Silk biomaterials

Abstract

The present invention relates, in particular, to various scaffold materials including scaffold materials comprising: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers comprising silk protein.

Description

Technology Field

[0001] The present invention relates to a scaffold material comprising silk protein. Such scaffold material is used as a wound dressing and an implant. Additionally, said scaffold material is also used in the field of tissue engineering, particularly as a scaffold for cell proliferation. Furthermore, a layered wound dressing, a layered implant, and a layered structure comprising silk protein and hyaluronic acid are also described. Background Technology

[0002] Silk fibroin is a protein found in silk produced by various insects, such as silkworm larvae (*Bombyx mori*). Silkworm larvae wrap their bodies in cocoons made of raw silk, which contains two major proteins: sericin and fibroin. Fibroin is a fibrous protein that forms the core of the raw silk fibers, while sericin forms a gum-like coating layer on the surface of the fibers, causing them to stick together.

[0003] Silk fibroin is biocompatible and can be used as a wound hemostatic agent and for various cosmetic applications. Additionally, it is soluble in aqueous solutions and can be easily reconfigured into various forms of materials, such as films, mats, hydrogels, and sponges, through manufacturing processes such as spin coating, electrospinning, freeze-drying, and physical and chemical crosslinking technologies.

[0004] Electrospinning is a method that uses electric force to draw threads from a charged polymer solution, producing fibers with nanometer-scale diameters. Generally, an electrospinning device consists of a power supply, a solution reservoir (e.g., a syringe) equipped with a blunt needle, a pump, and a collector. By applying a specific voltage, an electric field is formed between the needle and the collector. The pump causes the solution to flow at a constant speed, and charges accumulate on the liquid surface. When the electrostatic repulsion becomes greater than the liquid's surface tension, the liquid flow is ejected from the surface. This point of ejection is called the Taylor cone. Once the Taylor cone is formed, a charged liquid jet is ejected toward the collector. Because the surface area of ​​the ejected jet is large relative to its volume, the solvent evaporates efficiently; as the charge density increases due to the volume reduction, the jet splits to form solid fibers. A whipping phenomenon occurs between the Taylor cone and the collector plate, depositing a nonwoven fiber mat onto the collector plate.

[0005] This reconstructed silk fibroin material possesses structural properties that allow it to be used as a wound dressing and graft (e.g., vascular graft), and it can also be utilized in the field of tissue engineering as a scaffold material that supports cell regeneration and growth.

[0006] EP1408146A1 describes a nonwoven fabric containing silk fibroin and a method for manufacturing the said nonwoven fabric by dissolving silk fibroin in hexafluoroacetone and then electrospinning. The obtained material is said to be useful as a medical material.

[0007] EP2465472A2 describes a method for producing silk nanofibers by electrospinning a fibrous spinning solution onto an integrated plate coated with polyethylene oxide. These silk fibers are produced in a tubular shape and are used as nerve graft tubes for the treatment of nerve damage.

[0008] WO2004 / 000915A2 describes a fully hydrolytic process for the production of silk biomaterials. In this process, one or more biocompatible polymers, such as polyethylene oxide, are mixed with silk proteins and then processed, for example, by electrospinning. Biomaterials produced in this way are known to be useful for forming tissue engineering structures.

[0009] Although various scaffold materials formed using silk protein are known, the object of the present invention is to manufacture a new scaffold material with improved functional properties and potential therapeutic effects. Another object of the present invention is to manufacture wound dressings and grafts with improved functional properties and potential therapeutic effects.

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

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

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

[0013] According to another aspect of the present invention, a layered wound dressing or a layered graft comprising the following is provided:

[0014] i) the scaffold material defined above; and

[0015] ii) Support.

[0016] According to another aspect of the present invention, a layered structure for tissue engineering applications is provided, comprising:

[0017] i) A first layer containing silk protein;

[0018] ii) A second layer comprising a hydrogel and silk protein;

[0019] iii) A third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and

[0020] iv) A fourth layer comprising polycaprolactone and hydroxyapatite.

[0021] According to another aspect of the present invention, a tissue engineering structure comprising a support material, a layered wound dressing, a layered graft or layered structure and mammalian cells as described herein is provided.

[0022] According to another aspect of the present invention, a method for manufacturing a tissue engineering structure comprising the following steps is provided:

[0023] a) a step of manufacturing a scaffold material as described in the present specification;

[0024] b) A step of mixing scaffold material and mammalian cells in a suitable medium.

[0025] According to another aspect of the present invention, 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 protein is provided, the method comprising the following steps:

[0026] a) Step of preparing a silk protein solution;

[0027] b) a step of electrospinning the solution of step a) to form an electrospun fiber containing silk protein;

[0028] c) A step of forming a scaffold material by mixing electrospun fibers containing silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate.

[0029] According to another aspect of the present invention, 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 protein is provided, the method comprising the following steps:

[0030] a) Step of preparing a silk protein solution;

[0031] b) Prepare a mixture by mixing the silk protein solution from step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate;

[0032] c) A step of forming a scaffold material by electrospinning the mixture from step b).

[0033] According to another aspect of the present invention, a method for manufacturing a scaffold material comprising i) hydroxyapatite and ii) an electrospun fiber comprising a silk protein is provided, the method comprising the following steps:

[0034] a) Step of preparing a silk protein solution;

[0035] b) a step of electrospinning the solution of step a) to form an electrospun fiber containing silk protein;

[0036] c) A step of forming a scaffold material by mixing electrospun fibers containing silk protein with hydroxyapatite.

[0037] According to another aspect of the present invention, a method for manufacturing a scaffold material comprising i) hydroxyapatite and ii) an electrospun fiber comprising a silk protein is provided, the method comprising the following steps:

[0038] a) Step of preparing a silk protein solution;

[0039] b) A step of mixing the silk protein solution from step a) with hydroxyapatite to form a mixture;

[0040] c) A step of forming a scaffold material by electrospinning the mixture of step b).

[0041] According to another aspect of the present invention, a method for preparing a scaffold material comprising i) hyaluronic acid; and ii) an electrospun fiber comprising a silk protein is provided, the method comprising the following steps:

[0042] a) Step of preparing a silk protein solution;

[0043] b) A step of mixing the silk protein solution from step a) with hyaluronic acid to form a mixture;

[0044] c) A step of forming a scaffold material by electrospinning the mixture from step b).

[0045] The embodiments and preferences described below regarding the scaffold materials, tissue engineering structures, layered wound dressings, layered grafts, and layered structures of the present invention apply equally to the methods for manufacturing the scaffold materials, tissue engineering structures, layered wound dressings, layered grafts, and layered structures described herein. Brief explanation of the drawing

[0046] FIG. 1 shows a layered structure (“multiphase implant”) according to an embodiment of the present invention. Specific details for implementing the invention

[0047] The inventors of the present invention have discovered that a scaffold material composed of, in particular, i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) electrospun fibers containing silk protein can be used to form a material useful for tissue engineering.

[0048] The inventors have also discovered that a scaffold material composed of electrospun fibers comprising i) hyaluronic acid; and ii) silk protein can be used to form a material useful for tissue engineering.

[0049] The inventors have also discovered that a scaffold material composed of electrospun fibers comprising i) hydroxyapatite; and ii) silk protein can be used to form materials useful for tissue engineering, particularly to form scaffold materials with a more rigid structure.

[0050] In the present invention, “scaffold material” refers to a material or substance used, for example, for the repair and / or support (including reinforcement) of tissues. This scaffold material may promote or contribute to the formation of new body substances such as tissues, cartilage, tendons, nerves, and bones. This may be achieved by providing a supportive function or by enhancing cell growth, attachment, differentiation, distribution, etc. Preferably, the scaffold material takes the form of a film, membrane, mat, or mesh.

[0051] Platelet-rich plasma (PRP) is generally produced 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 may be used to remove platelet-deficient plasma. Since the cellular composition of PRP and the resulting therapeutic effects depend heavily on the methodology and equipment used to manufacture PRP, standardization of PRP production is essential.

[0052] Medical devices for obtaining standardized PRP and BMC have been developed, including devices used in automated procedures in a closed circuit. For example, there are centrifuge tubes produced by Regen Lab and described in WO2008 / 023026A2, WO2011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391A1, WO2021 / 198312A1, and WO2022 / 269035A1 (the whole of which is incorporated herein by reference).

[0053] The process of producing platelet-rich plasma or bone marrow concentrate generally includes the following steps:

[0054] a) A step of collecting body fluid or cell extract in a centrifuge vessel;

[0055] b) a step of centrifuging the centrifuge vessel of step a) to form a concentrated fraction; and

[0056] c) A step of recovering at least a portion of the concentrated fraction.

[0057] Centrifuge vessels are suitable as centrifuge tubes or centrifuge syringes, particularly centrifuge tubes such as blood fractionation tubes. Suitable materials and coatings for centrifuge vessels, and the use of density gradient media such as thixotropic gels are described in WO2008 / 023026A2, WO2011 / 110948A2, WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391A1, WO2021 / 198312A1 and WO2022 / 269035A1 (the whole of which is incorporated herein by reference).

[0058] The characteristics of the concentrated fraction depend on the material being centrifuged. Centrifugation separates the components of a solution according to density, causing denser particles to move toward the bottom of the container (distal end) and less dense particles to move toward the top (proximal end). Therefore, the “concentrated fraction” can be described as the fraction after centrifugation that has a higher concentration of specific cells than the centrifuged body fluid or cell extract. If the body fluid is whole blood, erythrocytes move to the bottom of the container (or below it if a density gradient medium is present), while platelets move above the erythrocytes, into the plasma, which has a higher concentration of platelets (and other cellular components) compared to the centrifuged whole blood. Thus, when the body fluid is whole blood, the concentrated fraction is platelet-rich plasma. In other words, the concentrated fraction is platelet-concentrated plasma. If the body fluid is bone marrow, the concentrated fraction is bone marrow concentrate. Preferably, the bone marrow concentrate is concentrated with stem cells such as mesenchymal stem cells. Preferably, the body fluid (particularly platelet-rich plasma and / or bone marrow concentrate) is autologous.

[0059] Centrifugation is performed with a force sufficient to form a barrier between the body fluid components to be separated for a specific period of time. For example, if the body fluid is whole blood, this is the force / time that causes the density gradient medium (if present) to separate red blood cells from platelets and other desired whole blood components. In one embodiment, centrifugation is performed with a centrifugal force between about 700g and about 2800g, for example, between about 1500g and about 2800g, between about 1500g and about 2500g, for example, between about 1500g and about 2000g. Optionally, centrifugation in step b) is performed for about 3 minutes to about 40 minutes, for example, between about 3 minutes and about 15 minutes.

[0060] When the body fluid is whole blood, the concentrated fraction is platelet-rich plasma (PRP). However, after centrifugation, the platelet-rich plasma (concentrated fraction) is not uniformly distributed, and platelets are generally concentrated just above the density gradient medium (if present), and a layer of platelet-poor plasma (PPP) forms above this layer, that is, at the proximal end of the centrifuge. It may be beneficial to remove some of this platelet-poor plasma. For example, by removing between about half and about one-third of the concentrated fraction (at the top end of the centrifuge, that is, the end furthest from the density gradient medium (if present), at the proximal end), some or all of the PPP layer can be removed to obtain a more concentrated or enriched concentrated fraction.

[0061] Accordingly, in one embodiment, step b) includes the following steps:

[0062] b-1) A step of centrifuging the centrifuge vessel (of step a) to form a concentrated fraction;

[0063] b-2) A step of removing and discarding at least a portion of the concentrated fraction (of step b-1).

[0064] At least a portion of the concentrated fraction is generally removed at the proximal end of the centrifuge vessel. In this embodiment, step c) is to collect at least a portion of the remaining concentrated fraction (i.e., the further concentrated or concentrated fraction).

[0065] Platelet lysate is a liquid obtained by exposing platelets to a freeze-thaw cycle. The freeze-thaw cycle lyses platelets, causing them to release large amounts of growth factors and cytokines. Platelet lysate is available commercially and can also be easily prepared in-house if donor platelets are available.

[0066] Platelet lysate may be in a freeze-dried (or lyophylisation) form. The freeze-drying process preserves the ability of platelets to release bioactive molecules such as growth factors and cytokines, ensuring stability and a long shelf life without refrigeration.

[0067] Platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate may be mixed with electrospun fibers containing silk protein. Accordingly, according to one aspect of the present invention, 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 containing silk protein is provided, the method comprising the following steps:

[0068] a) Step of preparing a silk protein solution;

[0069] b) a step of electrospinning the solution of step a) to form an electrospun fiber containing silk protein;

[0070] c) A step of forming a scaffold material by mixing electrospun fibers containing silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate.

[0071] As described in the background of the invention, silk proteins may be derived from silk produced by insects such as silkworms or spiders. As used herein, “silk protein” encompasses silk fibroin and related proteins. In one embodiment, the silk protein is silk fibroin. Specifically, silk fibroin is a high molecular weight copolymer composed of a heavy chain (about 370 kDa) and a light chain (about 26 kDa) connected by disulfide bonds. The silk protein is preferably obtained from a solution containing dissolved silkworm silk or spider silk. In one embodiment, the silk protein is obtained from a solution containing dissolved silk of a silkworm of the genus Bombyx mori. In another embodiment, the silk protein is obtained from a solution containing dissolved silk of a spider such as Nephila clavipes. In yet another embodiment, the silk protein is obtained from a solution containing genetically modified silk, such as that obtained from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants.

