A cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof

Abstract

The application relates to the technical field of biomaterials, in particular to a crosslinked amniotic membrane acellular matrix filling material and a preparation method thereof. The crosslinked amniotic membrane acellular matrix filling material is prepared by using a photo-crosslinking technology, and the specific preparation method is as follows: placing an acellular amniotic membrane matrix in a pretreatment agent phosphate buffer solution to perform pretreatment, then performing functionalization treatment on the acellular amniotic membrane extracellular matrix, preparing a photo-crosslinking solution, immersing the treated acellular amniotic membrane extracellular matrix in the photo-crosslinking solution, permeating at room temperature, and finally performing irradiation under a UV 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 application relates to the technical field of biological materials, in particular to a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof. BACKGROUND

[0002] The amniotic membrane acellular matrix is a biological material derived from the placental amniotic membrane and left after special treatment to remove the cellular components. Its core is to retain the extracellular matrix structure and biological active components of the amniotic membrane. The amniotic membrane acellular matrix is widely used in tissue repair and regenerative medicine due to its natural three-dimensional structure, low immunogenicity and rich biological active components such as collagen and laminin. However, the natural acellular matrix has low mechanical strength, fast in vivo degradation (usually 1-2 weeks), and is difficult to maintain long-term structural support, which limits its application in filling materials and other mechanical stable scenes.

[0003] To solve the foregoing problems, the prior art often uses cross-linking technology to improve the mechanical properties of the amniotic membrane decellularized matrix. For example, Chinese patent CN106860919B discloses a cross-linked decellularized amniotic membrane and its preparation method and application. By cross-linking treatment of the decellularized amniotic membrane with water-soluble carbodiimide, the problems of insufficient mechanical strength and biological stability are solved, more effective skin repair and cell proliferation are achieved, and the preparation efficiency and biocompatibility of the skin substitute are improved. The specific cross-linking method is as follows: water-soluble carbodiimide is added to the buffer solution (MES buffer solution) in which the soaked amniotic membrane is located, and the concentration of the carbodiimide is 0.01-0.1 mmol / mg, and the cross-linking reaction is carried out in a constant temperature incubator at 37±0.5℃ with a rotation speed of 28-32 rpm for 1-10 min; distilled water is used for cleaning, and vacuum freeze-drying is performed to obtain the cross-linked decellularized amniotic membrane. At the same time, the patent technology of document CN117159814A discloses a cross-linked amniotic membrane material and its preparation method and application. First, the fetal membrane chorion layer is separated by mechanical method, and the sponge layer and amniotic membrane layer are retained, denoted as amniotic membrane / sponge layer. The amniotic membrane / sponge layer is cleaned and dried, and then placed in a low-concentration cross-linking agent solution for cross-linking. The cross-linking agent used in the cross-linking agent solution is any one of an epoxide cross-linking agent, a dialdehyde cross-linking agent, EDC / NHS or genipin. The patent technology of document TWI324520B discloses a method for using cross-linked amniotic membrane as a bio-material, which includes the following steps: a, obtaining amniotic membrane, taking the amniotic membrane out of the frozen storage and thawing; cross-linking the amniotic membrane by using a cross-linking method, and the cross-linking agent used is EDC. Chinese patent CN120022424A discloses water-soluble carbodiimide and N-hydroxysuccinimide (EDC / NHS) as chemical cross-linking agents for cross-linking the amniotic membrane decellularized matrix. The specific method is as follows: the concentration of the cross-linking solution is 0.5-2%, the concentration of the water-soluble carbodiimide is 0.5-2%, the concentration of the N-hydroxysuccinimide is 0.125-0.5%, the mass ratio of the water-soluble carbodiimide to the N-hydroxysuccinimide is 4:1, the mass / volume ratio of the amniotic membrane to the cross-linking solution is 1:2, and the cross-linking time is 12-48 h, and the solution is replaced every 24 h. Although the above technologies solve the problem of low mechanical strength of the amniotic membrane material without cross-linking, they still have obvious defects, including but not limited to:

[0004] Firstly, the reaction conditions required for cross-linking are harsh (strong acid / high temperature), which destroys the natural biological active factors (such as growth factors, collagen structure) of the ECM;

[0005] Secondly, the cross-linking speed is slow (several hours to several days), which makes it difficult to achieve clinical instant operation;

[0006] Finally, residual toxicity or non-degradability causes inflammatory reactions.

