Multifunctional gradient structure artificial skin and preparation method and application thereof

Abstract

The application discloses a multifunctional gradient structure artificial skin and a preparation method and application thereof, and belongs to the technical field of biomaterials and biomedical engineering. The upper layer of the application is a silica gel layer, which is used for simulating epidermis to keep a moist environment of a wound and simultaneously plays a barrier role; the intermediate layer is a degradable polymer / natural bioactive protein composite layer, which is used for enhancing mechanical properties; and the lower layer is a hydrogel layer, which is formed by cross-linking hyaluronic acid and carboxymethyl chitosan to form a Schiff base bond and then cross-linking collagen through catalysis. The composition of the hydrogel layer is a component of an extracellular matrix, and the three-dimensional network structure formed is beneficial to cell growth, can simulate a dermis layer to play a function of promoting tissue regeneration and remodeling, and finally the artificial skin with good mechanical strength, capable of preventing wound infection and promoting wound healing is obtained.

Description

Technical Field

[0001] This invention belongs to the fields of biomaterials and biomedical engineering technology, and particularly relates to a multifunctional gradient structure artificial skin, its preparation method and application. Background Technology

[0002] The skin is the largest organ in the human body in terms of surface area, providing a physical barrier to protect the body from external environmental influences. After injury, the skin can complete its self-repair process through hemostasis, inflammation, proliferation, and remodeling phases. However, chronic traumas, such as burns, pressure ulcers, bedsores, and diabetic foot, are very difficult to repair and usually do not heal on their own, requiring external treatment.

[0003] Currently, there are many artificial skin products on the market and under development both domestically and internationally, which can effectively promote wound healing and tissue regeneration. Based on different functional attributes, artificial skin is mainly divided into three categories: epidermal substitutes, dermal substitutes, and full-skin substitutes. These can specifically target the regeneration of epidermal / dermal tissues, thus promoting wound healing. Although existing artificial skin products have achieved certain therapeutic effects in clinical applications, they still have limitations such as low mechanical strength, poor anti-infection properties, and sensitization.

[0004] Therefore, it is necessary to provide an artificial skin with good mechanical strength, which can prevent wound infection and promote wound healing. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a multifunctional gradient structure artificial skin, its preparation method, and its applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a multifunctional gradient structure artificial skin, comprising an upper layer, a middle layer, and a lower layer bonded together; the upper layer is a silicone layer, the middle layer is a biodegradable polymer / natural bioactive protein composite layer, and the lower layer is a hydrogel layer; the raw materials for preparing the hydrogel layer include oxidized hyaluronic acid, carboxymethyl chitosan, and collagen.

[0008] Technical Principle: This invention uses a silicone layer as the upper layer to simulate the epidermis, maintaining a moist environment for the wound and acting as a barrier. A biodegradable polymer / natural bioactive protein composite layer serves as the middle layer to enhance mechanical properties. A hydrogel layer forms the lower layer. This hydrogel layer is formed by cross-linking oxidized hyaluronic acid and carboxymethyl chitosan to form Schiff base bonds, which are then catalyzed to cross-link collagen. The components of the hydrogel layer are part of the extracellular matrix, and the resulting three-dimensional network structure promotes cell growth. It can simulate the dermis, promoting tissue regeneration and remodeling, ultimately resulting in an artificial skin with good mechanical strength, capable of preventing wound infection and promoting wound healing.

[0009] Furthermore, the raw materials for preparing the silicone layer include silicone liquid A and silicone liquid B; the components of silicone liquid A include silicon dioxide, vinyl silicone oil, methyl silicone oil and platinum water; the components of silicone liquid B include silicon dioxide, vinyl silicone oil, methyl silicone oil and hydrogen-containing silicone oil; the mass ratio of silicone liquid A to silicone liquid B is (0.5-2):1.

[0010] Furthermore, the degradable polymer in the biodegradable polymer / natural bioactive protein composite layer is selected from polycaprolactone, and the natural bioactive protein is selected from type I collagen.

[0011] Furthermore, the mass ratio of degradable polymer to natural bioactive protein in the degradable polymer / natural bioactive protein composite layer is (4-6):(6-4).

[0012] Furthermore, the preparation method of the biodegradable polymer / natural bioactive protein composite layer includes: using biodegradable polymer and natural bioactive protein as raw materials, and hexafluoroisopropanol as solvent, to prepare the biodegradable polymer / natural bioactive protein composite layer by electrospinning.

[0013] Furthermore, the electrospinning process parameters include: a spinning solution flow rate of 1-2 mL / h, a working voltage of 13-15 kV, a foil receiver rotation speed of 45-50 r / min, a syringe needle distance of 15-17 cm from the foil receiver, and a needle specification of 21 G.

[0014] Furthermore, the mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and collagen is 20:30:(10-30).

[0015] Furthermore, the raw materials for preparing the hydrogel layer also include transglutaminase; the mass ratio of transglutaminase to oxidized hyaluronic acid is 2.4:20.

