A biological amniotic membrane dressing and a method for preparing the same

Abstract

The present application relates to the technical field of medical dressings, and discloses a biological amniotic membrane dressing and a preparation method thereof. The biological amniotic membrane dressing is composed of an alginate non-woven fabric bottom layer, a polyacrylamide temperature-sensitive gel amniotic membrane copolymerization intermediate layer and an amniotic membrane surface layer. The biological amniotic membrane dressing is helpful for the adhesion and slow release of drugs and wounds, and the hydrophilicity and ion exchange capacity of the alginate non-woven fabric are combined by grafting acrylic acid initiated by cerium ammonium nitrate to construct a bottom layer support and exudate management system, which is beneficial to timely absorption of exudate and discharge in the repair process, thereby avoiding the phenomenon of fluid accumulation. The biological amniotic membrane dressing prepared by subjecting the amniotic membrane to enzymolysis to obtain an amniotic membrane sol has a better effect of promoting wound healing, closely fits the wound, reduces infection, and is suitable for covering acute and chronic wounds.

Description

Technical Field

[0001] The invention belongs to the technical field of medical dressings, and in particular relates to a biological amniotic membrane dressing and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body and is primarily responsible for protecting the body. After skin trauma occurs, blood vessels contract and fibrin clots form. Once bleeding is controlled, a large number of inflammatory cells migrate to the wound, clearing necrotic cells and bacterial debris, and releasing pro-inflammatory cytokines and growth factors. At the same time, epithelial cells proliferate, migrate, and secrete extracellular matrix to promote re-epithelialization. Fibroblasts secrete mucopolysaccharides and proteoglycans, which constitute the main components of the extracellular matrix (ECM). The ECM changes during the remodeling period and becomes a structure similar to normal tissue, achieving perfect healing of the wound. How to achieve optimal wound healing in the treatment of skin injuries such as burns, chemical burns, surgical operations, and diabetes has always been a focus of research.

[0003] The amniotic membrane (AM), the innermost layer of the fetal membrane, is rich in growth factors, cytokines, and structural proteins. These biological properties endow the AM with unique functionalities, such as antimicrobial, anti-inflammatory, anti-scarring, and anti-fibrotic properties. Consequently, the AM is widely used in medical fields such as tissue engineering and regenerative medicine. Human amniotic membrane (HAM) possesses antimicrobial, anti-tumor, anti-fibrotic, and anti-angiogenic properties, along with acceptable mechanical properties. It also reduces pain and inflammation, inhibits scarring, and exhibits virtually no immunogenicity, making it a promising treatment for wound healing and epithelialization. However, the application of AM presents several challenges. The AM must be processed into a thin sheet that is resistant to folding or tearing. During use, it must be secured to the wound with sutures or adhesives, which conform to the wound and require high surgical technique. Furthermore, the AM has poor mechanical properties and a high biodegradability rate. Conventional dressings are prone to adhesion to the wound surface, causing secondary wound damage during dressing changes, increasing patient pain and significantly complicating the operator. Hydrogels have good biocompatibility, flexibility and similarity to natural extracellular matrix, but ordinary hydrogels have problems such as low cell adhesion rate and poor tensile strength, which are not conducive to their use in joint motion areas. Summary of the Invention

[0004] The present invention aims to provide a biological amniotic membrane dressing and a preparation method thereof. The biological amniotic membrane dressing is a thermosensitive gel composited on an alginate non-woven fabric, and then the amniotic membrane sol is composited on the surface of the thermosensitive gel to form a thermosensitive responsive composite amniotic membrane biological dressing. The dressing adheres tightly to the wound, and the exudate management system limits bacterial activity and proliferation, reduces infection, and prolongs the dressing change time.

[0005] To achieve the above object, the present invention provides a method for preparing a biological amniotic membrane dressing, comprising the following steps:

[0006] S1. The amniotic membrane is virally inactivated, washed, and homogenized to obtain an amniotic membrane microparticle suspension. Buffer is added and vortexed to form a uniform amniotic membrane suspension. The suspension is incubated with a double-antibody solution, centrifuged, and the supernatant is collected. Collagenase I and CaCl2 solution are added and digested by stirring. After terminating the reaction, sodium carboxymethyl cellulose is added to obtain an amniotic membrane sol, which is stored at 4°C until further use.

[0007] S2. N-isopropylacrylamide and N,N'-methylenebisacrylamide were dissolved in deionized water and stirred in a water bath until completely dissolved. After cooling to room temperature, the amniotic membrane sol was added and ultrasonically dispersed to form a mixture. Ammonium persulfate powder was added and stirred to dissolve. Tetramethylethylenediamine was immediately added and mixed to obtain an N-isopropylacrylamide prepolymer solution.

