Self-supporting acellular matrix amniotic membrane composite material with interpenetrating network structure as well as preparation method and application of self-supporting acellular matrix amniotic membrane composite material

Abstract

The invention provides a self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The preparation method of the composite material comprises the following steps: immersing an acellular matrix amnion into a polyfunctional group polymer solution, then immersing into a cross-linking agent solution for cross-linking to obtain gel, and drying the gel to obtain the composite material. The obtained composite material has an interpenetrating network structure, and compared with an acellular matrix amniotic membrane, the mechanical property is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure, a preparation method thereof, and uses thereof. Background Art

[0002] Acellular matrix, also known as extracellular matrix, is a substance produced and secreted by cells outside the cell periphery. Composed primarily of proteins and polysaccharides, it supports, protects, and provides nutrition to tissue cells. It is also closely associated with fundamental cellular activities such as proliferation, differentiation, metabolism, recognition, adhesion, and migration. The acellular matrix (amniotic membrane) obtained by decellularizing the amniotic membrane has a unique structure and biological activity, promoting the adhesion, migration, differentiation, and proliferation of stem cells, effectively promoting tissue repair and regeneration.

[0003] Acellular amniotic membrane is highly biodegradable and can be excreted through metabolism. However, it is thin and lightweight, has poor mechanical properties, and is prone to rupture during use. Currently, this material is only used to repair open wounds on the skin's outer surface. Its application is limited to repairing soft tissue damage in vivo, where mechanical properties are critical.

[0004] Patent application publication number CN105194735A discloses a genipin-crosslinked acellular amniotic membrane matrix. This method involves decellularizing the amniotic membrane and then soaking it in a genipin solution for crosslinking. This approach overcomes the membrane's inherent shortcomings, including high immunogenicity, insufficient mechanical properties, and short degradation cycle. This patent involves autologously crosslinking the acellular matrix obtained after decellularization to enhance the membrane's strength, but the effect is limited. The maximum strength of the crosslinked acellular amniotic membrane matrix is equal to the maximum strength of its cytoskeleton.

[0005] Patent application number CN109718392A discloses a composite medical dressing that soaks pancreatic enzyme-treated amniotic membrane in a mixed solution containing carboxymethyl chitosan, alginate, silk fibroin, and sodium hyaluronate, and then adds carbodiimide for cross-linking to improve the mechanical properties of the amniotic membrane. This patent primarily relies on a polymer cross-linked network to wrap the amniotic membrane to build a biological scaffold to provide mechanical support. This single-network scaffold has limited mechanical performance improvements, and the amniotic membrane is wrapped in a polymer, preventing direct contact with the human body to play a repair role until the polymer is completely degraded. This patent coats the network scaffold with mesenchymal stem cells as "seed cells" to promote tissue repair. The repair properties of the amniotic membrane are not utilized, and the dressing is not suitable for use as a support material or soft tissue repair material in the body. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, a self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure and a preparation method and use thereof is provided, which has good mechanical strength and can be used as an in vivo support material for soft tissue repair.

[0007] The present invention provides a self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure, which is obtained by immersing the acellular matrix amniotic membrane in a multifunctional polymer solution, then immersing it in a crosslinking agent solution to crosslink to obtain a gel, and then drying the gel.

[0008] Furthermore, the concentration of the multifunctional polymer solution is 0.1 to 15 wt %; and / or the concentration of the cross-linking agent solution is 0.1 to 5 wt %.

[0009] Furthermore, the concentration of the multifunctional polymer solution is 0.5 to 2 wt %; and / or the concentration of the cross-linking agent solution is 0.1 to 2 wt %.

[0010] Further,

[0011] The multifunctional polymer is one or more of alginate, polyvinyl alcohol, polyethylene glycol, chitosan and its derivatives, cellulose and its derivatives;

[0012] And / or, the cross-linking agent is one or more of calcium chloride, glutaraldehyde, genipin, carbodiimide and its derivatives, succinic anhydride and its derivatives.

[0013] Further,

[0014] When the multifunctional polymer is alginate, the concentration of the multifunctional polymer solution is 1-2 wt%, the crosslinking agent is calcium chloride, and the concentration of the crosslinking agent solution is 2 wt%;

[0015] When the multifunctional polymer is polyvinyl alcohol, the concentration of the multifunctional polymer solution is 0.5 wt %, the crosslinking agent is glutaraldehyde, and the concentration of the crosslinking agent solution is 1 wt %.

[0016] Further,

[0017] The solvent of the multifunctional polymer solution is water;

[0018] And / or, the solvent of the cross-linking agent solution is water.

[0019] Further,

[0020] The time for immersing the decellularized amniotic matrix in the multifunctional polymer solution is 1 to 300 minutes;

[0021] And / or, the cross-linking time of immersing in the cross-linking agent solution is 1 to 300 minutes.

[0022] Further,

[0023] After obtaining the gel, soaking the gel in water to remove the cross-linking agent;

[0024] And / or, the drying is freeze-drying.

