Cross-linked amnion acellular matrix filling material and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a cross-linked amnion decellularized matrix filling material and a preparation method thereof.The cross-linked amnion decellularized matrix filling material is prepared through the photo-crosslinking technology, and the specific preparation method comprises the steps that a decellularized amnion matrix is placed in a phosphate buffer solution containing a pretreating agent to be pretreated; the method comprises the following steps: preparing a decellularized amnion extracellular matrix, then carrying out functionalization treatment on the decellularized amnion extracellular matrix, preparing a photo-crosslinking solution, soaking the treated decellularized amnion extracellular matrix in the photo-crosslinking solution, permeating at room temperature, and finally irradiating under an ultraviolet light source to form a stable three-dimensional network structure. The material has high mechanical strength, controllable degradability and excellent biocompatibility, and is suitable for tissue repair and regenerative medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. Background Art

[0002] Decellularized amniotic membrane matrix (AMM) is a biomaterial derived from the placental amniotic membrane, which has been specially processed to remove cellular components. Its core principle is to retain the extracellular matrix structure and bioactive components of the amniotic membrane. Due to its natural three-dimensional structure, low immunogenicity, and rich content of bioactive components such as collagen and laminin, AMM is widely used in tissue repair and regenerative medicine. However, natural decellularized matrix has low mechanical strength and rapid degradation in the body (typically within 1-2 weeks), making it difficult to maintain long-term structural support, limiting its application in applications requiring mechanical stability, such as filling materials.

[0003] To address these issues, existing technologies often employ cross-linking techniques to improve the mechanical properties of decellularized amniotic membrane matrices. For example, Chinese invention patent CN106860919B discloses a cross-linked decellularized amniotic membrane, its preparation method, and its application. By cross-linking the decellularized amniotic membrane with a water-soluble carbodiimide, the membrane overcomes its insufficient mechanical strength and biostability, achieving more effective skin repair and cell proliferation, and improving the preparation efficiency and biocompatibility of skin substitutes. The specific cross-linking method involves adding a water-soluble carbodiimide to a buffer (MES buffer) containing the soaked amniotic membrane at a concentration of 0.01-0.1 mmol / mg. The membrane is then placed on a thermostatic shaker at 37±0.5°C, with a rotation speed of 28-32 rpm, for a cross-linking reaction of 1-10 minutes. The membrane is then rinsed with distilled water and freeze-dried under vacuum to yield the cross-linked decellularized amniotic membrane. At the same time, the patent technology with document number CN117159814A discloses a cross-linked amniotic membrane material, its preparation method and application, which first uses a mechanical method to peel off the chorionic layer of the fetal membrane, retaining the sponge layer and the amniotic membrane layer, recorded as the amniotic membrane / sponge layer, and the amniotic membrane / sponge layer is cleaned and dried, and then placed in a low concentration cross-linker solution for cross-linking. The cross-linker used in the cross-linker solution is any one of an epoxide cross-linker, a dialdehyde cross-linker, EDC / NHS or genipin; the patent technology with document number TWI324520B discloses a method for cross-linking amniotic membrane as a biomedical material, the steps of which include: a. obtaining amniotic membrane, taking out the frozen amniotic membrane and thawing it; cross-linking the amniotic membrane using a cross-linking method, and the cross-linking agent used is EDC. Chinese invention patent CN120022424A discloses the use of water-soluble carbodiimide and N-hydroxysuccinimide (EDC / NHS) as chemical crosslinkers to crosslink amniotic membrane decellularized matrix. The specific method is as follows: the concentration of the crosslinking solution is 0.5-2%, of which the concentration of water-soluble carbodiimide is 0.5-2%, and the concentration of N-hydroxysuccinimide is 0.125-0.5%. The mass ratio of water-soluble carbodiimide to N-hydroxysuccinimide is 4:1, and the mass-to-volume ratio of amniotic membrane to crosslinking solution is 1:2. The crosslinking time is 12-48 hours, and the solution is replaced every 24 hours. Although the above technology solves the problem of low mechanical strength of uncrosslinked amniotic membrane materials, it still has significant drawbacks, including but not limited to: First, the reaction conditions required for cross-linking are harsh (strong acid / high temperature), which destroy the natural bioactive factors of the ECM (such as growth factors and collagen structure); Secondly, the cross-linking speed is slow (hours to days), making it difficult to achieve immediate clinical operation; Finally, residual toxicity or non-degradability triggers an inflammatory response.

