219] Gingival fibroblasts (GF).

[0220] Periodontal ligament cells (PDL).

[0221] Endothelial cells (HUVECs).

[0222] Assay Protocols:

[0223] Cells are cultured on the HA-Silk matrix for 15 days under the above conditions.

[0224] Evaluation Metrics:

[0225] Cell viability (Live / Dead assay (Invitrogen L3224) at Days 7 and 15).

[0226] Adhesion and elongation (phase-contrast microscopy).

[0227] Proliferation rates (MTT assay).

[0228] It is expected that the HA-Silk matrix will demonstrate optimal bioactivity with 0.5% (v / v) platelet lysate supplementation, supporting strong cell adhesion, elongation, and proliferation. Higher concentrations (1.0% PL) are expected to result in overcrowding effects, while lower concentrations (0.25% PL) are expected to insufficiently to enhance bioactivity. The findings of this experiment are expected to emphasize the importance of PL concentration optimization for tissue engineering applications.

[0229] Example 10 - Comparative Performance of Supporting Layers

[0230] The aim of this experiment is to evaluate the effects of different supporting materials on the structural integrity, cell viability, and handling of the HA-Silk matrix during biological assays. Electrospun HA-Silk matrices are fabricated as described in Example 5. The matrices are deposited onto different supporting materials during the electrospinning process.

[0231] Supporting Materials Tested:

[0232] LDPE / Cellulose Composite: High tensile strength, flexible support.

[0233] Polythene / Paper Composite: Moderately rigid, lightweight support with anti-adhesive properties.

[0234] Control Support: Standard anti-adhesive electrospinning paper.

[0235] Evaluation Metrics:

[0236] Structural Integrity: Assessed by observing matrix shrinkage or tearing during transfer and culture.

[0237] Peelability: Ease of removing the matrix from the support without damage, scored qualitatively. Cell Viability and Coverage: Measured using Live / Dead assay (Invitrogen L3224) and phasecontrast microscopy at Day 7 and Day 15.

[0238] Cell Types:

[0239] Gingival fibroblasts (GF).

[0240] Endothelial cells (HUVECs).

[0241] Among the tested supports, the Polythene / Paper composite is expected to demonstrate superior performance in terms of structural integrity, ease of handling, and cell viability. This support material is expected to be recommended for optimizing the HA-Silk matrix in future tissue engineering applications.

[0242] Example 11 - Long-Term Matrix Stability and Degradation

[0243] The degradation of the HA-Silk matrix over an extended period (up to 30 days) in PRP-HA culture conditions is monitored, focusing on macroscopic and microscopic changes. Electrospun HA-Silk matrices are fabricated and rehydrated as described in Example 5. The HA layer is oriented upwards in culture dishes for all experiments.

[0244] Experimental Conditions:

[0245] Control: DMEM supplemented with 10% FBS.

[0246] PRP Condition: DMEM supplemented with 10% platelet-rich plasma (PRP).

[0247] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1 % w / v). Evaluation Metrics:

[0248] Macroscopic Observations: Recorded shrinkage, discoloration, or surface irregularities at Days 7, 15, and 30.

[0249] Microscopic Analysis:

[0250] Phase-contrast microscopy for surface morphology.

[0251] Confocal microscopy for fiber degradation and cell coverage.

[0252] Mass Loss: Measured the percentage reduction in matrix weight over 30 days. The HA-Silk matrix is expected to demonstrate superior long-term stability in PRP-HA conditions, with minimal degradation and sustained structural integrity over 30 days. These findings are expected to emphasize its potential for prolonged tissue engineering applications.

[0253] Example 12 - Multi-Layered construct preparation

[0254] A multiphase construct mimicking osteochondral tissue using advanced techniques (electrospinning and 3D printing) is prepared and characterized (physicochemical and structural properties).

[0255] Cover Layer (Silk Fibroin):

[0256] Extracted from Bombyx mori cocoons following degumming and dissolution in 9.3 M lithium bromide. Known for its high tensile strength, biocompatibility, and ability to support cellular adhesion.

[0257] Hydrogel Layer:

[0258] Composition:

[0259] Hyaluronic acid (HA): Enhances water retention and facilitates cell proliferation.

[0260] Gelatin: A natural protein promoting cellular attachment and differentiation.

[0261] Silk fibroin: Provides structural integrity and mechanical reinforcement.

[0262] Platelet lysate proteins: Serve as a source of growth factors for chondrogenesis.

[0263] Electrospun Layer:

[0264] Polycaprolactone (PCL): A biodegradable polymer offering high mechanical strength and slow degradation.

[0265] Hydroxyapatite: Mimics the mineral content of the calcified cartilage layer.

[0266] FDM Layer:

[0267] A composite of PCL and hydroxyapatite to mimic the subchondral bone's load-bearing properties.

[0268] Fabrication Methods:

[0269] Cover Layer (50 pm):

[0270] Silk fibroin solution (7% w / v) is electrospun onto a rotating collector.

[0271] Electrospinning parameters:

[0272] Voltage: 15 kV.

[0273] Flow rate: 0.8 mL / h.

[0274] Collector distance: 10 cm.

[0275] The layer is stabilized by crosslinking with ethanol vapor.

[0276] Hydrogel Layer (1-2 mm):

[0277] HA (1 % w / v), gelatin (2% w / v), and silk fibroin (3% w / v) were mixed in PBS at 37 °C.

[0278] Platelet lysate proteins (0.5% v / v) were added to the mixture.

[0279] The hydrogel is cast into moulds and crosslinked with genipin for 24 hours to enhance stability. Electrospun Layer (100 pm):

[0280] A solution of PCL (10% w / v) and hydroxyapatite (2% w / v) in chloroform / DMF (1:1) is electrospun.

[0281] Electrospinning parameters:

[0282] Voltage: 18 kV.

[0283] Flow rate: 1.2 mL / h.

[0284] Collector distance: 12 cm.

[0285] FDM Layer (500 pm):

[0286] PCL and hydroxyapatite (70:30 ratio) are extruded using a fused deposition modelling (FDM) 3D printer. The base layer is printed at a nozzle temperature of 180 °C with a layer height of 100 pm.

[0287] Each layer is expected to demonstrate properties mimicking the respective zones of native osteochondral tissue, ensuring structural integrity and biocompatibility.

[0288] Example 13 - Multi-Layered Construct with HA-Silk Integration

[0289] The integration of the HA-Silk matrix into a multi-layered construct for cartilage repair and the assessment of its role in enhancing cell proliferation, matrix integration, and biomechanical stability is evaluated. A multi-layered construct is fabricated, comprising: Layer 1 (Cover Layer): Electrospun silk fibroin (SF).