[0016] This invention provides a method for preparing a multifunctional gradient structure artificial skin as described above, comprising the following steps: preparing a silicone layer, a biodegradable polymer / natural bioactive protein composite layer, and a hydrogel layer respectively; then using vacuum lamination to combine the silicone layer, the biodegradable polymer / natural bioactive protein composite layer, and the hydrogel layer to obtain the multifunctional gradient structure artificial skin; the vacuum lamination time is 24-72 hours.

[0017] The present invention also provides the application of the multifunctional gradient structure artificial skin as described above in the preparation of chronic wound repair materials.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] Carboxymethyl chitosan is obtained by methylation modification of chitosan. Chitosan oligosaccharides are small molecule oligosaccharides with amino groups on their side chains formed by the degradation of shrimp and crab shells. They have good biodegradability and biocompatibility. Methylation modification of chitosan improves its water solubility. Hyaluronic acid is a major component of the skin extracellular matrix. It has hydrophilicity, moisturizing properties, and can promote wound healing. Oxidized hyaluronic acid, obtained after oxidation, still retains the above advantages. Collagen can significantly reduce immune responses and is beneficial to the adhesion and proliferation of fibroblasts. The hydrogel layer prepared by the above raw materials in this invention is beneficial to promoting wound healing.

[0020] This invention uses synthetic biodegradable polymers and biologically derived natural bioactive proteins to prepare an intermediate layer. Synthetic biodegradable polymers (such as polycaprolactone) are commonly used biomedical materials that can be used as implantable materials and drug-controlled release materials. Biologically derived natural bioactive collagen has good biocompatibility and biodegradable safety, is a major component of most extracellular matrices, and has good immunogenicity.

[0021] This invention uses biodegradable polymers as raw materials to prepare the intermediate layer, resulting in a membrane with good mechanical properties. The addition of a certain proportion of collagen significantly improves the membrane's mechanical properties, altering the high toughness and low elasticity characteristics of polymer materials. The hydrogel layer in this invention possesses self-healing properties, allowing it to recover after breakage, thus enhancing its therapeutic effect. The carboxymethyl chitosan, hyaluronic acid, and recombinant human type III collagen in the hydrogel exhibit good biocompatibility and biodegradability, which is beneficial for the repair of diabetic foot wounds. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 The microstructure (A), filament diameter distribution (B), pore size distribution (D), filament diameter statistics (C), and pore size statistics (E) of the electrospun films prepared in Examples 1-3 and Comparative Example 1 are shown. PCL is Comparative Example 1, P6C4 is Example 2, P5C5 is Example 1, and P4C6 is Example 3 (the same applies below).

[0024] Figure 2 The water contact angles of the electrospun membranes prepared in Examples 1-3 and Comparative Example 1;

[0025] Figure 3 The mechanical properties of the electrospun films prepared in Examples 1-2 and Comparative Example 1 are shown, where A is the elastic modulus, B is the elongation at break, C is the tensile strength, and D is the ultimate load.

[0026] Figure 4 The mechanical properties of the silicone film and silicone-electrospun film prepared in step 2) of Example 2 are as follows: A is the elastic modulus, B is the elongation at break, C is the tensile strength, D is the ultimate load, Silica gel is the silicone film, and Bilayer is the silicone-electrospun film.

[0027] Figure 5 The microstructure (A), pore size distribution (B), and pore size statistics (C) of the hydrogels prepared in Examples 1, 4-5, and Comparative Example 2 are shown, where R0 is Comparative Example 2, R1 is Example 4, R2 is Example 1, and R3 is Example 5.

[0028] Figure 6 Cytotoxicity results of the silicone membrane (A) prepared for Example 1, the electrospun membrane (B) prepared for Examples 1-3 and Comparative Example 1, and the hydrogel (C) prepared for Examples 1, Examples 4-5 and Comparative Example 2;

[0029] Figure 7 The effects of hydrogels prepared for the control group and Comparative Example 2, as well as different concentrations of rhCol III, on cell migration and the statistical results of cell migration rate are presented. Among them, Control is the control group, R0 is Comparative Example 2, 10 mg / mL, 20 mg / mL, and 30 mg / mL are the concentrations of rhCol III, A is the effect of the control group and different concentrations of rhCol III on cell migration, B is the statistical results of cell migration rate of the control group and different concentrations of rhCol III, C is the effect of the hydrogels prepared for the control group and Comparative Example 2 on cell migration, and D is the statistical results of cell migration rate of the hydrogels prepared for the control group and Comparative Example 2. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] This invention provides a multifunctional gradient structure artificial skin, which includes an upper layer, a middle layer, and a lower layer bonded together; the upper layer is a silicone layer, the middle layer is a biodegradable polymer / natural bioactive protein composite layer, and the lower layer is a hydrogel layer; the raw materials for preparing the hydrogel layer include oxidized hyaluronic acid, carboxymethyl chitosan, and collagen.

