Silk protein biomaterials

Abstract

The present invention relates to a variety of scaffolds, including a scaffold consisting of: i) platelet rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers consisting of silk fibroin.

Description

Technical Field

[0001] This invention relates to scaffold materials comprising silk proteins. Such scaffold materials can be used as wound dressings and grafts. These scaffold materials can also be used in tissue engineering applications, particularly as scaffolds for cell growth. The invention also describes layered wound dressings, layered grafts, and layered constructs comprising silk proteins and hyaluronic acid. Background Technology

[0002] Silk protein is a protein found in the silk produced by various insects, including the larvae of the silkworm moth (Bombyx mori). Silkworm larvae enclose themselves in cocoons made of raw silk, which primarily contains two proteins: sericin and fibroin. Fibroin is a fibrous protein that forms the core of the raw silk fiber; while sericin forms a gel-like coating on the fiber surface, allowing the fibers to bond together.

[0003] Silk fibroin is biocompatible and can be used as a wound hemostatic agent and in various cosmetic fields. Furthermore, it is soluble in aqueous solutions and can be easily reconstructed into different forms of materials such as films, meshes, hydrogels, and sponges through preparation techniques such as spin coating, electrospinning, freeze drying, and physical and chemical crosslinking.

[0004] Electrospinning is a method of preparing fibers by stretching charged polymer solution torsions using an electric field, capable of producing fibers with diameters down to the nanometer scale. Broadly speaking, an electrospinning apparatus consists of a power source, a reservoir containing the solution (such as a syringe with a blunt needle at the tip), a pump, and a collector. An electric field is established between the needle and the collector by applying a specific voltage. The pump causes the solution to flow at a constant rate, and charges accumulate on the liquid surface. When the electrostatic repulsion exceeds the surface tension of the liquid, the liquid jets out from the surface. The jet point is called a Taylor cone. After the Taylor cone forms, the charged liquid jet is ejected towards the collector. Because the surface area of ​​the jet is much larger than its volume, the solvent evaporates efficiently; simultaneously, as the volume decreases, the charge density increases, causing the liquid jet to split and form solid fibers. A whipping motion occurs between the Taylor cone and the collector, depositing a nonwoven fiber pad on the collector.

[0005] These reconstructed silk fibroin materials possess unique structural properties that enable their use as wound dressings and grafts (such as vascular grafts), as well as in tissue engineering where they can serve as scaffold materials to support cell regeneration and growth.

[0006] EP1408146A1 discloses a nonwoven fabric composed of silk fibroin, and a method for preparing the nonwoven fabric by dissolving the silk fibroin in hexafluoroacetone and then electrospinning it. The resulting material is claimed to be usable as a medical material.

[0007] EP2465472A2 describes a method for preparing silk nanofibers by electrospinning a fiber spinning solution onto a polyethylene glycol-coated collector. The resulting silk fibers are in the shape of conduits and can be used as nerve conduits for the treatment of nerve injuries.

[0008] WO2004 / 000915A2 describes an all-aqueous process for producing silk biomaterials. In this process, at least one biocompatible polymer (e.g., polyethylene glycol) is mixed with silk fibroin and then processed by methods such as electrospinning. The prepared biomaterials are claimed to be used to construct tissue-engineered structures.

[0009] Although various scaffold materials made from silk fibroin are known, the object of this invention is to prepare novel scaffold materials with enhanced functional properties and potential therapeutic benefits. Another object of this invention is to prepare wound dressings and grafts with enhanced functional properties and potential therapeutic benefits. Summary of the Invention

[0010] According to one aspect of the invention, a scaffold material is provided comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers comprising silk proteins.

[0011] According to another aspect of the invention, a scaffold material is provided comprising: i) hydroxyapatite; and ii) electrospun fibers comprising silk protein.

[0012] According to another aspect of the invention, a scaffold material is provided comprising: i) hyaluronic acid; and ii) electrospun fibers comprising silk protein.

[0013] According to another aspect of the invention, a layered wound dressing or layered graft is provided, comprising: i) The support material as described above; and ii) A support material.

[0014] According to another aspect of the invention, a layered structure for tissue engineering applications is provided, comprising: i) The first layer containing silk proteins; ii) The second layer, which contains hydrogel and silk protein; iii) The third layer comprises polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) The fourth layer, which contains polycaprolactone and hydroxyapatite.

[0015] According to another aspect of the invention, a tissue-engineered construct is provided, comprising a scaffold material, a layered wound dressing, a layered graft or layered construct as described herein, and mammalian cells.

[0016] According to another aspect of the present invention, a method for preparing tissue-engineered constructs is provided, comprising the following steps: a) Preparation of the scaffold material described herein; b) Mix the scaffold material with mammalian cells in a suitable culture medium.

[0017] According to another aspect of the present invention, a method for preparing a scaffold material is provided, the scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers comprising silk proteins; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Mixing electrospun fibers containing silk proteins with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

[0018] According to another aspect of the present invention, a method for preparing a scaffold material is provided, the scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with platelet-rich plasma and / or platelet lysis buffer and / or bone marrow concentrate to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0019] According to another aspect of the present invention, a method for preparing a scaffold material is provided, the scaffold material comprising: i) hydroxyapatite, and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Electrospun fibers containing silk protein are mixed with hydroxyapatite to form a scaffold material.

[0020] According to another aspect of the present invention, a method for preparing a scaffold material is provided, the scaffold material comprising i) hydroxyapatite and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hydroxyapatite to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0021] According to another aspect of the present invention, a method for preparing a scaffold material is provided, the scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hyaluronic acid to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0022] The embodiments and preferred embodiments described below for the scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention are also applicable to the methods described herein for preparing the scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs. Attached Figure Description

[0023] Figure 1 A layered construct (“multiphase implant”) according to an embodiment of the present invention is shown.

[0024] Detailed description The inventors discovered that, Among other things In addition, a scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers containing silk proteins, can be used to prepare useful materials in tissue engineering.

[0025] The inventors have also discovered that a scaffold material comprising: i) hyaluronic acid; and ii) electrospun fibers containing silk fibroin can be used to prepare materials for use in tissue engineering.

[0026] The inventors have also discovered that a scaffold material composed of i) hydroxyapatite and ii) electrospun fibers containing silk proteins can be used to prepare materials for tissue engineering, particularly for preparing scaffold materials with stronger structures.

[0027] In this invention, "scaffold material" refers to a material or substance that can be used for, for example, tissue repair and / or support (including reinforcement). The scaffold material can promote or facilitate the formation of new body tissues, such as tissues, cartilage, tendons, nerves, and bones. Its mechanism of action may include providing support and / or promoting cell growth, attachment, differentiation, cell distribution, etc. Suitably, the scaffold material is in the form of a thin film, membrane, pad, or mesh structure.

[0028] Platelet-rich plasma (PRP) is typically prepared by centrifuging whole blood to separate platelets from red blood cells. Bone marrow concentrate (BMC) can be obtained through a similar process. Additional centrifugation or separation steps can be used to remove platelet-deficient plasma. Because the cellular composition of PRP (and consequently its therapeutic efficacy) depends heavily on the methods and equipment used to prepare it, standardization of PRP production is crucial.

[0029] Medical devices for the preparation of standardized PRP and BMC have been developed, including equipment for closed-loop automated processes. For example, centrifuge tubes manufactured by Regen Labs, whose related technologies are described in WO2008 / 023026A2, WO2011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391A1, WO2021 / 198312A1, and WO2022 / 269035A1 (all of which are cited in full hereafter).

[0030] The process for preparing platelet-rich plasma or bone marrow concentrate typically includes the following steps: a) Collect body fluids or cell extracts in a centrifuge container; b) Centrifuge the centrifuge container from step a) to form an enriched component; and c) Collect at least a portion of the enriched fraction.

[0031] Centrifuge containers are best suited to centrifuge tubes or centrifuge syringes, especially centrifuge tubes, such as blood fractionation tubes. Suitable materials and coatings for centrifuge containers, as well as the use of density gradient media such as thixotropic gels, have been described in WO2008 / 023026A2, WO2011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391A1, WO2021 / 198312A1, and WO2022 / 269035A1 (all of which are cited in full hereafter).

[0032] The properties of enriched fractions depend on the material being centrifuged. Centrifugation separates components based on their density; denser particles migrate towards the bottom (far end) of the container, while less dense particles migrate towards the top (proximal end). Therefore, an "enriched fraction" can be defined as a component whose specific cell concentration is higher than that of the centrifuged body fluid or cell extract. When the body fluid is whole blood, red blood cells (erythrocytes) settle to the bottom of the container (or below if a density gradient medium is present), while platelets are above them. In plasma, the concentration of platelets (and other cellular components) is higher than in centrifuged whole blood. Therefore, when the body fluid is whole blood, the enriched fraction is platelet-rich plasma. In other words, the enriched fraction is platelet-rich plasma. When the body fluid is bone marrow, the enriched fraction is bone marrow concentrate. Typically, this bone marrow concentrate is rich in stem cells, such as mesenchymal stem cells. Usually, this body fluid (especially platelet-rich plasma and / or bone marrow concentrate) is of autologous origin.

[0033] Centrifugation is performed at a sufficiently high centrifugal force for a specific duration to create a boundary layer between the fluid components to be separated; for example, when the fluid is whole blood, this refers to a combination of centrifugal force / time that allows a density gradient medium (if present) to separate red blood cells from platelets and other desired components in the whole blood. In one embodiment, centrifugation is performed at a centrifugal force of about 700 g to about 2800 g, for example, at a centrifugal force of about 1500 g to about 2800 g, about 1500 g to about 2500 g, or for example, about 1500 g to about 2000 g. Suitably, the duration of the centrifugation in step b) is between about 3 minutes and about 40 minutes, for example, between about 3 minutes and about 15 minutes.

[0034] When the body fluid is whole blood, the enriched fraction is platelet-rich plasma (PPP). However, after centrifugation, the PPP is not uniformly distributed; platelets are typically concentrated only directly above the density gradient medium (if present), with a layer of platelet-poor plasma (PPP) above them (i.e., near the centrifuge container). Removing part of this platelet-poor plasma can be beneficial, for example, removing about half to about one-third of the enriched fraction (from the top of the centrifuge container, i.e., the end furthest from the density gradient medium (if present), i.e., the near end), thereby removing part or all of the PPP layer and obtaining a more concentrated or enriched fraction.

[0035] Therefore, in one embodiment, step b) includes the following steps: b-1) Centrifuge the centrifuge container in (step a) to form an enriched fraction; b-2) Remove and discard at least a portion of the enriched fraction (from step b-1).

[0036] Typically, at least a portion of the enriched distillate is removed from the proximal end of the centrifuge container. In this embodiment, step c) is to collect at least a portion of the remaining enriched distillate (i.e., the distillate with a higher concentration or greater enrichment).

[0037] Platelet lysis buffer is a liquid obtained by subjecting platelets to freeze-thaw cycles. Freeze-thaw cycles cause platelets to lyse, releasing large amounts of growth factors and cytokines. Platelet lysis buffer is commercially available or can be easily prepared in-house if donor platelets are available.

[0038] Platelet lysate can be in lyophilized (or freeze-dried) form. The lyophilization process preserves the ability of platelets to release bioactive molecules (such as growth factors and cytokines), ensures its stability and extends its shelf life, and eliminates the need for refrigeration.

[0039] Platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate can be mixed with electrospun fibers containing silk proteins. Therefore, according to one aspect of the invention, a method for preparing a scaffold material is provided, the scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers containing silk proteins; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Mixing electrospun fibers containing silk proteins with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

[0040] As described in the background of the invention, silk fibroin can be derived from silk produced by insects such as silkworms and spiders. The term "silk fibroin" is intended to encompass sericin and related proteins in this specification. In one embodiment, silk fibroin is specifically fibroin. More specifically, fibroin is a high-molecular-weight copolymer composed of heavy chains (approximately 370 kDa) and light chains (approximately 26 kDa) linked by disulfide bonds. This silk fibroin is preferably obtained from a solution containing dissolved silkworm or spider silk. In one embodiment, the silk fibroin is obtained from a solution containing dissolved silk (e.g., from the silkworm *Bombyx mori*). In another embodiment, the silk fibroin is obtained from a solution containing dissolved spider silk (e.g., from the giant spider *Nephila clavipes*). In yet another embodiment, the silk fibroin is obtained from a solution containing transgenic silk (e.g., from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants).