[0290] Layer 2 (Hydrogel Layer): HA-Silk matrix combined with gelatin and platelet lysate proteins (0.5% v / v).

[0291] Layer 3 (Electrospun Transition Layer): Polycaprolactone (PCL) and hydroxyapatite.

[0292] Layer 4 (FDM Base Layer): 3D-printed PCL and hydroxyapatite.

[0293] HA-Silk Layer Integration:

[0294] The HA-Silk matrix is embedded in the hydrogel layer to mimic cartilage-like properties, with bioactivity enhanced by PRP-HA supplementation.

[0295] Cell Types:

[0296] Human chondrocytes are seeded onto the construct to assess integration and proliferation.

[0297] Assay Protocols:

[0298] Integration Testing: Assess inter-layer cohesion using mechanical shear tests.

[0299] Cell Proliferation: Measure via MTT assay at Days 7, 15, and 30.

[0300] Matrix Bioactivity: Analyze cellular network formation and growth factor release via ELISA.

[0301] Biomechanical Stability:

[0302] Evaluate compressive strength and modulus using a uniaxial testing machine.

[0303] The HA-Silk matrix is expected to successfully integrate into a multi-layered construct, and is expected to enhance bioactivity, cell proliferation, and biomechanical stability. This construct is expected to demonstrate potential for cartilage repair applications, particularly in degenerative or trauma-induced conditions.

[0304] Example 14 - Multilayer construct evaluation

[0305] The biocompatibility, cellular adhesion, proliferation, and differentiation on the multiphase construct prepared according to Example 12 is evaluated under different culture conditions using chondrocytes and mesenchymal stem cells (MSCs).

[0306] Cell Types:

[0307] Chondrocytes: Isolated from human articular cartilage biopsies.

[0308] MSCs: Harvested from bone marrow aspirates.

[0309] Culture Media Conditions:

[0310] Control: DMEM + 10% fetal bovine serum (FBS).

[0311] PRP: DMEM supplemented with 10% platelet-rich plasma.

[0312] PRP-HA: DMEM supplemented with 10% platelet-rich plasma and hyaluronic acid (1% w / v).

[0313] Matrix Preparation for Cell Culture:

[0314] Multiphase constructs are sterilized using ethylene oxide gas and rehydrated in culture media for 1 hour prior to cell seeding. Constructs are oriented with the cover layer (silk fibroin) upwards to promote initial cell attachment.

[0315] Assay Protocols:

[0316] Cell Adhesion (24 hours):

[0317] Cells are seeded on the constructs (1 x 105cells / cm2) and incubated for 24 hours.

[0318] Adhesion is assessed using phase-contrast microscopy and SEM.

[0319] Proliferation (7 and 14 days):

[0320] Cellular metabolic activity is evaluated using the WST-1 assay.

[0321] Chondrogenic Differentiation (21 days):

[0322] Constructs seeded with MSCs are cultured in chondrogenic induction media.

[0323] Glycosaminoglycan (GAG) production is assessed using Alcian Blue staining and quantified via a dimethylmethylene blue (DMMB) assay.

[0324] The PRP-HA condition is expected to significantly enhanced cellular adhesion, proliferation, and chondrogenic differentiation on the multiphase construct. The hydrogel and electrospun layers are expected to play a crucial role in supporting bioactivity, demonstrating the potential for use in osteochondral tissue regeneration. The multiphase construct is expected to support high levels of cellular bioactivity, particularly in the presence of PRP-HA. The findings are expected to underscore its suitability for cartilage repair applications.

[0325] Example 15 - Multilayer construct mechanical testing Multiphase constructs are fabricated and sterilized as described in Example 12. Samples are cut into discs (8 mm diameter, 2 mm thickness) for mechanical testing.

[0326] Mechanical Testing Protocols:

[0327] Compression Testing (Bulk Construct):

[0328] Performed using a universal testing machine.

[0329] Constructs are compressed at a rate of 1 mm / min until 50% deformation was achieved.

[0330] Parameters measured:

[0331] Compressive strength (MPa).

[0332] Compressive modulus (MPa).

[0333] Layer-Specific Tensile Testing:

[0334] Individual layers (cover, hydrogel, electrospun, FDM) are carefully isolated and subjected to uniaxial tensile testing.

[0335] Tensile strength and elastic modulus are recorded for each layer.

[0336] Layer Integration Testing:

[0337] Adhesion strength between layers is evaluated by subjecting samples to shear stress using a custom shear testing setup.

[0338] Dynamic Mechanical Analysis (DMA):

[0339] Performed to assess viscoelastic properties under cyclic loading.

[0340] Constructs are subjected to dynamic loading at 1 Hz frequency to simulate physiological conditions.

[0341] The constructs are expected to demonstrate excellent mechanical properties, with compressive and tensile strengths comparable to native osteochondral tissue. Layer integration testing is expected to confirm strong adhesion between layers, critical for maintaining structural stability under mechanical loading.

[0342] The multiphase construct is expected to exhibit biomechanical properties suitable for loadbearing applications, making it an ideal candidate for osteochondral tissue repair.

[0343] Example 16 - In vivo evaluation of multilayer construct

[0344] The performance of the multiphase construct in promoting osteochondral repair is assessed using a preclinical animal model.

[0345] Animal Model:

[0346] Species: New Zealand White Rabbits (n = 15).

[0347] Defect Model: Full-thickness osteochondral defects (4 mm diameter, 2 mm depth) are created in the femoral condyles under sterile conditions.

[0348] Construct Groups:

[0349] Group 1 : Control (no implant).

[0350] Group 2: Multiphase construct without PRP. Group 3: Multiphase construct with PRP-HA.

[0351] Surgical Procedure:

[0352] Constructs are sterilized using ethylene oxide and rehydrated in sterile PBS before implantation.

[0353] Defects are filled with constructs, ensuring complete coverage of the defect site. Constructs are secured using fibrin glue to maintain their position during healing. Postoperative Care:

[0354] Rabbits are monitored for 12 weeks post-surgery.

[0355] Weight-bearing and mobility are assessed weekly to evaluate functional recovery.

[0356] Evaluation Methods:

[0357] Histological Analysis:

[0358] At 4, 8, and 12 weeks, rabbits are euthanized, and femoral condyles were harvested for histological analysis. Sections are stained with Safranin O / Fast Green to assess cartilage regeneration and integration.

[0359] Micro-CT Imaging:

[0360] Performed at 12 weeks to evaluate bone regeneration and construct degradation.