[0025] The present invention also provides a method for preparing the aforementioned self-supporting acellular matrix amniotic membrane composite material, which comprises the following steps:

[0026] (1) immersing the decellularized amniotic membrane in a multifunctional polymer solution;

[0027] (2) immersing the acellular matrix amniotic membrane obtained in step (1) in a crosslinking agent solution to crosslink and obtain a gel;

[0028] (3) Drying the gel obtained in step (2) to obtain.

[0029] The present invention also provides use of the aforementioned self-supporting acellular matrix amniotic membrane composite material in preparing soft tissue repair materials.

[0030] In order to solve the problem that the amniotic membrane has low strength and weak self-supporting performance, and cannot be used as a soft tissue repair material in the body, the present invention provides a self-supporting decellularized matrix amniotic membrane composite material with an interpenetrating network structure. The decellularized matrix amniotic membrane is immersed in a multi-functional polymer solution, and the polymer molecular chains are immersed in the pores of the decellularized matrix amniotic membrane. It is then immersed in a cross-linker solution. After the polymer is cross-linked, a gel is formed. The gel network and the decellularized matrix amniotic membrane's own structure form an interpenetrating network. After the gel is dried, a polymer / decellularized matrix amniotic membrane composite material is obtained. This material has an interpenetrating network structure and has significantly improved mechanical properties compared to the decellularized matrix amniotic membrane.

[0031] The polymer / acellular matrix amniotic membrane composite material of the present invention has a loose porous structure, which is conducive to cell proliferation along its network structure, and can be directly used in the soft tissue defect position in the body, providing a cell proliferation scaffold while occupying the space and supporting it.

[0032] The present invention introduces a polymer network into the decellularized matrix amniotic membrane skeleton network to construct an interpenetrating network structure. The two networks simultaneously support the material. Different polymers can be selected for cross-linking. By selecting polymer materials and adjusting the degree of cross-linking, decellularized matrix amniotic membrane composite materials of different strengths can be obtained, thereby effectively broadening the application scope of the decellularized matrix amniotic membrane.

[0033] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0034] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. DETAILED DESCRIPTION

[0035] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0036] Acellular amniotic membrane was purchased from Chengdu Qingshan Likang Pharmaceutical Co., Ltd., with the registration number for Class III medical devices: National Medical Device Registration No. 20153132192.

[0037] Example 1. Preparation of the self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure of the present invention

[0038] The decellularized amniotic membrane was immersed in a 2wt% sodium alginate aqueous solution for 30 minutes to allow the sodium alginate aqueous solution to fully penetrate the network structure of the decellularized amniotic membrane. The decellularized amniotic membrane adsorbed with the sodium alginate aqueous solution was immersed in a 2wt% calcium chloride aqueous solution for another 30 minutes to complete the cross-linking of the sodium alginate in the pores of the decellularized amniotic membrane. The cross-linked decellularized amniotic membrane was washed and soaked in deionized water multiple times to remove excess calcium chloride to obtain a calcium alginate / decellularized amniotic membrane double network gel with an interpenetrating network structure. The calcium alginate / decellularized amniotic membrane double network gel was freeze-dried to obtain the corresponding calcium alginate / decellularized amniotic membrane composite material.

[0039] Mechanical properties of uncross-linked acellular amniotic membrane and the prepared calcium alginate / acellular amniotic membrane composite were tested using the GB / T1040.3-2006 standard. The tensile strength of the uncross-linked acellular amniotic membrane was 0.79 MPa, while the calcium alginate / acellular amniotic membrane composite had a tensile strength of 5.12 MPa, a 548% increase compared to the uncross-linked acellular amniotic membrane.

[0040] Example 2: Preparation of the self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure of the present invention

[0041] The decellularized amniotic membrane was immersed in a 1wt% sodium alginate aqueous solution for 30 minutes to allow the sodium alginate aqueous solution to fully penetrate the network structure of the decellularized amniotic membrane. The decellularized amniotic membrane adsorbed with the sodium alginate aqueous solution was immersed in a 2wt% calcium chloride aqueous solution for another 30 minutes to complete the cross-linking of the sodium alginate in the pores of the decellularized amniotic membrane. The cross-linked decellularized amniotic membrane was washed and soaked in deionized water multiple times to remove excess calcium chloride to obtain a calcium alginate / decellularized amniotic membrane double network hydrogel with an interpenetrating network structure. The calcium alginate / decellularized amniotic membrane double network hydrogel was freeze-dried to obtain the corresponding milky white calcium alginate / decellularized amniotic membrane composite material.

[0042] Mechanical properties of uncross-linked acellular amniotic membrane and the prepared calcium alginate / acellular amniotic membrane composite were tested according to the method described in Example 1. The tensile strength of the uncross-linked acellular amniotic membrane was 0.79 MPa, while the tensile strength of the calcium alginate / acellular amniotic membrane composite was 3.20 MPa, a 305% increase compared to the uncross-linked acellular amniotic membrane.