[0004] Therefore, the existing technology still needs to be improved. Summary of the Invention

[0005] To solve the above technical problems, the purpose of the present invention is to provide a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. In the first aspect, the present invention provides a cross-linked amniotic membrane acellular matrix filling material, which is prepared by a photocrosslinking method.

[0006] In a second aspect, the present invention further provides a method for preparing a cross-linked amniotic membrane acellular matrix filling material, which specifically comprises the following steps: S1: Pretreatment of decellularized amniotic membrane extracellular matrix (dAM-ECM): The decellularized amniotic membrane extracellular matrix is placed in a phosphate buffer solution containing a pretreatment agent and soaked (e.g., at 37° C. for 1-3 hours). After pretreatment, the dAM-ECM is washed to remove residual reagents; S2: Functionalization of decellularized amniotic membrane extracellular matrix (dAM-ECM): Soak the washed dAM-ECM in a buffer solution (e.g., sodium bicarbonate buffer) containing 0.5%-2% methacrylic anhydride for reaction (e.g., at room temperature for 2 hours), rinse thoroughly with PBS to remove unreacted reagents, and freeze-dry for later use; S3: Preparation of photocrosslinking solution: preparing a photocrosslinking solution, wherein the photocrosslinking solution comprises: polyethylene glycol diacrylate, acryloyl RGD peptide, a photoinitiator, and a buffer; S4: Photocrosslinking treatment: The freeze-dried dAM-ECM-MA is immersed in a photocrosslinking solution, infiltrated (e.g., infiltrated at room temperature for 5-20 minutes), excess liquid is drained, irradiated with an ultraviolet light source (e.g., placed under a 365-405 nm ultraviolet light source for 5-15 minutes), and rinsed (e.g., with PBS) to remove unpolymerized monomers to obtain a cross-linked amniotic membrane decellularized matrix filling material.

[0007] Preferably, the pretreatment agent in S1 includes at least one or more of hyaluronic acid and calcium chloride.

[0008] Preferably, the pretreatment agent in S1 is a combination of hyaluronic acid and calcium chloride, wherein the mass fraction of hyaluronic acid is 0.1%-0.5%; the mass fraction of calcium chloride is 0.001 mol / L-0.01 mol / L.

[0009] Preferably, in step S2, the concentration of methacrylic anhydride is 0.5-1.5%, preferably 1.0%.

[0010] Preferably, the photocrosslinking solution in S3 consists of the following components: Polyethylene glycol diacrylate (molecular weight 3000Da) 5-15% w / v, acryloyl-RGD peptide 0.1-5% w / v, photoinitiator (Irgacure 2959) 0.2-1% w / v, and phosphate buffered saline (PBS, pH 7.4).

[0011] Further preferably, the photocrosslinking solution in S3 consists of the following components: Polyethylene glycol diacrylate (molecular weight 3000Da) 10% w / v, acryloyl-RGD peptide 0.2-2% w / v, photoinitiator (Irgacure 2959) 0.5% w / v and phosphate buffered saline (PBS, pH 7.4).

[0012] Further preferably, the photocrosslinking solution in S3 is prepared by: Weigh polyethylene glycol diacrylate and photoinitiator in proportion, dissolve in an appropriate amount of phosphate buffer, stir at room temperature until completely dissolved, add acryloyl RGD peptide, continue stirring for 10-15 minutes, dilute to the target volume with phosphate buffer, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C in the dark until used.

[0013] Preferably, in step S4, the room temperature penetration time is 5-30 minutes, and the intensity of the ultraviolet light source is 5-10 mW / cm 2 , the irradiation time is 5-15 minutes.