[0007] Therefore, there is a need to improve the prior art. SUMMARY

[0008] To solve the above technical problems, the present application aims to provide a cross-linked amniotic membrane acellular matrix filler and a preparation method thereof. In the first aspect, the present application provides a cross-linked amniotic membrane acellular matrix filler prepared by a photo-crosslinking method.

[0009] In the second aspect, the present application further provides a preparation method of a cross-linked amniotic membrane acellular matrix filler, which specifically comprises the following steps:

[0010] S1: Pretreatment of decellularized amniotic membrane extracellular matrix (dAM-ECM):

[0011] The decellularized amniotic membrane extracellular matrix is placed in a pretreatment agent phosphate buffer solution, soaked (for example, soaked at 37℃ for 1-3 hours), and the dAM-ECM is washed after pretreatment to remove residual reagents;

[0012] S2: Functionalization treatment of decellularized amniotic membrane extracellular matrix (dAM-ECM):

[0013] The washed dAM-ECM is soaked in a buffer solution (for example, sodium bicarbonate buffer solution) containing 0.5%-2% methacrylic anhydride, reacted (for example, reacted at room temperature for 2 hours), and after reaction, the unreacted reagents are removed by thorough washing with PBS, and freeze-dried for standby;

[0014] S3: Preparation of photo-crosslinking solution:

[0015] The photo-crosslinking solution is prepared, and the composition of the photo-crosslinking solution is: polyethylene glycol diacrylate, acrylated RGD peptide, photoinitiator, and buffer solution;

[0016] S4: Photo-crosslinking treatment:

[0017] The freeze-dried dAM-ECM-MA is soaked in the photo-crosslinking solution, penetrated (for example, penetrated at room temperature for 5-20 minutes), drained of excess liquid, irradiated with an ultraviolet light source (for example, irradiated under a 365-405nm ultraviolet light source for 5-15 minutes), and washed (for example, washed with PBS) to remove un-polymerized monomers, thereby obtaining a cross-linked amniotic membrane acellular matrix filler.

[0018] Preferably, the pretreatment agent in S1 comprises at least any one or more of hyaluronic acid and calcium chloride.

[0019] 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%, and the mass fraction of calcium chloride is 0.001mol / L-0.01mol / L.

[0020] Preferably, in step S2, the concentration of the methacrylic anhydride is 0.5-1.5%, preferably 1.0%.

[0021] Preferably, the photo-crosslinking solution in S3 is composed of the following components:

[0022] Polyethylene glycol diacrylate (molecular weight 3000 Da) 5-15 % w / v, acrylated RGD peptide 0.1-5 % w / v, photoinitiator (Irgacure 2959) 0.2-1 % w / v, and phosphate buffer (PBS, pH 7.4).

[0023] Further preferably, the photo-crosslinking solution in S3 is composed of the following components:

[0024] Polyethylene glycol diacrylate (molecular weight 3000 Da) 10 % w / v, acrylated RGD peptide 0.2-2 % w / v, photoinitiator (Irgacure 2959) 0.5 % w / v, and phosphate buffer (PBS, pH 7.4).

[0025] Further preferably, the preparation method of the photo-crosslinking solution in S3 is:

[0026] Polyethylene glycol diacrylate, photoinitiator, and acrylated RGD peptide are weighed according to the proportion, dissolved in an appropriate amount of phosphate buffer, stirred at room temperature until completely dissolved, acrylated RGD peptide is added, and stirring is continued for 10-15 minutes, the target volume is set with phosphate buffer, sterilized by filtering through a 0.22 μm filter membrane, and stored at 4°C in the dark for standby.