[0033] In a preferred embodiment, the raw materials for preparing the silicone layer include silicone liquid A and silicone liquid B; the components of silicone liquid A include silicon dioxide, vinyl silicone oil, methyl silicone oil, and platinum water, and the mass ratio of silicon dioxide, vinyl silicone oil, methyl silicone oil, and platinum water is 0.35∶0.47∶0.178∶0.002; the components of silicone liquid B include silicon dioxide, vinyl silicone oil, methyl silicone oil, and hydrogen-containing silicone oil, and the mass ratio of silicon dioxide, vinyl silicone oil, methyl silicone oil, and hydrogen-containing silicone oil is 0.35∶0.47∶0.102∶0.078; the mass ratio of silicone liquid A to silicone liquid B is (0.5-2):1.

[0034] In a preferred embodiment, the thickness of the silicone layer is 0.3-0.5 mm. The silicone layer in this invention serves as the upper layer, simulating the epidermis to maintain a moist environment in the wound, while also acting as a barrier.

[0035] In a preferred embodiment, the degradable polymer in the biodegradable polymer / natural bioactive protein composite layer is selected from polycaprolactone, and the natural bioactive protein is selected from type I collagen; the type I collagen is derived from bovine Achilles tendon. Polycaprolactone is a commonly used biomedical material, suitable for in vivo implantation and drug release. Naturally derived bioactive collagen exhibits good biocompatibility and biodegradable safety, is a major component of most extracellular matrices, and possesses good immunogenicity. This invention, by adding a certain proportion of collagen to a biodegradable polymer, significantly improves the mechanical properties of the membrane, alters the high toughness and low elasticity characteristics of polymer materials, and enhances the mechanical properties of the multifunctional gradient structure artificial skin.

[0036] In a preferred embodiment, the mass ratio of degradable polymer to natural bioactive protein in the degradable polymer / natural bioactive protein composite layer is (4-6):(6-4).

[0037] In a preferred embodiment, the method for preparing the biodegradable polymer / natural bioactive protein composite layer includes: using biodegradable polymers and natural bioactive proteins as raw materials, and hexafluoroisopropanol as a solvent, to obtain the biodegradable polymer / natural bioactive protein composite layer by electrospinning. This invention prepares the biodegradable polymer / natural bioactive protein composite layer by electrospinning, eliminating the need for toxic substances such as chemical crosslinking agents. The resulting product is non-toxic and highly biocompatible. The provided preparation method has advantages such as simple process, mild conditions, wide availability of raw materials, and low cost, making it suitable for large-scale production applications.

[0038] In a preferred embodiment, the electrospinning process parameters include: a spinning solution flow rate of 1-2 mL / h, a working voltage of 13-15 kV, a foil receiver rotation speed of 45-50 r / min, a syringe needle distance of 15-17 cm from the foil receiver, and a needle specification of 21G.

[0039] In a preferred embodiment, the concentration of the spinning solution used in the electrospinning is 15% (w / v).

[0040] In a preferred embodiment, the thickness of the biodegradable polymer / natural bioactive protein composite layer is 0.1-0.2 mm.

[0041] In a preferred embodiment, the mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and collagen is 20:30:(10-30), more preferably 20:30:20. The hydrogel layer in this invention is formed by cross-linking oxidized hyaluronic acid and carboxymethyl chitosan to form Schiff base bonds, followed by catalytic cross-linking of collagen. The components of the hydrogel layer are part of the extracellular matrix, and the resulting three-dimensional network structure is conducive to cell growth, mimicking the dermis to promote tissue regeneration.

[0042] In a preferred embodiment, the collagen is selected from human recombinant type III collagen. Human recombinant type III collagen is obtained using genetic engineering technology and has a highly consistent amino acid sequence with human natural collagen. It has the characteristic structure of type III collagen. Therefore, human recombinant type III collagen has low immunogenicity, which can significantly reduce the immune response and is beneficial to the adhesion and proliferation of fibroblasts.

[0043] In a preferred embodiment, the preparation method of the oxidized hyaluronic acid includes: oxidizing hyaluronic acid with sodium periodate as an oxidant to obtain the oxidized hyaluronic acid; the ratio of sodium periodate to hyaluronic acid is 2.5 mmol:1 g; water is used as a solvent in the preparation of the oxidized hyaluronic acid; the oxidation specifically involves: stirring the reaction at room temperature in the dark for 2-6 hours; and quenching with ethylene glycol after oxidation. This invention uses sodium periodate to modify hyaluronic acid with aldehydes, and the aldehyde-modified oxidized hyaluronic acid can crosslink with carboxymethyl chitosan to form Schiff base bonds.

[0044] In a preferred embodiment, the raw materials for preparing the hydrogel layer further include transglutaminase; the mass ratio of transglutaminase to oxidized hyaluronic acid is 2.4:20. The transglutaminase in this invention is used to catalyze the cross-linking of collagen, thereby forming a hydrogel with a three-dimensional network structure.

[0045] In a preferred embodiment, the thickness of the hydrogel layer is 1-5 mm.

[0046] This invention provides a method for preparing a multifunctional gradient structure artificial skin as described above, comprising the following steps: preparing a silicone layer, a biodegradable polymer / natural bioactive protein composite layer, and a hydrogel layer respectively; then using vacuum lamination to combine the silicone layer, the biodegradable polymer / natural bioactive protein composite layer, and the hydrogel layer to obtain the multifunctional gradient structure artificial skin; the vacuum lamination time is 24-72 hours.