[0361] Biomechanical Testing:

[0362] Repaired tissue is subjected to indentation testing to measure stiffness and compare it to native cartilage.

[0363] The PRP-HA-enhanced multiphase construct is expected to significantly improved cartilage and bone regeneration compared to the control and non-PRP conditions. The layered design is expected to effectively mimic native osteochondral tissue, supporting cellular growth, matrix deposition, and mechanical functionality. The in vivo study is expected to demonstrate the potential of the multiphase construct for osteochondral repair, particularly when combined with PRP-HA. Its biomimetic properties and clinical-grade fabrication are expected to make it a promising candidate for cartilage repair in humans.

[0364] Example 17 - Controlled Growth Factor Release from the Hydrogel Layer

[0365] The hydrogel layer’s ability to release platelet-derived growth factors (PDGFs) in a sustained manner is evaluated.

[0366] Hydrogel Preparation:

[0367] Hydrogel layer composed of hyaluronic acid (HA, 1 % w / v), gelatin (2% w / v), silk fibroin (3% w / v), and platelet lysate proteins (0.5% v / v).

[0368] Crosslinked with genipin for 24 hours to stabilize the network and regulate protein release. Experimental Setup:

[0369] Hydrogel discs (5 mm diameter, 1 mm thickness) are fabricated and immersed in phosphate- buffered saline (PBS) at 37°C. Samples are transferred to fresh PBS at predefined intervals (1 , 3, 7, 14, and 21 days) to mimic physiological conditions.

[0370] Growth Factor Quantification:

[0371] Growth factors (e.g., PDGF, TGF-pi , VEGF) released into the PBS are quantified using enzyme-linked immunosorbent assays (ELISA). Release profiles are evaluated by plotting cumulative release percentages over time.

[0372] The hydrogel layer of the multiphase construct is expected to exhibit excellent growth factor release properties, delivering bioactive signals in a controlled manner over 21 days. This feature is expected to enhance the construct’s potential for promoting osteochondral regeneration.

[0373] Example 18: Preparation of Silk Fibroin / Hyaluronic Acid Biological Dressing

[0374] Silk fibroin was derived from Bombyx mori cocoons. The cocoons were degummed using a standard boiling method, followed by rinsing and air drying. The dried fibroin was dissolved in formic acid to create a solution with a final concentration of 7.5% w / v. Separately, hyaluronic acid, with a molecular weight of 1550 kDa, was dissolved in formic acid to prepare a solution with a final concentration of 0.5% w / v. The two solutions were combined in a controlled environment and stirred until a homogeneous mixture was achieved, resulting in a final composition of 93.75% silk fibroin and 6.25% hyaluronic acid by weight.

[0375] The mixture was then subjected to electrospinning to fabricate nanofibrous mats. The electrospinning process was conducted under conditions optimized for uniform fiber formation. The setup included a voltage of 15 kV, a flow rate of 1 mL / hour, and a collector distance of 10 cm. The nanofibers were deposited onto a medical-grade support layer, chosen for its compatibility with biological applications and ease of handling during subsequent assays.

[0376] After fabrication, the mats were allowed to dry under ambient conditions before being sterilized. No crosslinking agents were introduced during the process to ensure the bioactivity of the hyaluronic acid was preserved. The resulting dressings were stored under sterile conditions for subsequent testing.

[0377] Example 19: Protocol for Assessing Cell Viability on Silk Fibroin / Hyaluronic Acid Matrix The silk fibroin / hyaluronic acid matrix prepared as described in Example 18 was rehydrated prior to use in cell viability assays. The matrices were immersed in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for one hour at 37°C in a humidified incubator. After rehydration, the matrices were placed into individual wells of a six- well plate with the bioactive layer oriented upwards to maximize cell interaction.

[0378] Normal human dermal fibroblasts (NHDFs) were chosen for the assay. Cells were seeded at a density of 50,000 cells per cm2directly onto the surface of the rehydrated matrices. The culture medium, consisting of DMEM with 10% FBS, was carefully added to ensure complete coverage of the matrix. The seeded matrices were then incubated at 37°C with 5% CO2in a humidified atmosphere for up to seven days. Medium changes were performed every 48 hours to maintain optimal culture conditions.

[0379] At designated time points (e.g., Days 1 , 3, and 7), samples were removed from the incubator, washed gently with phosphate-buffered saline (PBS) to remove non-adherent cells, and prepared for further viability analysis using the Live / Dead assay protocol. This assay involved staining the matrices with calcein-AM to indicate live cells and ethidium homodimer-1 to mark dead cells. Staining solutions were prepared according to the manufacturer’s instructions, and the matrices were incubated with the staining solution for 30 minutes at room temperature in the dark.

[0380] The stained matrices were then visualized using a fluorescence microscope to capture images for subsequent analysis. This protocol ensures a standardized and reproducible method for assessing the ability of the matrix to support cell viability.

[0381] Example 20: Protocol for Rehydration of the HA-Silk Matrix

[0382] The silk fibroin / hyaluronic acid matrix prepared as described in Example 18 was rehydrated to render it suitable for biological assays. Square samples measuring 1 cm2were cut from the electrospun mats under sterile conditions. These samples were immersed in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere containing 5% CO2for one hour.

[0383] During the incubation, the matrices absorbed the medium, transitioning from their initial dry and rigid state to a hydrated and flexible form suitable for cell seeding. The bioactive side of the matrix was ensured to face upward throughout the rehydration process to maximize exposure for subsequent biological interaction. The rehydrated matrices were then transferred to sterile six-well plates using forceps, taking care to avoid physical damage to the delicate fibers.

[0384] This rehydration protocol provides a standardized and reproducible method for preparing the HA-silk fibroin matrix for use in cell-based assays or other biological applications.

[0385] Example 21 : Stability Evaluation of the HA-Silk Matrix in Culture Conditions

[0386] The stability of the HA-silk fibroin matrix was assessed under various culture media conditions to evaluate its physical integrity over time. Samples of the matrix were prepared as described in Example 18, rehydrated as outlined in Example 20, and placed into six-well plates. Each sample was exposed to one of the following conditions: (1) Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS (control), (2) DMEM supplemented with platelet-rich plasma (PRP), and (3) DMEM supplemented with PRP and hyaluronic acid (PRP-HA). The matrices were incubated at 37°C in a humidified atmosphere with 5% CO2. Observations of swelling, structural integrity, and detachment from the support layer were recorded at Days 1 , 3, 7, and 15. The matrices were gently rinsed with phosphate-buffered saline (PBS) before visual assessment to remove any unattached cells or debris.

