Radial gradient collagen composite sponge scaffold for ligament injury repair and preparation method

Abstract

The invention relates to a radial gradient collagen composite sponge scaffold for repairing ligament injury. The radial gradient collagen composite sponge scaffold comprises the following raw materials: an initial collagen composite sponge scaffold, a cross-linking solution and a bioactive solution, the initial collagen composite sponge scaffold is formed by freezing a collagen solution; the collagen solution comprises type I collagen powder with the concentration of 8 to 40 mg / mL, chitosan with the concentration of 1 to 3 mg / mL, sodium hyaluronate with the concentration of 1 to 5 mg / mL and sulfated silk fibroin with the concentration of 1 to 5 mg / mL; the cross-linking solution is a genipin solution; the concentration of the genipin is 45 to 55 mg / mL; the bioactive solution is an eucommia polysaccharide solution; the concentration of the eucommia polysaccharide is 9-11 mg / mL. The sponge scaffold has the advantages of high modulus, appropriate degradation rate and good biological activity.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical materials, in particular to a radial gradient collagen composite sponge scaffold for ligament injury repair and a preparation method thereof. BACKGROUND

[0002] The treatment of anterior cruciate ligament (ACL) injury aims to restore the stability of the knee joint, reduce secondary injury, and help patients return to sports function. For patients with complete rupture of the ACL and active patients, surgical reconstruction is the standard treatment. At present, the grafts used in anterior cruciate ligament reconstruction (ACLR) mainly include autologous tendon, allogeneic tendon and artificial synthetic materials.

[0003] However, these grafts have their own limitations. Autografts can cause complications in the donor area, allografts have the risk of disease transmission and immune rejection, and synthetic materials perform poorly in biological integration. More importantly, regardless of the type of graft used, the quality of the "interfacial healing" between the ligament and the bone tunnel is always the key bottleneck that determines the long-term success or failure of the surgery. The "ligament-bone" interface of the natural ACL is a complex structure transition zone that can achieve smooth transmission of stress; while the interface scar tissue after reconstruction often has poor mechanical properties, leading to an increased risk of bone tunnel expansion and graft relaxation.

[0004] Therefore, the research focus in the field of ACL reconstruction has shifted from simply pursuing the initial strength of the graft to promoting the biological integration and functional healing of the graft in the bone tunnel. In this context, collagen sponges with excellent biocompatibility and three-dimensional porous structure show great application potential.

[0005] Collagen sponges themselves as bioactive carriers or functional enhancement materials for grafts have unique advantages: they can simulate the extracellular matrix microenvironment, providing an ideal scaffold for the migration, proliferation and differentiation of seed cells (such as fibroblasts and mesenchymal stem cells); their porous structure is conducive to the transport of nutrients and the discharge of metabolic waste; in addition, they can also act as a slow-release carrier for growth factors (such as BMP and TGF-β), precisely promoting the regeneration of the fibrocartilage transition zone at the ligament-bone interface by constructing a bioactive microenvironment, thereby accelerating and enhancing the quality of interfacial healing.

[0006] Collagen sponge as a biological scaffold material is widely studied, but its homogeneous structure cannot simulate the gradient characteristics of the natural interface. In the prior art, although there are studies on regulating performance by changing the crosslinking degree, most of them are simply stacked after preparing multiple homogeneous scaffolds with different crosslinking degrees, or realizing the gradient through complex equipment, which is cumbersome and difficult to meet the different needs of mechanical support, moderate degradation rate and cell directional migration in the body. Therefore, there is an urgent need in the art for a scaffold preparation method that can be integrally formed, structurally stable, and accurately simulate the natural interface gradient, to realize the radial regulation of mechanical properties to biological functions through a simple process, so as to truly promote the high-quality integration of the ligament-bone interface. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to provide a radial gradient collagen composite sponge scaffold for ligament injury repair, which can meet the different needs of initial mechanical support, moderate degradation rate and cell directional migration.

[0008] In a first aspect of the present application, a radial gradient collagen composite sponge scaffold for ligament injury repair is provided, comprising the following raw materials: an initial collagen composite sponge scaffold, a crosslinking solution and a bioactive solution; the initial collagen composite sponge scaffold is formed by freezing a collagen solution; the collagen solution comprises type I collagen powder with a concentration of 8-40 mg / mL, chitosan with a concentration of 1-3 mg / mL, sodium hyaluronate with a concentration of 1-5 mg / mL and sulfated silk fibroin with a concentration of 1-5 mg / mL; the crosslinking solution is a genipin solution; the concentration of genipin is 45-55 mg / mL; the bioactive solution is a eucommia ulmoides polysaccharide solution; the concentration of eucommia ulmoides polysaccharide is 9-11 mg / mL.