[0014] In a third aspect, the present invention further provides a cross-linked amniotic membrane acellular matrix filling material prepared by the aforementioned preparation method and its application in tissue repair and regenerative medicine.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a cross-linked amniotic membrane decellularized matrix filling material and a preparation method thereof. The cross-linked amniotic membrane decellularized matrix filling material is prepared using photocrosslinking technology. The specific preparation method is: placing the decellularized amniotic membrane matrix in a phosphate buffer solution containing a pretreatment agent for pretreatment, and then functionalizing the decellularized amniotic membrane extracellular matrix; then preparing a photocrosslinking solution, immersing the treated decellularized amniotic membrane extracellular matrix in the photocrosslinking solution, infiltrating at room temperature, and finally irradiating under an ultraviolet light source to form a stable three-dimensional network structure.

[0016] The method provided by this invention achieves efficient crosslinking under mild conditions, avoiding the high-temperature / strong-acid environment of traditional chemical crosslinkers that damage the natural bioactive factors of the amniotic membrane (such as growth factors and collagen structure). Furthermore, the introduction of the RGD peptide enhances cell-matrix interactions, forming a dense three-dimensional network, significantly improving the material's mechanical strength and significantly slowing its degradation rate in vivo, making it suitable for applications such as soft tissue repair requiring long-term mechanical support. This process is simple, controllable, and reproducible, with the potential for large-scale production. It provides a novel filler material for tissue engineering that combines high mechanical strength, controlled degradability, and excellent biocompatibility. DETAILED DESCRIPTION

[0017] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are therefore only used as examples and are not intended to limit the scope of protection of the present invention.

[0018] For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. To better illustrate the present invention, numerous specific details are provided in the following detailed description.

[0019] It will be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention are all understood to include inevitable systematic errors in industrial production.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used without indicating the manufacturer are all reagents and materials that can be obtained from commercial channels. If no specific conditions are specified in the examples, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in this technical field.

[0021] It should be noted that the decellularized amniotic extracellular matrix used in the following examples was prepared according to the method disclosed in the literature Song Yongzhou, Cui Huixian, Wang Zhenxian, et al. Preparation of amniotic decellularized matrix and its biocompatibility [J]. Chinese Journal of Tissue Engineering Research and Clinical Rehabilitation, 2008, (01): 51-55. (For details, see the literature method - Preparation of amniotic decellularized matrix).

[0022] Example 1 The purpose of this embodiment is to provide a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. Specifically, the preparation method includes the following steps: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.1% hyaluronic acid and 0.001 mol / L calcium chloride and immersed at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0023] S2: dAM-ECM functionalization: The washed dAM-ECM was immersed in sodium bicarbonate buffer (pH 8.5) containing 0.5% (w / v, g:mL) methacrylic anhydride (MA) and reacted at room temperature for 2 hours. After the reaction, it was washed three times with PBS to thoroughly rinse and remove unreacted reagents, and then freeze-dried for later use.

[0024] S3: Preparation of photocrosslinking solution Weigh 10 g of 3000 Da polyethylene glycol diacrylate (PEG-DA) and 0.5 g of the photoinitiator Irgacure 2959 and dissolve them in an appropriate amount of phosphate buffered saline (PBS, pH 7.4) (approximately 1 / 2 of the total volume). Stir magnetically at room temperature until completely dissolved. Add 1 g of acryloyl-RGD peptide and continue stirring for 10 minutes. Dose the solution to 100 mL with PBS, sterilize it by filtering through a 0.22 μm filter, and store it at 4°C in the dark until ready for use.

[0025] S4: Photocrosslinking The freeze-dried dAM-ECM-MA was immersed in the photocrosslinking solution and infiltrated at room temperature for 5 minutes. The excess liquid was drained and the dAM-ECM was exposed to a 365 nm UV light source (intensity: 5 mW / cm²) for 5 minutes. The unpolymerized monomers were then rinsed with PBS to obtain the RGD-functionalized cross-linked dAM-ECM.