[0072] In step a), the silk protein solution can be prepared using methods well known in the field (see, e.g., Rockwood et al., Nat Protoc. Sep 22, 2011; 6(10):1612-31; Woltje et al., Int J Mol Sci. Sep 29, 2021; 22(19):10565; Melke et al., Acta Biomaterialia, Vol. 31, 2016, pp. 1-16; and Bucciarelli et al., Biomater Adv. Aug, 2022; 139:212982). For silk protein derived from silkworms, the cocoons are typically boiled in soap (e.g., sodium oleate) or an aqueous solution of sodium carbonate (called a heat bath) to remove the sericin protein and other fats covering the silk fibroin. This process is called “degumming.” Degumming can also be performed via a microwave-assisted method, which produces high-purity fibroin to preserve intrinsic properties and ensure excellent bioactivity. After degumming, the silk protein is washed and dried. Further purification through dialysis and / or filtration may be performed. An exemplary process for preparing the silk protein solution is specified in the General Methods.

[0073] The silk protein solution to be used in the electrospinning process of step b) is suitable for having a silk protein (especially silk fibroin) concentration of about 5 wt.% to about 30 wt.%, for example, between about 12 wt.% and about 18 wt.%. The higher the concentration of silk protein in the electrospinning solution, the larger the fiber diameter and the thicker the formed electrospun fiber layer. However, if the concentration of silk protein is too high, the viscosity of the solution becomes too high, making electrospinning impossible. The silk protein solution is generally prepared by dissolving it in water.

[0074] In step b), an electrospinning process is performed using the silk protein solution prepared in step a) via any method known in the art. A typical electrospinning device operates by applying a voltage (e.g., between about 5 kV and about 30 kV, e.g., between about 12 kV and about 18 kV) between a needle (also called a spinneret or capillary) into which the silk protein solution is injected (typically via a syringe) and a collector. The collector is positioned at a close distance from the needle, typically 5 to 20 cm (e.g., 8 to 12 cm). Preferably, a syringe pump is used to maintain a constant volumetric flow rate to maintain the flow rate while preventing the solution from dripping from the needle tip. In one embodiment, the flow rate is between about 0.8 mL / hour and about 1.2 mL / hour. To obtain a stable solution jet stream, the electric field, the solution flow rate, and the distance between the needle tip and the collector are adjusted. The collector can be any material capable of functioning as a counter electrode, such as wire mesh, polymer mesh, metal foil (e.g., aluminum foil), or a water bath. Preferably, the collector has a non-adhesive surface so that the electrospun fibers can be easily removed. For example, the collector screen may be Teflon-coated or non-adhesive paper. Alternatively, the collector may be a substrate into which the electrospun solution is injected to form a layered product. For example, the collector may be a sheet material or a support material (described below). Preferably, the collector rotates at a speed, for example, 100 to 300 rpm, to produce a uniform and highly aligned coating of electrospun fibers forming a scaffold material.

[0075] The diameter of the electrospun fiber is preferably between about 50 nm and about 1,000 nm, for example, between about 200 nm and about 500 nm. The electrospun fiber generally takes the form of a film, membrane, mat, or mesh. If the electrospinning solution contains only silk fibroin, the mesh produced after electrospinning becomes a solid. If the electrospinning solution contains additional components (details regarding this are described below), the resulting scaffold material may have semi-solid properties.

[0076] Once the electrospun fibers are collected, they may optionally undergo one or more additional processing steps, such as washing (e.g., in distilled water) or fiber stretching.

[0077] In step c) of the above example, the electrospun fiber containing silk protein is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material. Preferably, the scaffold material is a semi-solid scaffold material. In one embodiment, the scaffold material takes the form of a film, membrane, mat, or mesh.

[0078] In the present invention, “semi-solid” means that the scaffold material is in a flexible solid state, possesses physical properties sufficient to allow for suturing, and has sufficient strength to provide the mechanical and physiological support required for a scaffold.

[0079] i) in an alternative process for forming a scaffold material comprising platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; ii) in an alternative process for forming a scaffold material comprising electrospun fibers comprising silk protein, instead of mixing with electrospun fibers comprising already formed silk fibroin, platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate may be included in the silk protein solution of step a). That is, platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate may be included in the electrospinning solution. The mixture of platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and silk protein subsequently undergoes an electrospinning process.

[0080] Accordingly, an alternative method for preparing a scaffold material comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and ii) electrospun fibers comprising silk protein is provided, the method comprising the following steps:

[0081] a) Step of preparing a silk protein solution;

[0082] b) a step of mixing the silk protein solution from step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture;

[0083] c) A step of forming a scaffold material by electrospinning the mixture of step b).

[0084] In another aspect of the present invention, a scaffold material comprising: i) hydroxyapatite; and ii) an electrospun fiber comprising silk protein. The inventors have discovered that when a scaffold material comprising an electrospun fiber comprising silk protein is combined with hydroxyapatite, it forms a scaffold material having a more rigid structure.

[0085] Hydroxyapatite is a naturally occurring mineral form of calcium apatite with the chemical formula Ca5(PO4)3(OH), commonly denoted as Ca10(PO4)6(OH)2.

[0086] This scaffold material can be manufactured using a method comprising the following steps:

[0087] a) Step of preparing a silk protein solution;

[0088] b) a step of electrospinning the solution of step a) to form an electrospun fiber containing silk protein;

[0089] c) A step of forming a scaffold material by combining electrospun fibers containing silk protein with hydroxyapatite.

[0090] Steps a) and b) may be performed as described above. In step c), an electrospun fiber containing silk protein is combined with hydroxyapatite to form a scaffold material. Preferably, the scaffold material is a solid scaffold material. In one embodiment, the scaffold material takes the form of a film, membrane, mat, or mesh.

[0091] In another process for manufacturing a scaffold material composed of electrospun fibers containing hydroxyapatite and silk protein, instead of mixing with electrospun fibers containing already formed silk fibroin, hydroxyapatite may be included in the silk protein solution of step a), i.e., the electrospinning solution. Then, the mixture of hydroxyapatite and silk protein is introduced into the electrospinning process.

[0092] Accordingly, an alternative method for manufacturing scaffold materials comprising the following steps is provided:

[0093] a) Step of preparing a silk protein solution;

[0094] b) A step of mixing the silk protein solution from step a) with hydroxyapatite to form a mixture;

[0095] c) A step of forming a scaffold material by electrospinning the mixture of step b).

[0096] In another aspect of the present invention, a method for preparing a scaffold material comprising i) hyaluronic acid and ii) an electrospun fiber comprising a silk protein is provided, the method comprising the following steps:

[0097] a) Step of preparing a silk protein solution;

[0098] b) A step of mixing the silk protein solution from step a) with hyaluronic acid to form a mixture;

[0099] c) A step of forming a scaffold material by electrospinning the mixture from step b).

[0100] Step a) may be performed as described above. In one embodiment, the weight ratio of hyaluronic acid to silk protein in step b) is between about 1:2 and about 1:4, for example, between about 1:3. In one embodiment, the method further comprises step d), which crosslinks the scaffold material by reacting it with an aldehyde-based crosslinking agent, such as glutaraldehyde, for example (e.g., in the gas phase). In one embodiment, the method further comprises step d), which adds platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to the scaffold material of step c).

[0101] In all of the above methods for forming a scaffold material by combining an electrospun fiber containing silk protein with another material (e.g., platelet-rich plasma, platelet lysate, bone marrow concentrate, hydroxyapatite, hyaluronic acid, or additional components or mixtures thereof described below), the added material may be extruded. If desired, the electrospinning device may be combined with a micro-extrusion device such as that described in WO2019 / 211803A1 (incorporated herein by reference). An example of such a combined device is an “Electrospider” that combines electrospinning, melt additive modeling, and pneumatic hydrogel extrusion to manufacture (or “print”) 3D structures that may have multiple layers.

[0102] The scaffold material described above may include additional components, which may be included before electrospinning (i.e., included in the electrospinning solution containing the silk protein to be electrospun) or added to the electrospun fiber composed of silk protein. Alternatively, a combination of additional components may be added, some of which are included in the electrospinning solution and others of which are added to the scaffold material after electrospinning.

[0103] In the case of a scaffold material that does not already contain hyaluronic acid, in one embodiment, the scaffold material additionally contains hyaluronic acid.

[0104] Hyaluronic acid is a glycosaminoglycan. The biological effects of hyaluronic acid are related to its molecular weight. In one embodiment (referring to all possible substances containing the hyaluronic acid 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, e.g., about 1,400 kDa and about 1,600 kDa, e.g., about 1,500 kDa. In another embodiment, the hyaluronic acid is a 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 hyaluronic acid contained in the centrifuge vessel of the present invention has 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.

[0105] A mixture of hyaluronic acids with different molecular weights may also be used. Accordingly, in one embodiment, the hyaluronic acid is a mixture of hyaluronic acids comprising the following:

[0106] Low molecular weight hyaluronic acid (LMW-HA) with a molecular weight between approximately 400 kDa and approximately 1,000 kDa; and / or

[0107] Medium molecular weight hyaluronic acid with a molecular weight between about 1,000 kDa and about 1,800 kDa, e.g. between about 1,400 kDa and about 1,600 kDa, e.g. about 1,500 kDa; and / or

[0108] High molecular weight hyaluronic acid (HMW-HA) with a molecular weight greater than 1,800 kDa.

[0109] In one embodiment, the hyaluronic acid is cross-linked. In another embodiment, the hyaluronic acid is linear. Preferably, the hyaluronic acid is linear. It is preferable that the hyaluronic acid be added in the form of a water solution, wherein the concentration of hyaluronic acid in the solution (added to the centrifuge vessel) is between about 1 wt% and about 5 wt%, for example, between about 1.8% and about 2.2 wt%. Alternatively, if the scaffold material comprises platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, the hyaluronic acid may be included in the centrifuge vessel used to form the platelet-rich plasma / platelet lysate / bone marrow concentrate. Suitably, the centrifuge vessel 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. Suitablely, the filling volume of the centrifuge vessel is 10 mL or 15 mL, particularly 15 mL.

[0110] In one embodiment, the scaffold material further comprises an anti-fibrotic material.

[0111] An antifibrinolytic substance refers to a substance (e.g., a compound) that inhibits fibrinolysis. An antifibrinolytic substance prevents or reduces the activation of plasminogen to form plasmin, thereby preventing or reducing the breakdown of blood clots. An antifibrinolytic substance is generally tranexamic acid, which is a synthetic analog of the amino acid lysine. In one embodiment, the antifibrinolytic substance is selected from the group consisting of tranexamic acid, aminocaproic acid, and aprotinin, or any combination thereof. Preferably, the antifibrinolytic substance is a small molecule with a molecular weight of 500 Da or less, e.g. 400 Da or less, 300 Da or less, or 200 Da or less. In one embodiment, the antifibrinolytic substance is not an enzyme, 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. Suitablely, the anti-fibrinolytic substance is 5% by weight of tranexamic acid (suitably dissolved in water). The reference to “one” anti-fibrinolytic substance is intended to include “at least one” anti-fibrinolytic substance, and combinations of anti-fibrinolytic substances are also considered.

[0112] In one embodiment, the scaffold material further comprises a coagulation activator.

[0113] A coagulation promoter is an agent, such as a compound or enzyme, capable of inducing or activating plasma coagulation and platelet aggregation to form a coagulation that may have a gel-like viscosity. The reference to “one” coagulation promoter is intended to include “at least one” coagulation promoter, and a combination of coagulation promoters is also included. Typically, a coagulation promoter is a compound or moiety that is a thrombin activator and / or a fibrinogen activator. In one embodiment, the coagulation promoter is a compound selected from the group consisting of calcium salts and thrombin. Preferably, the calcium salt is selected from the group consisting of calcium gluconate, calcium carbonate, calcium sulfate, calcium saccharate, and calcium chloride, or any combination thereof, in particular calcium gluconate.

[0114] In one embodiment, the coagulation activator is a calcium salt (particularly calcium gluconate) contained in a water solution in an amount between about 1 wt% and about 20 wt%, for example between about 5 wt% and about 15 wt% (e.g., about 10 wt%). The calcium salt may include a mixture of calcium salts, for example, a combination of calcium gluconate and calcium saccharate.

[0115] In one embodiment, the scaffold material further comprises vitamin K2. In one embodiment, vitamin K2 is selected from the group consisting of menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone-12 (MK-12), menaquinone-13 (MK-13) and menaquinone-14 (MK-14), and mixtures thereof. References to “vitamin K2” and “menaquinone” are intended to include “at least one” menaquinone, and combinations of menaquinone are also included. In one embodiment, vitamin K2 is selected from the group consisting of MK-6, MK-7, and MK-8, and mixtures thereof. In another embodiment, vitamin K2 is selected from the group consisting of MK-4, MK-5, MK-6, and MK-7, and mixtures thereof. In one embodiment, vitamin K2 is MK-4. In one embodiment, vitamin K2 is MK-7.

[0116] In one embodiment, the scaffold material further comprises one or more therapeutic agents. In one embodiment, the therapeutic agent comprises a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, hemoglobin, anesthetic, analgesic, It is selected from the group consisting of opioids, saponins, and THC, or combinations thereof.

[0117] In the case of a scaffold material that does not yet contain hydroxyapatite, in one embodiment, the scaffold material further contains hydroxyapatite.

[0118] Particularly preferred scaffold materials also include hyaluronic acid. Accordingly, in a preferred embodiment, a scaffold material comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibers containing silk protein; and iii) hyaluronic acid is provided. In another preferred embodiment, a scaffold material comprising i) hydroxyapatite; ii) electrospun fibers containing silk protein; and iii) hyaluronic acid is provided. In a more preferred embodiment, a scaffold material comprising i) platelet-rich plasma and / or bone marrow concentrate; ii) electrospun fibers containing silk protein; iii) hyaluronic acid; and iv) hydroxyapatite is provided.

[0119] When hyaluronic acid is included in the scaffold material, the ratio (weight ratio) of silk protein to hyaluronic acid is preferably between about 50:50 and about 95:5.

[0120] The scaffold material of the present invention has therapeutic uses, in particular, as a wound dressing and a graft (e.g., a vascular graft).