[0009] By adopting the above technical solution, the present application provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which has high modulus, appropriate degradation rate and good bioactivity. This may be because type I collagen provides the scaffold with basic biocompatibility and cell adhesion sites, chitosan has good biocompatibility and antibacterial properties, which can enhance the mechanical properties and stability of the scaffold, sodium hyaluronate provides water retention and lubricity, promotes cell migration and proliferation, sulfated silk fibroin enhances the toughness and stability of the scaffold, genipin enhances the stability of the scaffold through crosslinking, and eucommia ulmoides polysaccharide promotes cell adhesion and proliferation through bioactivity.

[0010] Optionally, the concentration of the type I collagen powder is 18-30 mg / mL.

[0011] Optionally, in the collagen solution, the mass ratio of sodium hyaluronate to sulfated silk fibroin is (0.2-5):1.

[0012] By adopting the above technical solution and controlling the amount of sodium hyaluronate and sulfated silk fibroin according to the above mass ratio, when the mass ratio of sodium hyaluronate and sulfated silk fibroin is (0.2-5):1, the sponge scaffold can be guaranteed to have good toughness and stability and can meet the migration needs of cells.

[0013] Optionally, the preparation of the sulfated silk fibroin includes the following steps: a1: Add 50-100 times the weight of degumming agent to silkworm cocoon shells or waste silk, refine for 0.5-1.5 hours at a temperature of 98-99℃, filter, repeat refining and filtering 1-3 times, wash, soak, and dry to obtain fibroin; a2: Take the silk fibroin prepared in step a1 and add it to sulfuric acid solution. Hydrolyze it for 2-5 hours at 20-30℃ with stirring to obtain hydrolysate. Pour the hydrolysate into water, filter, wash with water until neutral, and dry to obtain silk fibroin microcrystalline fibers. a3: Under ice bath and stirring conditions, dichloromethane was added, and chlorosulfonic acid was added to dichloromethane under inert gas protection. The reaction temperature was adjusted to below 15°C, and the silk fibroin microcrystalline fiber prepared in step a2 was added. The reaction was carried out for 15-60 minutes. After the reaction was completed, the reaction solution was poured into sodium acetate-ethanol solution, filtered, and the precipitate was washed to obtain the product. a4: Dissolve the product obtained in step a3 in water, adjust the pH to 8-8.5, dialyze for 24-48 hours, and then freeze-dry the dialysate to obtain sulfated silk fibroin.

[0014] Optionally, in step a1, the degumming agent is a 0.4 wt% Na2CO3 solution.

[0015] Optionally, in step a2, the volume (mL) / mass (g) ratio of sulfuric acid solution to silk fibroin is (10-20):1.

[0016] Optionally, in the collagen solution, the mass ratio of type I collagen powder to chitosan is (6-15):1.

[0017] By adopting the above technical solution and adjusting the amount of type I collagen powder and chitosan according to the above mass ratio, when the mass ratio of type I collagen powder to chitosan is (6-15):1, the sponge scaffold can be guaranteed to have good biocompatibility and antibacterial properties.

[0018] Optionally, the preparation method of the initial collagen composite sponge scaffold includes the following steps: Type I collagen powder and chitosan were dissolved in acetic acid solution and stirred. Sodium hyaluronate and sulfated silk fibroin were added and stirred to obtain a collagen solution. The collagen solution was then injected into a mold, pre-frozen, and freeze-dried to obtain the initial collagen composite sponge scaffold.

[0019] Optionally, the concentration of acetic acid is 0.05-0.5M.

[0020] Optionally, the mass ratio of genipin to eucommia polysaccharide is (0.81-1.22):1.

[0021] By adopting the above technical solution and adjusting the dosage of genipin and eucommia polysaccharide according to the above mass ratio, when the mass ratio of genipin to eucommia polysaccharide is (0.81-1.22):1, the sponge scaffold can be guaranteed to have good stability and can promote cell adhesion and proliferation.