[0041] In step a), a silk fibroin solution can be prepared using methods known in the art (see, for example, Rockwood et al., Nature Protocols, Sep 22, 2011; 6(10):1612-31; Wöltje et al., International Journal of Molecular Sciences, Sep 29, 2021; 22(19):10565; Melke et al., Journal of Biomaterials, Vol. 31, 2016, pp. 1-16; and Bucciarelli et al., Progress in Biomaterials, Aug 2022; 139:212982). When the silk fibroin is derived from silkworms, the cocoons are typically boiled in an aqueous solution of soap (such as sodium oleate) or sodium carbonate (i.e., a so-called hot bath) to remove the gelatinous proteins and other fats covering the surface of the silk fibroin. This process is called "degumming". Degumming can also be achieved by microwave-assisted methods, which can produce high-purity silk fibroin while retaining its inherent properties and ensuring excellent bioactivity. After degumming, the silk fibroin is washed and dried. Further purification can be achieved through dialysis and / or filtration. An exemplary process for preparing silk fibroin solutions is detailed in the "General Methods" section.

[0042] The silk fibroin solution used in the electrospinning process of step b) has a suitable concentration of silk fibroin (especially fibroin) between about 5 wt.% and about 30 wt.%, for example, between about 12 wt.% and about 18 wt.%. Higher concentrations of silk fibroin in the electrospinning solution result in larger fiber diameters and thicker electrospinned fiber layers. However, if the silk fibroin concentration is too high, the solution viscosity will be too high, making electrospinning impossible. Silk fibroin solutions are typically prepared using water as a solvent.

[0043] In step b), the silk protein solution from step a) is electrospun using any method known in the art, thus completing the electrospinning process. A typical electrospinning apparatus operates by applying a voltage (e.g., from about 5 kV to about 30 kV, or from about 12 kV to about 18 kV) between a needle (also called a spinning nozzle or capillary) through which the silk protein solution passes (usually pumped by a syringe) and a collector. The collector is placed a short distance from the needle, typically 5-20 cm, for example 8-12 cm. Preferably, a syringe pump is used to maintain a constant volumetric flow rate to keep the flow rate constant while preventing the solution from dripping from the needle tip. In one embodiment, the flow rate is between about 0.8 mL / h and about 1.2 mL / h. A stable solution jet is obtained by adjusting the electric field, the solution flow rate, and the distance between the needle tip and the collector. The collector can be any material that can serve as a counter electrode, such as a wire mesh, a polymer mesh, a metal foil (e.g., aluminum foil), or a water bath. Suitably, the collector has a non-stick surface to facilitate the removal of electrospun fibers; for example, the collector screen may have a polytetrafluoroethylene coating or be made of release paper. Alternatively, the collector may be a substrate for casting the electrospun solution to form a layered product; for example, the collector may be a sheet or support material (described below). Suitably, the collector rotates at a speed of, for example, 100-300 rpm to produce a uniform, highly oriented coating of electrospun fibers that forms a support material.

[0044] Electrospun fibers are typically in the range of approximately 50 nanometers to approximately 1,000 nanometers, for example, between approximately 200 nanometers and approximately 500 nanometers. Electrospun fibers are usually in the form of films, membranes, pads, or webs. If the electrospinning solution contains only sericin, the resulting web will be solid. If the electrospinning solution contains other components (details are described below), the resulting scaffold material may have semi-solid properties.

[0045] After collecting the electrospun fibers, one or more subsequent processing steps may be selectively performed on them, such as washing (e.g., with distilled water) or stretching the fibers.

[0046] In step c) of the above embodiment, electrospun fibers containing silk protein are mixed 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, membrane, pad, or mesh structure.

[0047] In this invention, "semi-solid" refers to a scaffold material that has flexible solid properties, whose physical properties allow it to be sutured while possessing sufficient strength to provide the mechanical and physiological support required as a scaffold.

[0048] In an alternative process, the preparation comprises i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers containing silk fibroin. In this process, the platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate are not mixed with the already formed sericin-containing electrospun fibers, but are instead added to the silk fibroin solution of step a), i.e., the platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate are added to the electrospinning solution. The mixture of platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate with silk fibroin is then processed by an electrospinning process.

[0049] Therefore, an alternative method for preparing a scaffold material is provided, the scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibers containing silk proteins; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with platelet-rich plasma and / or platelet lysis buffer and / or bone marrow concentrate to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0050] Another aspect of the invention provides a scaffold material comprising: i) hydroxyapatite; and ii) electrospun fibers comprising silk fibroin. The inventors have discovered that the scaffold material comprising electrospun fibers of silk fibroin, when combined with hydroxyapatite, can form a more robust scaffold material.

[0051] Hydroxyapatite is a natural mineral form of calcium apatite, with the chemical formula Ca5( PO4 3(OH), often abbreviated as Ca 10 ( PO4 )6(OH)2.

[0052] The scaffold material can be prepared by the following method, which includes the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Combining electrospun fibers containing silk protein with hydroxyapatite to form a scaffold material.

[0053] Steps a) and b) can be performed as described above. In step c), electrospun fibers containing silk protein are combined with hydroxyapatite to form a scaffold material. Preferably, the scaffold material is a solid scaffold material. In one embodiment, the scaffold material is in the form of a film, membrane, pad, or mesh structure.

[0054] In an alternative process for preparing a scaffold material composed of hydroxyapatite and sericin-containing electrospun fibers, the hydroxyapatite does not need to be mixed with the prepared sericin-containing electrospun fibers, but can be added to the sericin solution in step a), i.e., the hydroxyapatite can be added to the electrospinning solution. Subsequently, the mixture of hydroxyapatite and sericin is treated with an electrospinning process.

[0055] Therefore, an alternative method for preparing scaffold materials is provided, which includes the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hydroxyapatite to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0056] Another aspect of the present invention provides a method for preparing a scaffold material comprising i) hyaluronic acid and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hyaluronic acid to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

[0057] Step a) can be performed as described above. In one embodiment, the weight ratio of hyaluronic acid to silk fibroin in step b) is between about 1:2 and about 1:4, for example, about 1:3. In one embodiment, the method further includes step d): crosslinking the scaffold material, for example by reacting it with an aldehyde crosslinking agent (such as glutaraldehyde) (e.g., gas phase method). In one embodiment, the method further includes step d): adding platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to the scaffold material of step c).

[0058] In all the methods described above, when electrospun fibers containing silk proteins are combined with another material (e.g., platelet-rich plasma, platelet lysate, bone marrow concentrate, hydroxyapatite, hyaluronic acid, or additional components described below, or mixtures thereof) to form a scaffold material, the added material can be extruded. If desired, the electrospinning device can be used in conjunction with a microextrusion device, as described, for example, in WO2019 / 211803A1 (incorporated herein by reference). An example of such a combined device is the “Electrospider,” which combines electrospinning, fused deposition modeling, and pneumatic hydrogel extrusion techniques for fabricating (or “printing”) 3D structures that can have multiple layers.

[0059] The aforementioned scaffold material may contain additional components that can be added before electrospinning (i.e., added to the electrospinning solution containing the silk protein to be electrospinned) or added to the electrospinned fibers composed of silk protein. Alternatively, a combination of multiple additional components may be added, some of which are contained in the electrospinning solution and others added to the scaffold material after electrospinning.

[0060] For a scaffold material that does not yet contain hyaluronic acid, in one embodiment, the scaffold material further contains hyaluronic acid.

[0061] 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 containing hyaluronic acid as described above), the hyaluronic acid is low molecular weight hyaluronic acid (LMW-HA) with a 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 medium molecular weight hyaluronic acid with a molecular weight between about 1,000 kDa and about 1,800 kDa, for example, between about 1,400 kDa and about 1,600 kDa, for example, about 1,500 kDa. In another embodiment, the hyaluronic acid is high molecular weight hyaluronic acid (HMW-HA) with a molecular weight between about 800 kDa and about 1,200 kDa, or greater than 1,800 kDa. Preferably, the molecular weight of the hyaluronic acid in the centrifuge container of the present invention is between about 1,000 kDa and about 1,800 kDa, for example between about 1,400 kDa and about 1,600 kDa, for example about 1,500 kDa.

[0062] Hyaluronic acid mixtures of different molecular weights can also be used. Therefore, in one embodiment, the hyaluronic acid is a mixture of hyaluronic acid comprising the following components: Low molecular weight hyaluronic acid (LMW-HA), with a molecular weight between approximately 400 kDa and approximately 1,000 kDa; and / or Medium molecular weight hyaluronic acid, with a molecular weight between approximately 1,000 kDa and approximately 1,800 kDa, for example, between approximately 1,400 kDa and approximately 1,600 kDa, for example, approximately 1,500 kDa; and / or High molecular weight hyaluronic acid (HMW-HA) has a molecular weight greater than 1,800 kDa.

[0063] In one embodiment, the hyaluronic acid is cross-linked. In another embodiment, the hyaluronic acid has a linear structure. Preferably, the hyaluronic acid has a linear structure. It is preferable to add the hyaluronic acid in the form of an aqueous solution, wherein the concentration of hyaluronic acid in the solution (added to the centrifuge container) is between about 1 wt.% and about 5 wt.%, for example, between about 1.8% and about 2.2 wt.%. Alternatively, when the scaffold material also comprises platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, the hyaluronic acid can be added to the centrifuge container used to prepare platelet-rich plasma / platelet lysate / bone marrow concentrate. Suitably, the centrifuge container contains about 1.0 ml to about 5.0 ml of hyaluronic acid, particularly about 1.5 ml to about 3 ml, for example, about 2 ml. Suitably, the filling volume of the centrifuge container is 10 ml or 15 ml, particularly 15 ml.

[0064] In one embodiment, the scaffold material further comprises an antifibrinolytic substance.

[0065] Antifibrinolytic substances are substances (e.g., compounds) capable of inhibiting fibrinolysis. Antifibrinolytic substances prevent or reduce the activation of plasminogen into plasmin, thereby preventing or reducing the degradation of blood clots. Antifibrinolytic substances are typically synthetic analogs of the amino acid lysine. In one embodiment, the antifibrinolytic substance is selected from tranexamic acid, aminocaproic acid, and alpratinine, or any combination thereof. Suitably, the antifibrinolytic substance is a small molecule, for example, with a molecular weight of 500 Da or less, such as 400 Da or less, 300 Da or less, or 200 Da or less. In one embodiment, the antifibrinolytic substance is not an enzyme, polypeptide, or 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 soluble in water). The reference to "one" antifibrinolytic substance is intended to cover "at least one" antifibrinolytic substance, and combinations of antifibrinolytic substances are also contemplated.

[0066] In one embodiment, the stent material further comprises a coagulation activator.

[0067] A coagulation activator is a preparation (e.g., a compound or enzyme) capable of triggering or activating plasma coagulation and platelet aggregation to form a clot, which may have a gel-like consistency. The expression "a" coagulation activator is intended to cover "at least one" coagulation activator, and combinations of coagulation activators are also considered. Typically, a coagulation activator is a compound or fragment that acts as a thrombin activator and / or fibrinogen activator. In one embodiment, the coagulation activator is a compound selected from the group consisting of calcium salts and thrombin. Suitably, the calcium salt is selected from the group consisting of calcium gluconate, calcium carbonate, calcium sulfate, calcium gluconate, and calcium chloride; or any combination thereof, particularly calcium gluconate.

[0068] In one embodiment, the coagulation activator is a calcium salt (particularly calcium gluconate) in an aqueous solution at a concentration between about 1 wt.% and about 20 wt.% (e.g., between about 5 wt.% and about 15 wt.%, such as about 10 wt.%). The calcium salt may comprise a mixture of calcium salts, such as a combination of calcium gluconate and calcium gluconate.