[0121] In another aspect of the present invention, a layered wound dressing or layered graft comprising the following is provided:

[0122] i) Scaffold material comprising the following:

[0123] ia) electrospun fibers containing silk protein; and

[0124] ii) Hyaluronic acid;

[0125] and

[0126] ii) Support material.

[0127] Combining electrospun fibers containing silk proteins with hyaluronic acid allows for the utilization of both the mechanical strength of silk and the hydrophilicity and cell-friendly environment provided by hyaluronic acid. This combination supports cell viability due to its biocompatibility and an environment favorable for cell growth. The hydrophilic properties of hyaluronic acid help create a moisture-retaining environment essential for cell survival. The presence of hyaluronic acid enhances cell adhesion through its role in cell signaling and interactions with cell surface receptors (e.g., CD44). Silk proteins, particularly silk fibroin, provide a stable and supportive structure for cell attachment.

[0128] In one embodiment, the scaffold material further comprises platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate (as described in detail above). In one embodiment, the scaffold material further comprises hyaluronic acid (as described in detail above). In one embodiment, the scaffold material further comprises an antifibrinolytic agent such as tranexamic acid (as described in detail above). In one embodiment, the scaffold material further comprises a coagulation promoter such as calcium gluconate (as described in detail above). In one embodiment, the scaffold material further comprises vitamin K2 (as described in detail above). In one embodiment, the scaffold material further comprises hydroxyapatite (as described in detail above).

[0129] The scaffold material can be any scaffold material that can generally be used for wound dressings or grafts. In one embodiment, the scaffold material comprises synthetic polymers such as poly(glycolic acid), poly(lactic acid), polyethylene glycol (PEG), low-density polyethylene (LDPE), and polycaprolactone (PCL), or natural polymers such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran, or albumin. The scaffold material of the present invention may be attached to the scaffold material using conventional techniques well known to those skilled in the art, including adhesion (e.g., using adhesive) or lamination.

[0130] In a preferred embodiment, the support material comprises a plastic material such as low-density polyethylene (LDPE) or polycaprolactone (PCL).

[0131] The scaffold material of the present invention is also used in the field of tissue engineering. In one embodiment, the scaffold material of the present invention may be combined with cells to form a tissue engineering structure used to regenerate or replace damaged or degenerated tissues or organs.

[0132] Tissue engineering structures can be manufactured in various ways. In one embodiment, the tissue engineering structure comprises i) a scaffold material as described herein; and ii) cells (e.g., mammalian cells). While specific cell types may vary depending on the intended use of the composition, suitable mammalian cells include hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal cells, cholangiocytes, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, cardiomyocytes, renal epithelial cells, renal tubular cells, renal basement membrane cells, neurons, vascular cells, bone and cartilage-forming cells, smooth muscle cells, skeletal muscle cells, oral cells, epithelial cells, bone marrow cells, keratinocytes, pluripotent cells, and stem cells, and combinations thereof, selected from the group consisting of cells selected from the group consisting of combinations thereof.

[0133] In one embodiment, a method for manufacturing a tissue engineering structure is provided, comprising the following steps:

[0134] a) a step of manufacturing a scaffold material as described in the present specification; and

[0135] b) A step of mixing the scaffold material with cells (e.g., mammalian cells) in a suitable medium.

[0136] The appropriate medium is a culture medium, which varies depending on the type of cell being cultured, and can be easily prepared by a person skilled in the art.

[0137] In another aspect of the present invention, a layered structure for tissue engineering applications is provided, comprising the following:

[0138] i) A first layer containing silk protein;

[0139] ii) A second layer comprising a hydrogel and silk protein;

[0140] iii) A third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and

[0141] iv) A fourth layer composed of polycaprolactone and hydroxyapatite.

[0142] A layered structure according to an embodiment of the present invention is illustrated in FIG. 1 ("Multiphase Implant"). This innovative structure is specifically designed for the treatment of cartilage damage caused by degenerative changes or trauma and is applied in various fields, including sports medicine. The structure mimics the structure and function of biological osteochondral tissue by utilizing a layered multiphase approach. It is manufactured using GMP-compliant 3D printing and electrospinning technologies to ensure clinical-grade quality.

[0143] Appropriately, the thickness of the first layer composed of silk protein is between about 40 μm and about 60 μm, for example, about 50 μm. Appropriately, the first layer composed of silk protein is a coating layer. Appropriately, the silk protein of the first layer consists of electrospun fibers of silk protein. In one embodiment, electrospun fibers of silk protein are electrospun from a silk fibroin solution having a concentration of about 5% (w / v) to about 15% (w / v), for example, about 7% (w / v), under a voltage of 12 to 18 kV (e.g., about 15 kV); at a flow rate between about 0.8 mL / hour and about 1.2 mL / hour (e.g., 0.8 mL / hour); and at a collection distance of 5 to 20 cm (e.g., about 10 cm). The first layer consists of silk protein (particularly silk fibroin, subject to the preferred conditions described above) and provides a protective barrier for the layers below. The first layer also provides structural stability to the laminated structure and prevents degradation during the early stages of implantation. Finally, the first layer offers excellent biocompatibility to promote cell adhesion.

[0144] In one embodiment, the hydrogel of the second layer comprises hyaluronic acid and / or gelatin, or is itself hyaluronic acid and / or gelatin. Preferably, the hyaluronic acid is a mixture of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid (e.g., a weight ratio of about 1:1). In one embodiment, the silk protein of the second layer consists of electrospun fibers of silk protein. In one embodiment, the electrospun fibers of silk protein are prepared by electrospinning a silk fibroin solution having a concentration of about 5% (w / v) to 15% (w / v), e.g., about 7% (w / v); under a voltage of 12 to 18 kV (e.g., about 15 kV); at a flow rate between about 0.8 mL / hour and about 1.2 mL / hour (e.g., 0.8 mL / hour); and at a collection distance of 5 to 20 cm (e.g., about 10 cm). The second layer may be formed by mixing hyaluronic acid and / or gelatin with the electrospun silk protein according to the method described above, or alternatively, the hyaluronic acid and / or gelatin may be mixed with the silk protein solution before electrospinning. Thus, in one embodiment, the second layer consists of electrospun fibers of a hydrogel (e.g., hyaluronic acid) and silk protein. In one embodiment, the electrospun fibers of hyaluronic acid and silk protein are electrospun from a hyaluronic acid and silk fibroin solution having a concentration of about 5% (w / v) to 15% (w / v), e.g., about 7% (w / v), under a voltage of 12 to 18 kV (e.g., about 15 kV); at a flow rate between about 0.8 mL / hour and about 1.2 mL / hour (e.g., 0.8 mL / hour) and a collection distance of 5 to 20 cm (e.g., about 10 cm). In one embodiment, the second layer has a thickness of 1 to 2 mm. In one embodiment, the second layer further comprises platelet-rich plasma, platelet lysate and / or bone marrow concentrate, particularly further comprises platelet lysate. The hydrogel-based second layer mimics the cartilage intermediate zone to enable cartilage formation.This facilitates the controlled delivery of growth factors for tissue regeneration and enhances water retention, contributing to the biomechanical properties of the structure.

[0145] In one embodiment, the third layer comprises polycaprolactone (PCL) and hydroxyapatite and is a layer formed by electrospinning. In one embodiment, the third layer comprises hyaluronic acid and hydroxyapatite and is a layer formed by electrospinning. Preferably, the third layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 80:20. In one embodiment, the third layer has a thickness of 90 μm to 110 μm. The third layer mimics the calcified layer of osteochondral tissue to provide a transitional boundary between the cartilage and the subchondral bone located beneath it. The third layer combines mechanical strength and bioactivity to ensure integration with the subchondral bone.

[0146] In one embodiment, a fourth layer composed of polycaprolactone and hydroxyapatite is formed using fused additive modeling (FDM). FDM is a 3D printing method in which a thermoplastic polymer is heated above its melting point and then fed through a nozzle at a precise speed. The molten polymer is deposited onto a build plate at a lower temperature and rapidly cooled and solidified. In one embodiment, the fourth layer is composed of polycaprolactone and hydroxyapatite in a weight ratio of 70:30. 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 to provide load-bearing function and support, thereby promoting long-term adhesion with biological bone tissue.

[0147] The scaffold materials, tissue engineering structures, layered wound dressings, layered grafts, and layered structures described herein have therapeutic utility. Accordingly, one embodiment provides a scaffold material, layered wound dressing, layered graft, layered structure, or tissue engineering structure described herein for use in treatment. The scaffold material is a medical scaffold material. In one embodiment, the scaffold material takes the form of a wound dressing or a graft.

[0148] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention can provide mechanical support to the damaged site and can also provide physiological support by promoting the recovery of the original tissue.

[0149] In one embodiment, the scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure described herein is provided to be used for wound healing, wound closure, 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.

[0150] Specific examples of wound healing and tissue restoration include the treatment of gynecological diseases (e.g., pelvic organ prolapse), intestinal injuries, bladder injuries, vascular injuries, other non-healing wounds, burns, and diabetic foot disease.

[0151] In one embodiment, a scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure described herein is provided for use in tissue augmentation.

[0152] In one embodiment, a scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure described herein is provided for use as a delivery medium for a therapeutic agent.

[0153] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention, which include hydroxyapatite, are particularly suitable for use in bone regeneration.

[0154] In one embodiment, the scaffold material, tissue engineering structure, multilayer wound dressing, multilayer graft, or multilayer structure described herein is provided for use in the treatment or prevention of joint disorders or diseases. Preferably, the joint disorder or disease is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia, and rheumatic polymyalgia. Arthritis includes osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, cervical spondylitis, psoriatic arthritis, enteropathic arthritis, oligoarthritis, polyarthritis, and secondary arthritis.

[0155] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention may be in any suitable form, including in the form of a film, membrane, mat, or mesh. The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention may be used, in particular, as a wound dressing, a graft such as a vascular graft, and a scaffold for cell growth.

[0156] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention have excellent biocompatibility and, in certain embodiments, include pores or niches that enable the exchange of body fluids within the structure. The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention are suitably biodegradable and are therefore reabsorbed after application (e.g., implantation).

[0157] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention may form a layer over an existing material (e.g., wound dressing material) to improve mechanical strength. In one embodiment, the scaffold material of the present invention forms a layer over a suitable sheet material (e.g., backing layer). In one embodiment, the scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure of the present invention is laminated over a sheet comprising a viscose polyester matrix. In one embodiment, the scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure of the present invention is laminated over a sheet comprising a synthetic polymer such as poly(glycolic acid), poly(lactic acid), polyethylene glycol (PEG), low-density polyethylene (LDPE), or polycaprolactone (PCL), or a natural polymer such as agar, alginate, carrageenan, chitosan, starch, cellulose, dextran, or albumin. The scaffold material, tissue engineering structure, layered wound dressing, layered graft, or layered structure of the present invention may be attached to a sheet material using conventional techniques well known to those skilled in the art, including bonding (e.g., using an adhesive) or lamination.

[0158] In another process of the present invention, a composition comprising i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) an electrospun fiber containing silk protein can be prepared by forming platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate using a centrifuge vessel already containing a silk protein solution. The silk protein solution can be prepared as described above. In this embodiment, body fluid or cell extract is collected in a centrifuge vessel already containing a silk protein solution. Then, the centrifuge vessel is centrifuged to form a concentrated fraction and a silk protein solution. This solution can be subjected to an electrospinning process as described herein to form the scaffold material of the present invention.

[0159] In one embodiment, a centrifuge vessel comprising a silk protein (details as described above) is provided. The centrifuge vessel may further comprise hyaluronic acid (as described in detail above) and / or an anti-fibrinolytic substance (as described in detail above) and / or a coagulation promoter (as described in detail above) and / or vitamin K2 and / or one or more therapeutic agents (as described in detail above) and / or hydroxyapatite. If the centrifuge vessel comprises hyaluronic acid, trehalose may be included in the vessel as a stabilizer for the hyaluronic acid. The centrifuge vessel may also comprise a density gradient medium (details as described above).

[0160] In one embodiment, a centrifuge vessel comprising silk protein, a density gradient medium, and hyaluronic acid is provided.

[0161] Using electrospinning to form the scaffold material of the present invention offers several advantages. First, it enables the production of standardized scaffold materials that can be mass-produced and then cut to desired dimensions, making them suitable for various clinical applications. Second, this technology also enables the fabrication of customized scaffold materials that can be precisely designed and manufactured to suit the specific pathological conditions and anatomical requirements of individual patients. Therefore, the method of the present invention possesses the dual functions of standardized mass production and customized fabrication.

[0162] In one embodiment, the scaffold material of the present invention has an expansion rate of 300% or more. The expansion rate is defined as the difference between the initial weight of the material in a dry state and the weight of the material in an expanded state, divided by the initial weight.

[0163] The scaffold material, tissue engineering structure, layered wound dressing, layered graft, and layered structure of the present invention are expected to have one or more of the following advantages / benefits in certain embodiments:

[0164] - Porous suitable for nutrient and / or cell exchange;

[0165] - Structure suitable for supporting cell adhesion and / or elongation;

[0166] - Facilitating the controlled delivery of bioactive substances such as growth factors;

[0167] - Promotes cell growth, thereby promoting healing;

[0168] - Biocompatibility;

[0169] - Anti-apoptotic effect.

[0170] Additional specific advantages of the layered structure of the present invention:

[0171] Biomimetic Design:

[0172] Each layer reproduces the structure and function of the natural osteochondral tissue region from the articular cartilage surface to the subchondral bone.

[0173] Controlled release of growth factors:

[0174] The hydrogel layer contains platelet lysate protein, which can continuously release growth factors to promote tissue regeneration.