[0008] S3 alginate nonwoven fabric was rinsed with deionized water, dehydrated with anhydrous ethanol, dried and cut into sheets, immersed in a solution of ammonium cerium nitrate, activated at room temperature, removed and dried with filter paper on the surface of the liquid, immersed in an acrylic acid solution, rinsed with deionized water, and dried to obtain a modified alginate nonwoven fabric for standby use;

[0009] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of glutaraldehyde and PBS. Then pour N-isopropylacrylamide prepolymer solution and apply it evenly. Cover with a layer of sterile nitrocellulose membrane. Evenly add amniotic membrane sol dropwise to the surface of the sterile nitrocellulose membrane. Let it stand in a constant temperature oven to obtain a composite dressing. Soak the composite dressing in ethanol solution, rinse with deionized water, vacuum freeze-dry, and irradiate to sterilize it to obtain a biological amniotic membrane dressing.

[0010] Preferably, in S1, the virus inactivation is to place the amniotic membrane in an ethanol aqueous solution with a volume concentration of 65% to 75% and keep it at 20 to 45°C for 20 to 40 minutes; the volume ratio of the buffer solution to the amniotic membrane microparticle suspension is 1: (0.5 to 1.5); the double antibody solution is a mixed solution of an aqueous solution containing 0.5% to 1.5% penicillin by mass concentration and 0.5% to 1.5% streptomycin by mass concentration, and the volume ratio of the double antibody solution to the amniotic membrane suspension is 1: (90 to 110).

[0011] Preferably, in S1, the amount of collagenase I added is 80-120 U / mL; the molar concentration of the CaCl2 solution is a 0.1M aqueous solution, and the volume ratio of the added amount to the supernatant is (0.5-1.5):10; the digestion temperature is 30-45°C, and the time is 1.5-2.5h; the termination reaction is to add an aqueous solution with a molar concentration of 0.1M EDTA to the digested solution, and the added amount is (0.5-1.5):10 to the supernatant; the ratio of the added amount of sodium carboxymethyl cellulose to the digested solution is (1-3) g:100mL.

[0012] Preferably, in S2, the ratio of N-isopropylacrylamide, N,N'-methylenebisacrylamide and water is (0.8-1.2) g: (20-30) mg: 2.5 mL; and the water bath temperature is 45-55°C.

[0013] Preferably, in S2, the volume ratio of the amniotic membrane sol and the N-isopropylacrylamide solution is (0.8-1.2):1; the ratio of the added amount of ammonium persulfate to the mixed solution is (0.08-0.12) g:100 mL; and the ratio of the added amount of tetramethylethylenediamine to the mixed solution is (0.05-0.15) g:100 mL.

[0014] Preferably, in S3, the ammonium cerium nitrate solution is an aqueous solution with a mass concentration of 0.5% to 1.5%, and the activation time at room temperature is 10 to 20 minutes; the acrylic acid solution is an aqueous solution with a mass concentration of 1.5% to 2.5%, the reaction temperature is 25 to 35° C., and the activation time is 1.5 to 2.5 hours.

[0015] Preferably, in S4, the volume concentration of glutaraldehyde is 2% to 3% aqueous solution, the volume ratio of glutaraldehyde to PBS in the mixed solution is 1: (8 to 10); the amount ratio of N-isopropylacrylamide prepolymer solution to alginate non-woven fabric is (0.8 to 1.2) mL: 10 cm 2 .

[0016] Preferably, in S4, the pore size of the nitrocellulose membrane is 0.45 μm; the ratio of the amount of the amniotic membrane sol to the amount of the alginate non-woven fabric is (0.4 to 0.6) mL:10 cm2; the temperature of the constant temperature box is 30 to 45°C, and the time is 20 to 40 minutes; the volume concentration of the ethanol solution is 70% to 80% aqueous solution; the immersion time is 10 to 20 minutes; the freeze-drying temperature is -50 to -60°C, and the time is 46 to 50 hours; the irradiation sterilization dose is 30 to 50 kGy.

[0017] The present invention also provides a biological amniotic membrane dressing, which is obtained by adopting the above-mentioned preparation method.

[0018] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0019] (1) The amniotic membrane is enzymatically dissolved and gelled, retaining the growth factors while solving the problem of activity loss after re-dissolution of traditional freeze-dried amniotic membrane. Low-temperature swelling combined with mild enzymatic hydrolysis by collagenase I preserves the collagen triple helix structure while exposing more cell binding sites, resulting in higher activity than traditional re-dissolution methods.