[0027] Preferably, in step S4, the room temperature penetration time is 5-30 minutes, the ultraviolet light source intensity is 5-10 mW / cm 2 , and the irradiation time is 5-15 minutes.

[0028] In a third aspect, the application also provides a cross-linked amniotic membrane acellular matrix filling material prepared by the above preparation method and its application in tissue repair and regenerative medicine.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The application provides a crosslinked amniotic membrane acellular matrix filling material and a preparation method thereof.

[0031] The method provided by the application has mild crosslinking conditions, realizes efficient crosslinking, avoids the damage of a high-temperature / strong-acid environment of a traditional chemical crosslinking agent to natural biological active factors (such as growth factors and collagen structures) of amniotic membrane, and the introduction of RGD peptides enhances the cell-matrix interaction, forms a dense three-dimensional network, significantly improves the mechanical strength of the material, and significantly delays the degradation rate in the body, so that the material is suitable for soft tissue repair scenes that require long-term mechanical support. The process is simple and controllable, has good repeatability, has the potential for large-scale production, and provides a new filling material with high mechanical strength, controllable degradability and excellent biocompatibility for tissue engineering. DETAILED DESCRIPTION

[0032] The following examples are only used to more clearly illustrate the technical solutions of the application, and therefore only serve as examples, and cannot be used to limit the protection scope of the application.

[0033] Any equivalent modifications and replacements of the examples described below made by those skilled in the art are also within the scope of the application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the application should be covered within the scope of the application. In order to better illustrate the application, numerous specific details are given in the following specific embodiments.

[0034] Those skilled in the art should understand that the application can also be implemented without certain specific details. In some embodiments, methods, means, apparatuses and steps familiar to those skilled in the art are not described in detail, in order to highlight the main idea of the application. Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as 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 application should be understood to include the systematic errors inevitable in industrial production.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents or instruments used are commercially available reagents and materials, and the specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. Meanwhile, the source of the raw materials used in the present application is not limited, and the raw materials used in the present application are commercially available products in the technical field unless otherwise specified.

[0036] It should be particularly noted that the decellularized amniotic membrane extracellular matrix used in the following examples is prepared according to the method disclosed in the reference Song Yongzhou, Cui Huixian, Wang Zhensheng, et al. Preparation of amniotic membrane decellularized matrix and its biocompatibility [J]. Chinese tissue engineering research and clinical rehabilitation, 2008, (01): 51-55. (see the method of the literature for details- preparation of amniotic membrane decellularized matrix).

[0037] Example 1

[0038] The purpose of the present embodiment is to provide a cross-linked amniotic membrane decellularized matrix filling material and a preparation method thereof, and specifically, the preparation method comprises the following steps:

[0039] S1: Pretreatment of dAM-ECM

[0040] The decellularized amniotic membrane extracellular matrix is placed in a phosphate buffer solution (PBS, pH 7.4) containing 0.1% hyaluronic acid and 0.001 mol / L calcium chloride, and soaked at 37°C for 1 hour. After pretreatment, the dAM-ECM is washed with PBS for 3 times to remove residual reagents.

[0041] S2: dAM-ECM functionalization treatment:

[0042] The washed dAM-ECM is soaked in a 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 reaction, the dAM-ECM is washed with PBS for 3 times to completely rinse and remove unreacted reagents, and then freeze-dried for standby.

[0043] S3: Preparation of photo-crosslinking solution

[0044] 10 g of polyethylene glycol diacrylate (PEG-DA) with a molecular weight of 3000 Da and 0.5 g of photoinitiator Irgacure 2959 are weighed, dissolved in an appropriate amount of phosphate buffer (PBS, pH 7.4) (about 1 / 2 of the total volume), and magnetically stirred at room temperature until completely dissolved. 1 g of acrylated RGD peptide is added, and stirring is continued for 10 minutes. The volume is made up to 100 mL with PBS, sterilized by filtering through a 0.22 μm filter membrane, and stored at 4°C in the dark for standby.