[0175] Manufacturing technology:

[0176] Electrospinning: Forms a nanoscale fiber structure on the electrospinning layer to improve cell adhesion and mechanical strength.

[0177] 3D Printing: Ensures precise and scalable production of FDM layers and entire structures.

[0178] GMP Compliance:

[0179] All processes are carried out under GMP conditions to ensure regulatory and clinical applicability.

[0180] Scalability and Customization:

[0181] Structures can be manufactured to meet the specific needs of each patient using advanced 3D printing technology.

[0182] abbreviation

[0183] BMC Bone marrow concentrate

[0184] CBD Cannabidiol

[0185] DMA Dynamic mechanical analysis

[0186] DMEM Dulbecco Modified Eagle Badge

[0187] DMMB Dimethylmethylene blue

[0188] DMF dimethylformamide

[0189] ELISA Enzyme-linked immunosorbent method

[0190] EtO ethylene oxide

[0191] FDM Fusion stacking modeling

[0192] GAG Glycosaminoglycans

[0193] GF gingival fibroblasts

[0194] GMP Good Manufacturing Practice Standards

[0195] HA Hyaluronic acid

[0196] HMW-HA high molecular weight hyaluronic acid

[0197] HUVEC human umbilical vein endothelial cells

[0198] LDPE low-density polyethylene

[0199] LMW-HA low molecular weight hyaluronic acid

[0200] MK Menaquinone

[0201] MPV Mean platelet volume

[0202] MSC Mesenchymal stem cells

[0203] NSAID Non-steroidal anti-inflammatory drugs

[0204] PBS Phosphate buffered saline solution

[0205] PCL Polycaprolactone

[0206] PDGF Platelet-derived growth factor

[0207] PDL Periodontal ligament cells

[0208] PEG polyethylene glycol

[0209] PL Platelet lysate

[0210] PPP Platelet-low plasma

[0211] PRP Platelet-rich plasma

[0212] rpm revolutions per minute

[0213] SDS Sodium dodecyl sulfate

[0214] SEM Scanning electron microscope

[0215] SF Silk fibroin

[0216] THC Tetrahydrocannabinol

[0217] VF vaginal fibroblasts

[0218] example

[0219] general method

[0220] Cell preparation - fibroblasts

[0221] Gum tissue was collected from the interdental papilla during tooth extraction in adult patients. After collection, the tissue was immediately stored in sterile saline for 1 to 4 hours prior to processing. To dilute the oral bacterial flora of the gum tissue, it was washed 10 times with phosphate-buffered saline (PBS). After PBS washing, the tissue was cut into small pieces measuring 1 to 2 mm² using a No. 10 surgical knife. The grafts were then inoculated into 100 mm tissue culture dishes and cultured undisturbed at 37°C for 48 hours in a humidified incubator supplied with 5% CO₂. The medium was replaced after 48 hours. Cells were cultured in complete culture medium supplemented with 10% FBS, 1% HEPES 1M 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 tissues can also be cultured through a similar process.

[0222] Preparation of silk fibroin solution

[0223] First, 5g of silkworm cocoons are cut into small pieces and subjected to a degumming process. Then, the pieces are placed in 2 L of a 0.02 M sodium bicarbonate solution and boiled for 30 minutes, after which they are removed and the excess moisture is squeezed out of the silk. Subsequently, the silk pieces are rinsed three times with 1 L of deionized water for 20 minutes each, and then squeezed again to remove excess moisture. The degummed silk fibroin pieces are dried overnight in a fume hood and can be stored indefinitely at room temperature.

[0224] The silk fibroin solution is prepared by dissolving degummed silk fibroin in a 9.3 M lithium bromide solution at a concentration of 20 w / v% and dissolving it at 60 °C for 4 hours until a clear, transparent solution is formed. This solution can be purified by removing salts through dialysis (e.g., adding 12 mL to 1 L of deionized water and changing the water volume 6 times) and then centrifuging it three times for 20 minutes at 9,000 rpm (approx. 12,700 g) at 4 °C. Generally, 25 mL of a 7–8 w / v% silk solution is obtained from 5 g of silkworm cocoons.

[0225] Example 1a - Preparation of Platelet-Rich Plasma (PRP)

[0226] Blood collected from a healthy donor in a centrifuge tube (CuteCell TM The collected blood was placed in a PRP (Regen Lab SA). The collected blood was centrifuged at 1,500 g for 5 minutes at room temperature in a standard laboratory centrifuge. As a result, red blood cells and white blood cells settled at the bottom of the tube below the separation gel, while plasma and platelets remained on top of the gel layer. The platelet-containing plasma was homogenized by inverting the tube 5 times. The 6 mL of platelet-rich plasma obtained in this way was transferred to a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for storage until use.

[0227] Example 1b - Preparation of platelet-rich plasma containing hyaluronic acid

[0228] Blood collected from a healthy donor is centrifuged in a hyaluronic acid-containing centrifuge tube (Cellular Matrix TMThe collected blood was placed in a PRP (Regen Lab SA). The collected blood was centrifuged at 1500g for 5 minutes using a standard laboratory centrifuge at room temperature. As a result, red blood cells and white blood cells settled at the bottom of the tube below the separation gel, while plasma and platelets remained on top of the gel layer. The platelet-containing plasma was homogenized by inverting the tube 5 times. The 6 mL of platelet-rich plasma and hyaluronic acid obtained in this way were transferred to a polypropylene tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for storage until use.

[0229] Example 2 - Preparation of a membrane-type scaffold material composed of electrospun fibers made of silk protein and hyaluronic acid

[0230] A 0.5% (w / v) 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. Subsequently, this solution was prepared for electrospinning using previously optimized parameters and non-adhesive paper as the aggregate. Silk fibroin / hyaluronic acid nanofibers were electrospun and directly integrated onto the substrate for a deposition time of approximately 25 minutes. The resulting film was cut using a laser cutter, and the cut specimens were sterilized using EtO.

[0231] Example 3 - Preparation of a scaffold material by laminating electrospun fibers composed of silk protein and hyaluronic acid onto a non-woven viscose / polyester substrate

[0232] 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. Subsequently, this solution was prepared for electrospinning using a viscose / polyester substrate sheet as an integral using previously optimized parameters. Silk fibroin / hyaluronic acid nanofibers were electrospun and directly integrated onto the substrate over a deposition time of approximately 12 minutes. The resulting sheet was cut using a laser cutter, and the cut samples were sterilized using ethylene oxide (EtO).

[0233] Example 4 - Preparation of a scaffold material composed of electrospun fibers containing silk protein and hyaluronic acid

[0234] 5 g of degummed silk fibroin was dissolved in a 9.3 M lithium bromide solution at 60°C for 4 hours. This solution was dialyzed for 48 hours using distilled water as a control and adjusted to a concentration of 6% (w / v). Hyaluronic acid was dissolved in PBS to prepare a 2% (w / v) solution, and LMWHA (50–100 kDa) and HMWHA (800–1200 kDa) were prepared separately. The silk fibroin and HA 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 conditions:

[0235] Voltage: 15 kV.

[0236] Flow rate: 1 mL / hour.

[0237] Collector distance: 10 cm.

[0238] Nanofibers are deposited on non-adhesive paper and then dried at room temperature. The resulting matrix is ​​crosslinked with gas-phase glutaraldehyde for 12 hours to stabilize the structure. The resulting scaffold material is expected to exhibit excellent mechanical properties, swelling ability, and a uniform fiber morphology, making it a promising scaffold for tissue engineering applications.

[0239] Example 5 - Application of Silk-HA Scaffold Materials in Cell Analysis

[0240] The electrospun HA-silk matrix (1 cm² sample) prepared according to Example 4 is immersed in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum for 1 hour to reduce hydrophobicity. The matrix is ​​expected to absorb the medium and sink to the bottom of the culture dish. Subsequently, the matrix is ​​positioned so that the bioactive HA-silk layer faces the top of the dish to maximize the contact area with the inoculated cells. A single drop of 100,000 cells suspended in 500 μL of culture medium is placed on the surface of the matrix. After allowing the cells to attach for 1 to 2 hours, the experimental medium is added as follows:

[0241] Experimental conditions:

[0242] Control group (medium only): DMEM + 10% FBS.

[0243] PRP Condition: DMEM supplemented with platelet-rich plasma (10% PRP).

[0244] PRP-HA Condition: DMEM supplemented with platelet-rich plasma and hyaluronic acid (10% PRP-HA).

[0245] Subsequently, the utility of the HA-silk matrix for biological experiments is evaluated. A Live / Dead Kit (Invitrogen L3224) is used to evaluate cell viability on days 7 and 15. Imaging systems are used to capture images to observe cell proliferation and apoptosis.

[0246] Morphological analysis:

[0247] Test cell types: Gingival fibroblasts (GF), periodontal ligament cells (PDL), and endothelial cells (HUVECs). Observation results are recorded using a phase-contrast microscope.

[0248] The bioactive HA-silk matrix exhibits biocompatibility under various experimental conditions and is expected to support cell adhesion, proliferation, and viability. The addition of PRP-HA is expected to enhance cellular responses, highlighting the potential for tissue engineering applications.

[0249] Example 6 - Cell Attachment and Elongation Analysis

[0250] The attachment and elongation ability of cells on the HA-silk matrix and the structural alignment along the fibers are evaluated. An electrospun HA-silk matrix is ​​prepared and rehydrated as described in Example 5. To optimize interaction with inoculated cells, the matrix is ​​positioned with the HA-silk side facing upward.

[0251] Cell type:

[0252] Primary cell line for testing:

[0253] Gingival fibroblasts (GF).

[0254] Periodontal ligament cells (PDL).

[0255] Endothelial cells (HUVECs).

[0256] Experimental conditions:

[0257] Control group: DMEM with 10% FBS added.

[0258] PRP Condition: DMEM with 10% Platelet-Rich Plasma (PRP) added.

[0259] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1% w / v).

[0260] Analysis Protocol:

[0261] Cells are cultured on an HA-silk matrix for 7 and 15 days. Phase-contrast microscopy is used to observe the alignment of cells along the fibers and to evaluate adhesion and elongation. The HA-silk matrix is ​​expected to support strong cell adhesion and promote structural alignment along the fibers. PRP-HA conditions are expected to enhance elongation and network formation, highlighting the potential for matrix utilization in the field of tissue engineering.

[0262] Example 7 - Stability of the Hyaluronic Acid Layer

[0263] The stability of the HA layer within the HA-silk matrix is ​​evaluated over time under various culture conditions (control, PRP, PRP-HA). Electrospun HA-silk matrices are prepared and rehydrated as described in Example 5. In all analysis processes, the HA layer is oriented upward.

[0264] Experimental conditions:

[0265] Control group: DMEM with 10% FBS added.

[0266] PRP Condition: DMEM with 10% Platelet-Rich Plasma (PRP) added.

[0267] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1% w / v).

[0268] Experimental Procedure:

[0269] The matrix is ​​cultured for up to 15 days under each condition. On days 7 and 15, observations are recorded with a focus on the following: the macroscopic integrity of the matrix; and the stability of the microlayer via phase-contrast microscopy. The stability of the HA layer is expected to improve under the PRP-HA condition, which demonstrates the importance of combining PRP and hyaluronic acid. This characteristic is expected to support the use of the HA-Silk matrix as a robust scaffold for long-term tissue engineering applications.

[0270] Example 8 - Synergistic Effects of PRP-HA

[0271] The combined effects of the binding of platelet-rich plasma (PRP) and hyaluronic acid (HA) on cell proliferation, network formation, and bioactivity upon culture on an HA-silk matrix are evaluated. An electrospun HA-silk matrix is ​​prepared and rehydrated as described in Example 5. During all analysis processes, the HA-silk layer is positioned facing upward.

[0272] Cell type:

[0273] Primary cell line for testing:

[0274] Gingival fibroblasts (GF).

[0275] Periodontal ligament cells (PDL).

[0276] Endothelial cells (HUVECs).

[0277] Experimental conditions:

[0278] Control group: DMEM with 10% FBS added.

[0279] PRP Condition: DMEM with 10% Platelet-Rich Plasma (PRP) added.

[0280] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1% w / v).

[0281] Experimental Protocol:

[0282] Cells are cultured on an HA-Silk matrix for up to 15 days. Cell proliferation and network formation are evaluated 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, thereby promoting robust cell proliferation and network formation. This is expected to demonstrate the potential for matrix applications in the fields of tissue engineering and regenerative medicine.

[0283] Example 9 - Optimization of Platelet Lysates for Enhanced Bioactivity

[0284] Evaluate the effects of various platelet lysate (PL) concentrations on the bioactivity of the HA-silk matrix, specifically on cell adhesion, proliferation, and elongation. Prepare and rehydrate an electrospun HA-silk matrix as described in Example 5.

[0285] Platelet lysate concentration:

[0286] The experimental group included a matrix cultured in a medium supplemented with the following:

[0287] 0.25% (v / v) PL.

[0288] 0.5% (v / v) PL.

[0289] 1.0% (v / v) PL.

[0290] Control group: DMEM supplemented with 10% FBS (no PL).

[0291] Cell type:

[0292] 1st Human Cell Line Test:

[0293] Gingival fibroblasts (GF).

[0294] Periodontal ligament cells (PDL).

[0295] Endothelial cells (HUVECs).

[0296] Experimental Protocol:

[0297] Cells are cultured on an HA-Silk matrix under the above conditions for 15 days.

[0298] Evaluation Indicators:

[0299] Cell viability (Live / Dead analysis performed on days 7 and 15 (Invitrogen L3224)).

[0300] Adhesion and elongation (phase contrast microscopy observation).

[0301] Growth rate (MTT analysis).

[0302] The HA-Silk matrix is ​​expected to exhibit optimal bioactivity when supplemented with 0.5% (v / v) platelet lysate, which will support robust cell adhesion, elongation, and proliferation. Higher concentrations (1.0% PL) are expected to result in a densification effect, while lower concentrations (0.25% PL) are not expected to sufficiently enhance bioactivity. These experimental results are expected to highlight the importance of optimizing platelet lysate concentrations for tissue engineering applications.