[0020] (2) Glutaraldehyde is used to mediate the reaction between the amino groups of amniotic membrane protein and the side chain amino groups of N-isopropylacrylamide to form a Schiff base covalent bond, thereby improving the interlayer peeling strength and solving the problem of easy delamination of multi-layer dressings. Amniotic membrane sol is copolymerized with N-isopropylacrylamide to form an interpenetrating network, and the phase change is triggered by body temperature to achieve dynamic adhesion and drug release regulation. The dressing has low hardness at room temperature, which is convenient for cutting operations. The hardness increases at body temperature, providing moderate support and suitable for joint movement. By initiating acrylic acid grafting with ammonium cerium nitrate, the hydrophilicity of alginate non-woven fabric is combined with its ion exchange capacity to construct a base support and exudate management system. The alginate base layer absorbs the exudate to form a gel, which keeps the wound moist. At the same time, it reduces the pH of the wound through ion exchange, inhibits protease activity, and creates a suitable environment for cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a scanning electron micrograph of the surface layer of the amniotic membrane in Example 1 of the present application;

[0022] Figure 2 This is a scanning electron micrograph of the polyacrylamide thermosensitive gel amniotic membrane copolymer intermediate layer of Example 1 of the present application;

[0023] Figure 3 This is a scanning electron micrograph of the alginate nonwoven fabric layer of Example 1 of the present application;

[0024] Figure 4 This is a longitudinal scanning electron micrograph of the biological amniotic membrane dressing of Example 1 of the present application;

[0025] Figure 5 This is a statistical graph of skin cell proliferation experiments of Examples 1 to 3 and Comparative Examples 1 to 4 of the present application;

[0026] Figure 6 These are pictures of full-thickness skin incisions on rats according to Examples 1 to 3 and Comparative Examples 1 to 4 of the present application. DETAILED DESCRIPTION

[0027] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0028] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0029] Example 1

[0030] A method for preparing a biological amniotic membrane dressing comprises the following steps:

[0031] S1. After decellularization, human or animal amniotic membrane is placed in a 70% ethanol solution at 37°C for 30 minutes, washed with sterile water, and homogenized to obtain an amniotic membrane microparticle suspension. 5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.1 mL of a 1% penicillin solution and a 1% streptomycin solution are added for incubation. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 1000 U of collagenase I and 1 mL of a 0.1 M CaCl2 solution are added. The mixture is stirred and digested in a 37°C water bath for 2 hours. 1 mL of a 0.1 M EDTA solution is added to terminate the reaction, followed by 0.24 g of sodium carboxymethyl cellulose to obtain an amniotic membrane sol. The sol is stored at 4°C until use.

[0032] S2. Dissolve 2 g of N-isopropylacrylamide and 50 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 5 mL of amniotic membrane sol, and ultrasonically disperse to form a mixture. Add 10 mg of ammonium persulfate powder and stir to dissolve. Immediately add 10 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0033] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 15 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2% aqueous solution of acrylic acid at 30°C for 2 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0034] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2.5% aqueous glutaraldehyde solution and 9 mL of PBS. Pour 10 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric, apply the coating evenly, and cover with a layer of sterile nitrocellulose membrane. Evenly add 0.5 mL of amniotic membrane sol to the surface of the sterile nitrocellulose membrane and place it in a 37°C incubator for 30 minutes. Soak the composite dressing in a 75% aqueous ethanol solution for 15 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry at -55°C in a vacuum freeze-drying chamber for 48 hours, and sterilize by irradiation at a dose of 40 kGy to obtain a biological amniotic membrane dressing.

[0035] Example 2

[0036] A method for preparing a biological amniotic membrane dressing comprises the following steps:

[0037] S1. After decellularization, human or animal amniotic membrane is placed in a 65% ethanol solution at 20°C for 20 minutes, washed with sterile water, and homogenized to obtain an amniotic membrane microparticle suspension. 2.5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.083 mL of a 0.5% penicillin solution and a 0.5% streptomycin solution are added for incubation. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 800 U of collagenase I and 0.05 mL of a 0.1 M CaCl2 solution are added. The mixture is stirred and digested in a 37°C water bath for 2 hours. 1 mL of a 0.1 M EDTA solution is added to terminate the reaction, followed by 0.086 g of sodium carboxymethyl cellulose to obtain an amniotic membrane sol. The sol is stored at 4°C until use.