[0043] Example 3: Preparation of the self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure of the present invention

[0044] The decellularized matrix amniotic membrane was immersed in a 0.5wt% polyvinyl alcohol (PVA 1799) aqueous solution for 30 minutes to allow the PVA to fully enter the pores of the decellularized matrix amniotic membrane. The decellularized matrix amniotic membrane that had absorbed the PVA aqueous solution was taken out and immersed again in a 1wt% glutaraldehyde aqueous solution for 120 minutes to cross-link the PVA. After repeated immersion and washing with deionized water to remove excess glutaraldehyde, a polyvinyl alcohol / decellularized matrix amniotic membrane double network hydrogel with an interpenetrating network structure was obtained. The polyvinyl alcohol / decellularized matrix amniotic membrane double network hydrogel was freeze-dried to obtain a polyvinyl alcohol / decellularized matrix amniotic membrane composite material.

[0045] Mechanical properties of the uncross-linked acellular amniotic membrane and the prepared polyvinyl alcohol / acellular amniotic membrane composite were tested according to the method described in Example 1. The tensile strength of the uncross-linked acellular amniotic membrane was 0.82 MPa, while the tensile strength of the polyvinyl alcohol / acellular amniotic membrane composite was 2.15 MPa, a 162% increase compared to the uncross-linked acellular amniotic membrane.

[0046] In summary, in order to solve the problem that the amniotic membrane has low strength and weak self-supporting performance, and cannot be used as a soft tissue repair material in the body, the present invention provides a self-supporting decellularized matrix amniotic membrane composite material with an interpenetrating network structure. The decellularized matrix amniotic membrane is immersed in a multi-functional polymer solution, and the polymer molecular chains are immersed in the pores of the decellularized matrix amniotic membrane. It is then immersed in a cross-linker solution. After the polymer is cross-linked, a gel is formed. The gel network and the decellularized matrix amniotic membrane's own structure form an interpenetrating network. After the gel is dried, a polymer / decellularized matrix amniotic membrane composite material is obtained. This material has an interpenetrating network structure, and its mechanical properties are significantly improved compared to the decellularized matrix amniotic membrane.

Claims

1. A self-supporting acellular matrix amniotic membrane composite material with an interpenetrating network structure, characterized in that: The method comprises immersing the decellularized matrix amniotic membrane into a multifunctional polymer solution, then immersing the decellularized matrix into a cross-linking agent solution to cross-link to obtain a gel, and then drying the gel.

2. The self-supporting acellular matrix amniotic membrane composite material according to claim 1, characterized in that: The concentration of the multifunctional polymer solution is 0.1 to 15 wt %; and / or the concentration of the cross-linking agent solution is 0.1 to 5 wt %.

3. The self-supporting acellular matrix amniotic membrane composite material according to claim 2, characterized in that: The concentration of the multifunctional polymer solution is 0.5 to 2 wt %; and / or the concentration of the cross-linking agent solution is 0.1 to 2 wt %.

4. The self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 3, characterized in that: The multifunctional polymer is one or more of alginate, polyvinyl alcohol, polyethylene glycol, chitosan and its derivatives, cellulose and its derivatives; And / or, the cross-linking agent is one or more of calcium chloride, glutaraldehyde, genipin, carbodiimide and its derivatives, succinic anhydride and its derivatives.

5. The self-supporting acellular matrix amniotic membrane composite material according to claim 4, characterized in that: When the multifunctional polymer is alginate, the concentration of the multifunctional polymer solution is 1-2 wt%, the crosslinking agent is calcium chloride, and the concentration of the crosslinking agent solution is 2 wt%; When the multifunctional polymer is polyvinyl alcohol, the concentration of the multifunctional polymer solution is 0.5 wt %, the crosslinking agent is glutaraldehyde, and the concentration of the crosslinking agent solution is 1 wt %.

6. The self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 3, characterized in that: The solvent of the multifunctional polymer solution is water; And / or, the solvent of the cross-linking agent solution is water.

7. The self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 3, characterized in that: The time for immersing the decellularized amniotic matrix in the multifunctional polymer solution is 1 to 300 minutes; And / or, the cross-linking time of immersing in the cross-linking agent solution is 1 to 300 minutes.

8. The self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 3, characterized in that: After obtaining the gel, soaking the gel in water to remove the cross-linking agent; And / or, the drying is freeze-drying.

9. A method for preparing the self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 8, characterized in that: It includes the following steps: (1) immersing the decellularized amniotic membrane in a multifunctional polymer solution; (2) immersing the acellular matrix amniotic membrane obtained in step (1) in a crosslinking agent solution to crosslink and obtain a gel; (3) Drying the gel obtained in step (2) to obtain.

10. Use of the self-supporting acellular matrix amniotic membrane composite material according to any one of claims 1 to 8 in the preparation of soft tissue repair materials.