[0045] S4: Photo-crosslinking treatment

[0046] The freeze-dried dAM-ECM-MA was soaked in the photo-crosslinking solution, permeated at room temperature for 5 minutes, drained the excess liquid, irradiated under a 365 nm ultraviolet light source (intensity: 5 mW / cm²) for 5 minutes, washed with PBS to remove the un-polymerized monomers, and obtained the RGD functionalized crosslinked dAM-ECM.

[0047] Example 2

[0048] The purpose of the present embodiment is to provide a crosslinked amniotic membrane decellularized matrix filling material and a preparation method thereof, and specifically, the preparation method comprises the following steps:

[0049] S1: Pretreatment of dAM-ECM

[0050] The decellularized amniotic membrane extracellular matrix was placed in a phosphate buffer solution (PBS, pH 7.4) containing 0.5% by mass of hyaluronic acid and 0.01 mol / L of calcium chloride, soaked at 37°C for 3 hours, and after pretreatment, the dAM-ECM was washed with PBS for 3 times to remove residual reagents.

[0051] S2: Functionalization treatment of dAM-ECM

[0052] The washed dAM-ECM was soaked in a sodium bicarbonate buffer (pH 8.5) containing 2% (w / v, g:mL) methacrylic anhydride (MA), reacted at room temperature for 2 hours, and after the reaction, the unreacted reagents were removed by washing with PBS, and then freeze-dried for standby.

[0053] S3: Preparation of photo-crosslinking solution

[0054] 10 g of polyethylene glycol diacrylate (PEG-DA) with a molecular weight of 3000 Da and 0.5 g of Irgacure 2959 photoinitiator were weighed, dissolved in an appropriate amount of phosphate buffer (about 1 / 3 of the total volume) (PBS, pH 7.4), and magnetically stirred at room temperature until completely dissolved. 0.5 g of acrylated RGD peptide was added, and stirring was continued for 15 minutes. The volume was made up to 100 mL with phosphate buffer, sterilized by filtering through a 0.22 μm filter, and stored at 4°C in the dark for standby.

[0055] S3: Photo-crosslinking treatment

[0056] The freeze-dried dAM-ECM-MA was soaked in the photo-crosslinking solution, permeated at room temperature for 20 minutes, drained the excess liquid, irradiated under a 405 nm ultraviolet light source (intensity: 10 mW / cm²) for 15 minutes, washed with PBS to remove the un-polymerized monomers, and obtained the RGD functionalized crosslinked dAM-ECM.

[0057] Example 3

[0058] The purpose of the present embodiment is to provide a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof, specifically, the preparation method comprises the following steps:

[0059] S1: Pretreatment of dAM-ECM

[0060] The acellular amniotic membrane extracellular matrix is placed in a phosphate buffer solution (PBS, pH 7.4) containing 0.3% by mass of hyaluronic acid and 0.008 mol / L of calcium chloride, soaked at 37°C for 2 hours, and after pretreatment, the dAM-ECM is washed with PBS for 3 times to remove residual reagents.

[0061] S2: Functionalization treatment of dAM-ECM

[0062] The washed dAM-ECM is soaked in a 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 reagents are removed by thorough washing with PBS, and the freeze-dried product is ready for use.

[0063] S3: Preparation of photo-crosslinking solution

[0064] 10 g of polyethylene glycol diacrylate (PEG-DA) with a molecular weight of 3000 Da and 0.5 g of photoinitiator Irgacure 2959 are weighed and dissolved in an appropriate amount of phosphate buffer (about 1 / 3 of the total volume) (PBS, pH 7.4) at room temperature with magnetic stirring until completely dissolved. 2 g of acrylated RGD peptide is added, and stirring is continued for 12 minutes. The volume is made up to 100 mL with PBS, sterilized by filtration through a 0.22 μm filter, and stored at 4°C in the dark for standby.