[0038] S2. Dissolve 1.6 g of N-isopropylacrylamide and 40 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 4 mL of amniotic membrane sol, and ultrasonically disperse to form a mixture. Add 7.2 mg of ammonium persulfate powder and stir to dissolve. Immediately add 4.5 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0039] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 0.5% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 20 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 1.5% aqueous solution of acrylic acid at 35°C for 2.5 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0040] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2% glutaraldehyde aqueous solution and 8 mL of PBS. Pour 8 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric, apply the coating evenly, and cover with a layer of sterile nitrocellulose membrane. Evenly add 0.6 mL of amniotic membrane sol to the surface of the sterile nitrocellulose membrane. Incubate the composite dressing in a 30°C incubator for 40 minutes. Soak the composite dressing in an 80% ethanol aqueous solution for 10 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry at -50°C in a vacuum freeze-drying machine for 50 hours, and sterilize by irradiation at a dose of 30 kGy to obtain a biological amniotic membrane dressing.

[0041] Example 3

[0042] A method for preparing a biological amniotic membrane dressing comprises the following steps:

[0043] S1. After decellularization, human or animal amniotic membrane is placed in a 75% ethanol solution at 45°C for 40 minutes, washed with sterile water, and homogenized to obtain an amniotic membrane microparticle suspension. 7.5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.11 mL of a 1.5% aqueous solution of penicillin and a 1.5% aqueous solution of streptomycin are added for incubation. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 1200 U of collagenase I and 1.5 mL of a 0.1 M CaCl2 solution are added. The mixture is stirred and digested in a 37°C water bath for 2 hours. The reaction is terminated by adding 1 mL of a 0.1 M EDTA solution, followed by 0.42 g of sodium carboxymethyl cellulose to obtain an amniotic membrane sol. The sol is stored at 4°C until use.

[0044] S2. Dissolve 2.4 g of N-isopropylacrylamide and 60 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 6 mL of amniotic membrane sol, and ultrasonically disperse to form a mixture. Add 13.2 mg of ammonium persulfate powder and stir to dissolve. Immediately add 16.5 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0045] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1.5% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 10 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2.5% aqueous solution of acrylic acid at 25°C for 1.5 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0046] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 3% aqueous glutaraldehyde solution and 10 mL of PBS. Pour 12 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric, evenly coat it, and cover it with a layer of sterile nitrocellulose membrane. Evenly add 0.4 mL of amniotic membrane sol to the surface of the sterile nitrocellulose membrane. Incubate the composite dressing in a 45°C incubator for 20 minutes. Soak the composite dressing in a 70% aqueous ethanol solution for 20 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry at -60°C in a vacuum oven for 46 hours, and sterilize it with 50 kGy irradiation to obtain a biological amniotic membrane dressing.

[0047] Comparative Example 1

[0048] The preparation method of the non-enzymatically hydrolyzed biological amniotic membrane dressing comprises the following steps:

[0049] S1. After decellularization, human or animal amniotic membrane is placed in a 70% ethanol aqueous solution and incubated at 37°C for 30 minutes. The amniotic membrane is then washed with sterile water and homogenized to obtain an amniotic membrane microparticle suspension. 5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.1 mL of a 1% penicillin aqueous solution and a 1% streptomycin aqueous solution are added and incubated. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 1 mL of a 0.1 M CaCl2 aqueous solution is added. The mixture is stirred and mixed. The mixture is incubated in a 37°C water bath for 2 hours. 1 mL of a 0.1 M EDTA aqueous solution is added, and 0.24 g of sodium carboxymethyl cellulose is added to obtain an amniotic membrane sol. The amniotic membrane sol is stored at 4°C until use.

[0050] S2. Dissolve 2 g of N-isopropylacrylamide and 50 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 5 mL of amniotic membrane sol, and ultrasonically disperse to form a mixture. Add 10 mg of ammonium persulfate powder and stir to dissolve. Immediately add 10 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0051] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 15 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2% aqueous solution of acrylic acid at 30°C for 2 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0052] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2.5% aqueous glutaraldehyde solution and 9 mL of PBS. Pour 10 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric, apply the coating evenly, and cover with a layer of sterile nitrocellulose membrane. Evenly add 0.5 mL of amniotic membrane sol to the surface of the sterile nitrocellulose membrane and place it in a 37°C incubator for 30 minutes. Soak the composite dressing in a 75% aqueous ethanol solution for 15 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry at -55°C in a vacuum freeze-drying chamber for 48 hours, and sterilize by irradiation at a dose of 40 kGy to obtain a biological amniotic membrane dressing.