[0069] In one embodiment, the scaffold material further comprises vitamin K2. In one embodiment, vitamin K2 is selected from menadione-4 (MK-4), menadione-5 (MK-5), menadione-6 (MK-6), menadione-7 (MK-7), menadione-8 (MK-8), menadione-9 (MK-9), menadione-10 (MK-10), menadione-11 (MK-11), menadione-12 (MK-12), menadione-13 (MK-13), and menadione-14 (MK-14), and mixtures thereof. The terms “vitamin K2” and “menadione” are intended to cover “at least one” menadione, and also include combinations of menadiones. 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 implementation, vitamin K2 is MK-7.

[0070] 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 steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof. In particular, another therapeutic agent is selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or a combination thereof.

[0071] For a scaffold material that does not yet contain hydroxyapatite, in one embodiment, the scaffold material also contains hydroxyapatite.

[0072] Particularly preferred scaffold materials also include hyaluronic acid. Therefore, in one preferred embodiment, a scaffold material is provided comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibers containing silk protein; and iii) hyaluronic acid. In another preferred embodiment, a scaffold material is provided comprising: i) hydroxyapatite; ii) electrospun fibers containing silk protein; and iii) hyaluronic acid. In a further preferred embodiment, a scaffold material is provided comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibers containing silk protein; iii) hyaluronic acid; and iv) hydroxyapatite.

[0073] When the scaffold material contains hyaluronic acid, the weight ratio (w / w) of silk protein to hyaluronic acid is suitably between about 50:50 and about 95:5.

[0074] The stent material of the present invention has therapeutic uses. For example As wound dressings and grafts (e.g., vascular grafts).

[0075] Another aspect of the present invention provides a layered wound dressing or layered graft comprising: i) A support material comprising: ia) Electrospun fibers containing silk proteins; and ii) Hyaluronic acid; as well as ii) A support material.

[0076] Combining electrospun fibers containing silk fibroin with hyaluronic acid leverages both the mechanical strength of silk and the hydrophilic, cell-friendly environment provided by hyaluronic acid. This combination, due to its biocompatibility and favorable environment for cell growth, helps maintain cell survival. The hydrophilicity of hyaluronic acid helps maintain a hydrated environment, which is crucial for cell survival. The presence of hyaluronic acid also enhances cell adhesion, thanks to its role in cell signaling and its interaction with cell surface receptors (such as CD44). Silk fibroin, in particular, provides a stable and supportive structure for cell attachment.

[0077] In one embodiment, the stent material further comprises platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate (as described above). In one embodiment, the stent material further comprises hyaluronic acid (as described above). In one embodiment, the stent material further comprises an antifibrinolytic agent, such as tranexamic acid (as described above). In one embodiment, the stent material further comprises a coagulation activator, such as calcium gluconate (as described above). In one embodiment, the stent material further comprises vitamin K2 (as described above). In one embodiment, the stent material further comprises hydroxyapatite (as described above).

[0078] The support material is any support material commonly used in wound dressings or grafts. In one embodiment, the support material comprises a synthetic polymer, such as poly(oxalic acid), poly(lactic acid), polyethylene glycol (PEG), low-density polyethylene (LDPE), polycaprolactone (PCL), or a natural polymer, such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran, or protein. The scaffold material of the present invention can be attached to a support material using conventional techniques known to those skilled in the art, including adhesion (e.g., using adhesives) or lamination.

[0079] In a preferred embodiment, the support material comprises a plastic material, such as low-density polyethylene (LDPE) or polycaprolactone (PCL).

[0080] The scaffold material of the present invention is also suitable for tissue engineering applications. In one embodiment, the scaffold material of the present invention can be bound to cells to form a tissue-engineered construct for regenerating or replacing damaged or degenerated tissues or organs.

[0081] Tissue-engineered constructs can be prepared by a variety of methods. In one embodiment, a tissue-engineered construct includes: i) the scaffold material described herein; and ii) cells, such as mammalian cells. The specific cell type depends on the intended application of the construct, but suitable mammalian cells include cells selected from the group consisting of: hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal cells, bile duct cells, parathyroid cells, thyroid cells, adrenal-hypothalamic-pituitary axis cells, cardiomyocytes, renal epithelial cells, renal tubular cells, renal basement membrane cells, nerve cells, vascular cells, bone and cartilage-forming cells, smooth muscle cells, skeletal muscle cells, cochlear cells, epidermal cells, bone marrow cells, keratinocytes, pluripotent cells, and stem cells, and combinations thereof.

[0082] In one embodiment, a method for preparing a tissue-engineered construct is provided, comprising the following steps: a) Preparation of scaffold materials as described herein; and b) Mix the scaffold material with cells (e.g., mammalian cells) in a suitable culture medium.

[0083] The suitable culture medium is a culture medium, the specific type of which depends on the type of cells to be cultured, and can be easily prepared by those skilled in the art.

[0084] Another aspect of the present invention provides a hierarchical construct for tissue engineering applications, comprising: i) The first layer containing silk proteins; ii) The second layer, which contains hydrogel and silk protein; iii) The third layer comprises polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) The fourth layer, which contains polycaprolactone and hydroxyapatite.

[0085] Figure 1 (The "Multiphase Implant") illustrates a layered construction in one embodiment of the present invention. This innovative construction is specifically designed to treat cartilage damage caused by degenerative changes or trauma, including applications in sports medicine. The construct mimics the structure and function of natural osteochondral tissue through a layered, multiphase approach. It is manufactured using GMP-compliant 3D printing and electrospinning techniques to ensure clinical-grade quality.

[0086] Suitably, the thickness of the first layer containing silk fibroin is between about 40 μm and about 60 μm, for example, about 50 μm. Suitably, the first layer containing silk fibroin is a capping layer. Suitably, the silk fibroin in the first layer is composed of silk fibroin electrospun fibers. In one embodiment, the silk fibroin electrospun fibers are prepared by electrospinning a sericin solution at a concentration of about 5% (w / v) to 15% (w / v) (e.g., about 7% (w / v)); at a voltage of 12-18 kV (e.g., about 15 kV); at a flow rate of about 0.8 mL / h to about 1.2 mL / h (e.g., 0.8 mL / h); and at a collector distance of 5-20 cm (e.g., about 10 cm). The first layer, composed of silk fibroin (particularly silk fibroin, with specific preferences as described above), provides a protective barrier for the underlying layer. The first layer also provides structural integrity for the layered structure and prevents degradation during early implantation. Finally, it has excellent biocompatibility, which helps promote cell attachment.

[0087] In one embodiment, the hydrogel of the second layer comprises or is composed of 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., a weight ratio of approximately 1:1). In one embodiment, the silk protein in the second layer comprises silk protein electrospun fibers. In one embodiment, the silk protein electrospun fibers are prepared by electrospinning a sericin solution at a concentration of approximately 5% (w / v) to 15% (w / v) (e.g., approximately 7% (w / v)); at a voltage of 12-18 kV (e.g., approximately 15 kV); at a flow rate of approximately 0.8 mL / h to approximately 1.2 mL / h (e.g., 0.8 mL / h); and at a collector distance of 5-20 cm (e.g., approximately 10 cm). The second layer can be formed by mixing hyaluronic acid and / or gelatin with the electrospun silk protein, or, according to the method described above, by mixing the hyaluronic acid and / or gelatin with the silk protein solution before electrospinning. Therefore, in one embodiment, the second layer consists of electrospun fibers of hydrogel (e.g., hyaluronic acid) and silk fibroin. In one embodiment, the electrospun fibers of hyaluronic acid and silk fibroin are prepared by electrospinning a solution of hyaluronic acid and silk fibroin at a concentration between about 5% (w / v) and 15% (w / v) (e.g., about 7% (w / v)); at a voltage of 12-18 kV (e.g., about 15 kV); at a flow rate of about 0.8 mL / h to about 1.2 mL / h (e.g., 0.8 mL / h); and at a collector distance of 5-20 cm (e.g., about 10 cm). In one embodiment, the thickness of the second layer is 1-2 mm. In one embodiment, the second layer also comprises platelet-rich plasma, platelet lysate, and / or bone marrow concentrate, particularly platelet lysate. The hydrogel-based second layer mimics the central region of cartilage, thereby promoting cartilage formation. It facilitates the controlled release of growth factors to promote tissue regeneration and enhances water retention capacity, thereby improving the biomechanical properties of the construct.

[0088] In one embodiment, the third layer comprises polycaprolactone (PCL) and hydroxyapatite, and is formed by electrospinning. In another embodiment, the third layer comprises hyaluronic acid and hydroxyapatite, and is formed by electrospinning. Suitably, the third layer comprises PCL and hydroxyapatite in a mass ratio of 80:20. In one embodiment, the thickness of the third layer is 90 μm to 110 μm. The third layer mimics the calcified layer of osteochondral tissue, providing a transition interface between the cartilage and the underlying periosteum. The third layer combines mechanical strength with bioactivity to ensure integration with the subchondral bone.

[0089] In one embodiment, the fourth layer, composed of polycaprolactone and hydroxyapatite, is fabricated using fused deposition modeling (FDM) technology. FDM is a 3D printing technique that involves heating a thermoplastic polymer above its melting point and then extruding it through a nozzle at a precise rate. The molten polymer is deposited onto a build plate at a lower temperature, where it rapidly cools and solidifies. In one embodiment, the fourth layer comprises polycaprolactone and hydroxyapatite in a 70:30 mass ratio. In one embodiment, the thickness of the fourth layer is 450 μm to 550 μm, for example, about 500 μm. The fourth layer mimics the subchondral bone layer and provides load-bearing function and support, thereby promoting long-term fusion with the native bone tissue.

[0090] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs described herein all have therapeutic uses. Therefore, in one embodiment, the scaffold materials, layered wound dressings, layered grafts, layered constructs, or tissue-engineered constructs described herein are provided for therapeutic purposes. The scaffold material is a medical scaffold material. In one embodiment, the scaffold material is in the form of a wound dressing or graft.

[0091] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention can provide mechanical support at the site of injury and provide physiological support by enhancing the repair of native tissue.

[0092] In one embodiment, a scaffold material, tissue-engineered construct, layered wound dressing, layered graft, or layered construct described herein is provided for 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. Tissues include muscle, skin, and organs.

[0093] Specific examples of wound healing and tissue repair include the treatment of gynecological conditions (such as pelvic organ prolapse), intestinal injuries, bladder injuries, vascular injuries, other refractory wounds, burns, and diabetic foot.

[0094] In one embodiment, a scaffold material, tissue-engineered construct, layered wound dressing, layered graft, or layered construct as described herein is provided for tissue augmentation.

[0095] In one embodiment, a scaffold material, tissue-engineered construct, layered wound dressing, layered graft, or layered construct as described herein is provided for use as a delivery carrier for therapeutic agents.

[0096] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs containing hydroxyapatite in this invention are particularly suitable for bone regeneration.

[0097] In one embodiment, the scaffold material, tissue-engineered construct, layered wound dressing, layered graft, or layered construct described herein is provided for the treatment or prevention of joint diseases or conditions. Suitably, the joint diseases or conditions are selected from the group comprising arthritis, gout, fibromyalgia, lupus erythematosus, polymyalgia, and rheumatic diseases. Arthritis includes osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, cervical spondylosis, psoriatic arthritis, enteropathic arthritis, oligoarthritis, polyarthritis, and secondary arthritis.

[0098] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention can take any suitable form, including films, membranes, pads, or mesh structures. The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention can be used in (…). Including but not limited to Wound dressings, grafts (such as vascular grafts), and scaffolds for cell growth.

[0099] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention exhibit excellent biocompatibility and, in some embodiments, include pores or gaps to allow for the exchange of bodily fluids within their structure. The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention also possess suitable biodegradability and are therefore absorbed after use (e.g., implantation).

[0100] The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention can be layered over conventional materials (e.g., wound dressing materials) to provide enhanced mechanical strength. In one embodiment, the scaffold material of the present invention is layered over a suitable sheet material, such as a backing layer. In one embodiment, the scaffold material, tissue-engineered construct, layered wound dressing, layered graft, or layered construct of the present 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 present invention is layered onto a sheet comprising a synthetic polymer, such as poly(hydroxyacetic acid), polylactic acid, polyethylene glycol (PEG), low-density polyethylene (LDPE), polycaprolactone (PCL), or a sheet of natural polymers (such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran, or protein). The scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, or layered constructs of the present invention can be attached to sheets using conventional techniques known to those skilled in the art, including bonding (e.g., using adhesives) or lamination.