[0065] S4: Photo-crosslinking treatment

[0066] The freeze-dried dAM-ECM-MA is soaked in the photo-crosslinking solution, permeated at room temperature for 10 minutes, drained of excess liquid, and irradiated under a 385 nm ultraviolet light source (intensity: 8 mW / cm²) for 10 minutes. The un-polymerized monomers are removed by washing with PBS, and the RGD-functionalized cross-linked dAM-ECM is obtained.

[0067] Comparative Example 1

[0068] Take the acellular amniotic membrane extracellular matrix, wash it only with PBS, and do not perform the subsequent pretreatment, functionalization treatment, photo-crosslinking solution preparation, and photo-crosslinking treatment steps, directly as a sample for standby.

[0069] Comparative Example 2

[0070] This comparative example provides a cross-linked amniotic membrane acellular matrix filling material and a preparation method thereof according to Example 1, specifically, the preparation method of the present example is different from Example 1 in that:

[0071] S3: Preparation of photocrosslinking solution

[0072] Preparation of photocrosslinking solution: 10 g of polyethylene glycol diacrylate (PEG-DA) with a molecular weight of 3000 Da and 0.5 g of photoinitiator Irgacure 2959 were weighed out, dissolved in an appropriate amount of phosphate buffer solution (PBS, pH 7.4), magnetically stirred at room temperature until completely dissolved, diluted to 100 mL with PBS, filtered through a 0.22 μm filter to remove bacteria, and stored at 4°C in the dark.

[0073] Comparative Example 3

[0074] This comparative example provides a crosslinked amniotic membrane decellularized matrix filler material and a preparation method thereof according to Example 1. Specifically, the difference between the preparation method and Example 1 is that:

[0075] S1: Pretreatment of dAM-ECM

[0076] The decellularized amniotic membrane extracellular matrix was placed in a phosphate buffer solution (PBS, pH 7.4) containing calcium chloride with a concentration of 0.001 mol / L, soaked at 37°C for 1 hour, and the dAM-ECM was washed with PBS 3 times after pretreatment to remove residual reagents.

[0077] Steps S2-S4 are the same as in Example 1.

[0078] Comparative Example 4

[0079] This comparative example provides a crosslinked amniotic membrane decellularized matrix filler material and a preparation method thereof according to Example 1. Specifically, the difference between the preparation method and Example 1 is that:

[0080] S1: Pretreatment of dAM-ECM

[0081] The decellularized amniotic membrane extracellular matrix was placed in a phosphate buffer solution (PBS, pH 7.4) containing calcium chloride with a concentration of 0.002 mol / L, soaked at 37°C for 1 hour, and the dAM-ECM was washed with PBS 3 times after pretreatment to remove residual reagents.

[0082] Steps S2-S4 are the same as in Example 1.

[0083] Comparative Example 5

[0084] This comparative example provides a crosslinked amniotic membrane decellularized matrix filler material and a preparation method thereof according to Example 1. Specifically, the difference between the preparation method and Example 1 is that:

[0085] S1: Pretreatment of dAM-ECM

[0086] The dAM-ECM was pre-treated by immersing in phosphate buffer solution (PBS, pH 7.4) containing 0.1% (w / v) of hyaluronic acid for 1 hour at 37°C. After pre-treatment, the dAM-ECM was washed with PBS for 3 times to remove residual reagents.

[0087] Steps S2-S4 were the same as in Example 1.

[0088] Comparative Example 6

[0089] This comparative example provides a cross-linked dAM-ECM filler material and a method for preparing the same according to Example 1, specifically, the difference between the present preparation method and Example 1 is that:

[0090] S1 : Pretreatment of dAM-ECM

[0091] The dAM-ECM was pre-treated by immersing in phosphate buffer solution (PBS, pH 7.4) containing 0.2% (w / v) of hyaluronic acid for 1 hour at 37°C. After pre-treatment, the dAM-ECM was washed with PBS for 3 times to remove residual reagents.

[0092] Steps S2-S4 were the same as in Example 1.