[0047] In a preferred embodiment, the method for preparing the silicone layer includes: stirring silicone liquid A and silicone liquid B evenly, and then curing them to obtain the silicone layer; the stirring time is 5-10 minutes; the curing temperature is room temperature, and the curing time is 4-6 hours.

[0048] In a preferred embodiment, the method for preparing the biodegradable polymer / natural bioactive protein composite layer includes: dissolving the biodegradable polymer and natural bioactive protein in hexafluoroisopropanol, stirring to obtain a spinning solution; and electrospinning the obtained spinning solution to obtain the biodegradable polymer / natural bioactive protein composite layer.

[0049] In a preferred embodiment, the method for preparing the hydrogel layer includes: mixing oxidized hyaluronic acid, carboxymethyl chitosan, collagen, and transglutaminase in water to obtain the hydrogel layer; more preferably: mixing transglutaminase in water and then adding oxidized hyaluronic acid to obtain mixed solution 1; mixing carboxymethyl chitosan and collagen in water to obtain mixed solution 2; and mixing mixed solution 1 and mixed solution 2 by shaking to obtain the hydrogel layer.

[0050] In a preferred embodiment, the order of bonding the silicone layer, the biodegradable polymer / natural bioactive protein composite layer, and the hydrogel layer can be either to bond the silicone layer, the biodegradable polymer / natural bioactive protein composite layer, and the hydrogel layer sequentially, or to bond the silicone layer and the biodegradable polymer / natural bioactive protein composite layer first, and then bond the hydrogel layer.

[0051] The present invention also provides the application of the multifunctional gradient structure artificial skin as described above in the preparation of chronic wound repair materials.

[0052] In this embodiment of the invention, room temperature refers to "25±2℃".

[0053] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0054] The abbreviations used in the following embodiments specifically refer to:

[0055] PCL: Polycaprolactone;

[0056] COLⅠ: Type I collagen derived from bovine Achilles tendon;

[0057] rhCol Ⅲ: Recombinant human type III collagen;

[0058] HFIP: Hexafluoroisopropanol;

[0059] HA: Hyaluronic acid;

[0060] OHA: Oxidized hyaluronic acid;

[0061] CMCS: Carboxymethyl chitosan;

[0062] TGase: Transglutaminase.

[0063] Example 1

[0064] A method for preparing a multifunctional gradient structure artificial skin, comprising the following steps:

[0065] 1) Preparation of the upper silicone layer

[0066] 1 mL of silica gel solution A and 1 mL of silica gel solution B were stirred and mixed for 10 min, poured into a mold and leveled, and allowed to stand at room temperature for 6 h to obtain a silica gel film. The components of silica gel solution A were silica, vinyl silicone oil, methyl silicone oil and platinum water, with a mass ratio of 0.35:0.47:0.178:0.002. The components of silica gel solution B were silica, vinyl silicone oil, methyl silicone oil and hydrogen-containing silicone oil, with a mass ratio of 0.35:0.47:0.102:0.078.

[0067] 2) Preparation of the intermediate layer P5C5 electrospun film

[0068] 0.75 g PCL and 0.75 g COLⅠ were dissolved in 10 mL HFIP and stirred thoroughly for 8 h to obtain a 15 wt% P5C5 solution with a PCL to COLⅠ mass ratio of 5:5. 4 mL of the above P5C5 solution was taken and electrospun using a 21G needle with the following settings: a spinning solution flow rate of 2 mL / h, a working voltage of 14 kV, a distance of 15 cm between the syringe needle and the aluminum foil receiver, and a aluminum foil receiver rotation speed of 50 r / min. After electrospinning, a P5C5 electrospun membrane was obtained.

[0069] 3) Preparation of oxidized hyaluronic acid

[0070] Dissolve 2g of HA in 200mL of deionized water, slowly add 5mmol (5mL) of sodium periodate aqueous solution to the above solution, stir at room temperature in the dark for 6h, add 1mL of ethylene glycol and quench at room temperature for 1h, dialyze in deionized water for 3 days, and then freeze dry under vacuum to obtain OHA.

[0071] 4) Preparation of the lower layer OHA / CMCS / rhCol Ⅲ hydrogel

[0072] Dissolve TGase in deionized water, then add OHA obtained in step 3) to obtain mixed solution 1; dissolve CMCS and rhCol Ⅲ in deionized water to prepare mixed solution 2; mix mixed solution 1 and mixed solution 2 under shaking to obtain OHA / CMCS / rhCol Ⅲ hydrogel; wherein, the mass-volume ratio of OHA to deionized water is 20mg:1mL, the mass-volume ratio of CMCS to deionized water is 30mg:1mL, the mass-volume ratio of rhCol Ⅲ to deionized water is 20mg:1mL, the mass-volume ratio of TGase to deionized water is 2.4mg:1mL, and the total amount of water used is 1mL.