[0026] Example 2 The purpose of this embodiment is to provide a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. Specifically, the preparation method includes the following steps: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.5% hyaluronic acid and 0.01 mol / L calcium chloride at 37°C for 3 hours. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0027] S2: dAM-ECM functionalization The washed dAM-ECM was immersed in sodium bicarbonate buffer (pH 8.5) containing 2% (w / v, g:mL) methacrylic anhydride (MA) and reacted at room temperature for 2 hours. After the reaction, the unreacted reagents were thoroughly rinsed with PBS and freeze-dried for later use.

[0028] S3: Preparation of photocrosslinking solution Weigh 10 g of 3000 Da polyethylene glycol diacrylate (PEG-DA) and 0.5 g of Irgacure 2959 photoinitiator and dissolve them in an appropriate amount of phosphate buffered saline (PBS, pH 7.4) (approximately 1 / 3 of the total volume). Stir magnetically at room temperature until completely dissolved. Add 0.5 g of acryloyl-RGD peptide and continue stirring for 15 minutes. Dose the solution to 100 mL with phosphate buffered saline. Sterilize the solution by filtering through a 0.22 μm filter and store at 4°C in the dark until ready for use.

[0029] S3: Photocrosslinking The freeze-dried dAM-ECM-MA was immersed in the photocrosslinking solution and infiltrated at room temperature for 20 minutes. The excess liquid was drained and the solution was irradiated under a 405 nm UV light source (intensity: 10 mW / cm²) for 15 minutes. The unpolymerized monomers were then rinsed with PBS to obtain the RGD-functionalized cross-linked dAM-ECM.

[0030] Example 3 The purpose of this embodiment is to provide a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. Specifically, the preparation method includes the following steps: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.3% hyaluronic acid and 0.008 mol / L calcium chloride at 37°C for 2 hours. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0031] S2: dAM-ECM functionalization The washed dAM-ECM was immersed in sodium bicarbonate buffer (pH 8.5) containing 1.0% (w / v, g:mL) methacrylic anhydride (MA) and reacted at room temperature for 2 hours. After the reaction, the unreacted reagent was thoroughly rinsed with PBS and freeze-dried for later use.

[0032] S3: Preparation of photocrosslinking solution Weigh 10 g of 3000 Da polyethylene glycol diacrylate (PEG-DA) and 0.5 g of Irgacure 2959 photoinitiator and dissolve them in an appropriate amount of phosphate buffered saline (PBS, pH 7.4) (approximately 1 / 3 of the total volume). Stir magnetically at room temperature until completely dissolved. Add 2 g of acryloyl-RGD peptide and continue stirring for 12 minutes. Dose to 100 mL with PBS, filter through a 0.22 μm filter, and store at 4°C in the dark until ready for use.

[0033] S4: Photocrosslinking The freeze-dried dAM-ECM-MA was immersed in the photocrosslinking solution and infiltrated at room temperature for 10 minutes. The excess liquid was drained and the dAM-ECM was exposed to a 385 nm UV light source (intensity: 8 mW / cm²) for 10 minutes. The unpolymerized monomers were then rinsed with PBS to obtain the RGD-functionalized cross-linked dAM-ECM.

[0034] Comparative Example 1 The decellularized amniotic extracellular matrix was obtained and washed only with PBS without subsequent pretreatment, functionalization, photocrosslinking solution preparation, and photocrosslinking treatment steps, and was directly used as a sample.

[0035] Comparative Example 2 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S3: Preparation of photocrosslinking solution Preparation of photocrosslinking solution: Weigh 10 g of polyethylene glycol diacrylate (PEG-DA) with a molecular weight of 3000 Da and 0.5 g of photoinitiator Irgacure 2959, dissolve them in an appropriate amount of phosphate buffer (PBS, pH 7.4), stir magnetically at room temperature until completely dissolved, make up to 100 mL with PBS, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C in the dark until used.

[0036] Comparative Example 3 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.001 mol / L calcium chloride and immersed at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0037] Steps S2-S4 are the same as those in Example 1.

[0038] Comparative Example 4 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.002 mol / L calcium chloride at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0039] Steps S2-S4 are the same as those in Example 1.

[0040] Comparative Example 5 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.1% hyaluronic acid at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0041] Steps S2-S4 are the same as those in Example 1.