[0303] Example 10 - Comparison of Support Layer Performance

[0304] The purpose of this experiment is to evaluate the influence of various support materials on the structural integrity, cell viability, and handling properties of the HA-silk matrix during the biological analysis process. An electrospun HA-silk matrix is ​​prepared as described in Example 5. The matrix is ​​deposited onto various support materials during the electrospinning process.

[0305] Tested support material:

[0306] LDPE / cellulose composite: A flexible support with high tensile strength.

[0307] Polyethylene / paper composite: A lightweight support with moderate rigidity and anti-adhesion properties.

[0308] Control group support: Standard non-adhesive electrospun paper.

[0309] Evaluation Indicators:

[0310] Structural integrity: Evaluated by observing shrinkage or rupture of the matrix during metastasis and culture processes.

[0311] Delamination: Qualitatively evaluates the degree to which the matrix is ​​easy to remove from the support without damage.

[0312] Cell viability and coverage: Measured on days 7 and 15 using the Live / Dead assay (Invitrogen L3224) and phase-contrast microscopy.

[0313] Cell type:

[0314] Gingival fibroblasts (GF).

[0315] Endothelial cells (HUVECs).

[0316] Among the tested scaffolds, the polyethylene / paper composite is expected to demonstrate superior performance in terms of structural integrity, ease of handling, and cell viability. This scaffold material is expected to be recommended for optimizing HA-Silk matrices in future tissue engineering applications.

[0317] Example 11 - Long-term stability and decomposition of the matrix

[0318] Observe the degradation of the HA-silk matrix over an extended period (up to 30 days) under PRP-HA culture conditions, focusing on macroscopic and microscopic changes. Prepare and rehydrate the electrospun HA-silk matrix as described in Example 5. In all experiments, the HA layer in the culture dish is positioned facing upward.

[0319] Experimental conditions:

[0320] Control group: DMEM with 10% FBS added.

[0321] PRP Condition: DMEM with 10% Platelet-Rich Plasma (PRP) added.

[0322] PRP-HA Condition: DMEM supplemented with 10% PRP and hyaluronic acid (1% w / v).

[0323] Evaluation Indicators:

[0324] Visual observation: Record shrinkage, discoloration, or surface irregularities on days 7, 15, and 30.

[0325] Microscopic analysis:

[0326] Phase contrast microscopy for observing surface morphology.

[0327] Confocal microscopy to check fiber degradation and cell covering.

[0328] Mass loss: Measure the percentage decrease in matrix weight over 30 days.

[0329] The HA-Silk matrix is ​​expected to exhibit excellent long-term stability with minimal degradation and maintained structural integrity for 30 days under PRP-HA conditions. These results are expected to highlight the potential for long-term tissue engineering applications.

[0330] Example 12 - Fabrication of Multilayer Structures

[0331] We manufacture multiphase structures that mimic osteochondral tissue using advanced technologies (electrospinning and 3D printing) and analyze their properties (physicochemical and structural properties).

[0332] Coating layer (silk fibroin):

[0333] It is extracted from silkworm cocoons, degummed, and dissolved in a 9.3 M lithium bromide solution. It is known for its high tensile strength, biocompatibility, and ability to support cell adhesion.

[0334] Hydrogel layer:

[0335] ingredient:

[0336] Hyaluronic Acid (HA): Increases moisture retention and promotes cell proliferation.

[0337] Gelatin: A natural protein that promotes cell adhesion and differentiation.

[0338] Silk fibroin: Provides structural stability and mechanical strength.

[0339] Platelet lysate protein: Serves as a source of growth factors for cartilage formation.

[0340] Electroradiating layer:

[0341] Polycaprolactone (PCL): A biodegradable polymer with high mechanical strength and a slow degradation rate.

[0342] Hydroxyapatite: Mimics the mineral components of the calcified cartilage layer.

[0343] FDM layer:

[0344] It is a complex of PCL and hydroxyapatite that mimics the load-bearing properties of subchondral bone.

[0345] Manufacturing method:

[0346] Cover layer (50 μm):

[0347] Electrospinning a silk fibroin solution (7% w / v) onto a rotating accumulator.

[0348] Electrospinning conditions:

[0349] Voltage: 15 kV.

[0350] Flow rate: 0.8 mL / h.

[0351] Collector distance: 10 cm.

[0352] The layer is stabilized through a cross-linking reaction using ethanol vapor.

[0353] Hydrogel layer (1-2 mm):

[0354] HA (1% w / v), gelatin (2% w / v), and silk fibroin (3% w / v) were mixed in PBS at 37°C.

[0355] Platelet lysate protein (0.5% v / v) was added to this mixture.

[0356] The hydrogel is injected into a mold and crosslinked with genipin for 24 hours to increase stability.

[0357] Electrospinning layer (100 μm):

[0358] Electrospinning a solution of PCL (10% w / v) and hydroxyapatite (2% w / v) dissolved in chloroform / DMF (1:1) solvent.

[0359] Electrospinning conditions:

[0360] Voltage: 18 kV.

[0361] Flow rate: 1.2 mL / h.

[0362] Collector distance: 12 cm.

[0363] FDM layer (500 μm):

[0364] PCL and hydroxyapatite (70:30 ratio) are extruded using a Fused Deposition Modeling (FDM) 3D printer. The base layer is printed at a nozzle temperature of 180°C and a layer height of 100μm.

[0365] Each layer exhibits characteristics that mimic each region of biological osteochondral tissue, and is expected to ensure structural integrity and biocompatibility.

[0366] Example 13 - Multilayer structure incorporating HA-silk

[0367] We incorporate an HA-silk matrix into a multilayer structure for cartilage repair and evaluate its role in cell proliferation, matrix integration, and enhancement of biomechanical stability. We fabricate a multilayer structure composed of the following:

[0368] Layer 1 (Cover Layer): Electrospun silk fibroin (SF).

[0369] Layer 2 (hydrogel layer): HA-silk matrix combined with gelatin and platelet lysate protein (0.5% v / v).

[0370] Layer 3 (Electrosion Transition Layer): Polycaprolactone (PCL) and hydroxyapatite.

[0371] Layer 4 (FDM base layer): 3D printed PCL and hydroxyapatite.

[0372] HA-Silk Layer Integration:

[0373] The HA-silk matrix is ​​embedded in a hydrogel layer to mimic cartilage-like properties, and its bioactivity is enhanced through the addition of PRP-HA.

[0374] Cell type:

[0375] Human chondrocytes are inoculated into the structure to evaluate the degree of integration and proliferation.

[0376] Analysis Protocol:

[0377] Fusion Test: Evaluates interlayer cohesion through mechanical shear testing.

[0378] Cell proliferation: Measured via MTT assay on days 7, 15, and 30.

[0379] Substrate bioactivity: Analyze cell network formation and growth factor release via ELISA.

[0380] Biomechanical stability:

[0381] Compressive strength and elastic modulus are evaluated using a uniaxial testing machine.

[0382] The HA-silk matrix is ​​expected to be successfully integrated into a multilayer structure and is anticipated to enhance bioactivity, cell proliferation, and biomechanical stability. This structure is expected to demonstrate potential for cartilage regeneration applications, particularly in degenerative or traumatic diseases.

[0383] Example 14 - Evaluation of Multilayer Structures

[0384] Biocompatibility, cell adhesion, proliferation, and differentiation of the multiphase structure prepared according to Example 12 are evaluated under various culture conditions using chondrocytes and mesenchymal stem cells (MSCs).

[0385] Cell type:

[0386] Chondrocytes: Isolated from human joint cartilage biopsy tissue.

[0387] MSC: Taken from bone marrow aspirate.

[0388] Culture medium conditions:

[0389] Control group: DMEM + 10% fetal bovine serum (FBS).

[0390] PRP: DMEM with 10% platelet-rich plasma added.

[0391] PRP-HA: DMEM supplemented with 10% platelet-rich plasma and hyaluronic acid (1% w / v).

[0392] Preparation of cell culture matrix:

[0393] The multiphase structure is sterilized using ethylene oxide gas and then rehydrated in the culture medium for 1 hour before cell inoculation. To promote initial cell attachment, the structure is positioned with the surface layer (silk fibroin) facing upward.

[0394] Analysis Protocol:

[0395] Cell adhesion (24 hours):

[0396] Inoculate cells into the structure (1 × 10 5 Incubate cells / cm² for 24 hours.

[0397] Evaluate attachment using relative phase microscopy and SEM.

[0398] Proliferation (Days 7 and 14):

[0399] Cellular metabolic activity is evaluated through WST-1 analysis.

[0400] Chondrodysm (21 days):

[0401] Cultures inoculated with MSCs are cultured in a chondrogenic differentiation induction medium.

[0402] Glycosaminoglycan (GAG) production is evaluated through Alcian blue staining and quantified through dimethylmethylene blue (DMMB) analysis.

[0403] PRP-HA conditions are expected to significantly enhance cell adhesion, proliferation, and chondrogenesis in the multiphase structure. The hydrogel and electrospun layers play a crucial role in supporting bioactivity, demonstrating potential for utilization in osteochondral tissue regeneration. This multiphase structure is expected to support high levels of cellular bioactivity, particularly in the presence of PRP-HA. These results are expected to highlight its suitability for cartilage regeneration applications.

[0404] Example 15 - Mechanical testing of a multilayer structure

[0405] The multiphase structure is manufactured and sterilized as described in Example 12. The specimen is cut into a disc shape (diameter 8 mm, thickness 2 mm) for mechanical testing.

[0406] Mechanical Test Protocol:

[0407] Compression test (entire structure):

[0408] It is performed using a general-purpose testing machine.

[0409] The specimen is compressed at a speed of 1 mm per minute until 50% deformation occurs.

[0410] Measurement items:

[0411] Compressive strength (MPa).

[0412] Compressive modulus of elasticity (MPa).

[0413] Layer-by-layer tensile test:

[0414] Carefully separate the individual layers (cover, hydrogel, electrospun, FDM) and perform a uniaxial tensile test.

[0415] Record the tensile strength and elastic modulus for each layer.

[0416] Interlayer bonding test:

[0417] Interlayer adhesion strength is evaluated by applying shear stress to the specimen using a customized shear testing device.

[0418] Dynamic Mechanical Analysis (DMA):

[0419] It is performed to evaluate viscoelastic properties under periodic loading.

[0420] To simulate physiological conditions, a dynamic load of 1 Hz frequency is applied to the specimen.

[0421] This structure is expected to exhibit excellent mechanical properties, including compressive and tensile strengths comparable to biological osteochondral tissue. Interlayer adhesion testing is expected to confirm strong interlayer adhesion, which is essential for maintaining structural stability under mechanical loads.

[0422] This multiphase structure is expected to exhibit biomechanical properties suitable for load-bearing applications, making it an ideal candidate for osteochondral tissue restoration.

[0423] Example 16 - In vivo evaluation of multilayer structures

[0424] The performance of multiphase structures in promoting osteochondrosis regeneration is evaluated using preclinical animal models.

[0425] Animal Model:

[0426] Species: New Zealand White Rabbit (n = 15).

[0427] Defect model: Create a full-thickness osteochondral defect (diameter 4 mm, depth 2 mm) in the femoral condyle under sterile conditions.

[0428] Implant material group:

[0429] Group 1: Control group (no graft material).

[0430] Group 2: PRP-free multi-stage implant material.

[0431] Group 3: Multistage implant material including PRP-HA.

[0432] Surgical procedure:

[0433] The graft is sterilized using ethylene dioxide and then rehydrated in a sterile PBS solution before transplantation.

[0434] Fill the defect area with construct so that the defect is completely covered.

[0435] The graft is secured using fibrous adhesive to maintain its position during the healing period.

[0436] Post-operative care:

[0437] Observe the rabbit for 12 weeks after surgery.

[0438] Weight-bearing and mobility are evaluated weekly to assess functional recovery.

[0439] Evaluation Method:

[0440] Histological analysis:

[0441] Rabbits were euthanized at 4, 8, and 12 weeks, and femoral condyles were harvested for histological analysis. Sections were stained with Safranin O / Fast Green to evaluate cartilage regeneration and adhesion.

[0442] Micro-CT Scan:

[0443] Bone regeneration and structural degradation are evaluated by performing the procedure at the 12-week mark.

[0444] Biomechanical testing:

[0445] An indentation test is performed on the restored tissue to measure stiffness, and this is compared to natural cartilage.

[0446] PRP-HA reinforced multiphase structures are expected to demonstrate significantly improved cartilage and bone regeneration compared to control and non-PRP conditions. These layered structures effectively mimic in vivo osteochondral tissue, and are anticipated to support cell growth, matrix deposition, and mechanical function. This in vivo study is expected to demonstrate the potential of multiphase structures for osteochondral repair, particularly when combined with PRP-HA. Equipped with biomimetic properties and clinical-grade manufacturing processes, these structures are expected to be promising candidates for human cartilage repair.

[0447] Example 17 - Controlled growth factor release from a hydrogel layer

[0448] Evaluate the ability of the hydrogel layer to release platelet-derived growth factor (PDGF) in a sustained manner.

[0449] Hydrogel manufacturing:

[0450] A hydrogel layer 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).

[0451] To stabilize the network and regulate protein release, it was cross-linked with genipin for 24 hours.

[0452] Experiment setup:

[0453] A hydrogel disc (diameter 5 mm, thickness 1 mm) is prepared and immersed in phosphate-buffered saline (PBS) at 37°C.

[0454] To mimic physiological conditions, transfer the sample to fresh PBS at predetermined intervals (days 1, 3, 7, 14, and 21).