[0073] 5) Fabrication of multifunctional gradient structure artificial skin

[0074] Take the silicone membrane prepared in step 1), the P5C5 electrospun membrane prepared in step 2), and the OHA / CMCS / rhCol Ⅲ hydrogel prepared in step 4), cut them into the same size, and stack them in the order of silicone membrane, P5C5 electrospun membrane, and OHA / CMCS / rhCol Ⅲ hydrogel. Then place them in a vacuum environment for 72 hours to obtain a multifunctional gradient structure artificial skin. The thickness of the silicone membrane in the multifunctional gradient structure artificial skin is 0.5 mm, the thickness of the P5C5 electrospun membrane is 0.1-0.2 mm, and the thickness of the OHA / CMCS / rhCol Ⅲ hydrogel is 4 mm.

[0075] Example 2

[0076] A method for preparing a multifunctional gradient structure artificial skin, comprising the following steps:

[0077] 1) Preparation of P6C4 electrospun membrane with intermediate layer

[0078] 0.9 g PCL and 0.6 g COLⅠ were dissolved in 10 mL HFIP and stirred thoroughly for 8 h to obtain a 15 wt% P6C4 solution with a PCL to COLⅠ mass ratio of 6:4. 4 mL of the above P6C4 solution was taken and electrospun using a 21G needle with the following settings: a spinning solution flow rate of 2 mL / h, a working voltage of 14 kV, a distance of 15 cm between the syringe needle and the aluminum foil receiver, and a aluminum foil receiver rotation speed of 50 r / min. After electrospinning, a P6C4 electrospun membrane was obtained.

[0079] 2) Preparation of silicone-electrospun membrane

[0080] 1 mL of silica gel solution A and 1 mL of silica gel solution B were stirred and mixed for 10 min, poured into a mold and leveled, and allowed to stand at room temperature for 6 h to react, thus obtaining a silica gel membrane. The P6C4 electrospun membrane obtained in step 1) was then applied to the silica gel membrane and bonded in a vacuum environment for 72 h to obtain a silica gel-electrospun membrane. The silica gel solution A consisted of silica, vinyl silicone oil, methyl silicone oil and platinum water, with a mass ratio of 0.35:0.47:0.178:0.002. The silica gel solution B consisted of silica, vinyl silicone oil, methyl silicone oil and hydrogen-containing silicone oil, with a mass ratio of 0.35:0.47:0.102:0.078.

[0081] 3) Preparation of oxidized hyaluronic acid

[0082] Dissolve 2g of HA in 200mL of deionized water, slowly add 5mmol (5mL) of sodium periodate aqueous solution to the above solution, stir at room temperature in the dark for 6h, add 1mL of ethylene glycol and quench at room temperature for 1h, dialyze in deionized water for 3 days, and then freeze dry under vacuum to obtain OHA.

[0083] 4) Preparation of OHA / CMCS / rhCol Ⅲ hydrogel

[0084] Dissolve TGase in deionized water, then add OHA obtained in step 3) to obtain mixed solution 1; dissolve CMCS and rhCol III in deionized water to prepare mixed solution 2; mix mixed solution 1 and mixed solution 2 under shaking to obtain OHA / CMCS / rhCol III hydrogel; wherein, the mass-volume ratio of OHA to deionized water is 20 mg:1 mL, the mass-volume ratio of CMCS to deionized water is 30 mg:1 mL, the mass-volume ratio of rhCol III to deionized water is 10 mg:1 mL, the mass-volume ratio of TGase to deionized water is 2.4 mg:1 mL, and the total amount of water used is 1 mL.

[0085] 5) Fabrication of multifunctional gradient structure artificial skin

[0086] The silicone-electrospun membrane obtained in step 2) was coated onto the OHA / CMCS / rhCol Ⅲ hydrogel obtained in step 4), and the mixture was bonded under vacuum for 72 hours to obtain a multifunctional gradient structure artificial skin. The thickness of the silicone membrane in the multifunctional gradient structure artificial skin was 0.5 mm, the thickness of the P6C4 electrospun membrane was 0.1-0.2 mm, and the thickness of the OHA / CMCS / rhCol Ⅲ hydrogel was 4 mm.

[0087] Example 3

[0088] A method for preparing a multifunctional gradient structure artificial skin, comprising the following steps:

[0089] 1) Preparation of P4C6 electrospun membrane with intermediate layer

[0090] 0.6 g PCL and 0.9 g COLⅠ were dissolved in 10 mL HFIP and stirred thoroughly for 8 h to obtain a 15 wt% P4C6 solution with a PCL to COLⅠ mass ratio of 4:6. 4 mL of the above P4C6 solution was taken and electrospun using a 21G needle with the following settings: the flow rate of the spinning solution was 2 mL / h, the working voltage was 14 kV, the distance between the syringe needle and the aluminum foil receiver was 15 cm, and the rotation speed of the aluminum foil receiver was 50 r / min. After electrospinning, a P4C6 electrospun membrane was obtained.

[0091] Steps 2) to 5) are the same as in Example 2.

[0092] Example 4

[0093] A method for preparing a multifunctional gradient structure artificial skin differs from Example 1 in that, in step 4), the mass-to-volume ratio of rhCol Ⅲ to deionized water is 10 mg: 1 mL, while the rest is the same as in Example 1.