[0042] Comparative Example 6 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.2% hyaluronic acid at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0043] Steps S2-S4 are the same as those in Example 1.

[0044] Comparative Example 7 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.1% hyaluronic acid and 0.02 mol / L calcium chloride at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0045] Steps S2-S4 are the same as those in Example 1.

[0046] Comparative Example 8 This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof with reference to Example 1. Specifically, the present preparation method differs from Example 1 in that: S1: Pretreatment of dAM-ECM The decellularized amniotic membrane extracellular matrix was placed in phosphate buffered saline (PBS, pH 7.4) containing 0.1% hyaluronic acid and 0.0005 mol / L calcium chloride at 37°C for 1 hour. After pretreatment, the dAM-ECM was washed three times with PBS to remove residual reagents.

[0047] Steps S2-S4 are the same as those in Example 1.

[0048] Test Example 1 The purpose of this example is to perform performance tests on the samples prepared in each example and comparative example, including: 1.1 Mechanical strength test The test was performed using a universal material testing machine (Instron 5967). According to the test method of the universal material testing machine (Instron 5967), the specific process of the mechanical strength test is as follows: The finished products of Examples 1-3 and Comparative Examples 1-8 were cut into uniform sizes (20 mm long × 5 mm wide × 1 mm thick). The number of samples in each group was 6 (n=6). During the test, the clamp spacing was set to 10 mm, and the stretching was performed at a constant rate of 5 mm / min. The core indicators tested included tensile strength (MPa), elongation at break (%), and elastic modulus (GPa). The experimental results are shown in Table 1.

[0049] Table 1 Mechanical strength test of each group of samples

[0050] The above experimental results show that: Examples 1-3 formed stable three-dimensional network structures due to the complete HA / CaCl2 pretreatment → MA functionalization → RGD-PEG cross-linking process. The tensile strength, elongation at break, and elastic modulus were significantly better than all the comparative examples. Among them, Example 2 formed a denser cross-linked network and had the best mechanical properties due to the use of a higher concentration of methacrylic anhydride (2% MA) and longer UV irradiation (405 nm, 15 minutes).

[0051] Comparative Example 1 has the worst mechanical properties due to the lack of cross-linking. Although Comparative Example 2 obtains a certain strength through PEG-DA cross-linking, it is still significantly lower than the example due to the lack of RGD-mediated cell-matrix interaction. The experimental results show that RGD peptide enhances network stability by participating in photopolymerization.

[0052] Comparative Examples 3-8 tested pretreatment conditions separately. Comparative Examples 3-4 and Comparative Examples 5-6 demonstrated that HA and CaCl2 synergistically optimized the functionalization efficiency of the amniotic membrane decellularized matrix by exposing collagen cross-linking sites and enhancing hydrophilicity during pretreatment, and that both HA and CaCl2 were essential for the pretreatment process. Comparative Examples 7 and 8 further tested calcium chloride concentrations in the presence of HA. The results demonstrated that a calcium chloride concentration of ≥0.001 mol / L was required to effectively activate the cross-linking sites. Furthermore, excessive calcium chloride concentrations also resulted in poor cross-linking effectiveness.

[0053] 1.2 In vivo degradation test Male SD rats (200 ± 20 g) were used as the animal model and randomly divided into 11 experimental groups (each group corresponding to one test material, with n = 6 samples within each group). Pre-prepared standardized material samples (uniformly sized 5 mm × 5 mm × 1 mm) were implanted into the subcutaneous tissue of the back of each rat group.

[0054] At two pre-set time points, 4 and 12 weeks after surgery, three rats from the corresponding groups were sacrificed and the implants were removed for morphological observation and degradation rate calculation, specifically: Morphological observation: Detailed record of changes in the physical state of the implant, including the structural integrity of the material (whether it is fragmented or dissolved) and the reaction of the surrounding tissue (such as the growth of blood vessels into the material).

[0055] Weigh the removed implants, accurately weigh the residual mass of the sample after removal, and calculate the degradation rate. The calculation method is: Degradation rate = ((initial implant mass - residual mass) / initial implant mass) × 100%.

[0056] The experimental results are shown in Table 2.