[0387] The evaluation included documenting physical changes such as swelling ratios, fiber morphology, and any delamination of the matrix under the tested conditions. This protocol provides insight into the suitability of the HA-silk fibroin matrix for extended culture periods in different bioactive environments.

[0388] Example 22: Preparation of Platelet Lysate-Supplemented HA-Silk Matrix

[0389] The silk fibroin / hyaluronic acid matrix was supplemented with platelet lysate (PL) to enhance its bioactive properties. Platelet lysate was prepared from commercially available sources at two concentrations: 0.25% and 0.5% (v / v). The PL was mixed with DM EM supplemented with 10% FBS to create the experimental media.

[0390] The HA-silk fibroin matrix samples, prepared as described in Example 18, were rehydrated with these PL-supplemented media under sterile conditions. The matrices were immersed in the PL-enriched medium for one hour at 37°C in a humidified atmosphere with 5% CO2. Following rehydration, the matrices were placed into six-well plates with the bioactive side oriented upwards.

[0391] This preparation method enables the investigation of the enhanced bioactivity of the matrix through the incorporation of platelet lysate while maintaining the integrity of the silk fibroin and hyaluronic acid components.

[0392] Example 23: Standard Preparation of Silk Fibroin Solution from Bombyx mori Cocoons Silk fibroin was extracted from Bombyx mori cocoons using a standard degumming and dissolution process. Initially, 5 g of cocoons were cut into small pieces and subjected to a boiling degumming step in 2 L of a 0.02 M sodium carbonate solution for 30 minutes. This step effectively removed sericin while preserving the fibroin fibers. The degummed silk fibers were rinsed three times in 1 L of deionized water for 20 minutes each rinse and then squeezed to remove excess water. The fibers were dried overnight in a fume hood and stored at room temperature for further use.

[0393] For dissolution, the dried silk fibroin fibers were dissolved in a 9.3 M lithium bromide solution at a 20% (w / v) concentration. This mixture was heated to 60°C and stirred for 4 hours until a clear and transparent silk fibroin solution was obtained. The solution was then desalted via dialysis against deionized water using a 12-14 kDa molecular weight cut-off membrane. The dialysis process involved renewing the water six times over a period of 48 hours. Following dialysis, the fibroin solution was centrifuged at 9,000 rpm (12,700 g) for 20 minutes at 4°C, repeated three times, to remove any insoluble particles.

[0394] The resulting silk fibroin solution, with a concentration of 7-8% (w / v), was stored at 4°C for further use in scaffold preparation. This process reliably yields high-quality silk fibroin suitable for applications in biomaterial research and tissue engineering.

[0395] Example 24: Microwave-Assisted Preparation of Silk Fibroin Solution

[0396] Silk fibroin was extracted from Bombyx mori cocoons using a microwave-assisted degumming process. Two grams of cocoons were cut into small pieces and subjected to three cycles of microwave-assisted degumming. Each cycle involved immersing the cocoon pieces in a 0.02 M sodium carbonate solution containing 0.25% sodium dodecyl sulfate (SDS) and microwaving the solution for 1 minute at 800 W, followed by a 10-minute resting period (approximate temperature: 80°C). After each cycle, the fibers were rinsed in deionized water for 1 minute and then squeezed to remove excess liquid. Following three cycles, the degummed fibers were either dried overnight or used directly for dissolution.

[0397] For dissolution, 0.1 g of degummed silk fibers was submerged in 10 mL of 56% (w / w) aqueous zinc chloride (ZnCI2) solution at 45°C for 1 hour. The resulting 1% (w / v) silk fibroin solution was then subjected to desalting using a CentriPure P50 column to remove zinc chloride. The process was repeated to ensure the complete elimination of ZnCI2, verified using a zinc test kit measuring absorbance at 560 nm. The eluted fibroin solution was either used immediately or dried via lyophilization for storage.

[0398] This microwave-assisted method provides a rapid and reliable process for producing silk fibroin solutions suitable for a range of biomedical applications.

[0399] Example 25: Preparation of Platelet Lysate-Supplemented HA-Silk Matrix

[0400] Platelet lysate (PL) was incorporated into the silk fibroin / HA matrix at concentrations of 0.25% and 0.5% (v / v). Platelet lysate was prepared from commercially available PLT MAX kits. The silk fibroin / HA blend (7.5% w / v silk fibroin and 0.5% w / v HA) was rehydrated in DMEM supplemented with the respective PL concentrations. Rehydration was performed at 37°C for one hour, ensuring the matrix absorbed the PL-supplemented medium fully.

[0401] The rehydrated platelet lysate-supplemented HA-silk matrices were subsequently used in cell viability and proliferation assays to assess their bioactivity.

[0402] Example 26: Preparation of PRP and PRP-HA

[0403] Peripheral blood samples were collected from healthy donors following ethical approval (CCER Geneva ethics committee, ID 2017-00700). A total of 30 mL of blood was collected into specific medical devices, including CuteCell PRP and CellularMatrix ACP-HA tubes from Regen Lab. Blood samples were centrifuged at 1500 g for 5 minutes at room temperature. After centrifugation, red and white blood cells settled below the separator gel, leaving plasma and platelets in the upper layer.

[0404] The plasma and platelets were homogenized by inverting the tube five times. Each CuteCell PRP tube yielded 6 mL of PRP, while each CellularMatrix ACP-HA tube provided 3 mL of PRP mixed with 2 mL of HA. Platelet, red blood cell, and white blood cell concentrations, as well as mean platelet volume (MPV), were confirmed to align with previously reported ranges in the literature.

[0405] The prepared PRP and PRP-HA solutions were stored in polypropylene tubes until use in biological assays.

[0406] Example 27: Protocol for Cell Viability Assessment on Silk Fibroin / Hyaluronic Acid Biological Dressings

[0407] Silk fibroin / hyaluronic acid biological dressings were fabricated as described in prior examples, incorporating platelet lysates at concentrations of 0.25% (v / v) and 0.5% (v / v). These dressings were prepared without a supporting membrane and sterilized before use. The cell attachment side of the dressings was uniform due to the homogeneous mixture of silk fibroin and hyaluronic acid during fabrication.

[0408] The cell viability assessment was conducted using periodontal ligament cells (PDL, passage 3), gingival fibroblasts (GF, passage 3), and myoblasts (passage 3). Cells were harvested through an implant technique and preserved in liquid nitrogen prior to the experiment. The Live / Dead assay kit was used to assess cell viability at four time points: 1 , 2, 3, and 4 weeks. For the assay, biological dressings were placed into a p-Plate 24 well black plate (uncoated) at one dressing per well. Each dressing was rehydrated with 500 pL of DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C for 30 minutes. After rehydration, the medium was aspirated, ensuring the dressings remained wet. A suspension containing 150,000 cells in 500 pL of medium was seeded onto each dressing, with the drop deposited centrally to ensure uniform spreading. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2to allow for cell attachment.