[0094] Example 5

[0095] A method for preparing a multifunctional gradient structure artificial skin differs from Example 1 in that, in step 4), the mass-to-volume ratio of rhCol Ⅲ to deionized water is 30 mg: 1 mL, while the rest is the same as in Example 1.

[0096] Comparative Example 1

[0097] A method for preparing a PCL electrospun membrane includes the following steps: 1.5g of PCL is dissolved in 10mL of HFIP and stirred thoroughly for 8h to obtain a PCL solution; 4mL of the above PCL solution is taken, and electrospinning is performed using a 21G needle with the following settings: the flow rate of the spinning solution is 2mL / h, the working voltage is 14kV, the distance between the syringe needle and the aluminum foil receiver is 15cm, and the rotation speed of the aluminum foil receiver is 50r / min. After electrospinning, a PCL electrospun membrane is obtained.

[0098] Comparative Example 2

[0099] A method for preparing a multifunctional gradient structure artificial skin differs from Example 1 in that, in step 4), the mass-to-volume ratio of rhCol Ⅲ to deionized water is 0 mg: 1 mL, while the rest is the same as in Example 1.

[0100] I. Observation of the surface morphology of electrospun films

[0101] The microstructure of the electrospun films prepared in Examples 1-3 and Comparative Example 1 was observed by SEM, and the results are shown in the figure. Figure 1 .

[0102] Figure 1 The microstructure (A), filament diameter distribution (B), pore size distribution (D), filament diameter statistics (C), and pore size statistics (E) of the electrospun membranes prepared in Examples 1-3 and Comparative Example 1 are shown below. PCL represents Comparative Example 1, P5C5 represents Example 1, P6C4 represents Example 2, and P4C6 represents Example 3 (the same applies below). Figure 1As can be seen, the fibers of PCL, P6C4, P5C5, and P4C6 in the four electrospun membranes all exhibit smooth, dense, and randomly oriented morphologies. The fiber diameter in the pure PCL electrospun membrane is 1.68±0.05 μm, while the fiber diameter is significantly reduced after incorporation of COLⅠ. There is no significant difference in fiber diameter among the P6C4 (0.67±0.03 μm), P5C5 (0.87±0.06 μm), and P4C6 (0.63±0.02 μm) electrospun membranes. With the incorporation of collagen, compared to PCL, the fiber diameter is significantly reduced, the electrospun membrane becomes more dense, the pore size is significantly reduced, and a denser structure is formed. The pore size of the PCL electrospun membrane is 11.03±0.57μm. The pore sizes of the P6C4 electrospun membrane (3.93±0.17μm), P5C5 electrospun membrane (4.10±0.12μm), and P4C6 electrospun membrane (4.51±0.28μm) do not differ significantly, indicating that the electrospun membrane prepared by this invention forms a dense barrier layer, which can effectively prevent bacteria from migrating into the material and meets the basic requirements of a barrier membrane.

[0103] II. Hydrophilicity of electrospun membranes

[0104] Figure 2 The water contact angle is for the electrospun membranes prepared in Examples 1-3 and Comparative Example 1. From... Figure 2 As can be seen, when water droplets come into contact with the material surface, the water contact angle of the PCL electrospun membrane prepared in Comparative Example 1 is 121.41±3.18°, the water contact angle of the P6C4 electrospun membrane prepared in Example 2 is 65.77±8.56°, the water contact angle of the P5C5 electrospun membrane prepared in Example 1 is 49.25±3.98°, and the water contact angle of the P4C6 electrospun membrane prepared in Example 3 is 41.75±3.52°. It is evident that the higher the content of COL I, the smaller the water contact angle of the electrospun membrane and the better its hydrophilicity. This is because COL I contains abundant hydrophilic groups, such as amino, hydroxyl, and carboxyl groups, which effectively improve the hydrophilicity of the material and enable it to quickly absorb water droplets falling onto the material surface. The good hydrophilicity of the electrospun membrane can promote cell adhesion and proliferation, effectively accelerating the wound healing process.

[0105] III. Mechanical Properties of Electrospun Films

[0106] Good mechanical properties can maintain the shape of artificial skin during application. Tensile mechanical property tests on various groups of materials revealed that incorporating an appropriate amount of COL Ⅰ can improve the elastic modulus, tensile strength, and ultimate load of the materials.

[0107] Figure 3The mechanical properties of the electrospun films prepared in Examples 1-2 and Comparative Example 1 are shown, where A is the elastic modulus, B is the elongation at break, C is the tensile strength, and D is the ultimate load. Figure 3 As shown in Part A, the PCL / COL I electrospun film with an appropriate amount of COL I incorporated exhibits a higher elastic modulus than the PCL electrospun film. The elastic modulus of the PCL electrospun film is 2.44±0.16 MPa, the P6C4 electrospun film is 40.62±1.22 MPa, and the P5C5 electrospun film is 24.74±2.10 MPa. Figure 3 As shown in Part B, the elongation at break of the PCL electrospun film is 522.70±18.14%, the elongation at break of the P6C4 electrospun film is 58.89±1.32%, and the elongation at break of the P5C5 electrospun film is 50.07±2.55%. Figure 3 As can be seen from section C, the tensile strength of the PCL electrospun film is 2.96±0.05MPa, the tensile strength of the P6C4 electrospun film is 6.71±0.19MPa, and the tensile strength of the P5C5 electrospun film is 2.19±0.12MPa. (From...) Figure 3 As shown in section D, the ultimate load of the PCL electrospun film is 6.04±0.10N, the ultimate load of the P6C4 electrospun film is 13.31±0.20N, and the ultimate load of the P5C5 electrospun film is 3.12±0.01N. In summary, it can be seen that the electrospun film prepared by incorporating an appropriate amount of COLⅠ has superior mechanical properties.