[0057] Table 2 In vivo degradation test results of each group of samples

[0058] The above experimental results demonstrate that the synergistic crosslinking systems of Examples 1-3 effectively slow degradation, with degradation rates significantly lower than those of the other examples over 12 weeks. Comparative Example 1 was almost completely degraded over 12 weeks, and the degradation rates of all comparative examples exceeded 50% over 12 weeks. This type of crosslinked acellular wool matrix presents significant limitations in applications requiring long-term structural support.

[0059] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and such modifications or equivalents shall be encompassed by the claims of the present invention. Any techniques, shapes, and structures not described in detail herein are well known.

Claims

1. A cross-linked amniotic membrane acellular matrix filling material, characterized in that: It is prepared by a photocrosslinking method.

2. The method for preparing a cross-linked amniotic membrane acellular matrix filling material according to claim 1, wherein: The following steps are involved: S1: Pretreatment of decellularized amniotic membrane extracellular matrix (dAM-ECM): The decellularized amniotic membrane extracellular matrix is immersed in a phosphate buffer solution containing a pretreatment agent, and the dAM-ECM is washed after pretreatment to remove residual agents; S2: Functionalization of decellularized amniotic membrane extracellular matrix (dAM-ECM): The washed dAM-ECM is immersed in a buffer solution containing 0.5%-2% methacrylic anhydride for reaction, and then rinsed to remove unreacted reagents and freeze-dried for later use; S3: Preparation of photocrosslinking solution: preparing a photocrosslinking solution, wherein the photocrosslinking solution comprises: polyethylene glycol diacrylate, acryloyl RGD peptide, a photoinitiator, and a buffer; S4: Photocrosslinking treatment: The freeze-dried dAM-ECM-MA was immersed in a photocrosslinking solution, infiltrated, excess liquid was drained, and the material was irradiated under an ultraviolet light source and washed to remove unpolymerized monomers, thereby obtaining a cross-linked amniotic membrane decellularized matrix filling material.

3. The method for preparing a cross-linked amniotic membrane acellular matrix filling material according to claim 2, wherein: The pretreatment agent in S1 includes at least one or more of hyaluronic acid and calcium chloride.

4. The method for preparing a cross-linked amniotic membrane acellular matrix filling material according to claim 3, wherein: The pretreatment agent in S1 is a combination of hyaluronic acid and calcium chloride, wherein the mass fraction of hyaluronic acid is 0.1%-0.5%; the mass fraction of calcium chloride is 0.001 mol / L-0.01 mol / L.

5. The cross-linked amniotic membrane acellular matrix filling material and the preparation method thereof according to claim 2, characterized in that: In step S2, the concentration of methacrylic anhydride is 0.5-1.5%.

6. The cross-linked amniotic membrane acellular matrix filling material and the preparation method thereof according to claim 1, characterized in that: The photocrosslinking solution in S3 is composed of the following components: Polyethylene glycol diacrylate (molecular weight 3000Da) 5-15% w / v, acryloyl-RGD peptide 0.1-5% w / v, photoinitiator (Irgacure 2959) 0.2-1% w / v, and phosphate buffered saline (PBS, pH 7.4).

7. The method for preparing a cross-linked amniotic membrane acellular matrix filling material according to claim 2, wherein: The method for preparing the photocrosslinking solution in S3 is: Weigh polyethylene glycol diacrylate and photoinitiator in proportion, dissolve in an appropriate amount of phosphate buffer, stir, add acryloyl RGD peptide, continue stirring, dilute to the target volume with phosphate buffer, filter sterilize with filter membrane, and store in the dark for later use.

8. The method for preparing a cross-linked amniotic membrane acellular matrix filling material according to claim 2, wherein: In step S4, the penetration time is 5-30 minutes, and the intensity of the ultraviolet light source is 5-10 mW / cm 2 , the irradiation time is 5-15 minutes.

9. A cross-linked amniotic membrane acellular matrix filling material prepared by the preparation method according to any one of claims 1 to 8. 10 . Use of the cross-linked amniotic membrane acellular matrix filling material according to claim 9 in tissue repair and regenerative medicine.

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