[0022] Optionally, the preparation of the Eucommia ulmoides polysaccharide includes the following steps: b1: Dry the Eucommia ulmoides, pulverize it, extract it by reflux with ethanol, filter it, collect the filter residue, and obtain the Eucommia ulmoides residue; b2: Add the Eucommia ulmoides residue obtained in step b1 to water, heat in a water bath for 2-4 hours, cool, filter, collect the supernatant to obtain the preliminary extract of Eucommia ulmoides polysaccharides. b3: Concentrate the preliminary extract of Eucommia ulmoides polysaccharide prepared in step b2, add ethanol solution, let stand, centrifuge, collect the precipitate, and obtain crude Eucommia ulmoides polysaccharide. b4: Dissolve the crude polysaccharide of Eucommia ulmoides prepared in step b3 into crude polysaccharide solution a, mix the crude polysaccharide solution a with Sevag reagent, shake, let stand, centrifuge, collect the upper aqueous phase solution, and obtain crude polysaccharide solution b. b5: Mix the crude polysaccharide solution b obtained in step b4 with macroporous resin, shake, and filter to obtain the polysaccharide solution of Eucommia ulmoides. b6: Freeze-dry the Eucommia ulmoides polysaccharide solution prepared in step b5 to obtain Eucommia ulmoides polysaccharide.

[0023] Optionally, in step b4, the Sevag reagent is chloroform and n-butanol in a volume ratio of 4:1.

[0024] Optionally, the volume ratio of the crosslinking solution to the collagen solution is (1-1.5):25; the volume ratio of the bioactive solution to the collagen solution is (1-1.5):5.

[0025] In a second aspect, this application provides a method for preparing a radial gradient collagen composite sponge scaffold for ligament injury repair as described in the first aspect of this application, comprising the following steps: S1: Partial wetting: Spray the cross-linked solution onto the outer surface of the initial collagen composite sponge scaffold, react for 12-36 hours, wash, freeze dry, and obtain a highly stable gradient collagen composite sponge scaffold. S2: Bioactive modification: The highly stable gradient collagen composite sponge scaffold prepared in step S1 is immersed in the bioactive solution, pumped to 0.5 kPa or below, maintained for 5-15 min, depressurized, left to stand at 3-5℃ for 8-16 h, freeze-dried, and sterilized to obtain a radial gradient collagen composite sponge scaffold for ligament injury repair.

[0026] In a third aspect of this application, this application provides the application of the radial gradient collagen composite sponge scaffold for ligament injury repair described in the first aspect of this application in the preparation of a functionalized load scaffold.

[0027] In summary, the present invention has at least one of the following beneficial technical effects: 1. This application provides a radially gradient collagen composite sponge scaffold for ligament injury repair. This sponge scaffold possesses high modulus, a suitable degradation rate, and good bioactivity. Type I collagen provides the basic biocompatibility and cell attachment sites for the scaffold. Chitosan has good biocompatibility and antibacterial properties, which can enhance the mechanical properties and stability of the scaffold. Sodium hyaluronate provides water retention and lubrication, promoting cell migration and proliferation. Sulfated silk fibroin enhances the toughness and stability of the scaffold. Genipin enhances the stability of the scaffold through cross-linking. Eucommia ulmoides polysaccharide promotes cell adhesion and proliferation through bioactivity.

[0028] 2. This application provides a method for preparing a radial gradient collagen composite sponge scaffold for ligament injury repair. The method involves immersing the scaffold in a genipin solution, which induces diffusion and cross-linking reactions. Utilizing the high permeability and cross-linking efficiency of genipin in ethanol, a high cross-linking density region is formed on the outside of the scaffold, creating a gradient towards the inside. By immersing the scaffold in a Eucommia ulmoides polysaccharide solution, the Eucommia ulmoides polysaccharide is stably loaded onto the scaffold network through physical adsorption and possible intermolecular forces, promoting cell adhesion and proliferation. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] Type I collagen powder was purchased from Guangzhou Chuang'er Biotechnology Co., Ltd. (YC0012), silk fibroin from Hubei Hongxin Ruiyu Fine Chemical Co., Ltd., sodium alginate from Zhengzhou Yuhe Food Additives Co., Ltd., and D101 macroporous adsorption resin from Tianjin Yunkai Resin Technology Co., Ltd.