[0101] In another process of the present invention, a composition comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and electrospun fibers containing silk protein, can be prepared by using a centrifuge container already containing a silk protein solution; and ii) electrospun fibers containing silk protein. The silk protein solution can be prepared as described above. In this embodiment, body fluids or cell extracts are collected in a centrifuge container already containing a silk protein solution. The centrifuge container is then centrifuged to form an enriched component and a silk protein solution. This solution can then be electrospun as described herein to form the scaffold material of the present invention.

[0102] In one embodiment, a centrifuge container is provided containing silk protein (details as described above). The centrifuge container may also contain hyaluronic acid (details as described above) and / or an antifibrinolytic substance (details as described above) and / or a coagulation activator (details as described above) and / or vitamin K2 and / or one or more therapeutic agents (details as described above) and / or hydroxyapatite. When the centrifuge container contains hyaluronic acid, trehalose may also be added to the container as a stabilizer for the hyaluronic acid. The centrifuge container may also contain a density gradient medium (details as described above).

[0103] In one embodiment, a centrifuge container comprising silk protein, a density gradient medium, and hyaluronic acid is provided.

[0104] The use of electrospinning technology to prepare the scaffold material of this invention has several advantages. First, it enables the preparation of standardized scaffold materials that can be mass-produced and subsequently cut to the required size for various clinical applications. Second, this technology also allows for the manufacture of customized scaffold materials that can be precisely designed and manufactured to meet the specific pathological and anatomical requirements of individual patients. Therefore, the method of this invention combines the dual capabilities of standardized mass production and personalized manufacturing.

[0105] In one embodiment, the expansion rate of the stent material of the present invention is at least 300%. The expansion rate is defined as the difference between the initial dry weight and the expanded weight of the material divided by the initial weight.

[0106] In some embodiments, the scaffold materials, tissue-engineered constructs, layered wound dressings, layered grafts, and layered constructs of the present invention are expected to have one or more of the following advantages: Porosity suitable for nutrient and / or cell exchange; It has a suitable structure to support cell adhesion and / or extension; Promotes the controlled release of bioactive substances such as growth factors; It promotes cell growth, thereby promoting healing; Biocompatibility; Anti-apoptotic effect.

[0107] Other specific advantages of the layered structure of this invention: Bionic design: Each layer simulates the structure and function of the natural osteochondral tissue region from the surface of the articular cartilage to the subchondral bone.

[0108] Controlled release of growth factors: The hydrogel layer incorporates platelet lysate proteins, enabling the continuous release of growth factors and thus promoting tissue regeneration.

[0109] Preparation technology: Electrospinning: Nanoscale fiber structures are formed in the electrospun layer, thereby enhancing cell adhesion and mechanical strength.

[0110] 3D printing: Ensuring precise and scalable production of FDM layers and the overall structure.

[0111] Compliant with GMP standards: All processes are carried out under GMP conditions to ensure compliance with regulatory requirements and clinical application value.

[0112] Scalability and Customization: Advanced 3D printing technology can be used to customize constructs according to the specific needs of patients.

[0113] abbreviation BMC Bone Marrow Concentrate CBD (cannabidiol) DMA Dynamic Mechanical Analysis DMEM (Durbeco Modified Eagle's Medium) DMMB (Dimethyl Methylene Blue) DMF (dimethylformamide) ELISA (Enzyme-Linked Immunosorbent Assay) Ethylene oxide (EtO) FDM fused deposition modeling GAG glycosaminoglycans GF gingival fibroblasts GMP (Good Manufacturing Practice) HA Hyaluronic Acid HMW-HA High Molecular Weight Hyaluronic Acid HUVECs (human umbilical vein endothelial cells) LDPE (Low-density polyethylene) LMW-HA Low Molecular Weight Hyaluronic Acid MK Menaquinone MPV (Mean Platelet Volume) MSC mesenchymal stem cells NSAIDs (nonsteroidal anti-inflammatory drugs) PBS (Phosphate Buffered Saline) PCL (polycaprolactone) PDGF (platelet-derived growth factor) PDL periodontal ligament cells PEG (Polyethylene Glycol) PL platelet lysis fluid PPP low platelet plasma PRP (platelet-rich plasma) rpm (revolutions per minute) SDS Sodium lauryl sulfate SEM (Scanning Electron Microscope) SF sericin THC Tetrahydrocannabinol VF vaginal fibroblasts Example General Method Cell preparation—fibroblasts Gingival tissue was harvested from the interdental papilla at the time of tooth extraction in adult patients. Immediately after harvesting, the tissue was preserved in sterile saline for 1 to 4 hours before processing. The gingival tissue was rinsed 10 times with phosphate-buffered saline (PBS) to dilute the oral bacterial flora in the gingival tissue. After PBS washing, the tissue was cut into 1 to 2 mm pieces using a No. 10 scalpel. ² Small pieces of tissue were then seeded into 100 mm tissue culture dishes and placed in a humidified incubator containing 5% CO2 at 37 °C for 48 hours. The culture medium was changed after 48 hours. Cells were cultured in complete growth medium (Dulbecco's Modified Eagle's medium [DMEM]; Life Technologies, Paisley, UK), which was supplemented with 10% fetal bovine serum (FBS), 1% HEPES 1 M buffer (Life Technologies), 1% non-essential amino acid mixture 100× (Life Technologies), 1% L-glutamine 100x concentrate (Life Technologies), 1% penicillin / streptomycin 100x concentrate (Life Technologies), and 1% sodium pyruvate 100x concentrate (Life Technologies). Fibroblasts from other tissue sources can be cultured using a similar method.

[0114] Preparation of silk fibroin solution First, degumming is performed: 5 grams of silkworm cocoons are cut into small pieces. The pieces are then boiled in 2 L of 0.02 M sodium carbonate solution for 30 minutes, removed, and squeezed to remove excess water from the silk. Next, it is rinsed three times with 1 L of deionized water, 20 minutes each time, and then squeezed to remove excess water again. The degummed silk fibroin pieces are dried overnight in a fume hood and can be stored indefinitely at room temperature.

[0115] To prepare the silk fibroin solution, degummed silk fibroin is dissolved in 9.3 M lithium bromide solution to prepare a 20% (w / v) solution. The solution is then dissolved at 60 °C for 4 hours until a clear, transparent solution is formed. This solution can be desalted by dialysis, for example, by placing 12 mL of the solution in 1 L of deionized water (changing the water 6 times), and then centrifuging at 9,000 rpm (approximately 12,700 g) for 20 minutes at 4 °C, repeated three times. 5 grams of silkworm cocoons typically yield 25 mL of a 7-8 w / v silk solution.

[0116] Example 1a – Preparation of platelet-rich plasma Blood samples are collected from healthy donors and injected into centrifuge tubes (CuteCell). ™ PRP (Regen Lab SA). Collected blood was centrifuged at 1500 g for 5 minutes at room temperature in a standard laboratory centrifuge. Subsequently, red blood cells and white blood cells accumulated below the separating gel at the bottom of the tube, while plasma and platelets remained above the gel layer. The platelet-rich plasma was thoroughly mixed by inverting the centrifuge tube five times. The resulting 6 mL of platelet-rich plasma was transferred to a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for later use.

[0117] Example 1b – Preparation of platelet-rich plasma containing hyaluronic acid A blood sample is collected from a healthy donor and injected into a centrifuge tube containing hyaluronic acid. CellularMatrix™ PRP (Regen Lab SA). Collected blood was centrifuged at 1500 g for 5 minutes at room temperature in a standard laboratory centrifuge. Red blood cells and white blood cells then settled below the separating gel at the bottom of the tube, while plasma and platelets remained on top of the gel layer. The platelet-rich plasma was thoroughly mixed by inverting the centrifuge tube five times. The resulting 6 mL of platelet-rich plasma was then transferred with hyaluronic acid to a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for later use.

[0118] Example 2 – Preparation of an electrospun fiber membrane scaffold material composed of silk fibroin and hyaluronic acid A 0.5% (w / v) formic acid-hyaluronic acid solution was prepared by dissolving hyaluronic acid in formic acid at 55 °C for 3 hours. After cooling the solution, 7.5% (w / v) silk fibroin was added, and the mixture was stirred for 30 minutes. The solution was then ready for electrospinning using previously optimized parameters and an anti-sticking paper collector. The silk fibroin / hyaluronic acid nanofibers were directly collected on the substrate after electrospinning, with a deposition time of approximately 25 minutes. The resulting membranes were cut using a laser cutter, and the cut samples were sterilized using ethylene oxide (EtO).

[0119] Example 3 – Preparation of electrospun fibers composed of silk protein and hyaluronic acid, and layering them onto a nonwoven viscose / polyester substrate as a scaffold material. A 0.5% (w / v) formic acid-hyaluronic acid solution was prepared by dissolving hyaluronic acid in formic acid at 55 °C for 3 hours. After cooling the solution, 7.5% (w / v) silk fibroin was added, and the resulting mixture was stirred for 30 minutes. The solution was then subjected to electrospinning using previously optimized parameters, with a viscose / polyester substrate as the collection substrate. The silk fibroin / hyaluronic acid nanofibers were directly collected on the substrate after electrospinning, with a deposition time of approximately 12 minutes. The resulting sheets were cut using a laser cutter, and the cut samples were sterilized with ethylene oxide (EtO).

[0120] Example 4 - Preparation of electrospun fiber scaffold material composed of silk fibroin and hyaluronic acid 5 g of degummed silk fibroin was dissolved in 9.3 M lithium bromide solution and reacted at 60 °C for 4 hours. The solution was dialyzed against distilled water for 48 hours and adjusted to 6% (w / v). Hyaluronic acid was dissolved in PBS to prepare a 2% (w / v) solution, and low molecular weight hyaluronic acid (50–100 kDa) and high molecular weight hyaluronic acid (800–1200 kDa) were prepared separately. The silk fibroin and hyaluronic acid solutions were mixed in a 3:1 ratio with gentle stirring to form a homogeneous mixture. The SF-HA mixture was electrospun under the following parameters: Voltage: 15 kV.

[0121] Flow rate: 1 mL / hour.

[0122] Collector distance: 10 cm.

[0123] Nanofibers were deposited on release paper and dried at room temperature. The resulting matrix was cross-linked with vapor-phase glutaraldehyde for 12 hours to stabilize its structure. The resulting scaffold material is expected to exhibit excellent mechanical properties, water absorption and swelling capacity, and uniform fiber morphology, making it a promising scaffold material for tissue engineering applications.

[0124] Example 5 – Application of silk-HA scaffold materials in cell experiments The electrospun HA-silk protein matrix (1) prepared according to Example 4 was used. cm ²The sample was immersed in Durbeco modified Eagle's medium supplemented with 10% fetal bovine serum for 1 hour to reduce its hydrophobicity. It was expected that the substrate would absorb the medium and settle to the bottom of the culture dish. The substrate was then positioned in the culture dish with the bioactive HA-silk layer facing upwards to maximize contact with the seeded cells. 100,000 cells suspended in 500 µL of medium were added dropwise to the substrate surface. Cells were allowed to attach for 1–2 hours before adding the experimental medium, as follows: Experimental conditions: Control group (culture medium only): DMEM + 10% FBS.

[0125] PRP group: DMEM supplemented with platelet-rich plasma (10% PRP).

[0126] PRP-HA group: DMEM supplemented with platelet-rich plasma and hyaluronic acid (10% PRP-HA).

[0127] The effectiveness of the HA-silk matrix in biological experiments was then evaluated. Cell viability was assessed on days 7 and 15 using the Live / Dead Kit (Invitrogen L3224). Cell proliferation and death were observed using an imaging system.

[0128] Morphological analysis: Cell types tested: gingival fibroblasts (GF), periodontal ligament cells (PDL), and endothelial cells (HUVECs). Observations were recorded using phase-contrast microscopy.