[0053] Comparative Example 2

[0054] The preparation method of the non-thermosensitive gel biological amniotic membrane dressing comprises the following steps:

[0055] S1. After decellularization, human or animal amniotic membrane is placed in a 70% ethanol aqueous solution and incubated at 37°C for 30 minutes. The membrane is then washed with sterile water and homogenized to obtain an amniotic membrane microparticle suspension. 5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.1 mL of a 1% penicillin aqueous solution and a 1% streptomycin aqueous solution are added for incubation. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 1000 U of collagenase I and 1 mL of a 0.1 M CaCl2 aqueous solution are added. The mixture is stirred and digested in a 37°C water bath for 2 hours. 1 mL of a 0.1 M EDTA aqueous solution is added to terminate the reaction, followed by 0.24 g of sodium carboxymethyl cellulose to obtain an amniotic membrane sol. The amniotic membrane sol is stored at 4°C for later use.

[0056] S2. Dissolve 0.4 g of sodium alginate in 4 mL of deionized water and stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 5 mL of amniotic membrane sol, and ultrasonically disperse the mixture. Add 1 mL of 0.1 g / mL calcium chloride and mix rapidly to obtain a sodium alginate gel solution.

[0057] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 15 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2% aqueous solution of acrylic acid at 30°C for 2 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0058] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2.5% glutaraldehyde aqueous solution and 9 mL of PBS. Pour 10 mL of freshly prepared sodium alginate gel solution onto the alginate nonwoven fabric, evenly coating it. Cover it with a layer of sterile nitrocellulose membrane. Evenly add 0.5 mL of amniotic membrane sol to the surface of the sterile nitrocellulose membrane. Incubate the composite dressing in a 37°C incubator for 30 minutes. Soak the composite dressing in a 75% ethanol aqueous solution for 15 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry it at -55°C in a vacuum freeze-drying machine for 48 hours, and sterilize it with 40 kGy irradiation to obtain a biological amniotic membrane dressing.

[0059] Comparative Example 3

[0060] A method for preparing a biological amniotic membrane dressing without supporting the cross-linking of the surface layer of the amniotic membrane with a nitrocellulose membrane comprises the following steps:

[0061] S1. After decellularization, human or animal amniotic membrane is placed in a 70% ethanol aqueous solution and incubated at 37°C for 30 minutes. The membrane is then washed with sterile water and homogenized to obtain an amniotic membrane microparticle suspension. 5 mL of the amniotic membrane microparticle suspension is added to 5 mL of Tris-HCl buffer (pH 7.4) and vortexed to form a uniform amniotic membrane suspension. 0.1 mL of a 1% penicillin aqueous solution and a 1% streptomycin aqueous solution are added for incubation. The supernatant is centrifuged at 4000 rpm for 15 minutes, and 1000 U of collagenase I and 1 mL of a 0.1 M CaCl2 aqueous solution are added. The mixture is stirred and digested in a 37°C water bath for 2 hours. 1 mL of a 0.1 M EDTA aqueous solution is added to terminate the reaction, followed by 0.24 g of sodium carboxymethyl cellulose to obtain an amniotic membrane sol. The amniotic membrane sol is stored at 4°C for later use.

[0062] S2. Dissolve 2 g of N-isopropylacrylamide and 50 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 5 mL of amniotic membrane sol, and ultrasonically disperse to form a mixture. Add 10 mg of ammonium persulfate powder and stir to dissolve. Immediately add 10 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0063] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 15 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2% aqueous solution of acrylic acid at 30°C for 2 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0064] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2.5% aqueous glutaraldehyde solution and 9 mL of PBS. Pour 10 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric and apply it evenly. Evenly add 0.5 mL of amniotic membrane sol dropwise onto the surface of the N-isopropylacrylamide prepolymer. Place the composite dressing in a 37°C incubator for 30 minutes. Soak the composite dressing in a 75% aqueous ethanol solution for 15 minutes, rinse three times with deionized water, freeze-dry at -55°C in a vacuum oven for 48 hours, and sterilize by irradiation at a dose of 40 kGy to obtain a biological amniotic membrane dressing.

[0065] Comparative Example 4

[0066] The preparation method of the non-amniotic gel dressing comprises the following steps:

[0067] S1. Add 5 mL of sterile water to 5 mL of Tris-HCl buffer (pH 7.4) and vortex. Add 0.1 mL of 1% penicillin and 1% streptomycin in water, incubate, centrifuge at 4000 rpm for 15 minutes, remove the supernatant, add 1000 U of collagenase I and 1 mL of 0.1 M CaCl₂ in water, stir to mix, and digest in a 37°C water bath for 2 hours. Terminate the reaction by adding 1 mL of 0.1 M EDTA in water, then add 0.24 g of sodium carboxymethyl cellulose to the resulting mixture. Store at 4°C until needed.