[0031] Preparation Example 1 Preparation of sulfated silk fibroin a1: Add 75 times the mass of 0.4wt% Na2CO3 solution to waste silk, refine at 99℃ for 1 hour, filter, repeat the refining and filtration process twice, wash thoroughly with distilled water, then soak in 0.5mg / mL acetic acid aqueous solution for 5 minutes, filter, and dry in an oven to obtain silk fibroin. a2: Take 10g of the silk fibroin prepared in step a1 and dissolve it in 150mL of 45wt% sulfuric acid solution. Hydrolyze it at 25℃ under stirring for 3.5h to obtain hydrolysate. Then slowly pour the hydrolysate into 90mL of distilled water, filter, wash with water until neutral, and dry to obtain silk fibroin microcrystalline fibers. a3: Under ice bath and stirring conditions, dichloromethane was added to a three-necked flask. Chlorosulfonic acid was added to the dichloromethane under nitrogen protection. The molar ratio of dichloromethane to chlorosulfonic acid was 1:1, and the total volume of dichloromethane and chlorosulfonic acid was 20 mL. The reaction temperature was adjusted to below 15 °C. Then, 1 g of the silk fibroin microcrystalline fiber prepared in step a2 was added. The reaction was allowed to proceed for 30 min. After the reaction was completed, the reaction solution was poured into 4.5 times the volume of sodium acetate ethanol solution. The concentration of sodium acetate in the sodium acetate ethanol solution was 3 mg / mL. The solution was filtered, and the precipitate was washed with anhydrous ethanol to obtain the product. a4: Dissolve the product in 5 mL of distilled water and adjust the pH of the product to 8.3 with 2 mol / L sodium hydroxide. Finally, dialyze the product in distilled water for 36 h and freeze-dry the dialysate to obtain sulfated silk fibroin.

[0032] Preparation Example 2 Preparation of Eucommia ulmoides polysaccharides b1: After drying and pulverizing 500g of Eucommia ulmoides, it was extracted three times by reflux with 3L of 95% ethanol at 75℃, with each extraction time being 1h. After filtration, the residue was collected to obtain Eucommia ulmoides residue. b2: Add the Eucommia ulmoides residue obtained in step b1 to 10L of distilled water, heat in a water bath at 80℃ for 3 hours, cool to room temperature, filter, collect the supernatant, repeat the process of adding the Eucommia ulmoides residue to distilled water, heating, cooling, filtering, and collecting the supernatant, and combine the supernatants obtained from the three extractions to obtain the preliminary extract of Eucommia ulmoides polysaccharides. b3: The preliminary extract of Eucommia ulmoides polysaccharide prepared in step b2 was concentrated to 20% of its original volume using a rotary evaporator. Four times the volume of 60% ethanol was added, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation to obtain crude Eucommia ulmoides polysaccharide. b4: Dissolve the crude polysaccharide obtained in step b3 in distilled water to form a crude polysaccharide solution a with a concentration of 1 mg / mL. Mix the crude polysaccharide solution a with Sevag reagent (chloroform: n-butanol = 4:1 (v / v)) at a ratio of 5:1, shake for 3 min, let stand for 15 min, centrifuge at 4000 rpm for 10 min, and collect the upper aqueous phase solution. Repeat the mixing, shaking, standing, centrifugation, and collection of the upper aqueous phase solution of the crude polysaccharide solution a and Sevag reagent for 9 times to obtain crude polysaccharide solution b. b5: Mix the crude polysaccharide solution b obtained in step b4 with D101 macroporous adsorption resin at a volume ratio of 4:1, place it in a constant temperature shaker and shake for 16 hours, and separate the D101 macroporous adsorption resin by vacuum filtration to obtain the polysaccharide solution of Eucommia ulmoides. b6: Freeze-dry the Eucommia ulmoides polysaccharide solution prepared in step b5 for 36 hours to obtain Eucommia ulmoides polysaccharide.

[0033] The sulfated silk fibroin used in the following examples and comparative examples were all derived from Preparation Example 1, and the Eucommia polysaccharide was derived from Preparation Example 2.

[0034] Example 1

[0035] Example 1 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, comprising the following raw materials: type I collagen powder, chitosan, sodium hyaluronate, sulfated silk fibroin, acetic acid, cross-linking solution, and bioactive solution. The cross-linking solution contains genipin at a concentration of 50 mg / mL and is in 60% ethanol. The bioactive solution contains Eucommia ulmoides polysaccharide at a concentration of 10 mg / mL and is in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) as the solvent.