[0129] This bioactive HA-silk matrix is ​​expected to exhibit biocompatibility and support cell adhesion, proliferation, and survival under various experimental conditions. The addition of PRP-HA is anticipated to enhance cellular responses, highlighting its potential for tissue engineering applications.

[0130] Example 6 - Cell Adhesion and Spreading Experiment The cell attachment and extension capabilities on the HA-Silk matrix were evaluated, as well as the structural alignment along the fiber direction. The HA-Silk matrix was prepared and rehydrated and electrospun as described in Example 5. The matrix was placed with the HA-Silk side facing up to optimize the interaction with the seeded cells.

[0131] Cell type: Primary cell lines used in the test: Gingival fibroblasts (GF).

[0132] Periodontal ligament cells (PDL).

[0133] Endothelial cells (HUVECs).

[0134] Experimental conditions: Control group: DMEM medium containing 10% fetal bovine serum (FBS).

[0135] PRP group: DMEM medium containing 10% platelet-rich plasma (PRP).

[0136] PRP-HA group: DMEM medium containing 10% PRP and 1% (w / v) hyaluronic acid.

[0137] Testing plan: Cells were cultured on the HA-Silk matrix for 7 and 15 days. Cell attachment and elongation were assessed using phase-contrast microscopy, with a focus on cell alignment along the fibers. The HA-Silk matrix is ​​expected to support strong cell attachment and promote structural alignment of cells along the fibers. PRP-HA treatment is expected to enhance cell elongation and network formation, highlighting the matrix's potential applications in tissue engineering.

[0138] Example 7 – Stability of the hyaluronic acid layer The stability of the HA layer in the HA-Silk matrix over time was evaluated under different culture conditions (control group, PRP group, and PRP-HA group). The HA-Silk matrix was prepared and electrospun using water as described in Example 5. In all experiments, the HA layer was facing upwards.

[0139] Experimental conditions: Control group: DMEM medium containing 10% fetal bovine serum (FBS).

[0140] PRP conditions: DMEM containing 10% platelet-rich plasma (PRP).

[0141] PRP-HA group: DMEM medium containing 10% PRP and 1% (w / v) hyaluronic acid.

[0142] Experimental plan: Under various culture conditions, the longest substrate culture time was 15 days. Observations were recorded on days 7 and 15, focusing on: the macroscopic integrity of the substrate; and the stability of the microstructure observed by phase contrast microscopy. Enhanced stability of the HA layer was expected under PRP-HA conditions, demonstrating the importance of combining PRP with hyaluronic acid. This property is expected to support the use of HA-Silk substrate as a robust scaffold for long-term tissue engineering applications.

[0143] Example 8 – Synergistic Effect of PRP-HA The combined effects of platelet-rich plasma (PRP) and hyaluronic acid (HA) cultured on HA-Silk matrix on cell proliferation, network formation, and bioactivity were evaluated. The HA-Silk matrix was prepared and rehydrated by electrospinning as described in Example 5. In all experiments, the HA-Silk layer was placed face up.

[0144] Cell type: Primary cell lines used in the test: Gingival fibroblasts (GF).

[0145] Periodontal ligament cells (PDL).

[0146] Endothelial cells (HUVECs).

[0147] Experimental conditions: Control group: DMEM medium containing 10% fetal bovine serum (FBS).

[0148] PRP group: DMEM medium containing 10% platelet-rich plasma (PRP).

[0149] PRP-HA group: DMEM medium containing 10% PRP and 1% (w / v) hyaluronic acid.

[0150] Experimental plan: Cells were cultured on the HA-Silk matrix for up to 15 days. Cell proliferation and network formation were assessed using high-throughput imaging and phase-contrast microscopy. The synergistic effect of PRP and HA is expected to significantly enhance the bioactivity of the HA-Silk matrix, promoting robust cell proliferation and network formation. This is expected to highlight the matrix's potential applications in tissue engineering and regenerative medicine.

[0151] Example 9 - Optimizing platelet lysis buffer to enhance bioactivity The effects of different platelet lysis buffer (PL) concentrations on the bioactivity of the HA-silk matrix were evaluated, with a focus on cell adhesion, proliferation, and elongation. The HA-silk matrix was prepared and electrospun using the method described in Example 5.

[0152] Platelet lysis buffer concentration: The experimental group consisted of substrates cultured in the following supplemental culture media: 0.25% (by volume) PL.

[0153] 0.5% (by volume) PL.

[0154] 1.0% (v / v) PL.

[0155] Control group: DMEM (without PL) with 10% FBS added.

[0156] Cell type: Testing was conducted using primary human cell lines: Gingival fibroblasts (GF).

[0157] Periodontal ligament cells (PDL).

[0158] Endothelial cells (HUVECs).

[0159] Experimental plan: Under the above conditions, the cells were cultured on HA-Silk substrate for 15 days.

[0160] Evaluation indicators: Cell viability (live and dead cell detection on days 7 and 15 (Invitrogen L3224)).

[0161] Adhesion and stretching (observed under a phase-contrast microscope).

[0162] Proliferation rate (MTT method).

[0163] The HA-Silk matrix is ​​expected to exhibit optimal bioactivity with the addition of 0.5% (v / v) platelet lysis buffer, thus supporting strong cell adhesion, extension, and proliferation. Higher concentrations (1.0% PL) are expected to lead to cell overcrowding, while lower concentrations (0.25% PL) are insufficient to enhance bioactivity. These results are expected to highlight the importance of optimizing platelet lysis buffer concentration for tissue engineering applications.

[0164] Example 10 - Performance Comparison of Support Layers This experiment aimed to evaluate the effects of different support materials on the structural integrity, cell viability, and manipulability of the HA-Silk matrix during bioassay. The electrospun HA-Silk matrix was prepared according to the method described in Example 5. During electrospinning, the matrix was deposited on different support materials.

[0165] Supporting materials for the test: LDPE / cellulose composites: high tensile strength, flexible support.

[0166] Polyethylene / paper composite material: a support material with medium rigidity, lightweight and anti-adhesion properties.

[0167] Reference substrate: standard anti-stick electrospun paper.

[0168] Evaluation indicators: Structural integrity: assessed by observing the shrinkage or tearing of the matrix during transfer and culture.

[0169] Peelability: Assess the ease with which the matrix can be peeled off the carrier without damage, with a qualitative score.

[0170] Cell viability and coverage were measured on days 7 and 15 using a live / dead cell assay kit (Invitrogen L3224) and a phase contrast microscope.

[0171] Cell type: Gingival fibroblasts (GF).

[0172] Endothelial cells (HUVECs).

[0173] Among the tested substrates, the polyethylene / paper composite material is expected to demonstrate superior performance in terms of structural integrity, ease of handling, and cell viability. This substrate is anticipated to be recommended for optimizing HA-Silk matrices in future tissue engineering applications.

[0174] Example 11 - Long-term stability and degradation of the matrix Under PRP-HA culture conditions, the long-term degradation (up to 30 days) of the HA-Silk matrix was monitored, with a focus on macroscopic and microscopic changes. The HA-Silk matrix was prepared and electrospun using the method described in Example 5. In all experiments, the HA layer was placed face up in the culture dish.

[0175] Experimental conditions: Control group: DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0176] PRP group: DMEM medium containing 10% platelet-rich plasma (PRP).

[0177] PRP-HA group: DMEM medium containing 10% PRP and hyaluronic acid (1% w / v).

[0178] Evaluation indicators: Macroscopic observation: Record shrinkage, discoloration, or surface unevenness on days 7, 15, and 30.

[0179] Microscopic analysis: Surface morphology was observed using a phase contrast microscope.

[0180] Fiber degradation and cell coverage were observed using confocal microscopy.

[0181] Mass loss: Measured percentage reduction in matrix weight over 30 days.

[0182] The HA-Silk matrix is ​​expected to exhibit excellent long-term stability under PRP-HA conditions, with extremely low degradation and maintained structural integrity within 30 days. These findings highlight its potential for long-term tissue engineering applications.

[0183] Example 12 - Preparation of multilayer constructs A multiphase construct simulating osteochondral tissue was prepared and characterized (physicochemical and structural properties) using advanced technologies (electrospinning and 3D printing).

[0184] Covering layer (silk protein): Extracted from silkworm cocoons, it is dissolved in a 9.3 M lithium bromide solution after degumming. It is known for its high tensile strength, biocompatibility, and ability to support cell adhesion.

[0185] Hydrogel layer: Element: Hyaluronic acid (HA): Enhances water retention and promotes cell proliferation.

[0186] Gelatin: A natural protein that promotes cell attachment and differentiation.

[0187] Silk protein: Provides structural integrity and enhances mechanical strength.

[0188] Platelet lysate proteins: a source of growth factors required for cartilage formation.

[0189] Electrospun layer: Polycaprolactone (PCL): A biodegradable polymer with high mechanical strength and slow degradation.

[0190] Hydroxyapatite: A mineral component that mimics the calcified cartilage layer.

[0191] FDM layer: A composite material composed of PCL and hydroxyapatite was used to simulate the load-bearing characteristics of subchondral bone.

[0192] Preparation method: Cover layer (50 μm): Electrospin a 7% (w / v) silk protein solution onto a rotating collector.

[0193] Electrospinning parameters: Voltage: 15 kV.

[0194] Flow rate: 0.8 mL / h.

[0195] Collector distance: 10 cm.

[0196] The layer is stabilized by crosslinking with ethanol vapor.

[0197] Hydrogel layer (1–2 mm): Mix HA (1% w / v), gelatin (2% w / v) and silk fibroin (3% w / v) in PBS at 37 °C.

[0198] Add platelet lysate protein (0.5% v / v) to the mixture.

[0199] The hydrogel was injected into the mold and crosslinked with amygdalin for 24 hours to enhance stability.

[0200] Electrospun layer (100 μm): Electrospinning was performed on a chloroform / dimethyl sulfoxide (DMF, 1:1) solution of polycaprolactone (PCL, 10% w / v) and hydroxyapatite (2% w / v).

[0201] Electrospinning parameters: Voltage: 18 kV.

[0202] Flow rate: 1.2 mL / h.

[0203] Collector distance: 12 cm.

[0204] FDM layer (500 μm): Polycaprolactone (PCL) and hydroxyapatite (in a 70:30 ratio) were extruded using a fused deposition modeling (FDM) 3D printer. The bottom layer was printed at a nozzle temperature of 180 °C and a layer height of 100 μm.

[0205] Each layer is expected to exhibit properties that mimic the characteristics of different regions of natural osteochondral tissue, thereby ensuring structural integrity and biocompatibility.

[0206] Example 13 - Multilayer constructs integrating hydroxyapatite-silk protein This study evaluated the efficacy of integrating HA-silk matrix into a multilayer construct for cartilage repair, and assessed its role in promoting cell proliferation, matrix integration, and biomechanical stability. A multilayer construct consisting of the following components was prepared: Layer 1 (Covering Layer): Electrospun silk protein (SF).

[0207] Layer 2 (hydrogel layer): a mixture of HA-silk protein matrix, gelatin, and platelet lysate protein (0.5% v / v).

[0208] The third layer (electrospinning transition layer): polycaprolactone (PCL) and hydroxyapatite.

[0209] Layer 4 (FDM base layer): 3D printed PCL and hydroxyapatite.

[0210] Integration of HA-silk protein layer: HA-silk protein matrix was embedded in the hydrogel layer to mimic cartilage properties, and its bioactivity was enhanced by supplementing it with PRP-HA.

[0211] Cell type: Human chondrocytes were seeded onto the construct to assess cell integration and proliferation.

[0212] Testing plan: Integration test: Interlayer adhesion was evaluated using a mechanical shear test.

[0213] Cell proliferation: measured by MTT assay at days 7, 15 and 30.

[0214] Matrix bioactivity: ELISA was used to analyze the formation of cell networks and the release of growth factors.

[0215] Biomechanical stability: Compressive strength and modulus are evaluated using a uniaxial testing machine.

[0216] The HA-Silk matrix is ​​expected to be successfully integrated into the multilayer construct, potentially enhancing bioactivity, cell proliferation, and biomechanical stability. This construct holds promise for cartilage repair applications, particularly in cases of degenerative or traumatic lesions.