[0455] Growth Factor Quantification:

[0456] Growth factors (e.g., PDGF, TGF-β1, VEGF) released into PBS are quantified using enzyme-linked immunosorbent assay (ELISA). The release profile is evaluated by graphing the cumulative release rate over time.

[0457] The hydrogel layer of the multiphase structure is expected to deliver bioactive signals in a controlled manner for 21 days and exhibit excellent growth factor release characteristics. This characteristic is expected to enhance the structure's potential to promote osteochondral regeneration.

[0458] Example 18: Preparation of Silk Fibroin / Hyaluronic Acid Biological Dressing

[0459] Silk fibroin was extracted from silkworm cocoons. The silkworm cocoons were degummed using a standard heat dissolution method, rinsed, and air-dried. The dried fibroin was 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 was dissolved in formic acid to prepare a solution with a final concentration of 0.5% (w / v). The two solutions were mixed in a controlled environment and stirred until a homogeneous mixture was obtained, finally yielding a composition consisting of 93.75% silk fibroin and 6.25% hyaluronic acid by weight.

[0460] Subsequently, a nanofiber mat was prepared by electrospinning this mixture. The electrospinning process was performed under optimized conditions to ensure uniform fiber formation. The equipment conditions were set to a voltage of 15 kV, a flow rate of 1 mL / hour, and a dust collector distance of 10 cm. The nanofibers were deposited onto a medical support layer selected for compatibility with biological applications and ease of handling during subsequent analysis.

[0461] After manufacturing, the mat was dried at room temperature before sterilization. To preserve the bioactivity of hyaluronic acid, no crosslinking agent was added during the process. The manufactured dressings were stored under sterile conditions for subsequent testing.

[0462] Example 19: Protocol for Evaluating Cell Viability on Silk Fibroin / Hyaluronic Acid Matrix

[0463] The silk fibroin / hyaluronic acid matrix prepared as described in Example 18 was rehydrated before use in the cell viability assay. The matrix was immersed in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and rehydrated for 1 hour in a humidity-controlled incubator at 37°C. After rehydration, the matrix was placed in individual wells of a 6-well plate with the bioactive layer facing upward to maximize cell interactions.

[0464] Normal human dermal fibroblasts (NHDFs) were selected for this analysis. Cells were inoculated directly onto a rehydrated matrix surface at a density of 50,000 cells per cm². A culture medium consisting of DMEM containing 10% FBS was carefully poured over the matrix to completely cover it. The inoculated matrix was then cultured at 37°C for up to 7 days in a humid environment containing 5% CO₂. To maintain optimal culture conditions, the medium was replaced every 48 hours.

[0465] At specified time points (e.g., days 1, 3, and 7), samples were removed from the incubator and gently washed with phosphate-buffered saline (PBS) to remove unattached cells, after which additional viability analysis was performed using the Live / Dead assay protocol. In this assay, the matrix was stained with Calcein-AM to indicate live cells and with Ethidium homodimer-1 to indicate dead cells. The staining solution was prepared according to the manufacturer's instructions, and the matrix was incubated with the staining solution in a dark place at room temperature for 30 minutes.

[0466] The stained matrix was then observed under a fluorescence microscope to capture images for subsequent analysis. This protocol ensures a standardized and reproducible method for evaluating the matrix's ability to maintain cell viability.

[0467] Example 20: Rehydration Protocol of HA-Silk Matrix

[0468] The silk fibroin / hyaluronic acid matrix prepared as described in Example 18 was rehydrated to be suitable for biological analysis. Square samples measuring 1 cm² were cut from an electrospinning mat under sterile conditions. These samples were immersed in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C for 1 hour in a humid atmosphere containing 5% CO₂.

[0469] During the culture process, the matrix absorbed the medium, transforming from an initially dry and rigid state into a hydrated and flexible form suitable for cell inoculation. To maximize exposure for subsequent biological interactions, the bioactive side of the matrix was positioned facing upward throughout the rehydration process. Subsequently, the rehydrated matrix was transferred to a sterile 6-well plate using tweezers, taking care to avoid physical damage to the delicate fibers.

[0470] This rehydration protocol provides a standardized and reproducible method for preparing an HA-silk fibroin matrix for use in cell-based analysis or other biological applications.

[0471] Example 21: Evaluation of HA-silk matrix stability under culture conditions

[0472] To evaluate the stability of the HA-silk fibroin matrix, physical integrity over time was assessed under various culture medium conditions. Matrix samples were prepared as described in Example 18, rehydrated as specified in Example 20, and placed in 6-well plates. Each sample was exposed to one of the following conditions: (1) Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (control), (2) DMEM supplemented with platelet-rich plasma (PRP), and (3) DMEM supplemented with PRP and hyaluronic acid (HA) (PRP-HA).

[0473] The matrix was cultured at 37°C in a humid atmosphere containing 5% CO₂. On days 1, 3, 7, and 15, the degree of swelling, structural integrity, and detachment from the support layer were observed and recorded. Before visual evaluation, the matrix was gently rinsed with phosphate-buffered saline (PBS) to remove unattached cells or debris.

[0474] The evaluation included recording physical changes such as expansion rate, fiber morphology, and matrix delamination under test conditions. This protocol provides insights into the suitability of HA-silk fibroin matrix during long-term culture in various bioactive environments.

[0475] Example 22: Preparation of HA-Silk Matrix Added to Platelet Lysate

[0476] To enhance bioactivity, platelet lysate (PL) was added to a silk fibroin / hyaluronic acid matrix. Platelet lysate was prepared at two concentrations, 0.25% and 0.5% (v / v), from commercially available raw materials. An experimental medium was prepared by mixing PL with DMEM containing 10% FBS.

[0477] HA-silk fibroin matrix samples prepared as described in Example 18 were rehydrated in a medium supplemented with this PL under sterile conditions. The matrices were immersed for 1 hour in a PL-enriched medium maintained at 37°C in a humid atmosphere containing 5% CO₂. After rehydration, the matrices were placed in a 6-well plate with the bioactive side facing upward.

[0478] Through this preparation method, the enhanced bioactivity of the matrix can be investigated by introducing platelet lysate (PL) while maintaining the integrity of the silk fibroin and hyaluronic acid components.

[0479] Example 23: Standard method for preparing silk fibroin solution from silkworm cocoons

[0480] Silk fibroin was extracted from silkworm cocoons using a standard degumming and dissolution process. First, 5 g of silkworm cocoons were cut into small pieces and subjected to a degumming step in which they were boiled in 2 L of 0.02 M sodium carbonate solution for 30 minutes. This step effectively removed sericin while preserving the fibroin fibers. The degummed silk fibers were rinsed three times for 20 minutes each in 1 L of deionized water, and then squeezed to remove excess moisture. The fibers were dried overnight in a fume hood and then stored at room temperature for future use.

[0481] For the dissolution process, dried silk fibroin fibers were dissolved in a 9.3 M lithium bromide solution at a concentration of 20% (w / v). This mixture was heated to 60°C and stirred for 4 hours until a clear and transparent silk fibroin solution was obtained. Subsequently, salts were removed from the solution by dialysis using a 12-14 kDa molecular weight cutoff membrane with deionized water as a control. The dialysis process was carried out over 48 hours with the water being replaced 6 times. After dialysis, the fibroin solution was centrifuged at 9,000 rpm (12,700 g) at 4°C for 20 minutes, and this process was repeated 3 times to remove insoluble particles.

[0482] The silk fibroin solution with a concentration of 7-8% (w / v) obtained in this way was stored at 4°C for use in scaffold fabrication. Through this process, high-quality silk fibroin suitable for application in biomaterials research and tissue engineering can be stably obtained.

[0483] Example 24: Preparation of silk fibroin solution using microwaves

[0484] Silk fibroin was extracted from silkworm cocoons using a microwave-assisted degumming process. 2 g of silkworm cocoons were cut into small pieces and subjected to three microwave-assisted degumming cycles. Each cycle involved immersing the cocoon pieces in a 0.02 M sodium carbonate solution containing 0.25% sodium dodecyl sulfate (SDS), heating at 800 W for 1 minute, and allowing a resting period of 10 minutes (approximate temperature: 80°C). After each cycle, the fibers were rinsed with deionized water for 1 minute and then squeezed to remove excess liquid. After three cycles, the degummed fibers were dried overnight or used directly in dissolution experiments.

[0485] For dissolution, 0.1 g of degummed silk fiber was immersed in 10 mL of a 56% (w / w) aqueous zinc chloride (ZnCl₂) solution at 45°C for 1 hour. The resulting 1% (w / v) silk fibroin solution was subjected to salt removal using a CentriPure P50 column to remove zinc chloride. This process was repeated to confirm the complete removal of ZnCl₂, verified using a zinc test kit that measures absorbance at 560 nm. The eluted fibroin solution was used immediately or dried via freeze-drying for storage.

[0486] This microwave-assisted method provides a rapid and reliable process for producing silk fibroin solutions suitable for various biomedical applications.

[0487] Example 25: Preparation of HA-Silk Matrix with Added Platelet Lysate

[0488] Platelet lysate (PL) was added to a silk fibroin / HA matrix at concentrations of 0.25% and 0.5% (v / v). The platelet lysate was prepared using a commercially available PLT MAX kit. The silk fibroin / HA mixture (silk fibroin 7.5% w / v and HA 0.5% w / v) was rehydrated in DMEM solutions to which each PL concentration was added. Rehydration was performed at 37°C for 1 hour to allow the matrix to completely absorb the PL-added medium.

[0489] The rehydrated platelet lysate-added HA-silk matrix was subsequently used for cell viability and proliferation analysis to evaluate bioactivity.

[0490] Example 26: Preparation of PRP and PRP-HA

[0491] Peripheral blood samples were collected from healthy donors after obtaining ethical approval (CCER Geneva Ethics Committee, ID 2017-00700). A total of 30 mL of blood was collected into specific medical devices, 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 at the bottom of the separation gel, while plasma and platelets remained in the supernatant.

[0492] The tubes were inverted five times to homogenize the plasma and platelets. 6 mL of PRP was obtained from each CuteCell PRP tube, and 3 mL of PRP mixed with 2 mL of HA was obtained from each CellularMatrix ACP-HA tube. The concentrations of platelets, erythrocytes, and leukocytes, as well as the mean platelet volume (MPV), were found to be within the ranges reported in the literature.

[0493] The prepared PRP and PRP-HA solutions were stored in polypropylene tubes until they were used for biological analysis.

[0494] Example 27: Protocol for Evaluating Cell Viability of Silk Fibroin / Hyaluronic Acid Biodressings

[0495] Silk fibroin / hyaluronic acid bio-dresses were prepared containing platelet lysates at concentrations of 0.25% (v / v) and 0.5% (v / v), as described in the previous example. These dressings were prepared without a protective membrane and sterilized prior to use. Because the silk fibroin and hyaluronic acid were uniformly mixed during the preparation process, the cell adhesion surface of the dressing was uniform.

[0496] Cell viability was evaluated using periodontal ligament cells (PDL, 3rd generation), gingival fibroblasts (GF, 3rd generation), and myoblasts (3rd generation). Cells were harvested via a transplantation technique and stored in liquid nitrogen prior to the experiment. Cell viability was evaluated at four time points, at 1, 2, 3, and 4 weeks, using a Live / Dead analysis kit.

[0497] For testing, one biological dressing was placed per well on an uncoated 24-well μ-Plate assay plate. Each dressing was rehydrated in 500 μL of DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C for 30 minutes. After rehydration, the medium was removed to keep the dressing wet. A suspension containing 150,000 cells in 500 μL of medium was inoculated onto each dressing, and the droplets were placed in the center for uniform distribution. To induce cell attachment, the plates were incubated at 37°C in a humid environment containing 5% CO₂.

[0498] Live / dead staining was performed at each time point by incubating the dressing with the staining solution according to the manufacturer's instructions. The stained samples were observed using a Cytation 7 confocal microscope. In addition, scanning electron microscopy (SEM) was performed on fixed samples to observe the integrity and degradation of the matrix over time. Quantification of green and red signals was performed using image analysis software, and matrix degradation was evaluated visually and via SEM.

[0499] Example 28: Evaluation of HUVEC cell viability and adhesion on silk fibroin / hyaluronic acid nanofibers

[0500] Silk fibroin / hyaluronic acid nanofibers were prepared using the same method as described in the previous example, and cell viability and adhesion were evaluated using human umbilical vein endothelial cells (HUVECs, P5). Four types of scaffolds coated with SF-HA nanofibers were tested: 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.

[0501] Each sample was exposed to five different culture conditions, including a control medium, 10% PRP, 10% (w / v) PRP-HA, and 10% (v / v) PL. Dressings were observed using Cytation imaging technology, and samples were placed upside down to evaluate cell interactions with nanofibers.

[0502] Sample 1, an LDPE / cellulose composite with nanofibers formed on the cellulose side, floated on the medium due to the foaming properties of the support layer and failed to support the attachment of endothelial cells under any conditions. Similarly, Sample 2, an LDPE / cellulose composite with nanofibers formed on the LDPE side, also exhibited a floating phenomenon, and no significant endothelial cell attachment was observed. On the other hand, Sample 3, composed of polyethylene paper with nanofibers attached to the polyethylene side, demonstrated endothelial cell attachment and viability when cultured in PRP, PRP-HA, and PL media. However, no significant cell attachment was observed under control medium conditions. Polyethylene paper with nanofibers formed on the paper side (Sample 4) showed strong endothelial cell attachment and viability under PRP, PRP-HA, and PL conditions, and the cell layer remained attached even after the peelability test.

[0503] In conclusion, samples 3 and 4 provided optimal conditions for endothelial cell adhesion and survival, particularly in the presence of PRP, PRP-HA, and PL. Samples 1 and 2 were unsuitable due to floating phenomena and a lack of cell adhesion.