[0093] Comparative Example 7

[0094] This comparative example provides a cross-linked dAM-ECM filler material and a method for preparing the same according to Example 1, specifically, the difference between the present preparation method and Example 1 is that:

[0095] S1 : Pretreatment of dAM-ECM

[0096] The dAM-ECM was pre-treated by immersing in phosphate buffer solution (PBS, pH 7.4) containing 0.1% (w / v) of hyaluronic acid and 0.02 mol / L of calcium chloride for 1 hour at 37°C. After pre-treatment, the dAM-ECM was washed with PBS for 3 times to remove residual reagents.

[0097] Steps S2-S4 were the same as in Example 1.

[0098] Comparative Example 8

[0099] This comparative example provides a cross-linked dAM-ECM filler material and a method for preparing the same according to Example 1, specifically, the difference between the present preparation method and Example 1 is that:

[0100] S1 : Pretreatment of dAM-ECM

[0101] The decellularized amniotic membrane extracellular matrix was immersed in a phosphate buffer solution (PBS, pH 7.4) containing 0.1% by mass of hyaluronic acid and 0.0005 mol / L of calcium chloride at 37°C for 1 hour, and after the pretreatment, the dAM-ECM was washed with PBS three times to remove residual reagents.

[0102] The S2-S4 steps were the same as in Example 1.

[0103] Test Example 1

[0104] The purpose of this example is to test the performance of the samples prepared in each example and comparative example, including:

[0105] 1.1 Mechanical strength test

[0106] A universal material testing machine (Instron 5967) was used for testing, and according to the test method of the universal material testing machine (Instron 5967), the specific process of the mechanical strength test was as follows:

[0107] The finished products of Examples 1-3 and Comparative Examples 1-8 were cut into uniform sizes (20 mm long x 5 mm wide x 1 mm thick), and the number of samples in each group was 6 (n=6). The clamp distance was set to 10 mm during testing, and the stretching was performed at a constant rate of 5 mm / min. The core indicators detected included tensile strength (MPa), elongation at break (%), and elastic modulus (GPa). The experimental results are shown in Table 1.

[0108] Table 1 Mechanical strength test of each group of samples

[0109]

[0110] The above experimental results show that:

[0111] Examples 1-3 formed a stable three-dimensional network structure due to the complete HA / CaCl2 pretreatment, MA functionalization, and RGD-PEG crosslinking process, and the tensile strength, elongation at break, and elastic modulus were significantly better than all the comparative examples. Among them, Example 2 used a higher concentration of methacrylic anhydride (2% MA) and longer ultraviolet irradiation (405 nm, 15 minutes) to form a denser crosslinked network, and the mechanical properties were the best.

[0112] Comparative Example 1 had the worst mechanical properties due to the lack of crosslinking, and Comparative Example 2 obtained a certain strength through PEG-DA crosslinking, but due to the lack of RGD-mediated cell-matrix interaction, it was still significantly lower than the examples. This experimental result shows that RGD peptides enhance the stability of the network by participating in photopolymerization.

[0113] Comparative Examples 3-8 were tested for the conditions of pretreatment, wherein Comparative Examples 3-4 and Comparative Examples 5-6 proved that both HA and CaCl2 synergistically optimized the functionalization efficiency of the amniotic membrane acellular matrix by exposing the collagen crosslinking sites and enhancing the hydrophilicity during the pretreatment process, and HA and CaCl2 were indispensable during the pretreatment process. Comparative Example 7 and Comparative Example 8 further tested the calcium chloride concentration in the presence of HA, and the experimental results showed that the calcium chloride concentration needed to be ≥0.001 mol / L to effectively activate the crosslinking sites, and at the same time, too high a concentration of calcium chloride would also lead to deterioration of the crosslinking effect.

[0114] 1.2 In vivo degradation test

[0115] SD rats were used as animal models (male, 200 ± 20 g), which were randomly divided into 11 experimental groups (each group corresponds to one test material, and the sample number n = 6 in each group). The pre-prepared standardized material samples (uniform size of 5 mm × 5 mm × 1 mm) were implanted into the subcutaneous tissue of the back of each group of rats.