[0217] Example 14 – Evaluation of Multi-Layer Constructs The biocompatibility, cell adhesion, proliferation and differentiation of the multiphase constructs prepared according to Example 12 were evaluated under different culture conditions using chondrocytes and mesenchymal stem cells (MSCs).

[0218] Cell type: Chondrocytes: obtained from human articular cartilage biopsy samples.

[0219] MSCs: Obtained from bone marrow aspiration fluid.

[0220] Culture medium conditions: Control group: DMEM + 10% fetal bovine serum (FBS).

[0221] PRP: 10% platelet-rich plasma added to DMEM culture medium.

[0222] PRP-HA: DMEM medium supplemented with 10% platelet-rich plasma and 1% (w / v) hyaluronic acid.

[0223] Preparation of cell culture substrate: The multiphase constructs were sterilized with ethylene oxide gas and rehydrated in the culture medium for 1 hour before cell inoculation. The constructs were placed with the capping layer (silk fibroin) facing upwards to promote initial cell attachment.

[0224] Testing plan: Cell adhesion (24 hours): Cells were seeded onto the construct (1 × 10⁻⁶). 5 (cells / cm²), and cultured for 24 hours.

[0225] Cell adhesion was assessed using phase contrast microscopy and scanning electron microscopy (SEM).

[0226] Proliferation (7 days and 14 days): Cellular metabolic activity was assessed using the WST-1 assay.

[0227] Chondrogenesis (day 21): The constructs inoculated with mesenchymal stem cells (MSCs) were cultured in chondrogenic medium.

[0228] Glycosaminoglycan (GAG) formation was assessed by Alsin blue staining and quantitative analysis was performed using the dimethyl methylene blue (DMMB) method.

[0229] PRP-HA treatment is expected to significantly enhance cell adhesion, proliferation, and chondrogenesis on the multiphase construct. The hydrogel and electrospun layer are anticipated to play key roles in maintaining bioactivity, demonstrating its potential application in osteochondral tissue regeneration. This multiphase construct is expected to support high levels of cellular bioactivity, especially in the presence of PRP-HA. The findings are expected to highlight its applicability in cartilage repair applications.

[0230] Example 15 – Mechanical Testing of Multilayer Structures The preparation and sterilization methods for the multiphase constructs are as described in Example 12. The samples were cut into discs (8 mm in diameter and 2 mm in thickness) for mechanical testing.

[0231] Mechanical testing plan: Compression test (monolithic construct): The test was conducted using a universal testing machine.

[0232] The structure is compressed at a rate of 1 mm / min until 50% deformation is achieved.

[0233] Measurement parameters: Compressive strength (MPa).

[0234] Compressive modulus (MPa).

[0235] Layered tensile test: Each layer (cover layer, hydrogel layer, electrospun layer, FDM layer) was carefully separated and subjected to uniaxial tensile tests.

[0236] Record the tensile strength and elastic modulus of each layer.

[0237] Inter-layer integration testing: The adhesive strength between the layers was evaluated by applying shear stress to the sample using a custom shear test apparatus.

[0238] Dynamic Mechanical Analysis (DMA): Used to evaluate the viscoelastic properties of materials under cyclic loading.

[0239] The construct is subjected to dynamic loads at a frequency of 1 Hz to simulate physiological conditions.

[0240] The construct is expected to exhibit excellent mechanical properties, with compressive and tensile strengths comparable to those of natural osteochondral tissue. Interlayer integration testing aims to verify strong adhesion between the layers, which is crucial for maintaining structural stability under mechanical loads.

[0241] This multiphase construct is expected to exhibit biomechanical properties suitable for load-bearing applications, making it an ideal candidate material for osteochondral tissue repair.

[0242] Example 16 – In vivo evaluation of multilayer constructs The performance of multiphase constructs in promoting osteochondral repair was evaluated using preclinical animal models.

[0243] Animal models: Species: New Zealand White Rabbit (n = 15).

[0244] Defect model: A full-thickness osteochondral defect (4 mm in diameter and 2 mm in depth) was prepared at the femoral condyle under aseptic conditions.

[0245] Builder group: Group 1: Control group (no implant).

[0246] Group 2: Multiphase constructs without PRP.

[0247] Group 3: Multiphase constructs containing PRP-HA.

[0248] Surgical procedure: Tissue-engineered materials are sterilized with ethylene oxide and rehydrated in sterile PBS before implantation.

[0249] Fill the defect with tissue engineering material to ensure complete coverage of the defect area.

[0250] Fibrin glue is used to fix the tissue in place to ensure it remains in position during the healing process.

[0251] Postoperative care: The rabbits were observed for 12 weeks after the surgery.

[0252] Assess weight-bearing and activity levels weekly to evaluate functional recovery.

[0253] Evaluation methods: Histological analysis: Rabbits were euthanized at weeks 4, 8, and 12, and the femoral condyles were removed for histological analysis. Sections were stained with Safranin O / Fast Green to assess cartilage regeneration and integration.

[0254] MicroCT Imaging: The study was conducted in week 12 to assess bone regeneration and implant degradation.

[0255] Biomechanical testing: Indentation tests were performed on the repaired tissue to measure its stiffness and compare it with that of the original cartilage.

[0256] Compared to the control group and the group without PRP, the PRP-HA-enhanced multiphase construct is expected to significantly improve cartilage and bone regeneration. The layered design is expected to effectively mimic native osteochondral tissue, supporting cell growth, matrix deposition, and mechanical function. This in vivo study is expected to demonstrate the potential of multiphase constructs in osteochondral repair, especially when combined with PRP-HA. Its biomimetic properties and clinical-grade fabrication process make it a promising candidate for human cartilage repair.

[0257] Example 17 - Controlled release of growth factors from hydrogel layers To assess the ability of the hydrogel layer to continuously release platelet-derived growth factors (PDGFs).

[0258] Hydrogel preparation: The hydrogel layer is composed of hyaluronic acid (HA, 1% w / v), gelatin (2% w / v), silk fibroin (3% w / v), and platelet lysate protein (0.5% v / v).

[0259] Cross-linking with amygdalin for 24 hours stabilized the network structure and regulated protein release.

[0260] Experimental setup: Prepare hydrogel discs (5 mm in diameter and 1 mm in thickness) and immerse them in phosphate-buffered saline (PBS) at 37°C.

[0261] Samples were transferred to fresh PBS at preset time points (1, 3, 7, 14 and 21 days) to simulate physiological conditions.

[0262] Quantitative analysis of growth factors: Growth factors (such as PDGF, TGF-β1, and VEGF) released into PBS were quantitatively determined using enzyme-linked immunosorbent assay (ELISA). The release characteristics were assessed by plotting a cumulative release percentage curve over time.

[0263] The hydrogel layer of the multiphase construct is expected to exhibit excellent growth factor release properties, releasing bioactive signals in a controlled manner over 21 days. This property is expected to enhance the construct's potential to promote osteochondral regeneration.

[0264] Example 18: Preparation of silk fibroin / hyaluronic acid bio-dressing Silk fibroin is derived from silkworm cocoons. The cocoons are degummed using a standard boiling method, followed by rinsing and air-drying. The dried silk fibroin is dissolved in formic acid to prepare a solution with a final concentration of 7.5% (w / v). Separately, hyaluronic acid with a molecular weight of 1550 kDa is dissolved in formic acid to prepare a solution with a final concentration of 0.5% (w / v). The two solutions are mixed and stirred under controlled conditions until a homogeneous mixture is formed. The final composition, by weight, is 93.75% silk fibroin and 6.25% hyaluronic acid.

[0265] The mixture was then electrospun to prepare nanofiber membranes. The electrospinning process was performed under optimized conditions to ensure uniform fiber formation. Experimental settings included a 15 kV voltage, a flow rate of 1 mL / h, and a collector distance of 10 cm. The nanofibers were deposited on a medical-grade support layer, which was chosen for its compatibility with biological applications and ease of handling in subsequent assays.

[0266] After preparation, the fiber membrane was dried at room temperature and then sterilized. No cross-linking agents were added throughout the process to ensure the preservation of the hyaluronic acid's bioactivity. The resulting dressing was stored under sterile conditions for subsequent testing.

[0267] Example 19: Cell Viability Assessment Protocol on Silk Fibroin / Hyaluronic Acid Matrix The silk fibroin / hyaluronic acid matrix prepared according to Example 18 was rehydrated before use for cell viability assay. The matrix was immersed in Durbeco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified incubator at 37°C for one hour. After rehydration, the matrix was placed in each well of a six-well plate with the bioactive layer facing upwards to maximize cell interaction.

[0268] This experiment used normal human dermal fibroblasts (NHDFs) for detection. Cells were directly seeded onto the rehydrated substrate surface at a density of 50,000 cells per square centimeter. A medium consisting of DMEM and 10% FBS was then carefully added to ensure complete coverage of the substrate surface. The seeded substrate was then cultured in a humidified environment of 37°C and 5% CO2 for up to seven days. The medium was changed every 48 hours to maintain optimal culture conditions.

[0269] At designated time points (e.g., days 1, 3, and 7), samples were removed from the incubator, gently washed with phosphate-buffered saline (PBS) to remove unattached cells, and then subjected to subsequent viability analysis according to the "live / dead" assay protocol. This assay involves staining the matrix with calcein-AM to label live cells and labeling dead cells with pyrimidine dimer-1. Staining solutions were prepared according to the manufacturer's instructions, and the matrix was incubated with the staining solutions at room temperature in the dark for 30 minutes.

[0270] The stained matrix was then observed using a fluorescence microscope, and images were acquired for subsequent analysis. This protocol ensures a standardized and reproducible method for assessing the viability of matrix-supported cells.

[0271] Example 20: Rehydration scheme for HA-silk fibroin matrix The silk fibroin / hyaluronic acid matrix prepared according to the description in Example 18 was rehydrated to make it suitable for bioassays. Under aseptic conditions, 1 cm² square samples were cut from the electrospun fiber membrane. These samples were immersed in Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C for 1 hour in a humidified environment containing 5% CO2.

[0272] During incubation, the substrate absorbs the culture medium, transforming from an initially dry and rigid state into a moist and flexible form suitable for cell seeding. Throughout the rehydration process, ensure the bioactive side of the substrate is facing upwards to maximize the contact area for subsequent biological interactions. The rehydrated substrate is then transferred to sterile six-well plates using forceps, taking care to avoid physical damage to the delicate fibers.

[0273] This rehydration protocol provides a standardized and reproducible method for preparing HA-silk fibroin matrices for cell-based assays or other biological applications.

[0274] Example 21: Stability assessment of HA-silk fibroin matrix under culture conditions To assess the physical integrity of the glycolic acid-silk fibroin matrix over time, its stability under various culture conditions was evaluated. Matrix samples were prepared as described in Example 18, rehydrated as described in Example 20, and placed in six-well plates. Each sample was placed under one of the following conditions: (1) Durbeco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (control group), (2) DMEM supplemented with platelet-rich plasma (PRP), and (3) DMEM supplemented with both PRP and hyaluronic acid (HA) (PRP-HA).

[0275] The substrate was cultured at 37°C in a humidified environment containing 5% CO2. Swelling, structural integrity, and detachment from the support layer were recorded on days 1, 3, 7, and 15. Prior to visual evaluation, the substrate was gently rinsed with phosphate-buffered saline (PBS) to remove any unattached cells or debris.

[0276] The evaluation included recording physical changes, such as swelling rate, fiber morphology, and any stratification of the matrix under test conditions. This protocol helps to understand the suitability of HA-silk fibroin matrix for long-term culture in different bioactive environments.

[0277] Example 22: Preparation of HA-silk fibroin matrix with platelet lysis buffer Platelet lysis buffer (PL) was added to the silk fibroin / hyaluronic acid matrix to enhance its bioactivity. The platelet lysis buffer was prepared using a commercially available product at concentrations of 0.25% and 0.5% (v / v). The experimental culture medium was prepared by mixing PL with DMEM containing 10% fetal bovine serum (FBS).

[0278] The HA-silk fibroin matrix samples prepared according to Example 18 were rehydrated under aseptic conditions using PL-enriched culture media. The matrix was immersed in PL-enriched culture medium and soaked for one hour in a humidified environment at 37°C with 5% CO2. After rehydration, the matrix was placed in a six-well plate with the bioactive side facing up.