[0504] Example 29: Evaluation of Silk Fibroin / Hyaluronic Acid Matrix Using Vaginal Fibroblasts and Periodontal Ligament Cells

[0505] Silk fibroin and hyaluronic acid matrices were prepared as previously described. These matrices were subjected to cell viability, adhesion, and proliferation tests using vaginal fibroblasts (VF) and periodontal ligament cells (PDL). The experiments included various culture conditions, such as control medium, 10% platelet-rich plasma (PRP), and PRP supplemented with 10% (w / v) hyaluronic acid (PRP-HA).

[0506] For evaluation using vaginal fibroblasts, 1 cm² pieces of SF-HA matrix were placed in tissue culture dishes. Cells were seeded at a density of 100,000 cells per matrix and cultured under specified conditions. After 15 days of culture, cell viability was assessed using a Live / Dead Kit. Viable cells were identified by calcein fluorescence, and dead cells were detected by ethidium homodimer-1 fluorescence. Microscopic observation revealed strong adhesion and proliferation of vaginal fibroblasts under the 10% PRP-HA condition, along with significant elongation and alignment along the matrix fibers. Minimal dead cells were observed under all conditions. However, under the control condition, fibroblast proliferation was low and no significant elongation was observed. Moderate proliferation and elongation were observed under the 10% PRP (w / v) condition.

[0507] Similar experiments were performed using periodontal ligament cells. In this experiment, the SF-HA matrix was evaluated under identical conditions. Observations after 15 days revealed strong adhesion and proliferation, along with clear cell elongation and alignment, under the PRP-HA 10% (w / v) condition. In contrast, cell adhesion was rare under the control condition, while moderate cell proliferation without significant elongation was observed under the PRP 10% condition. The PRP-HA condition proved to be particularly effective in maintaining the structural integrity of the HA layer within the matrix, as evidenced by consistent cell coverage and the absence of delamination of the electrospun HA layer.

[0508] Overall, the data clearly demonstrated that 10% (w / v) PRP-HA provides an optimal environment for cell adhesion, proliferation, and alignment for both vaginal fibroblasts and periodontal ligament cells. This highlights the bioactive potential of the SF-HA matrix reinforced with PRP and HA.

[0509] Example 30: Evaluation of Myoblast Differentiation in Silk Fibroin / Hyaluronic Acid Biodressings Supplemented with Platelet Lysate and Platelet-Rich Plasma

[0510] Silk fibroin / hyaluronic acid bio-dressings (SF-HA) were prepared as previously reported, and their ability to promote myoblast differentiation under various culture conditions was evaluated. This study compared differentiation results using 10% (v / v) platelet lysate (PL), 10% (w / v) platelet-rich plasma (PRP), and PRP conjugated with 10% (w / v) hyaluronic acid (HA) (HA-PRP) with standard differentiation medium (DM) as the reference control. Primary human myoblasts (P5) were inoculated into the SF-HA matrix and cultured under these conditions for 3 weeks.

[0511] Experiment setup

[0512] The matrix was first rehydrated for 30 minutes in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified CO₂ incubator at 37°C. After rehydration, 150,000 myoblasts suspended in 500 μL of medium were inoculated onto each dressing and allowed to attach for 30 minutes. Subsequently, 500 μL of each treatment medium containing DM, PL 10% (v / v), PRP 10% (w / v), and HA-PRP 10% (w / v) was added.

[0513] Results after 3 weeks

[0514] At the 3-week mark, samples were fixed with 4% paraformaldehyde for immunofluorescence staining and confocal microscopy. Immunostaining was performed using markers specific to myoblast differentiation, such as Mef2C and alpha-actinin.

[0515] 1. HA-PRP 10% (w / v): The most potent differentiation results were observed in the HA-PRP group. Confocal microscopy confirmed the extensive formation of aligned multinucleated myocanalities with excellent structural integrity. The myocanalities showed significant elongation and alignment along the SF-HA matrix, reflecting enhanced myogenic differentiation. The presence of HA appeared to provide a synergistic effect that maximized differentiation ability by enhancing the bioactivity of PRP.

[0516] 2. PRP 10% (w / v): Samples treated with PRP alone showed strong differentiation ability along with clear evidence of canal formation and alignment. However, the degree of elongation and structural organization were somewhat reduced compared to the HA-PRP 10% condition, suggesting that while PRP effectively supports myogenesis, optimal differentiation may be limited if the structural benefits of HA are lacking.

[0517] 3. PL 10% (v / v): Although differentiation was observed, it was significantly less effective compared to PRP and HA-PRP conditions. The root canals were less aligned and showed reduced structural integrity. These limitations observed in PL may be attributed to the use of formic acid in the preparation process. As previously described, formic acid likely inhibited the bioactivity of growth factors within PL through protein denaturation, pH instability, and peptide bond hydrolysis.

[0518] 4. Differentiation medium (DM):The control condition exhibited the lowest differentiation ability. Confocal microscopy images showed that the muscle tubes were sparsely distributed and poorly aligned, confirming that the SF-HA matrix alone is insufficient to promote effective muscle formation without supplemental bioactive factors.

[0519] conclusion

[0520] This study demonstrates that HA-PRP 10% (w / v) treatment provides the most effective environment for myoblast differentiation and exhibits significantly superior performance compared to PRP 10% (w / v), PL 10% (v / v), and control differentiation media. The combination of HA and PRP enhances myoblast alignment, elongation, and tube formation by creating a synergistic microenvironment that supports cell adhesion and differentiation. Conversely, the use of platelet lysates prepared with formic acid resulted in reduced differentiation outcomes, highlighting the importance of preserving the physiological activity of growth factors during the preparation process. These findings demonstrate that HA-PRP 10% (w / v) is an excellent therapeutic agent for myoblast differentiation and emphasize the need to optimize platelet lysate preparation to prevent the loss of physiological activity.

Claims

Claim 1 As a scaffold material, the following: i) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate; and ii) a scaffold material composed of electrospun fibers containing silk protein. Claim 2 A scaffold material according to claim 1, additionally comprising hyaluronic acid. Claim 3 A scaffold material according to claim 1 or 2, further comprising an anti-fibrolytic substance such as tranexamic acid. Claim 4 A scaffold material according to any one of claims 1 to 3, further comprising a coagulation promoter such as calcium gluconate. Claim 5 A scaffold material according to any one of claims 1 to 4, wherein the scaffold material further comprises vitamin K2. Claim 6 A scaffold material according to any one of claims 1 to 5 comprises a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, wherein, in particular, additional therapeutic agents include corticosteroids, NSAIDs, cartogenin, A scaffold material selected from the group consisting of hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 7 A scaffold material according to any one of paragraphs 1 to 6, wherein the scaffold material is a semi-solid scaffold material. Claim 8 A scaffold material according to any one of claims 1 to 7, wherein the scaffold material further comprises hydroxyapatite. Claim 9 A scaffold material according to paragraph 8, which is a solid scaffold material. Claim 10 A scaffold material comprising the following components: i) hydroxyapatite; and ii) electrospun fibers containing silk protein. Claim 11 A scaffold material according to paragraph 10, further comprising platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate. Claim 12 A scaffold material according to claim 10 or 11, wherein the scaffold material further comprises hyaluronic acid. Claim 13 A scaffold material according to any one of claims 10 to 12, further comprising an anti-fibrolytic substance such as tranexamic acid. Claim 14 A scaffold material according to any one of claims 10 to 13, wherein the scaffold material further comprises a coagulation promoter such as calcium gluconate. Claim 15 A scaffold material according to any one of paragraphs 10 to 14, wherein the scaffold material further comprises vitamin K2. Claim 16 A scaffold material according to any one of claims 10 to 15 comprising a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, wherein, in particular, additional therapeutic agents include corticosteroids, NSAIDs, cartogenin, A scaffold material selected from the group consisting of hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 17 A scaffold material according to any one of paragraphs 10 to 16, wherein the scaffold material is a solid scaffold material. Claim 18 A scaffold material comprising the following components: i) hyaluronic acid; and ii) an electrospun fiber comprising silk protein. Claim 19 A scaffold material according to paragraph 18, stabilized through cross-linking using an aldehyde-based cross-linking agent such as glutaraldehyde (e.g., gaseous form). Claim 20 A scaffold material according to claim 18 or 19, wherein the weight ratio of electrospun fibers comprising hyaluronic acid and silk protein is about 1:2 to about 1:4, for example, about 1:

3. Claim 21 A scaffold material according to any one of claims 18 to 20, further comprising platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate. Claim 22 A scaffold material according to any one of claims 18 to 21, wherein the scaffold material further comprises hydroxyapatite. Claim 23 A scaffold material according to any one of paragraphs 18 to 22, further comprising an anti-fibrolytic substance such as tranexamic acid. Claim 24 A scaffold material according to any one of claims 18 to 23, wherein the scaffold material further comprises a coagulation promoter such as calcium gluconate. Claim 25 A scaffold material according to any one of paragraphs 18 to 24, wherein the scaffold material further comprises vitamin K2. Claim 26 A scaffold material according to any one of claims 18 to 25, comprising a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, in particular additional therapeutic agent is a corticosteroid, NSAID, cartogenin, A scaffold material selected from the group consisting of hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 27 A layered wound dressing or layered graft comprising: i) a scaffold material defined in any one of claims 1 to 26; and ii) a support. Claim 28 A layered wound dressing or layered graft according to paragraph 27, comprising: i) a scaffold material comprising: ia) electrospun fibers comprising silk protein; and ii) hyaluronic acid; and ii) a scaffold material comprising a layered wound dressing or layered graft. Claim 29 As a layered structure for tissue engineering applications, the following: i) ii) a first layer containing silk protein; A second layer comprising a hydrogel and silk protein; iii) A third layer comprising polycaprolactone and / or hyaluronic acid; and hydroxyapatite; and iv) A layered structure comprising a fourth layer composed of polycaprolactone and hydroxyapatite. Claim 30 A layered structure according to claim 29, wherein the thickness of the first layer containing silk protein is about 40 μm to about 60 μm, for example, about 50 μm. Claim 31 A layered structure according to paragraph 29 or 30, wherein the first layer containing silk protein is a coating layer. Claim 32 A layered structure according to any one of paragraphs 29 to 31, wherein the silk protein in the first layer is composed of electrospun fibers of silk protein. Claim 33 A layered structure according to claim 32, wherein the silk protein electrospun fiber is produced by using a silk fibroin solution having a concentration of about 5% (w / v) to 15% (w / v), for example about 7% (w / v), under a voltage of 12 to 18 kV, for example about 15 kV; at a flow rate of about 0.8 mL / hour to about 1.2 mL / hour, for example 0.8 mL / hour; and at a collector plate distance of 5 to 20 cm, for example about 10 cm. Claim 34 A layered structure according to any one of claims 29 to 33, wherein the hydrogel of the second layer comprises hyaluronic acid and / or gelatin. Claim 35 A layered structure according to paragraph 34, wherein the hyaluronic acid is a mixture of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid (e.g., a weight ratio of about 1:1). Claim 36 A layered structure according to any one of claims 29 to 35, wherein the silk protein of the second layer is composed of electrospun fibers of silk protein. Claim 37 A layered structure according to claim 36, wherein the silk protein electrospun fiber is produced by using a silk fibroin solution having a concentration of about 5% (w / v) to 15% (w / v), for example about 7% (w / v), under a voltage of 12 to 18 kV, for example about 15 kV; at a flow rate between about 0.8 mL / hour and about 1.2 mL / hour, for example 0.8 mL / hour; and at a current collection distance of 5 to 20 cm, for example about 10 cm. Claim 38 A layered structure according to any one of claims 29 to 37, wherein the thickness of the second layer is 1 to 2 mm. Claim 39 A 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 further comprising platelet lysate. Claim 40 A 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. Claim 41 A 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. Claim 42 A layered structure according to paragraph 40, wherein the third layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 80:

20. Claim 43 A layered structure according to any one of claims 29 to 42, wherein the thickness of the third layer is 90 μm to 110 μm. Claim 44 A layered structure according to any one of claims 29 to 43, wherein a fourth layer comprising polycaprolactone and hydroxyapatite is formed using melt additive modeling (FDM). Claim 45 A layered structure according to any one of claims 29 to 44, wherein the fourth layer comprises polycaprolactone and hydroxyapatite in a weight ratio of 70:

30. Claim 46 A 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. Claim 47 A scaffold material, a layered wound dressing, a layered graft, or a layered structure according to any one of claims 1 to 46, wherein the silk protein is a silk fibroin suitably obtained from a solution of dissolved silkworm silk such as Bombyx mori. Claim 48 A scaffold material, a layered wound dressing, a layered graft, or a layered structure according to any one of claims 1 to 47, characterized in that the diameter of the electrospun fiber is between about 50 nm and about 1,000 nm, for example, between about 200 nm and about 500 nm. Claim 49 A scaffold material according to any one of paragraphs 1 through 26, 47, or 48, wherein the scaffold material is in the form of a film, membrane, mat, or mesh. Claim 50 A scaffold material according to any one of claims 1 to 26 or claims 47 to 49, characterized by having a swelling rate of 300% or more. Claim 51 A tissue engineering structure comprising: i) a scaffold material, a stacked wound dressing, a stacked graft, or a stacked structure according to any one of claims 1 to 50; and ii) a tissue engineering structure composed of mammalian cells. Claim 52 A tissue engineering structure according to claim 51, wherein the mammalian cells comprise cells selected from the group consisting of hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal-derived cells, cholangiocarcinocytes, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, myocardial cells, renal epithelial cells, renal tubular cells, renal basement membrane cells, neurons, vascular cells, bone and cartilage-forming cells, smooth muscle cells, skeletal muscle cells, oral cells, epithelial cells, bone marrow cells, keratinocytes, pluripotent cells, and stem cells, and combinations thereof. Claim 53 A method for manufacturing a tissue engineering structure, comprising the following steps: a) manufacturing a scaffold material according to any one of claims 1 to 50; and b) mixing the scaffold material with mammalian cells in a suitable medium. Claim 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 containing silk protein, comprising the following steps: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibers containing silk protein; and c) mixing the electrospun fibers containing silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a scaffold material. Claim 55 A method according to paragraph 54, further comprising the step of adding hyaluronic acid to a silk protein solution after step a) and before step b). Claim 56 A method according to claim 54 or 55, wherein in step c) an electrospun fiber containing silk protein is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and hyaluronic acid to form a scaffold material. Claim 57 A method according to any one of claims 54 to 56, comprising the step of adding an anti-fibrolytic substance (such as tranexamic acid) to a silk protein solution after step a) and before step b). Claim 58 A method according to any one of claims 54 to 57, comprising, in step c), the step of forming a scaffold material by combining an electrospun fiber containing a silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and an antifibrinolytic substance (tranexamic acid, etc.). Claim 59 A method according to any one of claims 54 to 58, comprising the step of adding a coagulation activator (e.g., calcium gluconate) to a silk protein solution after step a) and before step b). Claim 60 A method according to any one of claims 54 to 59, characterized in that, in step c), an electrospun fiber containing silk protein is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and a coagulation promoter (e.g., calcium gluconate) to form a scaffold material. Claim 61 A method according to any one of claims 54 to 60, comprising the additional step of adding vitamin K2 to a silk protein solution after step a) and before step b). Claim 62 A method according to any one of claims 54 to 61, comprising, in step c), the step of combining an electrospun fiber containing silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and vitamin K2 to form a scaffold material. Claim 63 A method according to any one of claims 54 to 62, comprising the additional step of adding an additional therapeutic agent to a silk protein solution after step a) and before step b). Claim 64 A method according to any one of claims 54 to 63, wherein in step c) an electrospun fiber comprising a silk protein is combined with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and one or more additional therapeutic agents to form a scaffold material. Claim 65 A method according to paragraph 63 or 64, wherein one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, A method selected from the group consisting of hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 66 A method according to any one of claims 54 to 65, comprising the additional step of adding hydroxyapatite to a silk protein solution after step a) and before step b). Claim 67 A method according to any one of claims 54 to 66, comprising, in step c), combining an electrospun fiber containing silk protein with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate and hydroxyapatite to form a scaffold material. Claim 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 containing silk protein, comprising the following steps: a) preparing a silk protein solution; b) mixing the silk protein solution of step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture; and c) electrospinning the mixture of step b) to form a scaffold material. Claim 69 A method according to claim 68 comprising, in step b), mixing the silk protein mixture of step a) with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and with hyaluronic acid to form a mixture. Claim 70 A method according to claim 68 or claim 69, wherein in step b), the silk protein mixture of step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and an antifibrinolytic substance (tranexamic acid, etc.) to form a mixture. Claim 71 A method according to any one of claims 68 to 70, wherein in step b), the silk protein mixture of step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and a coagulation promoter (e.g., calcium gluconate) to form a mixture. Claim 72 A method according to any one of claims 68 to 71, wherein in step b), the silk protein mixture of step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and vitamin K2 to form a mixture. Claim 73 A method according to any one of claims 68 to 72, wherein in step b), the silk protein mixture of step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and one or more additional therapeutic agents to form a mixture. Claim 74 As a method according to paragraph 73, the one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, hemoglobin, anesthetic, A method selected from the group consisting of analgesics, opioids, saponins and THC, or combinations thereof. Claim 75 A method according to any one of claims 68 to 74, wherein in step b), the silk protein mixture of step a) is mixed with platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate, and hydroxyapatite to form a mixture. Claim 76 A method according to any one of claims 68 to 75, further comprising step d), wherein in step d), hyaluronic acid is added to the scaffold material of step c). Claim 77 A method according to any one of claims 68 to 76, further comprising the step of adding an anti-fibrotic substance (tranexamic acid, etc.) to the scaffold material of step c). Claim 78 A method according to any one of claims 68 to 77, further comprising the step of adding a coagulation accelerator (e.g., calcium gluconate) to the scaffold material of step c). Claim 79 A method according to any one of claims 68 to 78, further comprising the step of adding vitamin K2 to the scaffold material of step c). Claim 80 A method according to any one of claims 68 to 79, comprising the step of adding one or more additional therapeutic agents to the scaffold material of step c). Claim 81 As a method according to paragraph 80, the one or more therapeutic agents are steroids, corticosteroids, glucocorticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), cartogenins, anesthetics, antimicrobial 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, neuroleptics, proteins, peptides, lipoproteins, tumor-killing compounds, tumor-stopping compounds, toxins, vitamins (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle fillers, or combinations thereof; in particular, additional therapeutic agents are corticosteroids, NSAIDs, cartogenins, hemoglobin, anesthetics, A method selected from the group consisting of analgesics, opioids, saponins and THC, or combinations thereof. Claim 82 A method according to any one of claims 68 to 81, further comprising the step of adding hydroxyapatite to the scaffold material of step c). Claim 83 A method for preparing a scaffold material comprising i) hydroxyapatite and ii) an electrospun fiber comprising silk protein, comprising the following steps: a) preparing a silk protein solution; b) mixing the silk protein solution of step a) with hydroxyapatite to form a mixture; and c) electrospinning the mixture of step b) to form a scaffold material. Claim 84 A method according to claim 83, characterized in that in step b), the silk protein solution of step a) is mixed with hydroxyapatite and platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture. Claim 85 A method according to 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. Claim 86 A method according to any one of claims 83 to 85, wherein in step b), the silk protein solution of step a) is mixed with hydroxyapatite and an anti-fibrolytic substance (tranexamic acid, etc.) to form a mixture. Claim 87 A method according to any one of claims 83 to 86, wherein in step b), the silk protein solution of step a) is mixed with hydroxyapatite and a coagulation accelerator (e.g., calcium gluconate) to form a mixture. Claim 88 A method according to any one of claims 83 to 87, wherein in step b), the silk protein solution of step a) is mixed with hydroxyapatite and vitamin K2 to form a mixture. Claim 89 A method according to any one of claims 83 to 88, wherein in step b), the silk protein solution of step a) is mixed with hydroxyapatite and one or more additional therapeutic agents to form a mixture. Claim 90 As a method according to claim 89, the one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, hemoglobin, anesthetic, A method selected from the group consisting of analgesics, opioids, saponins and THC, or combinations thereof. Claim 91 A method according to any one of claims 83 to 90, comprising the step of adding platelet-rich plasma and / or bone marrow concentrate to the scaffold material of step c). Claim 92 A method according to any one of claims 83 to 91, further comprising the step of adding hyaluronic acid to the scaffold material of step c). Claim 93 A method according to any one of claims 83 to 92, further comprising the step of adding an anti-fibrotic substance (tranexamic acid, etc.) to the scaffold material of step c). Claim 94 A method according to any one of claims 83 to 93, further comprising the step of adding a coagulation accelerator (e.g., calcium gluconate) to the scaffold material of step c). Claim 95 A method according to any one of claims 83 to 94, further comprising the step of adding vitamin K2 to the scaffold material of step c). Claim 96 A method according to any one of claims 83 to 95, comprising the step of adding one or more additional therapeutic agents to the scaffold material of step c). Claim 97 As a method according to claim 96, the one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, cytokine enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, A method selected from the group consisting of cartogenin, hemoglobin, anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 98 A method for preparing a scaffold material composed of i) hydroxyapatite and ii) electrospun fibers containing silk protein, comprising the following steps: a) preparing a silk protein solution; b) electrospinning the solution of step a) to form electrospun fibers containing silk protein; and c) combining the electrospun fibers containing silk protein with hydroxyapatite to form a scaffold material. Claim 99 A method according to claim 98, comprising additionally adding d) platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to the scaffold material of step c). Claim 100 A method according to claim 98 or claim 99, further comprising the step of adding hyaluronic acid to the scaffold material of step c). Claim 101 A method according to any one of claims 98 to 100, further comprising the step of adding an anti-fibrotic substance (tranexamic acid, etc.) to the scaffold material of step c). Claim 102 A method according to any one of claims 98 to 101, further comprising the step of adding a coagulation accelerator (e.g., calcium gluconate) to the scaffold material of step c). Claim 103 A method according to any one of claims 98 to 102, further comprising the step of adding vitamin K2 to the scaffold material of step c). Claim 104 A method according to any one of claims 98 to 103, comprising the step of adding one or more additional therapeutic agents to the scaffold material of step c). Claim 105 As a method according to claim 104, the one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, hemoglobin, A method selected from the group consisting of anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 106 A method for preparing a scaffold material comprising i) hyaluronic acid and ii) an electrospun fiber comprising a silk protein, comprising the following steps: a) preparing a silk protein solution; b) mixing the silk protein solution of step a) with hyaluronic acid to form a mixture; and c) electrospinning the mixture of step b) to form a scaffold material. Claim 107 A method according to claim 106, wherein in step b), the weight ratio of hyaluronic acid to silk protein is about 1:2 to about 1:4, for example, about 1:

3. Claim 108 A method according to claim 106 or 107, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and platelet-rich plasma and / or platelet lysate and / or bone marrow concentrate to form a mixture. Claim 109 A method according to any one of claims 106 to 108, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and hydroxyapatite to form a mixture. Claim 110 A method according to any one of claims 106 to 109, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and an anti-fibrolytic substance (tranexamic acid, etc.) to form a mixture. Claim 111 A method according to any one of claims 106 to 110, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and a coagulation accelerator (such as calcium gluconate) to form a mixture. Claim 112 A method according to any one of claims 106 to 111, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and vitamin K2 to form a mixture. Claim 113 A method according to any one of claims 106 to 112, wherein in step b), the silk protein solution of step a) is mixed with hyaluronic acid and one or more additional therapeutic agents to form a mixture. Claim 114 As a method according to claim 113, the one or more therapeutic agents comprise a steroid, corticosteroid, glucocorticosteroid, non-steroidal anti-inflammatory drug (NSAID), cartogenin, anesthetic, antimicrobial compound, antibiotic, antifungal compound, antiparasitic compound, enzyme, enzyme inhibitor, glycoprotein, growth factor, hormone, antiviral compound, analgesic, opioid, saponin, hemoglobin, tetrahydrocannabinol (THC), cannabidiol (CBD), anti-angiogenic agent, anti-melanogenic agent, immunomodulator, immunoglobulin, mineral, neuroleptic, protein, peptide, lipoprotein, tumor-killing compound, tumor-stopping compound, toxin, vitamin (vitamin A, vitamin E, vitamin B, vitamin C, vitamin D; or derivatives thereof) or wrinkle filler, or a combination thereof, and in particular, additional therapeutic agents comprise a corticosteroid, NSAID, cartogenin, hemoglobin, A method selected from the group consisting of anesthetics, analgesics, opioids, saponins and THC, or combinations thereof. Claim 115 A method according to any one of claims 106 to 114, comprising the step of further comprising: step d) crosslinking a scaffold material by reacting it with an aldehyde-based crosslinking agent, such as glutaraldehyde (e.g., in the gas phase). Claim 116 A method according to any one of claims 106 to 115, comprising the step of adding platelet-rich plasma and / or bone marrow concentrate to the scaffold material of step c). Claim 117 A method according to any one of claims 54 to 116, further comprising the step of combining a mixture / scaffold material with mammalian cells to form a tissue engineering structure. Claim 118 A method according to claim 117, wherein the mammalian cells comprise cells selected from the group consisting of hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, endothelial cells, exocrine cells, intestinal-derived cells, cholangiocarcinocytes, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary axis, myocardial cells, renal epithelial cells, renal tubular cells, renal basement membrane cells, neurons, vascular cells, bone and cartilage-forming cells, smooth muscle cells, skeletal muscle cells, epithelial cells, bone marrow cells, keratinocytes, pluripotent cells and stem cells, and combinations thereof. Claim 119 A method according to any one of claims 54 to 118, wherein the electrospinning process is performed using a voltage of about 12 kV to about 18 kV. Claim 120 A method according to any one of claims 54 to 119, wherein the electrospinning process is performed at a flow rate of about 0.8 mL to about 1.2 mL per hour. Claim 121 A method according to any one of claims 54 to 120, wherein the electrospinning process is performed using a collector spacing of about 8 cm to about 12 cm. Claim 122 A scaffold material, a layered wound dressing, a layered graft, a layered structure, or a tissue engineering structure for use in treatment, according to any one of claims 1 to 52. Claim 123 A scaffold material, a layered wound dressing, a layered graft, a layered structure, or a tissue engineering structure according to paragraph 122, which is intended for use in wound healing, wound closure, tissue repair, tissue regeneration, cartilage repair, cartilage regeneration, tendon repair, tendon regeneration, nerve repair, nerve regeneration, bone repair and / or bone regeneration. Claim 124 A scaffold material, a stacked wound dressing, a stacked graft, a stacked structure, or a tissue engineering structure according to paragraph 122, used for the treatment or prevention of joint disorders or diseases. Claim 125 A scaffold material, a laminated wound dressing, a laminated graft, a laminated structure, or a tissue engineering structure for use according to paragraph 124, wherein the joint disorder or disease is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia and rheumatic myalgia. Claim 126 A scaffold material, a laminated wound dressing, a laminated graft, a laminated structure, or a tissue engineering structure for use in accordance with paragraph 123, wherein wound healing and tissue restoration include the treatment of gynecological diseases (e.g., pelvic organ prolapse), bowel injury, bladder injury, vascular injury, other non-healing wounds, burns, and diabetic foot. Claim 127 A scaffold material, stacked wound dressing, stacked graft, stacked structure, or tissue engineering structure according to paragraph 122, used for tissue augmentation, scaffold material, stacked wound dressing, stacked graft, stacked structure, or tissue engineering structure.

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