[0108] IV. Mechanical Properties of Silicone-Electrospun Film

[0109] Figure 4 The mechanical properties of the silicone film and silicone-electrospun film prepared in step 2) of Example 2 are shown, where A is the elastic modulus, B is the elongation at break, C is the tensile strength, D is the ultimate load, Silica gel is the silicone film, and Bilayer is the silicone-electrospun film. Figure 4 It can be seen that, compared with silicone film, the elastic modulus of silicone-electrospun film is higher (see...). Figure 4 Part A of the text), tensile strength (see ... Figure 4 Part C in the middle) and ultimate load ( Figure 4 The D portion of the membrane was significantly improved, indicating that the composite of silicone membrane and electrospun membrane has superior mechanical properties.

[0110] V. Observation of hydrogel surface morphology

[0111] The surface morphology and structure of the hydrogels prepared in Examples 1, 4-5, and Comparative Example 2 were observed by SEM. The results are shown in the figure. Figure 5 .

[0112] Figure 5 The microstructure (A), pore size distribution (B), and pore size statistics (C) of the hydrogels prepared in Examples 1, 4-5, and Comparative Example 2 are shown, where R0 is Comparative Example 2, R1 is Example 4, R2 is Example 1, and R3 is Example 5. Figure 5 As shown in Part A, R0, R1, R2, and R3 all exhibit a porous, interconnected three-dimensional network structure, which is beneficial for cell migration and metabolism. Statistical analysis of the hydrogel pore sizes reveals that R0 has a pore size of 374.82 ± 6.16 μm, R1 has a pore size of 300.38 ± 5.43 μm, R2 has a pore size of 311.96 ± 8.58 μm, and R3 has a pore size of 262.89 ± 5.03 μm. It can be observed that the pore size decreases to some extent with the incorporation of rhCol III. The porous structure of the hydrogel facilitates fibroblast migration and promotes wound healing.

[0113] VI. Biocompatibility of Artificial Skin

[0114] This study investigated the effect of material extracts on cell proliferation, simulating the influence of the materials on the activity of surrounding tissues in vivo. Following ISO 10993-12 standards, sterilized silica gel or lyophilized hydrogels were immersed in fresh culture medium at a concentration of 0.1 g / mL at 37°C for 24 hours. After centrifugation, the supernatant was collected and filtered through a 0.22 μm filter membrane for thorough sterilization to obtain silica gel or hydrogel extracts. Sterilized electrospun membranes were then filtered through a 6 cm... 2 The standard solution was soaked in fresh culture medium at 37°C for 24 hours. The supernatant was then aspirated and filtered through a 0.22 μm filter to obtain the electrospun membrane extract. L929 cells were seeded at a density of 3000 cells / well in 96-well plates. After 24 hours of culture, the culture medium for the experimental groups was changed to silica gel extract, electrospun membrane extract, or hydrogel extract, respectively. The blank control group used 1640 medium. The absorbance of each group was measured by CCK-8 assay on days 1, 2, and 3 to analyze the biocompatibility of each group. The results are shown in [Figure number missing]. Figure 6 .

[0115] Figure 6 Cytotoxicity results for the silicone membrane (A) prepared for Example 1, the electrospun membrane (B) prepared for Examples 1-3 and Comparative Example 1, and the hydrogel (C) prepared for Examples 1, 4-5, and Comparative Example 2. From Figure 6 As can be seen from Part A, on day 2 of culture, the silica gel group significantly promoted the proliferation of L929 cells; from Figure 6 As can be seen from Part B, on days 1, 2, and 3, there was no significant difference in absorbance between the electrospun membrane and the control group; from Figure 6As shown in section C, the R2 group significantly promoted the proliferation of L929 cells on the second and third days of culture, indicating that the addition of rhCol Ⅲ increased the biocompatibility of the hydrogel. In summary, all three layers of the artificial skin material can maintain cell viability, indicating that the material has good biocompatibility and no cytotoxicity.