[0279] This preparation method, while maintaining the integrity of the silk protein and hyaluronic acid components, allows us to study the enhancement of matrix bioactivity by adding platelet lysis buffer.

[0280] Example 23: Standard method for preparing silk fibroin solution from silkworm cocoons Silk fibroin was extracted from silkworm cocoons using a standard degumming and dissolving process. First, 5 grams of cocoon were cut into small pieces and degummed in 2 liters of 0.02 M sodium carbonate solution for 30 minutes using boiling water. This step effectively removed the sericin while preserving the silk fibers. The degummed silk fibers were then rinsed three times with 1 L of deionized water for 20 minutes each time, followed by squeezing to remove excess water. The fibers were dried overnight in a fume hood and stored at room temperature for later use.

[0281] To facilitate dissolution, dried silk fibroin fibers were dissolved in a 20% (w / v) 9.3 M lithium bromide solution. The mixture was heated to 60°C and stirred for 4 hours until a clear silk fibroin solution was obtained. Subsequently, the solution was desalted using a dialysis membrane with a molecular weight cutoff of 12-14 kDa through deionized water. The dialysis process involved changing the water six times over 48 hours. After dialysis, the sericin solution was centrifuged at 9,000 rpm (12,700 g) for 20 minutes at 4°C, repeated three times, to remove any insoluble particles.

[0282] The resulting silk fibroin solution, with a concentration of 7–8% (w / v), was stored at 4°C for subsequent scaffold preparation. This process can stably produce high-quality silk fibroin, suitable for applications in biomaterials research and tissue engineering.

[0283] Example 24: Microwave-assisted preparation of silk fibroin solution Silk fibroin was extracted from silkworm cocoons using a microwave-assisted degumming process. Two grams of cocoon were cut into small pieces and subjected to three rounds 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 microwave-heating at 800 W for 1 minute, followed by standing for 10 minutes (at approximately 80°C). After each cycle, the fibers were rinsed in deionized water for 1 minute and then squeezed to remove excess liquid. After three cycles, the degummed fibers were either dried overnight or used directly for dissolution.

[0284] For dissolution, 0.1 g of degummed silk was immersed in 10 mL of a 56% (w / w) aqueous solution of zinc chloride (ZnCl2) at 45°C for 1 hour. Subsequently, the resulting 1% (w / v) silk fibroin solution was desalted using a CentriPure P50 column to remove zinc chloride. This process was repeated to ensure complete removal of ZnCl2, and verified by measuring absorbance at 560 nm using a zinc assay kit. The eluted silk fibroin solution could be used immediately or lyophilized for storage.

[0285] This microwave-assisted method provides a rapid and reliable approach for preparing silk fibroin solutions suitable for a variety of biomedical applications.

[0286] Example 25: Preparation of HA-Silk Fiber Matrix with Platelet Lysis Buffer Platelet lysis buffer (PL) was added to the silk fibroin / HA matrix at concentrations of 0.25% and 0.5% (v / v). The platelet lysis buffer was prepared using a commercially available PLT MAX kit. The silk fibroin / HA mixture (7.5% (w / v) silk fibroin and 0.5% (w / v) HA) was rehydrated in DMEM containing the appropriate concentration of PL. The rehydration was performed at 37°C for one hour to ensure complete absorption of the platelet lysis buffer by the matrix.

[0287] Subsequently, the rehydrated HA-silk fibroin matrix, supplemented with platelet lysis buffer, was used for cell viability and proliferation assays to evaluate its bioactivity.

[0288] Example 26: Preparation of PRP and PRP-HA Peripheral blood samples were collected from healthy donors after obtaining ethical approval (Geneva CCER Ethics Committee, ID 2017-00700). A total of 30 ml of blood was collected into specialized medical equipment, including Regen Lab's CuteCell PRP and CellularMatrix ACP-HA tubes. The blood samples were centrifuged at 1500 g for 5 minutes at room temperature. After centrifugation, red blood cells and white blood cells settled below the separating gel, while plasma and platelets remained on the upper layer.

[0289] Plasma and platelets were thoroughly mixed by inverting the test tubes five times. Each CuteCell PRP tube yielded 6 mL of PRP, while each CellularMatrix ACP-HA tube provided a mixture of 3 mL PRP and 2 mL hyaluronic acid (HA). Platelet, erythrocyte, and leukocyte concentrations, as well as mean platelet volume (MPV), were confirmed to be within the ranges previously reported in the literature.

[0290] The prepared PRP and PRP-HA solutions were stored in polypropylene tubes for use in biological assays.

[0291] Example 27: Cell viability assessment protocol for silk fibroin / hyaluronic acid bio-dressing Silk fibroin / hyaluronic acid bio-dresses were prepared according to the method described in the previous example, with the addition of platelet lysis buffer at concentrations of 0.25% (v / v) and 0.5% (v / v). These dressings were not fitted with a supporting membrane and were sterilized before use. Due to the uniform mixing of silk fibroin and hyaluronic acid during preparation, the cell attachment surface of the dressings exhibited a uniform state.

[0292] Cell viability was assessed using periodontal ligament cells (PDL, passage 3), gingival fibroblasts (GF, passage 3), and myoblasts (passage 3). Cells were harvested via engraftment and stored in liquid nitrogen prior to the experiment. Cell viability was assessed at four time points: 1, 2, 3, and 4 weeks using a Live / Dead cell viability assay kit.

[0293] In the experiment, the biological dressings were placed in 24-well black microplates (uncoated), one piece per well. Each dressing was rehydrated for 30 minutes at 37°C with 500 µL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. After rehydration, the culture medium was aspirated, ensuring the dressing remained moist. A suspension of 150,000 cells was seeded onto each dressing, with the droplet placed in the center to ensure even spreading. The culture plates were incubated in a humid environment at 37°C and 5% CO2 to promote cell attachment.

[0294] At each time point, the dressing was incubated with the staining solution according to the manufacturer's instructions for live / dead staining. Stained samples were observed using a Cytation 7 confocal microscope. Additionally, samples were fixed for scanning electron microscopy (SEM) observation to monitor matrix integrity and degradation over time. Quantitative analysis of green and red signals was performed using image analysis software, and matrix degradation was assessed by visual observation and SEM.

[0295] Example 28: Survival and adhesion assessment of human umbilical vein endothelial cells (HUVECs) on silk fibroin / hyaluronic acid nanofibers Silk fibroin / hyaluronic acid nanofibers were prepared as described in previous examples, and their cell viability and adhesion were evaluated using human umbilical vein endothelial cells (HUVECs, P5). Four support layers were tested, each coated with SF-HA nanofibers: LDPE / cellulose with nanofibers coated on the cellulose side, LDPE / cellulose with nanofibers coated on the LDPE side, polyethylene paper with nanofibers coated on the polyethylene side, and polyethylene paper with nanofibers coated on the paper side.

[0296] Each sample was placed under five different culture conditions: control medium, 10% PRP, 10% (w / v) PRP-HA, and 10% (v / v) PL. The dressings were observed using Cytation imaging, and the samples were inverted to assess cell-nanofiber interactions.

[0297] Sample 1, composed of LDPE / cellulose composite material with nanofibers attached to its cellulose side, floated in the culture medium due to the foaming properties of the support layer and failed to support endothelial cell adhesion under any conditions. Similarly, Sample 2, also composed of LDPE / cellulose composite material with nanofibers attached to its LDPE side, exhibited floating behavior and no significant endothelial cell adhesion was observed. In contrast, Sample 3, composed of polyethylene paper with nanofibers on its polyethylene side, supported endothelial cell adhesion and survival under PRP, PRP-HA, and PL culture conditions. However, no significant cell adhesion was observed under control culture conditions. Sample 4, composed of polyethylene paper with nanofibers on its paper side, exhibited strong endothelial cell adhesion and survival under PRP, PRP-HA, and PL conditions, and the cell sheet remained attached after a peel test.

[0298] In summary, samples 3 and 4 provided optimal conditions for endothelial cell attachment and survival, especially in the presence of PRP, PRP-HA, and PL. Samples 1 and 2 were unsuitable due to their buoyancy and lack of cell attachment ability.

[0299] Example 29: Evaluation of silk fibroin / hyaluronic acid matrix in vaginal fibroblasts and periodontal ligament cells Silk fibroin and hyaluronic acid matrices were prepared as previously described. Cell viability, adhesion, and proliferation of these matrices were tested using vaginal fibroblasts (VF) and periodontal ligament cells (PDL). Experiments included various culture conditions: control medium, 10% platelet-rich plasma (PRP), and PRP supplemented with 10% (w / v) hyaluronic acid (PRP-HA).

[0300] In the evaluation of vaginal fibroblasts, 1 cm² fragments of SF-HA matrix were placed in tissue culture dishes. Each matrix fragment was seeded with 100,000 cells and cultured under specified conditions. After 15 days of culture, cell viability was assessed using a Live / Dead Kit. Calcein fluorescence was used to identify live cells, while pyrimidine dimer-1 fluorescence was used to detect dead cells. Microscopic observation showed that vaginal fibroblasts exhibited strong adhesion and proliferation at 10% PRP-HA, exhibiting significant elongation and alignment along the matrix fibers. Very few dead cells were observed under all experimental conditions. However, fibroblasts in the control group showed weaker proliferation and no significant elongation. At 10% PRP (w / v), fibroblasts exhibited moderate proliferation and elongation.

[0301] Researchers also conducted similar experiments using periodontal ligament cells. In this experiment, the SF-HA matrix was evaluated under the same conditions. Observations after 15 days showed that cell adhesion and proliferation were good under the 10% (w / v) PRP-HA condition, with cells elongating and aligning neatly. In contrast, cell adhesion was sparse in the control group, while the 10% PRP group showed moderate cell proliferation but no significant cell elongation. Notably, the PRP-HA group was particularly effective in maintaining the structural integrity of the HA layer within the matrix, as evidenced by uniform cell coverage and no shedding of the electrospun HA layer.

[0302] Synthetic data indicate that 10% (w / v) PRP-HA provides an optimal environment for cell adhesion, proliferation, and alignment of vaginal fibroblasts and periodontal ligament cells. This highlights the bioactivity potential of the SF-HA matrix after PRP and HA supplementation.

[0303] Example 30: Evaluation of myoblast differentiation on silk fibroin / hyaluronic acid bio-dress supplemented with platelet lysate and platelet-rich plasma. Silk fibroin / hyaluronic acid bio-dressing (SF-HA) was prepared according to previously described methods, and its ability to support myoblast differentiation under different culture conditions was evaluated. This study compared differentiation outcomes obtained using 10% (v / v) platelet lysis buffer (PL), 10% (w / v) platelet-rich plasma (PRP), and a combination of 10% (w / v) hyaluronic acid and platelet-rich plasma (HA-PRP), with standard differentiation medium (DM) as a reference control. Primary human myoblasts (P5) were seeded on the SF-HA matrix and cultured for three weeks under the aforementioned conditions.

[0304] Experimental setup First, the substrate was rehydrated for 30 minutes in a humidified CO2 incubator using Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After rehydration, 150,000 myoblasts suspended in 500 µL of medium were seeded onto each dressing and allowed to attach for 30 minutes. Subsequently, 500 µL of the appropriate treatment medium was added, including DM, 10% PL (v / v), 10% PRP (w / v), and 10% HA-PRP (w / v).

[0305] Results three weeks later At week 3, samples were fixed with 4% paraformaldehyde for immunofluorescence staining and confocal microscopy. Immunostaining was performed using myoblast differentiation-specific markers, such as Mef2C and α-actin.

[0306] 1.10% HA-PRP (weight / volume): The HA-PRP group showed the most significant differentiation results. Confocal microscopy revealed the formation of numerous well-arranged, multinucleated, and structurally intact myotubes. The myotubes exhibited significant elongation and alignment along the SF-HA matrix, reflecting enhanced myogenic differentiation. The presence of hyaluronic acid appeared to enhance the bioactivity of PRP, producing a synergistic effect that maximized differentiation capacity.