[0116] At two preset time points of 4 weeks and 12 weeks after the operation, 3 rats corresponding to each group were sacrificed and the implants were removed, and morphological observation and degradation rate calculation were performed, specifically:

[0117] Morphological observation: detailed record of the physical state change of the implant, including material structural integrity (whether fragmentation or dissolution) and surrounding tissue reaction (such as neovascularization and growth into the material inside).

[0118] The removed implants were weighed, the residual mass of the sample after removal was accurately weighed, and the degradation rate was calculated, and the calculation method was:

[0119] Degradation rate = ((initial implant mass - residual mass) / initial implant mass) × 100%.

[0120] The experimental results are shown in Table 2.

[0121] Table 2 In vivo degradation test results of samples in each group

[0122]

[0123] The above experimental results show that the synergistic crosslinking system of Examples 1-3 effectively delays degradation, and the 12-week degradation rate is significantly lower than that of the other example groups. Among them, Comparative Example 1 is almost completely degraded at 12 weeks, and the degradation rate of each comparative example within 12 weeks is more than 50%, and such crosslinked wool acellular matrix has significant defects in applications requiring long-term structural support.

[0124] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are all known technologies.

Claims

1. A cross-linked amniotic membrane decellularized matrix filling material, characterized in that, It is prepared using a photocrosslinking method and includes the following steps: S1: Pretreatment of decellularized amniotic extracellular matrix dAM-ECM: The decellularized amniotic extracellular matrix was immersed in a phosphate buffer solution containing pretreatment agent, and then the dAM-ECM was washed to remove residual reagents. S2: Functionalization of decellularized amniotic extracellular matrix dAM-ECM: The cleaned dAM-ECM was immersed in a buffer solution containing 0.5%-2% w / v methacrylic anhydride and reacted. After the reaction, the unreacted reagents were rinsed off and the product was freeze-dried for later use. S3: Preparation of photocrosslinking solution: A photocrosslinking solution is prepared, the photocrosslinking solution comprising: polyethylene glycol diacrylate, acrylamide RGD peptide, photoinitiator, and buffer solution; S4: Photocrosslinking treatment: The lyophilized dAM-ECM-MA was immersed in a photocrosslinking solution, allowed to permeate, and the excess liquid was drained. It was then irradiated under a UV light source and rinsed to remove unpolymerized monomers, resulting in a crosslinked amniotic membrane decellularized matrix filling material. 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% and the mass fraction of calcium chloride is 0.001mol / L-0.01mol / L.

2. The cross-linked amniotic membrane decellularized matrix filling material as described in claim 1, characterized in that: In step S2, the concentration of the methacrylic anhydride is 0.5-1.5% w / v.

3. The cross-linked amniotic membrane decellularized matrix filling material as described in claim 1, characterized in that, The photocrosslinking solution in S3 consists of the following components: The mixture contains 5-15% w / v polyethylene glycol diacrylate, 0.1-5% w / v acrylated RGD peptide, 0.2-1% w / v photoinitiator Irgacure2959, and PBS phosphate buffer, wherein the polyethylene glycol diacrylate has a molecular weight of 3000 Da and the PBS phosphate buffer has a pH of 7.

4.

4. The cross-linked amniotic membrane decellularized matrix filling material as described in claim 1, characterized in that, The method for preparing the photocrosslinking solution in S3 is as follows: Weigh out polyethylene glycol diacrylate and photoinitiator according to the specified proportions, dissolve them in an appropriate amount of phosphate buffer, stir, add acrylated RGD peptide, continue stirring, and bring the volume up to the target volume with phosphate buffer. Filter through a filter membrane for sterilization and store in the dark for later use.

5. The cross-linked amniotic membrane decellularized matrix filling material as described in claim 1, characterized in that, In step S4, the permeation time is 5-30 minutes, and the intensity of the ultraviolet light source is 5-10 mW / cm². 2 The irradiation time is 5-15 minutes.

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