[0116] VII. Cell migration promotion ability of hydrogel layers

[0117] During injury repair, endogenous fibroblasts migrate to the injured site, which is crucial for tissue regeneration. This study investigated the effects of different concentrations of rhCol III on the horizontal migration of cells to simulate the influence of materials on surrounding cells in vivo. Following ISO 10993-12 standards, sterilized lyophilized RO hydrogel (Comparative Example 2) was immersed in fresh culture medium at a concentration of 0.1 g / mL at 37°C for 24 h. The supernatant was then centrifuged and filtered through a 0.22 μm filter to obtain a serum-free RO hydrogel extract. rhCol III was dissolved in fresh culture medium at concentrations of 10 mg / mL, 20 mg / mL, and 30 mg / mL, respectively. The supernatant was then filtered through a 0.22 μm filter to obtain serum-free culture media containing different concentrations of rhCol III. L929 cells were cultured at 30 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100% per well in six-well plates. When the cell density reached 100%, the cells were streaked evenly in the center of each well using a 200 μL pipette tip. Floating cells were washed away with PBS. Cells were then cultured in serum-free 1640 medium (blank control), serum-free hydrogel extract, or different concentrations of rhCol III serum-free medium. Cells were photographed at 0 h, 12 h, and 24 h using an optical microscope. Results are shown in the figure. Figure 7 .

[0118] Figure 7 The effects of hydrogels prepared for the control group and Comparative Example 2, as well as different concentrations of rhCol III, on cell migration and the statistical results of cell migration rate were presented. Wherein, Control represents the control group, R0 represents Comparative Example 2, 10 mg / mL, 20 mg / mL, and 30 mg / mL represent the concentrations of rhCol III, A represents the effect of the control group and different concentrations of rhCol III on cell migration, B represents the statistical results of cell migration rate of the control group and different concentrations of rhCol III, C represents the effect of the hydrogels prepared for the control group and Comparative Example 2 on cell migration, and D represents the statistical results of cell migration rate of the hydrogels prepared for the control group and Comparative Example 2. Figure 7As shown in Parts A and B, after 12 hours, the cell migration rate of the control group was 20.75±2.79%, and after 24 hours, it was 43.45±5.16%. The group containing recombinant human type III collagen promoted cell migration, with the 10 mg / mL group showing the most significant effect. After 12 hours, the cell migration rate was 39.72±1.52%, and after 24 hours, it was 65.65±3.03%, with the scratch wound nearly closed. This indicates that the hydrogel containing recombinant human type III collagen can promote cell migration. Figure 7 As shown in sections C and D, after 12 hours of experimentation, the cell migration rate of the control group was 18.44±2.26%, and the cell migration rate of the R0 group was 21.69±3.32%. After 24 hours, the cell migration rate of the control group was 31.70±3.32%, and the cell migration rate of the R0 group was 37.87±2.35%, indicating that hydrogels without rhCol III had no significant effect on cell migration.

[0119] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A multifunctional gradient structure artificial skin, characterized in that, The multifunctional gradient structure artificial skin comprises an upper, middle, and lower layer bonded together. The upper layer is a silicone layer, the middle layer is a biodegradable polymer / natural bioactive protein composite layer, and the lower layer is a hydrogel layer. The raw materials for preparing the hydrogel layer include oxidized hyaluronic acid, carboxymethyl chitosan, and collagen. The biodegradable polymer in the biodegradable polymer / natural bioactive protein composite layer is selected from polycaprolactone, and the natural bioactive protein is selected from type I collagen. The mass ratio of biodegradable polymer to natural bioactive protein in the biodegradable polymer / natural bioactive protein composite layer is (4-6):(6-4). The mass ratio of oxidized hyaluronic acid, carboxymethyl chitosan, and collagen is 20:30:(10-30). The collagen is selected from recombinant human type III collagen. The raw materials for preparing the hydrogel layer also include transglutaminase. The mass ratio of transglutaminase to oxidized hyaluronic acid is 2.4:

20.

2. The multifunctional gradient structure artificial skin according to claim 1, characterized in that, The raw materials for preparing the silicone layer include silicone liquid A and silicone liquid B; the components of silicone liquid A include silicon dioxide, vinyl silicone oil, methyl silicone oil and platinum water; the components of silicone liquid B include silicon dioxide, vinyl silicone oil, methyl silicone oil and hydrogen-containing silicone oil; the mass ratio of silicone liquid A to silicone liquid B is (0.5-2):

1.

3. The multifunctional gradient structure artificial skin according to claim 1, characterized in that, The method for preparing the biodegradable polymer / natural bioactive protein composite layer includes: using biodegradable polymers and natural bioactive proteins as raw materials, and hexafluoroisopropanol as a solvent, the biodegradable polymer / natural bioactive protein composite layer is prepared by electrospinning.

4. The multifunctional gradient structure artificial skin according to claim 3, characterized in that, The electrospinning process parameters include: spinning solution flow rate of 1-2 mL / h, working voltage of 13-15 kV, aluminum foil receiver rotation speed of 45-50 r / min, distance between syringe needle and aluminum foil receiver of 15-17 cm, and needle specification of 21G.

5. A method for preparing a multifunctional gradient structure artificial skin as described in any one of claims 1-4, characterized in that, Includes the following steps: A silicone layer, a biodegradable polymer / natural bioactive protein composite layer, and a hydrogel layer are prepared separately; then, the silicone layer, the biodegradable polymer / natural bioactive protein composite layer, and the hydrogel layer are bonded together by vacuum lamination to obtain the multifunctional gradient structure artificial skin; the vacuum lamination time is 24-72 hours.

6. The application of a multifunctional gradient structure artificial skin as described in any one of claims 1-4 in the preparation of chronic wound repair materials.

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