[0307] 2. PRP 10% (w / v): Samples treated with PRP alone showed strong differentiation capacity, with clear evidence of myotube formation and alignment. However, compared with the HA-PRP 10% condition, the elongation and structural organization were slightly reduced, suggesting that while PRP effectively supports myogenesis, the lack of structural advantage from HA may limit optimal differentiation.

[0308] 3. PL 10% (v / v): Although signs of differentiation were visible, the effect was significantly lower than that under PRP and HA-PRP conditions. Myotube arrangement was less regular, and structural integrity was reduced. The above limitations of PL can be attributed to the use of formic acid in the preparation process. As previously mentioned, formic acid may weaken the biological activity of growth factors in PL through protein denaturation, pH instability, and peptide bond hydrolysis.

[0309] 4. Differentiation medium (DM): The control group showed the lowest differentiation capacity. Confocal microscopy images showed sparse and irregularly arranged myotubes, confirming that SF-HA matrix alone could not effectively promote myogenesis in the absence of supplemental bioactive factors.

[0310] in conclusion This study demonstrates that 10% (w / v) HA-PRP treatment provides the most effective environment for myoblast differentiation, significantly outperforming 10% (w / v) PRP, 10% (v / v) PL, and the control differentiation medium. The combination of HA and PRP enhances myoblast alignment, elongation, and myotube formation by creating a synergistic microenvironment that supports cell attachment and differentiation. In contrast, platelet lysate prepared with formic acid reduces differentiation, highlighting the importance of preserving growth factor bioactivity during preparation. These findings establish 10% (w / v) HA-PRP as the preferred treatment for promoting myoblast differentiation and underscore the necessity of optimizing platelet lysate preparation to avoid loss of bioactivity.

Claims

1. A scaffold material comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers comprising silk proteins.

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

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

4. The stent 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 those selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or combinations thereof, particularly another therapeutic agent selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof.

7. The support material according to any one of claims 1 to 6, wherein it is a semi-solid support material.

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

9. The support material according to claim 8 is a solid support material.

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

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

14. The stent 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 stent material according to any one of claims 10 to 15, further comprising one or more therapeutic agents, such as those selected from steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, and tetrahydrocannabinol (THC). Cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly another therapeutic agent selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captocopherol, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof.

17. The stent material according to any one of claims 10 to 16, wherein it is a solid stent material.

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

19. The scaffold material according to claim 18, which has been stabilized by crosslinking, for example using an aldehyde crosslinking agent (such as glutaraldehyde, for example, gas phase method).

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

3.

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

24. The stent 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 stent material according to any one of claims 18 to 25, further comprising one or more therapeutic agents, such as those selected from steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, and tetrahydrocannabinol (THC). Cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly selected from the group comprising corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captocopherol, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof, as another therapeutic agent.

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

28. The layered wound dressing or layered graft of claim 27, comprising: i) A support material comprising: ia) Electrospun fibers containing silk proteins; as well as ii) Hyaluronic acid; as well as ii) A support material.

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

30. The layered structure of claim 29, wherein the thickness of the first layer comprising silk protein is between about 40 μm and about 60 μm, for example about 50 μm.

31. The layered structure according to claim 29 or claim 30, wherein the first layer comprising silk fibroin is a covering layer.

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

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

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

36. The layered structure according to any one of claims 29 to 35, wherein the silk fibroin in the second layer is composed of silk fibroin electrospun fibers.

37. The layered structure according to claim 36, wherein the sericin electrospun fibers are prepared by electrospinning a sericin solution at a concentration of about 5% (w / v) to 15% (w / v) (e.g., about 7% (w / v)); at a voltage of 12-18 kV, for example about 15 kV; at a flow rate of about 0.8 mL / h to about 1.2 mL / h, for example 0.8 mL / h; and at a collector distance of 5-20 cm, for example about 10 cm.

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

39. The layered structure 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, particularly platelet lysate.

40. The layered structure 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 structure 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 structure according to claim 40, wherein the third layer comprises polycaprolactone and hydroxyapatite in a mass ratio of 80:

20.

43. The layered structure according to any one of claims 29 to 42, wherein the thickness of the third layer is 90 μm to 110 μm.

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

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

30.

46. ​​The layered structure according to any one of claims 29 to 45, wherein the thickness of the fourth layer is 450 μm to 550 μm, for example about 500 μm.

47. The scaffold material, layered wound dressing, layered graft or layered structure according to any one of claims 1 to 46, wherein the silk fibroin is fibroin, suitably obtained from a solution containing dissolved silk (e.g., silkworm silk).

48. The scaffold material, layered wound dressing, layered graft, or layered structure according to any one of claims 1 to 47, wherein the diameter of the electrospun fibers is between about 50 nm and about 1,000 nm, for example, between about 200 nm and about 500 nm.

49. The support material according to any one of claims 1 to 26, 47 or 48, wherein it is in the form of a thin film, membrane, pad or mesh structure.

50. The stent material according to any one of claims 1 to 26, or claims 47 to 49, wherein the expansion rate is at least 300%.

51. A tissue engineering construct, comprising: i) The scaffold material, layered wound dressing, layered graft, or layered structure according to any one of claims 1 to 50; as well as ii) Mammalian cells.

52. The tissue-engineered construct according to claim 51, wherein the mammalian cells are selected from hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal cells, bile duct cells, parathyroid cells, thyroid cells, adrenal-hypothalamic-pituitary axis cells, cardiomyocytes, renal epithelial cells, renal tubular cells, renal basement membrane cells, nerve cells, vascular cells, bone and cartilage forming cells, smooth muscle cells, skeletal muscle cells, cochlear cells, epidermal cells, bone marrow cells, keratinocytes, pluripotent cells, and stem cells, and combinations thereof.

53. A method for preparing tissue-engineered constructs, comprising the following steps: a) Prepare a scaffold material according to any one of claims 1 to 50; b) Mix the scaffold material with mammalian cells in a suitable culture 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 fibers comprising silk proteins; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Mixing electrospun fibers containing silk proteins with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material.

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

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

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

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

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

60. The method of any one of claims 54 to 59, wherein in step c), the electrospun fibers containing silk protein are mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and a coagulation activator (e.g., calcium gluconate) to form a scaffold material.

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

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

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

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

65. The method of claim 63 or claim 64, wherein the one or more therapeutic agents are selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly the other therapeutic agent being selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof.

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

67. The method according to any one of claims 54 to 66, wherein in step c), the electrospun fibers containing silk protein are combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and 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 fibers comprising silk proteins; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

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

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

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

72. The method according to any one of claims 68 to 71, wherein in step b), the silk protein mixture from step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and 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 from step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and one or more other therapeutic agents to form a mixture.

74. The method of claim 73, wherein the one or more therapeutic agents are selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly the other therapeutic agent being selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof.

75. The method according to any one of claims 68 to 74, wherein in step b), the silk protein mixture from step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and 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), namely, adding an antifibrinolytic substance (e.g., 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 (e.g., calcium gluconate) to the stent 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 other therapeutic agents to the stent material in step c).

81. The method of claim 80, wherein the one or more therapeutic agents are selected from the group comprising steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), kartogenin, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, and vitamins (such as vitamin A, vitamin E, vitamin B1, etc.). Vitamin C, vitamin D; or derivatives thereof) or a wrinkle-reducing filler, or a combination thereof, particularly another therapeutic agent selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captocopherol, hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof.

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

83. A method for preparing a scaffold material comprising i) hydroxyapatite and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hydroxyapatite to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

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

85. The method of claim 83 or claim 84, wherein in step b), the silk protein solution of step a) is mixed 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 from step a) is mixed with hydroxyapatite and an antifibrinolytic substance (e.g., 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 from step a) is mixed with hydroxyapatite and a coagulation activator (e.g., 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 from step a) is mixed 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 from step a) is mixed with hydroxyapatite and one or more other therapeutic agents to form a mixture.

90. The method of claim 89, wherein the one or more therapeutic agents are selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly the other therapeutic agent being selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations 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 in 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 (e.g., 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 (e.g., calcium gluconate) to the stent 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 other therapeutic agents to the stent material of step c).

97. The method of claim 96, wherein the one or more therapeutic agents are selected from the group comprising steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), kartogenin, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, and vitamins (such as vitamin A, vitamin E, vitamin B1, etc.). Vitamin C, vitamin D; or derivatives thereof) or anti-wrinkle fillers, or combinations thereof, particularly further therapeutic agents selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captocopherol, hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof.

98. A method for preparing a scaffold material comprising: i) hydroxyapatite, and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Electrospin the solution from step a) to form electrospun fibers containing silk protein; c) Combining electrospun fibers containing 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 in 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 (e.g., 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 (e.g., calcium gluconate) to the stent 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 other therapeutic agents to the stent material of step c).

105. The method of claim 104, wherein the one or more therapeutic agents are selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly the other therapeutic agent being selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or a combination thereof.

106. A method for preparing a scaffold material comprising i) hyaluronic acid and ii) electrospun fibers comprising silk fibroin; the method comprising the following steps: a) Preparation of silk fibroin solution; b) Mix the silk protein solution from step a) with hyaluronic acid to form a mixture; c) Electrospin the mixture from step b) to form a scaffold material.

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

3.

108. The method according to claim 106 or claim 107, wherein in step b), the silk protein solution from step a) is mixed 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 from step a) is mixed 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 from step a) is mixed with hyaluronic acid and an antifibrinolytic substance (e.g., 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 from step a) is mixed with hyaluronic acid and a coagulation activator (e.g., 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 from step a) is mixed 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 from step a) is mixed with hyaluronic acid and one or more other therapeutic agents to form a mixture.

114. The method of claim 113, wherein the one or more therapeutic agents are selected from the group consisting of steroids, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, anesthetics, antibacterial compounds, antibiotics, antifungal compounds, antiparasitic compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors, hormones, antiviral compounds, analgesics, opioids, saponins, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agents, anti-melanogenic agents, immunomodulators, immunoglobulins, minerals, antipsychotics, proteins, peptides, lipoproteins, antitumor compounds, tumor-suppressing compounds, toxins, vitamins (such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof), or a wrinkle-reducing filler, or a combination thereof, particularly the other therapeutic agent being selected from the group consisting of corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), captopril, hemoglobin, anesthetics, analgesics, opioids, saponins, and THC, or combinations thereof.

115. The method according to any one of claims 106 to 114, further comprising step d): crosslinking the scaffold material, for example by reacting it with an aldehyde crosslinking agent (such as glutaraldehyde) (e.g., in the gas 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 stent material in step c).

117. The method of 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 of claim 117, wherein the mammalian cells are selected from hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal cells, bile duct cells, parathyroid cells, thyroid cells, adrenal-hypothalamic-pituitary axis cells, cardiomyocytes, renal epithelial cells, renal tubular cells, renal basement membrane cells, nerve cells, vascular cells, bone and cartilage forming cells, smooth muscle cells, skeletal muscle cells, cochlear cells, epidermal 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 process is carried out at a voltage of about 12 kV to about 18 kV.

120. The method according to any one of claims 54 to 119, wherein electrospinning is carried out at a flow rate of about 0.8 mL / h to about 1.2 mL / h.

121. The method according to any one of claims 54 to 120, wherein, Electrospinning is carried out at a collector distance of approximately 8 cm to approximately 12 cm.

122. The scaffold material, layered wound dressing, layered graft, layered structure, or tissue-engineered structure according to any one of claims 1 to 52, for therapeutic purposes.

123. The scaffold material, layered wound dressing, layered graft, layered structure or tissue engineering structure 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, layered wound dressing, layered graft, layered structure or tissue-engineered structure according to claim 122, for the treatment or prevention of joint diseases or conditions.

125. The scaffold material, layered wound dressing, layered graft, layered structure, or tissue-engineered structure according to claim 124, for the treatment or prevention of joint diseases or conditions, wherein the joint diseases or conditions are selected from the group consisting of arthritis, gout, fibromyalgia, lupus erythematosus, polymyalgia, and rheumatic diseases.

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

127. The scaffold material, layered wound dressing, layered graft, layered structure, or tissue engineering structure according to claim 122, for tissue augmentation.

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