[0409] At each time point, Live / Dead staining was performed by incubating the dressings with the staining solution according to the manufacturer’s instructions. The stained samples were visualized using a Cytation 7 confocal microscope. Additionally, samples were fixed for scanning electron microscopy (SEM) to observe matrix integrity and degradation over time. Quantification of green and red signals was performed using image analysis software, and matrix degradation was assessed visually and through SEM. Example 28: Evaluation of HUVEC Cell Viability and Attachment on Silk Fibroin / Hyaluronic Acid Nanofibers

[0410] The silk fibroin / hyaluronic acid nanofibers were prepared as described in earlier examples and evaluated for cell viability and attachment using human umbilical vein endothelial cells (HLIVECs, P5). Four types of supportive layers were tested, each coated with the SF-HA nanofibers: LDPE / Cellulose with nanofibers on the cellulose side, LDPE / Cellulose with nanofibers on the LDPE side, Polythene Paper with nanofibers on the polythene side, and Polythene Paper with nanofibers on the paper side.

[0411] Each sample was exposed to five different culture conditions: control medium, PRP at 10% concentration, PRP-HA at 10% (w / v) concentration, and PL at 10% (v / v) concentration. The dressings were visualized using Cytation imaging technology, with the samples placed upside down to evaluate cell interaction with the nanofibers.

[0412] Sample 1 , consisting of LDPE / Cellulose with nanofibers on the cellulose side, floated in the medium due to foam properties of the support layer and did not support endothelial cell adhesion under any conditions. Similarly, Sample 2, consisting of LDPE / Cellulose with nanofibers on the LDPE side, exhibited floating behavior and no significant endothelial cell adhesion was observed. In contrast, Sample 3, consisting of Polythene Paper with nanofibers on the polythene side, supported endothelial cell adhesion and viability when cultured with PRP, PRP-HA, and PL. However, no significant cell adhesion was observed under control medium conditions. Sample 4, consisting of Polythene Paper with nanofibers on the paper side, demonstrated strong endothelial cell attachment and viability under PRP, PRP-HA, and PL conditions, and sheets of cells remained adhered after peelability testing.

[0413] In conclusion, Samples 3 and 4 provided optimal conditions for endothelial cell attachment and viability, particularly in the presence of PRP, PRP-HA, and PL. Samples 1 and 2 were unsuitable due to their floating behavior and lack of cell adherence.

[0414] Example 29: Evaluation of Silk Fibroin / Hyaluronic Acid Matrices with Vaginal Fibroblasts and Periodontal Ligament Cells

[0415] Silk fibroin and hyaluronic acid matrices were prepared as previously described. These matrices were tested for cell viability, adhesion, and proliferation using vaginal fibroblasts (VF) and periodontal ligament cells (PDL). The experiments included multiple culture conditions: control medium, platelet-rich plasma (PRP) at 10%, and PRP supplemented with hyaluronic acid (PRP-HA) at 10% (w / v).

[0416] For the assessment with vaginal fibroblasts, 1 cm2fragments of the SF-HA matrices were placed into tissue culture dishes. Cells were seeded at a density of 100,000 per matrix and cultured under the specified conditions. After 15 days of culture, cell viability was assessed using a Live / Dead Kit. Calcein fluorescence identified live cells, and ethidium homodimer-1 fluorescence detected dead cells. Microscopic observations revealed robust adhesion and proliferation of vaginal fibroblasts in the PRP-HA 10% condition, with significant elongation and alignment along the matrix fibers. Minimal dead cells were observed across all conditions. However, the control condition exhibited less proliferation and no substantial elongation of fibroblasts. The PRP 10% (w / v) condition supported moderate proliferation and elongation. Similar experiments were conducted using periodontal ligament cells. Here, the SF-HA matrices were evaluated under the same conditions. Observations after 15 days indicated strong adhesion and proliferation in the PRP-HA 10% (w / v) condition, with well-defined cellular elongation and alignment. In contrast, the control condition showed sparse cellular adhesion, and the PRP 10% condition demonstrated moderate cellular proliferation without significant elongation. It was noted that the PRP-HA condition was particularly effective in maintaining the structural integrity of the HA layer within the matrix, as evidenced by consistent cell coverage and no detachment of the electrospun HA layer.

[0417] The data collectively highlighted that PRP-HA at 10% (w / v) provided the optimal environment for cell adhesion, proliferation, and alignment for both vaginal fibroblasts and periodontal ligament cells. This underscores the bioactive potential of the SF-HA matrix when supplemented with PRP and HA.

[0418] Example 30: Assessment of Myoblast Differentiation on Silk Fibroin / Hyaluronic Acid Biological Dressings Supplemented with Platelet Lysates and Platelet-Rich Plasma The silk fibroin / hyaluronic acid biological dressings (SF-HA) were prepared as previously described and evaluated for their ability to support myoblast differentiation under various culture conditions. The study compared the differentiation outcomes using platelet lysates (PL) at 10% (v / v), platelet-rich plasma (PRP) at 10% (w / v), and PRP combined with hyaluronic acid (HA-PRP) at 10% (w / v), with standard differentiation medium (DM) serving as a reference control. Primary human myoblasts (P5) were seeded onto the SF-HA matrices and cultured for three weeks under these conditions.

[0419] Experimental Setup

[0420] The matrices were first rehydrated in Dulbecco’s Modified Eagle Medium (DM EM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified CO2incubator for 30 minutes. After rehydration, 150,000 myoblasts suspended in 500 pL of medium were seeded onto each dressing and allowed to attach for 30 minutes. Subsequently, 500 pL of the respective treatment media were added, including DM, PL 10% (v / v), PRP 10% (w / v), and HA-PRP 10% (w / v).

[0421] Results at Three Weeks At the three-week time point, samples were fixed in 4% paraformaldehyde for immunofluorescence staining and confocal microscopy. Immunostaining was performed using markers specific for myoblast differentiation, such as Mef2C and alpha-actinin.

[0422] 1. HA-PRP 10% (w / v): The most robust differentiation outcomes were observed in the HA-PRP group. Confocal microscopy revealed extensive formation of aligned, multinucleated myotubes with excellent structural integrity. Myotubes exhibited significant elongation and alignment along the SF-HA matrix, reflecting enhanced myogenic differentiation. The presence of HA appeared to enhance PRP's bioactivity, providing a synergistic effect that maximized differentiation capacity.