[0036] Preparation method S1: Preparation of collagen solution: Weigh 2.4g of type I collagen powder and 0.2g of chitosan, and dissolve them together in 90mL of 0.2M acetic acid solution. Stir mechanically at room temperature for 12h until completely swollen. Then, add 0.3g of sodium hyaluronate and 0.3g of sulfated silk fibroin in sequence, and then add 0.2M acetic acid solution to make up to 100mL. Continue stirring for 2h to obtain a homogeneous collagen solution with a type I collagen concentration of 24mg / mL. S2: Freeze-forming: The homogeneous composite solution obtained in step S1 is injected into a cylindrical mold and pre-frozen at -50°C. Then it is transferred to a freeze dryer and freeze-dried for 36 hours to obtain an initial collagen composite sponge scaffold with a three-dimensional interconnected porous structure. S3: Partial impregnation: The initial collagen composite sponge scaffold obtained in step S2 is horizontally fixed on a porous support frame. Using a precision spray gun, 5 mL of crosslinking solution (the concentration of genipin in the crosslinking solution is 50 mg / mL, and the solvent is 60% ethanol) is evenly sprayed onto the outer surface of the initial collagen composite sponge scaffold. Then, the scaffold is immediately placed in a closed environment with >95%RH to carry out diffusion and crosslinking reactions for 24 hours to obtain a gradient collagen composite sponge scaffold. S4: Cleaning and post-treatment: After the cross-linking reaction is completed, the gradient collagen composite sponge scaffold prepared in step S3 is cleaned with a large amount of deionized water three times. Then, it is freeze-dried again to obtain a highly stable gradient collagen composite sponge scaffold. S5: Bioactive Modification: The highly stable gradient collagen composite sponge scaffold prepared in step S4 was immersed in 25 mL of bioactive solution (the concentration of Eucommia ulmoides polysaccharide in the bioactive solution was 10 mg / mL, and the solvent was PBS solution). The vacuum was drawn to 0.5 kPa and maintained for 10 min. The pressure was released and the vacuuming and depressurization were repeated 3 times. The scaffold was allowed to stand at 4 °C for 12 hours for adsorption. After removing the excess liquid on the surface with filter paper, the scaffold was finally freeze-dried. Finally, it was sterilized by cobalt-60 irradiation to obtain a radial gradient collagen composite sponge scaffold for ligament injury repair.

[0037] The mass ratio of sodium hyaluronate to sulfated silk fibroin is 1:1; the mass ratio of genipin to Eucommia ulmoides polysaccharide is 1:1; and the mass ratio of type I collagen powder to chitosan is 12:1.

[0038] Example 2

[0039] Example 2 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the total mass of sodium hyaluronate and sulfated silk fibroin remains unchanged, and the mass ratio of sodium hyaluronate to sulfated silk fibroin is 1:5.

[0040] The preparation method is the same as in Example 1.

[0041] Example 3

[0042] Example 3 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the total mass of sodium hyaluronate and sulfated silk fibroin remains unchanged, and the mass ratio of sodium hyaluronate to sulfated silk fibroin is 5:1.

[0043] The preparation method is the same as in Example 1.

[0044] Example 4

[0045] Example 4 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the total mass of genipin and eucommia polysaccharide remains unchanged, and the mass ratio of genipin to eucommia polysaccharide is 0.81:1.

[0046] Specifically, the raw materials differ. This embodiment provides a radial gradient collagen composite sponge scaffold for ligament injury repair, comprising the following raw materials: type I collagen powder, chitosan, sodium hyaluronate, sulfated silk fibroin, acetic acid, a cross-linking solution, and a bioactive solution. The cross-linking solution contains genipin at a concentration of 45 mg / mL, and the solvent is 60% ethanol. The bioactive solution contains Eucommia ulmoides polysaccharide at a concentration of 11 mg / mL, and the solvent is phosphate-buffered saline (PBS, 0.01 M, pH 7.4).

[0047] The preparation method is the same as in Example 1.

[0048] Example 5

[0049] Example 5 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the total mass of genipin and eucommia polysaccharide remains unchanged, and the mass ratio of genipin to eucommia polysaccharide is 1.22:1.