[0068] S2. Dissolve 2 g of N-isopropylacrylamide and 50 mg of N,N'-methylenebisacrylamide in 5 mL of deionized water. Stir in a 50°C waterbath until completely dissolved. Cool to room temperature, add 5 mL of the mixed solution from S1, and ultrasonically disperse to form a mixture. Add 10 mg of ammonium persulfate powder and stir to dissolve. Immediately add 10 μL of tetramethylethylenediamine and mix rapidly to obtain an N-isopropylacrylamide prepolymer solution.

[0069] S3. The alginate nonwoven fabric was rinsed three times with deionized water, dehydrated twice with anhydrous ethanol, and dried at 50°C for 2 h. The fabric was then cut into 10 cm × 10 cm sheets and immersed in 10 mL of a 1% aqueous solution of cerium ammonium nitrate. The activated sheet was then activated at room temperature for 15 min. The surface liquid was then blotted with filter paper and immersed in 10 mL of a 2% aqueous solution of acrylic acid at 30°C for 2 h. The modified alginate nonwoven fabric was then rinsed three times with deionized water and dried at 50°C.

[0070] S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of 1 mL of a 2.5% glutaraldehyde aqueous solution and 9 mL of PBS. Pour 10 mL of N-isopropylacrylamide prepolymer onto the alginate nonwoven fabric, apply a uniform coating, and cover with a layer of sterile nitrocellulose membrane. Evenly add 0.5 mL of the mixed solution in S1 to the surface of the sterile nitrocellulose membrane. Incubate the composite dressing in a 37°C incubator for 30 minutes. Soak the composite dressing in a 75% ethanol aqueous solution for 15 minutes to dissolve the sterile nitrocellulose membrane. Rinse with deionized water three times, freeze-dry in a vacuum at -55°C for 48 hours, and sterilize by irradiation at a dose of 40 kGy to obtain a biological amniotic membrane dressing.

[0071] Experimental Example 1

[0072] Scanning electron microscopy characterization analysis

[0073] Example 1 was observed using a Hitachi FlexSEM 1000 II scanning electron microscope. Figure 1 The figure shows a scanning electron micrograph of the amniotic membrane surface, which forms a smooth surface layer on the gel. Figure 2 The figure shows the scanning electron micrograph of the polyacrylamide thermosensitive gel amniotic membrane copolymer intermediate layer. The polyacrylamide thermosensitive gel forms a porous structure, which not only has a good supporting effect, but also can achieve the effects of dynamic adhesion and drug release regulation. Figure 3 Shown is a scanning electron micrograph of alginate nonwoven fabric, which is in the form of long fibers. Figure 4 This is a longitudinal scanning electron microscope image of a biological amniotic membrane dressing, where 1 is the surface layer, 2 is the middle layer, and 3 is the bottom layer. The structural characteristics of the three-layer biological amniotic membrane dressing can be clearly seen in the figure.

[0074] Experimental Example 2

[0075] Skin cell proliferation assay

[0076] The dressings of Examples 1-3 and Comparative Examples 1-4 were respectively cut into circular shapes with a diameter of 1.5 cm under sterile conditions. 900 μL of sterile physiological saline was dripped onto the surface of each circular dressing and incubated at 37°C for 3 min. HaCaT cells were seeded in a 24-well plate at 4000 cells / well. The incubated dressings were placed in the upper chamber of a Transwell (pore size 0.4 μm), and the upper chamber was placed in a 24-well plate. DMEM complete medium was added to the upper and lower chambers, respectively, and the cells were cultured in an incubator for 24 h. The group without dressing was used as the 100% control group, and the cell activity in each well was detected by MTT. The results are shown as follows: Figure 5 shown.

[0077] The cell activity of the groups of examples 1-3 is significantly higher than that of the groups of comparative examples 1-4, and the cell activity of the group of example 1 is the highest. The cell activity of the groups of examples 1-3 and comparative examples 1-3 is higher than that of the control group, and the activity of the group of comparative example 4 is not significantly different from that of the control group, which indicates that the amniotic membrane has a promoting effect on the proliferation of HaCaT cells, and the amniotic membrane dressings prepared by examples 1-3 have a better promoting effect on the cell activity of HaCaT cells. It is proved that the biological amniotic membrane dressing prepared by the method of the application has the effect of promoting the proliferation of skin cells.