[0423] 2. PRP 10% (w / v): Samples treated with PRP alone demonstrated strong differentiation, with clear evidence of myotube formation and alignment. However, the extent of elongation and structural organization was slightly reduced compared to the HA-PRP 10% condition, indicating that while PRP supports myogenesis effectively, the absence of HA's structural benefits may limit optimal differentiation.

[0424] 3. PL 10% (v / v): Differentiation was evident but significantly less effective than in the PRP and HA-PRP conditions. Myotubes were less organized and exhibited reduced structural integrity. The observed limitations of PL can be attributed to the use of formic acid during its preparation. Formic acid likely compromised the bioactivity of growth factors in PL through protein denaturation, pH instability, and peptide bond hydrolysis, as previously described.

[0425] 4. Differentiation Medium (DM): The control condition exhibited the lowest differentiation capacity. Confocal images showed sparse and poorly aligned myotubes, confirming that SF-HA matrices alone are insufficient to promote effective myogenesis without supplemental bioactive factors.

[0426] Conclusion

[0427] This study demonstrates that the HA-PRP 10% (w / v) treatment provides the most effective environment for myoblast differentiation, significantly outperforming PRP 10% (w / v), PL 10% (v / v), and control differentiation medium. The combination of HA and PRP enhances myoblast alignment, elongation, and myotube formation by creating a synergistic microenvironment that supports cellular attachment and differentiation. By contrast, the use of platelet lysates prepared with formic acid is shown to diminish differentiation outcomes, underscoring the importance of preserving growth factor bioactivity in preparation processes. These findings establish HA-PRP 10% (w / v) as a superior treatment for myoblast differentiation and highlight the need to optimize platelet lysate preparation to avoid bioactivity loss.

Claims

CLAIMS1. A scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein.

2. The scaffold material according to claim 1 , further comprising hyaluronic acid.

3. The scaffold material according to claim 1 or claim 2, further comprising an antifibrinolytic substance, such as tranexamic acid.

4. The scaffold material according to any one of claims 1 to 3, further comprising a coagulation activator, such as calcium gluconate.

5. The scaffold material according to any one of claims 1 to 4, further comprising vitamin K2.

6. The scaffold material according to any one of claims 1 to 5, further comprising one or more therapeutic agents, such as selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

7. The scaffold material according to any one of claims 1 to 6, which is a semi-solid scaffold material.

8. The scaffold material according to any one of claims 1 to 7, further comprising hydroxyapatite.

9. The scaffold material according to claim 8, which is a solid scaffold material.

10. A scaffold material comprising: i) hydroxyapatite; and ii) electrospun fibres comprising silk protein.

11. The scaffold material according to claim 10, further comprising platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate.

12. The scaffold material according to claim 10 or claim 11 , further comprising hyaluronic acid.

13. The scaffold material according to any one of claims 10 to 12, further comprising an antifibrinolytic substance, such as tranexamic acid.

14. The scaffold material according to any one of claims 10 to 13, further comprising a coagulation activator, such as calcium gluconate.

15. The scaffold material according to any one of claims 10 to 14, further comprising vitamin K2.

16. The scaffold material according to any one of claims 10 to 15, further comprising one or more therapeutic agents, such as selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

17. The scaffold material according to any one of claims 10 to 16, which is a solid scaffold material.

18. A scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibres comprising silk protein.

19. The scaffold material according to claim 18, which has been stabilized by crosslinking, e.g. using an aldehyde-based crosslinking agent such as glutaraldehyde (e.g. vapour phase).

20. The scaffold material according to claim 18 or claim 19, wherein the weight ratio of hyaluronic acid to electrospun fibres comprising silk protein is between about 1 :2 and about 1 :4 e.g. about 1 :3.

21. The scaffold material according to any one of claims 18 to 20, further comprising platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate.

22. The scaffold material according to any one of claims 18 to 21 , further comprising hydroxyapatite.

23. The scaffold material according to any one of claims 18 to 22, further comprising an antifibrinolytic substance, such as tranexamic acid.

24. The scaffold material according to any one of claims 18 to 23, further comprising a coagulation activator, such as calcium gluconate.

25. The scaffold material according to any one of claims 18 to 24, further comprising vitamin K2.

26. The scaffold material according to any one of claims 18 to 25, further comprising one or more therapeutic agents, such as selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selectedfrom the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

27. A layered wound dressing or layered graft comprising: i) a scaffold material as defined in any one of claims 1 to 26; and ii) a support material.

28. A layered wound dressing or layered graft according to claim 27, comprising: i) a scaffold material comprising: ia) electrospun fibres comprising silk protein; and ii) hyaluronic acid; and ii) a support material.

29. A layered construct for tissue engineering applications, comprising: i) a first layer comprising silk protein; ii) a second layer comprising a hydrogel and silk protein; iii) a third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) a fourth layer comprising polycaprolactone and hydroxyapatite.

30. The layered construct according to claim 29, wherein the first layer comprising silk protein has a thickness between about 40 pm and about 60 pm, e.g. about 50 pm.

31. The layered construct according to claim 29 or claim 30, wherein the first layer comprising silk protein is a cover layer.

32. The layered construct according to any one of claims 29 to 31 , wherein the silk protein in the first layer is composed of electrospun fibres of silk protein.

33. The layered construct according to claim 32, wherein the electrospun fibres of silk protein are prepared by electrospinning silk fibroin solution at a concentration between about 5% (w / v) and 15% (w / v) e.g. about 7% (w / v); under a voltage of 12-18 kV e.g. about 15 kV; at a flow rate of between about 0.8 mL / hour to about 1.2 mL / hour, e.g. at 0.8 mL / hour; and a collector distance of 5-20 cm, e.g. about 10 cm.

34. The layered construct according to any one of claims 29 to 33, wherein the hydrogel of the second layer comprises hyaluronic acid and / or gelatin.

35. The layered construct according to claim 34, wherein the hyaluronic acid is a mixture of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid (e.g. in a weight ratio of about 1 :1).

36. The layered construct according to any one of claims 29 to 35, wherein the silk protein in the second layer is composed of electrospun fibres of silk protein.

37. The layered construct according to claim 36, wherein the electrospun fibres of silk protein are prepared by electrospinning silk fibroin solution at a concentration between about 5% (w / v) and 15% (w / v) e.g. about 7% (w / v); under a voltage of 12-18 kV e.g. about 15 kV; at a flow rate of between about 0.8 mL / hour to about 1.2 mL / hour; e.g. at 0.8 mL / hour, and a collector distance of 5-20 cm, e.g. about 10 cm.