[0050] Specifically, the raw materials differ. This embodiment provides a radial gradient collagen composite sponge scaffold for ligament injury repair, comprising the following raw materials: type I collagen powder, chitosan, sodium hyaluronate, sulfated silk fibroin, acetic acid, a cross-linking solution, and a bioactive solution. The cross-linking solution contains genipin at a concentration of 55 mg / mL and is in 60% ethanol. The bioactive solution contains Eucommia ulmoides polysaccharide at a concentration of 9 mg / mL and is in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) as the solvent.

[0051] The preparation method is the same as in Example 1.

[0052] Example 6

[0053] Example 6 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the mass of chitosan remains unchanged, and the mass ratio of type I collagen powder to chitosan is 9:1.

[0054] The preparation method is the same as in Example 1.

[0055] Example 7

[0056] Example 7 provides a radial gradient collagen composite sponge scaffold for ligament injury repair. The difference from Example 1 is that the mass of chitosan remains unchanged, and the mass ratio of type I collagen powder to chitosan is 15:1.

[0057] The preparation method is the same as in Example 1.

[0058] Comparative Example 1 Comparative Example 1 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that sodium hyaluronate and other similar substances are replaced with sulfated silk fibroin.

[0059] The preparation method is the same as in Example 1.

[0060] Comparative Example 2 Comparative Example 2 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that sulfated silk fibroin is replaced by sodium hyaluronate.

[0061] The preparation method is the same as in Example 1.

[0062] Comparative Example 3 Comparative Example 3 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that Eucommia polysaccharide and other ingredients are replaced with genipin.

[0063] The preparation method is the same as in Example 1.

[0064] Comparative Example 4 Comparative Example 4 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that genipin and other similar materials are replaced with Eucommia ulmoides polysaccharide.

[0065] The preparation method is the same as in Example 1.

[0066] Comparative Example 5 Comparative Example 5 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that sulfated silk fibroin is replaced with silk fibroin.

[0067] The preparation method is the same as in Example 1.

[0068] Comparative Example 6 Comparative Example 6 provides a radial gradient collagen composite sponge scaffold for ligament injury repair, which differs from Example 1 in that the mass of Eucommia ulmoides polysaccharide is replaced with sodium alginate.

[0069] The preparation method is the same as in Example 1.

[0070] Performance testing The radial gradient collagen composite sponge scaffolds prepared in Examples 1-7 and Comparative Examples 1-6 were subjected to compression tests using a universal testing machine to obtain the compression modulus. Referring to YY / T 1985-2025, collagenase was soaked in PBS solution for 28 days, and the mass loss was measured to evaluate its in vitro degradation rate. The formula for calculating the in vitro degradation rate is as follows: In vitro degradation rate = (m0 - m1) / m0 × 100%; In the formula, m0 is the mass of the radial gradient collagen composite sponge scaffolds prepared in Examples 1-7 and Comparative Examples 1-6 before soaking in collagenase PBS solution, and m1 is the mass of the radial gradient collagen composite sponge scaffolds prepared in Examples 1-7 and Comparative Examples 1-6 after soaking in collagenase PBS solution for 28 days. The biological activity of the cells was evaluated for cytotoxicity according to GB / T 16886.5 (ISO 10993-5). Three parallel experiments were set up for each group of tests, and the final results were averaged. The results are shown in Table 1.

[0071] Table 1

[0072] Results Analysis and Summary Based on Examples 1-3 and Table 1, it can be seen that when the mass ratio of sodium hyaluronate to sulfated silk fibroin in Example 1 is 1:1, the prepared radial gradient collagen composite sponge scaffold can achieve a balance between high modulus and moderate in vitro degradation rate, achieving the best balance between structural stability and biodegradability. In Example 2, the proportion of sodium hyaluronate decreased, and in Example 3, the proportion of sodium hyaluronate increased, both of which led to a decrease in mechanical properties. At the same time, the in vitro degradation rate was higher, and the bioactivity also decreased.

[0073] Based on Examples 1, 4-5 and Table 1, it can be seen that when the mass ratio of genipin to Eucommia polysaccharide in Example 1 is 1:1, the prepared radial gradient collagen composite sponge scaffold exhibits excellent bioactivity while maintaining excellent mechanical properties. In Example 4, the proportion of genipin decreased, and the mechanical properties were somewhat reduced. In Example 5, the proportion of genipin increased, and although the mechanical properties were improved, the in vitro degradation rate slowed down, which was not conducive to the replacement of newly formed tissues in the later stage.