[0078] Experimental example 3

[0079] Rat full-thickness skin defect wound model

[0080] The 70 rats were divided into 7 groups according to examples 1-3 and comparative examples 1-4. The 70 rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (45 mg / kg), the back was shaved and disinfected, and after the rats were fixed, a square incision with a side length of 1 cm was marked on the back, the incision was parallel to the spine, and the full-thickness skin was cut along the marked line to the fascia layer to form a wound. The dressings of examples 1-3 and comparative examples 1-4 were cut into squares with a side length of 1 cm under sterile conditions, 900 μL of sterile normal saline was added to the surface of each dressing, and after 3 min of incubation at 37°C, the dressing was fixed to the skin incision of the rat. The wound area of each mouse was measured on the 7th and 14th day (as shown in Figure 6 Table 1).

[0081]

[0082] The wound healing rate of the groups of examples 1-3 is significantly higher than that of the groups of comparative examples 1-4, and the wound healing rate of the group of example 1 is the highest. The wound healing rate of the groups of examples 1-3 and comparative examples 1-3 is higher than that of the group of comparative example 4, which indicates that the amniotic membrane composition has a good promoting effect on the wound healing rate, and the amniotic membrane dressing prepared by examples 1-3 has a better promoting effect on the wound healing rate. It is proved that the biological amniotic membrane dressing prepared by the method of the application has a better effect of promoting wound healing. The wound healing rate of examples 1-3 is higher than that of comparative example 1, which is prepared by not using the enzymatic method to treat the amniotic membrane, which indicates that the use of enzymatic treatment of amniotic membrane can improve the wound healing efficiency, because the enzymatic amniotic membrane retains the triple helix structure of collagen while exposing more cell binding sites. The wound healing rate of examples 1-3 is higher than that of comparative example 2 using conventional gel dressing, which indicates that the warm-sensitive gel helps to fix and release the active ingredients in the amniotic membrane. The conventional gel may cause the release of the active ingredients in the gel to be too slow due to the absorption effect, which cannot control the release speed, resulting in slower wound healing. The wound healing rate of examples 1-3 is higher than that of comparative example 3 without using nitrocellulose membrane, which indicates that the nitrocellulose membrane helps to cross-link the amniotic sol on the surface of the warm-sensitive gel, preventing the waste caused by excessive penetration of active ingredients.

[0083] Experimental Example 4

[0084] Rabbit knee joint wound experiment

[0085] Six New Zealand jackrabbit rabbits were anesthetized and a 1 cm diameter defect was created on the medial side of the knee joint, reaching deep into the fascia. The rabbits were divided into two groups. Circular shapes with a diameter of 1.5 cm were cut from the dressings of Example 1 and Comparative Example 2 under sterile conditions. 900 μL of sterile saline was dripped onto the surface of each circular dressing. After incubation at 37°C for 3 minutes, the wound was covered and the knee joint was lightly secured with an elastic bandage. The position was marked with a marker, allowing 0°-90° of movement. The range of motion of the knee joint, the distance of dressing displacement (displacement was determined if the dressing edge was >4 mm away from the mark), and the fit (visual observation of gaps) were observed daily after surgery. The experimental results are shown in Table 2.

[0086]

[0087] Experimental results show that the biological amniotic membrane dressing prepared by the method of the present invention has a better fitting effect and is less likely to be displaced when used on joints.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a biological amniotic membrane dressing, characterized in that: The following steps are involved: S1. The amniotic membrane is virally inactivated, washed, and homogenized to obtain an amniotic membrane microparticle suspension. Buffer is added and vortexed to form a uniform amniotic membrane suspension. The suspension is incubated with a double-antibody solution, centrifuged, and the supernatant is collected. Collagenase I and CaCl2 solution are added and digested by stirring. After terminating the reaction, sodium carboxymethyl cellulose is added to obtain an amniotic membrane sol, which is stored at 4°C until further use. S2. N-isopropylacrylamide and N,N'-methylenebisacrylamide were dissolved in deionized water and stirred in a water bath until completely dissolved. After cooling to room temperature, the amniotic membrane sol was added and ultrasonically dispersed to form a mixture. Ammonium persulfate powder was added and stirred to dissolve. Tetramethylethylenediamine was immediately added and mixed to obtain an N-isopropylacrylamide prepolymer solution. S3 alginate nonwoven fabric was rinsed with deionized water, dehydrated with anhydrous ethanol, dried and cut into sheets, immersed in a solution of ammonium cerium nitrate, activated at room temperature, removed and dried with filter paper on the surface of the liquid, immersed in an acrylic acid solution, rinsed with deionized water, and dried to obtain a modified alginate nonwoven fabric for standby use; S4. Lay the modified alginate nonwoven fabric flat and evenly spray the surface with a mixture of glutaraldehyde and PBS. Then pour N-isopropylacrylamide prepolymer solution and apply it evenly. Cover with a layer of sterile nitrocellulose membrane. Evenly add amniotic membrane sol dropwise to the surface of the sterile nitrocellulose membrane. Let it stand in a constant temperature oven to obtain a composite dressing. Soak the composite dressing in ethanol solution, rinse with deionized water, vacuum freeze-dry, and irradiate to sterilize it to obtain a biological amniotic membrane dressing.