38. The layered construct according to any one of claims 29 to 37, wherein the second layer has thickness of 1-2 mm.

39. The layered construct according to any one of claims 29 to 38, wherein the second layer further comprises platelet-rich plasma, platelet lysate and / or bone marrow concentrate, and in particular further comprises platelet lysate.

40. The layered construct according to any one of claims 29 to 39, wherein the third layer comprises polycaprolactone and hydroxyapatite, and is a layer formed by electrospinning.

41. The layered construct according to any one of claims 29 to 40, wherein the third layer comprises hyaluronic acid and hydroxyapatite, and is a layer formed by electrospinning.

42. The layered construct according to claim 40, wherein the third layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 80:20.

43. The layered construct according to any one of claims 29 to 42, wherein the third layer has thickness of 90 pm to 110 pm.

44. The layered construct according to any one of claims 29 to 43, wherein the fourth layer comprising polycaprolactone and hydroxyapatite is formed using fused deposition modelling (FDM).

45. The layered construct according to any one of claims 29 to 44, wherein the fourth layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 70:30.

46. The layered construct according to any one of claims 29 to 45, wherein the fourth layer has thickness of 450 pm to 550 pm, e.g. about 500 pm.

47. The scaffold material, the layered wound dressing, the layered graft or the layered construct according to any one of claims 1 to 46, wherein the silk protein is silk fibroin, which is suitably obtained from a solution containing dissolved silkworm silk, such as Bombyx mori.

48. The scaffold material, the layered wound dressing, the layered graft or the layered construct according to any one of claims 1 to 47, wherein the electrospun fibres have diameter between about 50 nm and about 1 ,000 nm, such as between about 200 nm and about 500 nm.

49. The scaffold material according to any one of claims 1 to 26, claim 47 or claim 48, in the form of a film, a membrane, a mat, or a mesh.

50. The scaffold material according to any one of claims 1 to 26, or claims 47-49, which has a swelling ratio of at least 300%.

51. A tissue engineered construct comprising: i) a scaffold material, the layered wound dressing, the layered graft or the layered construct according to any one of claims 1 to 50; and ii) mammalian cells.

52. The tissue engineered construct according to claim 51 , wherein the mammalian cells comprise cells selected from the group consisting of hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal- hypothalamic- pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smoothmuscle cells, skeletal muscle cells, oscular cells, integumentary cells, bone marrow cells, keratinocytes, pluripotent cells and stem cells and combinations thereof.

53. A method for preparing a tissue engineered construct comprising the steps of: a) preparing a scaffold material according to any one of claims 1 to 50; b) combining the scaffold material with mammalian cells in a suitable medium.

54. A method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

55. The method according to claim 54, further comprising a step after step a) but before step b) of adding hyaluronic acid to the silk protein solution.

56. The method according to claim 54 or claim 55, wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with hyaluronic acid, to form a scaffold material.

57. The method according to any one of claims 54 to 56, further comprising a step after step a) but before step b) of adding an antifibrinolytic substance (such as tranexamic acid) to the silk protein solution.

58. The method according to any one of claims 54 to 57, wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with an antifibrinolytic substance (such as tranexamic acid), to form a scaffold material.

59. The method according to any one of claims 54 to 58, further comprising a step after step a) but before step b) of adding a coagulation activator (such as calcium gluconate) to the silk protein solution.

60. The method according to any one of claims 54 to 59, wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysateand / or bone marrow concentrate, and with a coagulation activator (such as calcium gluconate), to form a scaffold material.

61. The method according to any one of claims 54 to 60, further comprising a step after step a) but before step b) of adding vitamin K2 to the silk protein solution.

62. The method according to any one of claims 54 to 61 , wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with vitamin K2, to form a scaffold material.

63. The method according to any one of claims 54 to 62, further comprising a step after step a) but before step b) of adding a further therapeutic agent to the silk protein solution.

64. The method according to any one of claims 54 to 63, wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with one or more further therapeutic agents, to form a scaffold material.

65. The method according to claim 63 or claim 64, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a non-steroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti- melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

66. The method according to any one of claims 54 to 65, further comprising a step after step a) but before step b) of adding hydroxyapatite to the silk protein solution.

67. The method according to any one of claims 54 to 66, wherein in step c) the electrospun fibres comprising silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with hydroxyapatite, to form a scaffold material.

68. A method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

69. The method according to claim 68, wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with hyaluronic acid, to form a mixture.

70. The method according to claim 68 or claim 69, wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with an antifibrinolytic substance (such as tranexamic acid), to form a mixture.

71. The method according to claim any one of claims 68 to 70, wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with a coagulation activator (such as calcium gluconate), to form a mixture.

72. The method according to claim any one of claims 68 to 71 , wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with vitamin K2, to form a mixture.

73. The method according to any one of claims 68 to 72, wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with one or more further therapeutic agents, to form a mixture.

74. The method according to claim 73, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterialcompound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

75. The method according to any one of claims 68 to 74, wherein in step b) the silk protein mixture of step a) is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with hydroxyapatite, to form a mixture.

76. The method according to any one of claims 68 to 75, further comprising step d) adding hyaluronic acid to the scaffold material of step c).

77. The method according to any one of claims 68 to 76, further comprising step d) adding an antifibrinolytic substance (such as tranexamic acid) to the scaffold material of step c).

78. The method according to any one of claims 68 to 77, further comprising step d) adding a coagulation activator (such as calcium gluconate) to the scaffold material of step c).

79. The method according to any one of claims 68 to 78, further comprising step d) adding vitamin K2 to the scaffold material of step c).

80. The method according to any one of claims 68 to 79, further comprising step d) adding one or more further therapeutic agents to the scaffold material of step c).

81. The method according to claim 80, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, animmunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

82. The method according to any one of claims 68 to 81 , further comprising step d) adding hydroxyapatite to the scaffold material of step c).

83. A method for preparing a scaffold material comprising i) hydroxyapatite, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hydroxyapatite, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

84. The method according to claim 83, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, to form a mixture.

85. The method according to claim 83 or claim 84, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and hyaluronic acid, to form a mixture.

86. The method according to any one of claims 83 to 85, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and an antifibrinolytic substance (such as tranexamic acid), to form a mixture.

87. The method according to any one of claims 83 to 86, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and a coagulation activator (such as calcium gluconate), to form a mixture.

88. The method according to any one of claims 83 to 87, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and vitamin K2, to form a mixture.