[0074] Based on Examples 1, 6-7 and Table 1, it can be seen that in Example 1, when the mass ratio of type I collagen powder to chitosan is 12:1, the prepared radial gradient collagen composite sponge scaffold has the best mechanical properties and a moderate in vitro degradation rate; in Examples 6 and 7, both increasing and decreasing the proportion of chitosan will reduce the mechanical properties of the sponge scaffold.

[0075] Based on Examples 1, Comparative Examples 1-2, and Table 1, it can be seen that in Comparative Example 1, replacing sodium hyaluronate with sulfated silk fibroin significantly reduced the water retention and lubricity of the sponge scaffold, resulting in poor bioactivity. Furthermore, the in vitro degradation rate was too slow, hindering the replacement of newly formed tissue. Similarly, in Comparative Example 2, replacing sulfated silk fibroin with sodium hyaluronate significantly reduced the mechanical properties of the sponge scaffold, also leading to poor bioactivity. Additionally, the in vitro degradation rate was too fast, preventing long-term repair. This indicates that sodium hyaluronate and sulfated silk fibroin work synergistically, enabling the radial gradient collagen composite sponge scaffold to achieve a balance between high modulus and a moderate in vitro degradation rate, while better meeting the needs of bioactivity and cell migration.

[0076] Based on Examples 1, Comparative Examples 3-4, and Table 1, it can be seen that in Comparative Example 3, replacing the eucommia polysaccharide with genipin significantly reduced the bioactivity of the prepared radial gradient collagen composite sponge scaffold. In Comparative Example 4, replacing genipin with eucommia polysaccharide significantly reduced the mechanical properties of the prepared radial gradient collagen composite sponge scaffold, resulting in an excessively rapid in vitro degradation rate, which is detrimental to long-term repair. This may be because eucommia polysaccharide promotes cell adhesion and proliferation through bioactivity, while genipin enhances scaffold stability through cross-linking. The two complement each other synergistically, thereby achieving a balance between high modulus and a moderate in vitro degradation rate, while better meeting the needs of bioactivity and cell migration.

[0077] Based on Example 1, Comparative Example 5, and Table 1, it can be seen that in Comparative Example 5, after replacing sulfated silk fibroin with an equal mass of silk fibroin, the mechanical properties and bioactivity of the prepared radial gradient collagen composite sponge scaffold both deteriorated. Furthermore, the in vitro degradation rate was too rapid, which is detrimental to long-term repair. This may be because sulfation treatment endows silk fibroin with more negative charge and higher hydrophilicity, enabling it to form a tighter and more stable electrostatic interaction with sodium hyaluronate. This interaction not only enhances the cross-linking strength and mechanical properties of the composite material but also promotes the formation of a uniform porous structure, providing a better environment for cell growth and thus improving bioactivity.

[0078] Based on Example 1, Comparative Example 6, and Table 1, it can be seen that in Comparative Example 6, after replacing the eucommia polysaccharide with sodium alginate by the same mass, the mechanical properties and bioactivity of the prepared radial gradient collagen composite sponge scaffold both deteriorated. Furthermore, the in vitro degradation rate was too rapid, which is detrimental to long-term repair. This may be because, on the one hand, the network formed by the cross-linking of sodium alginate and genipin is relatively soft, leading to poor mechanical properties of the sponge scaffold and hindering cell growth; on the other hand, eucommia polysaccharide is an essential component for producing significant bioactivity and cannot be replaced by sodium alginate or any other polysaccharide.

[0079] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the products, methods and principles of this application should be covered within the scope of protection of this application.

Claims

1. A radially gradient collagen composite sponge scaffold for ligament injury repair, characterized in that, The raw materials include: initial collagen composite sponge scaffold, cross-linking solution, and bioactive solution; The initial collagen composite sponge scaffold was frozen and molded from a collagen solution; The collagen solution comprises type I collagen powder at a concentration of 8-40 mg / mL, chitosan at a concentration of 1-3 mg / mL, sodium hyaluronate at a concentration of 1-5 mg / mL, and sulfated silk fibroin at a concentration of 1-5 mg / mL. The crosslinking solution is a genipin solution; the concentration of the genipin is 45-55 mg / mL; The bioactive solution is an Eucommia ulmoides polysaccharide solution; the concentration of the Eucommia ulmoides polysaccharide is 9-11 mg / mL.

2. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, In the collagen solution, the mass ratio of sodium hyaluronate to sulfated silk fibroin is (0.2-5):

1.

3. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, The preparation of the sulfated silk fibroin includes the following steps: a1: Add 50-100 times the weight of degumming agent to silkworm cocoon shells or waste silk, refine for 0.5-1.5 hours at a temperature of 98-99℃, filter, repeat refining and filtering 1-3 times, wash, soak, and dry to obtain fibroin; a2: Take the silk fibroin prepared in step a1 and add it to sulfuric acid solution. Hydrolyze it for 2-5 hours at 20-30℃ with stirring to obtain hydrolysate. Pour the hydrolysate into water, filter, wash with water until neutral, and dry to obtain silk fibroin microcrystalline fibers. a3: Under ice bath and stirring conditions, dichloromethane was added, and chlorosulfonic acid was added to dichloromethane under inert gas protection. The reaction temperature was adjusted to below 15°C, and the silk fibroin microcrystalline fiber prepared in step a2 was added. The reaction was carried out for 15-60 minutes. After the reaction was completed, the reaction solution was poured into sodium acetate-ethanol solution, filtered, and the precipitate was washed to obtain the product. a4: Dissolve the product obtained in step a3 in water, adjust the pH to 8-8.5, dialyze for 24-48 hours, and then freeze-dry the dialysate to obtain sulfated silk fibroin.

4. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, In the collagen solution, the mass ratio of type I collagen powder to chitosan is (6-15):

1.

5. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, The method for preparing the initial collagen composite sponge scaffold includes the following steps: Type I collagen powder and chitosan were dissolved in acetic acid solution and stirred. Sodium hyaluronate and sulfated silk fibroin were added and stirred to obtain a collagen solution. The collagen solution was then injected into a mold, pre-frozen, and freeze-dried to obtain the initial collagen composite sponge scaffold.

6. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, The mass ratio of genipin to eucommia polysaccharide is (0.81-1.22):

1.

7. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, The preparation of the Eucommia ulmoides polysaccharide includes the following steps: b1: Dry the Eucommia ulmoides, pulverize it, extract it by reflux with ethanol, filter it, collect the filter residue, and obtain the Eucommia ulmoides residue; b2: Add the Eucommia ulmoides residue obtained in step b1 to water, heat in a water bath for 2-4 hours, cool, filter, collect the supernatant to obtain the preliminary extract of Eucommia ulmoides polysaccharides. b3: Concentrate the preliminary extract of Eucommia ulmoides polysaccharide prepared in step b2, add ethanol solution, let stand, centrifuge, collect the precipitate, and obtain crude Eucommia ulmoides polysaccharide. b4: Dissolve the crude polysaccharide of Eucommia ulmoides prepared in step b3 into crude polysaccharide solution a, mix the crude polysaccharide solution a with Sevag reagent, shake, let stand, centrifuge, collect the upper aqueous phase solution, and obtain crude polysaccharide solution b. b5: Mix the crude polysaccharide solution b obtained in step b4 with macroporous resin, shake, and filter to obtain the polysaccharide solution of Eucommia ulmoides. b6: Freeze-dry the Eucommia ulmoides polysaccharide solution prepared in step b5 to obtain Eucommia ulmoides polysaccharide.

8. The radial gradient collagen composite sponge scaffold for ligament injury repair according to claim 1, characterized in that, The volume ratio of the crosslinking solution to the collagen solution is (1-1.5):25; the volume ratio of the bioactive solution to the collagen solution is (1-1.5):

5.

9. A method for preparing a radially gradient collagen composite sponge scaffold for ligament injury repair as described in any one of claims 1-8, characterized in that, Includes the following steps: S1: Partial wetting: The cross-linking solution is sprayed onto the outer surface of the initial collagen composite sponge scaffold, reacted for 12-36 hours, washed, and freeze-dried to obtain a highly stable gradient collagen composite sponge scaffold. S2: Bioactive modification: The highly stable gradient collagen composite sponge scaffold prepared in step S1 is immersed in the bioactive solution, vacuumed to 0.5 kPa or below, maintained for 5-15 min, depressurized, and left to stand at 3-5℃ for 8-16 h, freeze-dried, and sterilized to obtain a radial gradient collagen composite sponge scaffold for ligament injury repair.

10. The use of the radial gradient collagen composite sponge scaffold for ligament injury repair as described in any one of claims 1-8 in the preparation of a functionalized load scaffold.

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