2. The preparation method of the biological amniotic membrane dressing according to claim 1, characterized in that: In S1, the virus inactivation is to place the amniotic membrane in an ethanol aqueous solution with a volume concentration of 65% to 75% and keep it at 20 to 45°C for 20 to 40 minutes; the volume ratio of the buffer solution to the amniotic membrane microparticle suspension is 1: (0.5 to 1.5); the double antibody solution is a mixed solution of an aqueous solution containing a mass concentration of 0.5% to 1.5% penicillin and an aqueous solution containing a mass concentration of 0.5% to 1.5% streptomycin, and the volume ratio of the double antibody solution to the amniotic membrane suspension is 1: (90 to 110).

3. The preparation method of the biological amniotic membrane dressing according to claim 1, characterized in that: In S1, the amount of collagenase I added is 80-120 U / mL; the molar concentration of the CaCl2 solution is a 0.1M aqueous solution, and the volume ratio of the added amount to the supernatant is (0.5-1.5):10; the digestion temperature is 30-45°C, and the time is 1.5-2.5 h; the termination reaction is to add a 0.1M EDTA aqueous solution to the digested solution, and the added amount is (0.5-1.5):10 to the supernatant; the ratio of the added amount of sodium carboxymethyl cellulose to the digested solution is (1-3) g:100 mL.

4. The method for preparing the biological amniotic membrane dressing according to claim 1, wherein In S2, the ratio of N-isopropylacrylamide, N,N'-methylenebisacrylamide and water is (0.8-1.2) g: (20-30) mg: 2.5 mL; and the water bath temperature is 45-55°C.

5. The preparation method of the biological amniotic membrane dressing according to claim 1, characterized in that: In S2, the volume ratio of the amniotic membrane sol and the N-isopropylacrylamide solution is (0.8-1.2):1; the ratio of the added amount of ammonium persulfate to the mixed solution is (0.08-0.12) g:100 mL; and the ratio of the added amount of tetramethylethylenediamine to the mixed solution is (0.05-0.15) g:100 mL.

6. The method for preparing the biological amniotic membrane dressing according to claim 1, characterized in that: In S3, the ammonium cerium nitrate solution is an aqueous solution with a mass concentration of 0.5% to 1.5%, and the activation time at room temperature is 10 to 20 minutes; the acrylic acid solution is an aqueous solution with a mass concentration of 1.5% to 2.5%, the reaction temperature is 25 to 35°C, and the activation time is 1.5 to 2.5 hours.

7. The method for preparing the biological amniotic membrane dressing according to claim 1, characterized in that: In S4, the volume concentration of glutaraldehyde is 2% to 3% in aqueous solution, the volume ratio of glutaraldehyde to PBS in the mixed solution is 1: (8 to 10); the amount ratio of N-isopropylacrylamide prepolymer solution to alginate non-woven fabric is (0.8 to 1.2) mL: 10 cm 2 .

8. The method for preparing the biological amniotic membrane dressing according to claim 1, wherein: In S4, the pore size of the sterile nitrocellulose membrane is 0.45 μm; the ratio of the amount of the amniotic membrane sol to the amount of the alginate non-woven fabric is (0.4-0.6) mL:10 cm 2 The static temperature of the thermostat is 30 to 45°C, and the time is 20 to 40 minutes; the volume concentration of the ethanol solution is 70% to 80% aqueous solution; the soaking time is 10 to 20 minutes; the freeze-drying temperature is -50 to -60°C, and the time is 46 to 50 hours; the irradiation sterilization dose is 30 to 50 kGy.

9. A biological amniotic membrane dressing, characterized in that: Obtained according to the preparation method according to any one of claims 1 to 8.

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