89. The method according to any one of claims 83 to 88, wherein in step b) the silk protein solution of step a) is combined with hydroxyapatite and one or more further therapeutic agents, to form a mixture.

90. The method according to claim 89, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

91. The method according to any one of claims 83 to 90, further comprising step d) adding platelet-rich plasma and / or bone marrow concentrate to the scaffold material of step c).

92. The method according to any one of claims 83 to 91 , further comprising step d) adding hyaluronic acid to the scaffold material of step c).

93. The method according to any one of claims 83 to 92, further comprising step d) adding an antifibrinolytic substance (such as tranexamic acid) to the scaffold material of step c).

94. The method according to any one of claims 83 to 93, further comprising step d) adding a coagulation activator (such as calcium gluconate) to the scaffold material of step c).

95. The method according to any one of claims 83 to 94, further comprising step d) adding vitamin K2 to the scaffold material of step c).

96. The method according to any one of claims 83 to 95, further comprising step d) adding one or more further therapeutic agents to the scaffold material of step c).

97. The method according to claim 96, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, anenzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

98. A method for preparing a scaffold material comprising i) hydroxyapatite, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibres comprising silk protein; c) combining the electrospun fibres comprising silk protein with hydroxyapatite to form a scaffold material.

99. The method according to claim 98, further comprising step d) adding platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to the scaffold material of step c).

100. The method according to claim 98 or claim 99, further comprising step d) adding hyaluronic acid to the scaffold material of step c).

101. The method according to any one of claims 98 to 100, further comprising step d) adding an antifibrinolytic substance (such as tranexamic acid) to the scaffold material of step c).

102. The method according to any one of claims 98 to 101 , further comprising step d) adding a coagulation activator (such as calcium gluconate) to the scaffold material of step c).

103. The method according to any one of claims 98 to 102, further comprising step d) adding vitamin K2 to the scaffold material of step c).

104. The method according to any one of claims 98 to 103, further comprising step d) adding one or more further therapeutic agents to the scaffold material of step c).

105. The method according to claim 104, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

106. A method for preparing a scaffold material comprising i) hyaluronic acid, and ii) electrospun fibres comprising silk protein; comprising the steps of: a) preparing a silk protein solution; b) combining the silk protein solution of step a) with hyaluronic acid, to form a mixture; c) electrospinning the mixture of step b) to form a scaffold material.

107. The method according to claim 106, wherein the weight ratio of hyaluronic acid to silk protein in step b) is between about 1 :2 and about 1 :4 e.g. about 1 :3.

108. The method according to claim 106 or claim 107, wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, to form a mixture.

109. The method according to any one of claims 106 to 108, wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and hydroxyapatite, to form a mixture.

110. The method according to any one of claims 106 to 109, wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and an antifibrinolytic substance (such as tranexamic acid), to form a mixture.

111. The method according to any one of claims 106 to 110, wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and a coagulation activator (such as calcium gluconate), to form a mixture.

112. The method according to any one of claims 106 to 111 , wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and vitamin K2, to form a mixture.

113. The method according to any one of claims 106 to 112, wherein in step b) the silk protein solution of step a) is combined with hyaluronic acid and one or more further therapeutic agents, to form a mixture.

114. The method according to claim 113, wherein the one or more therapeutic agents are selected from the group consisting of a steroid, a corticosteroid, a glucocorticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), kartogenin, an anaesthetic, and antibacterial compound, an antibiotic, an antifungal compound, an antiparasitic compound, an enzyme, an enzyme inhibitor, a glycoprotein, a growth factor, a hormone, an antiviral compound, an analgesic, an opioid, a saponin, a haemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), an anti-angiogenetic agent, anti-melanogenetic agent, an immunomodulator, an immunoglobulin, a mineral, a neuroleptic, a protein, a peptide, a lipoprotein, a tumouricidal compound, a tumourstatic compound, a toxin, a vitamin (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or a derivative thereof) or a wrinkle filler, or a combination thereof, in particular a further therapeutic agent is selected from the group consisting of a corticosteroid, an NSAID, kartogenin, a haemoglobin, an anaesthetic, an analgesic, an opioid, a saponin and THC, or a combination thereof.

115. The method according to any one of claims 106 to 114, further comprising step d) cross-linking the scaffold material e.g. by reacting it with an aldehyde-based crosslinking agent such as glutaraldehyde (e.g. vapour phase).

116. The method according to any one of claims 106 to 115, further comprising step d) adding platelet-rich plasma and / or bone marrow concentrate to the scaffold material of step c).

117. The method according to any one of claims 54 to 116, further comprising the step of combining the mixture / scaffold material with mammalian cells to form a tissue engineered construct.

118. The method according to claim 117, wherein the mammalian cells comprise cells selected from the group consisting of hepatocytes, pancreatic Islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal- hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, smooth muscle cells, skeletal muscle cells, oscular cells, integumentary cells, bone marrow cells, keratinocytes, pluripotent cells and stem cells and combinations thereof.

119. The method according to any one of claims 54 to 118, wherein the electrospinning is carried out using a voltage of between about 12 kV and about 18 kV.

120. The method according to any one of claims 54 to 119, wherein the electrospinning is carried out at a flow rate of between about 0.8 mL / hour to about 1 .2 mL per hour.

121. The method according to any one of claims 54 to 120, wherein the electrospinning is carried out using a collector distance of between about 8 cm and about 12 cm.

122. The scaffold material, the layered wound dressing, the layered graft, the layered construct or the tissue engineered construct according to any one of claims 1 to 52, for use in therapy.

123. The scaffold material, the layered wound dressing, the layered graft, the layered construct or tissue engineered construct according to claim 122, for use in wound healing, wound sealing, tissue repair, tissue regeneration, cartilage repair, cartilage regeneration, tendon repair, tendon regeneration, nerve repair, nerve regeneration, bone repair and / or bone regeneration.

124. The scaffold material, the layered wound dressing, the layered graft, the layered construct or tissue engineered construct according to claim 122, for use in treating or preventing a joint disorder or condition.

125. The scaffold material, the layered wound dressing, the layered graft, the layered construct or tissue engineered construct for use according to claim 124, wherein the joint disorder or condition is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia and rheumatica.

126. The scaffold material, the layered wound dressing, the layered graft, the layered construct or tissue engineered construct for use according to claim 123, wherein wound healing and tissue repair include the treatment of gynaecological conditions (e.g. pelvic organ prolapse), bowel injuries, bladder injuries, vascular injuries, other non-healing wounds, burns, and diabetic foot.

127. The scaffold material, the layered wound dressing, the layered graft, the layered construct or tissue engineered construct according to claim 122, for use in tissue augmentation.

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