An injectable multi-crosslinked composite hydrogel scaffold with in-situ repair anterior cruciate ligament function and a preparation method thereof

Abstract

The application discloses an injectable multi-crosslinking composite hydrogel scaffold with an in-situ anterior cruciate ligament repair function and a preparation method thereof. A composite solution is obtained by using dopamine grafted oxidized chondroitin sulfate to reduce magnesium ions, and after being mixed with polylysine and ferric chloride by using a Schiff base reaction and metal chelation, a stable hydrogel is quickly formed through multi-layer crosslinking reaction; biological active substances such as stem cells, PRP, growth factors, and tranexamic acid are mixed into the hydrogel to promote the repair of the anterior cruciate ligament. The hydrogel scaffold prepared by the application can be in-situ gelled, is suitable for irregular anterior cruciate ligament wound repair and can integrally repair the ligament and the bone; meanwhile, the hydrogel scaffold has good antibacterial property and wet adhesion; in addition, the multi-crosslinking network makes the degradation performance and the mechanical property of the hydrogel scaffold suitable for the complex growth environment of the anterior cruciate ligament and the long repair period, thereby effectively improving the anterior cruciate ligament repair effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an injectable multi-crosslinked composite hydrogel scaffold with in-situ repair function of the anterior cruciate ligament and its preparation method. Background Technology

[0002] Approximately 2 million people worldwide suffer anterior cruciate ligament (ACL) injuries each year. With increasing demand for sports, the number of ACL injuries is rising annually. Current clinical treatments primarily include ACL reconstruction and ACL repair. However, ACL reconstruction methods using tendon grafts or artificial ligament substitutes are often accompanied by problems such as a 30% ligament re-rupture rate, poor healing of the bone marrow tract post-surgery, and severe osteoarthritis.

[0003] Anterior cruciate ligament (ACL) repair uses in-situ suture closure, which is minimally invasive and preserves the natural ACL and its proprioceptors, helping to monitor knee joint position and dynamic stability. It holds promise as a more advantageous and promising treatment method. However, the difficulty of suture closure increases dramatically when dealing with proximal tears and irregular wounds. Furthermore, the large amount of synovial fluid and enzymes in the joint cavity can erode the ligament fracture surface, hindering healing. Therefore, suture surgery alone is insufficient for effective ligament repair. In addition, the bone tunnel created during suturing can cause bone damage, harming the body and hindering functional recovery.

[0004] The natural interface between the anterior cruciate ligament (ACL) and bone is a specialized tissue called the "attachment point," which comprises four different types of tissue: ligament, uncalcified fibrocartilage, calcified fibrocartilage, and bone. Statistics show that inadequate bone tunnel healing is one of the main reasons for unsatisfactory clinical outcomes in ACL treatment. Studies have shown that magnesium ions (Mg...) 2+ Magnesium ions can regulate the crystallization and formation of minerals in newly formed bone, participate in the expression of cytokines related to osteogenic differentiation of bone-derived stem cells, and thus promote bone repair. Simultaneously, magnesium ions can also increase the expression of vascular endothelial growth factor, promoting local blood perfusion. However, in the past, people have only used magnesium ions to promote bone healing, or focused solely on ligament healing while neglecting bone repair. Therefore, the repair of the anterior cruciate ligament (ACL) requires not only the healing of the ligament itself but also the promotion of bone healing.

[0005] In view of this, the present invention designs an integrated hydrogel scaffold capable of simultaneously repairing ligaments and bone. Its in-situ gelling properties can adapt to various irregular anterior cruciate ligament (ACL) injury surfaces; the loaded magnesium ions promote bone healing; the hydrogel scaffold protects the ligament from the invasion of synovial fluid and enzymes within the joint cavity; and the composite bioactive substances further enhance cell migration, adhesion, and angiogenesis. In summary, the hydrogel scaffold of the present invention can promote ligament tissue and bone interface regeneration, maximizing the restoration of the patient's original limb function, thereby improving the clinical efficacy of ligament repair and meeting significant clinical needs. Summary of the Invention

[0006] The present invention aims to provide an injectable multi-crosslinked composite hydrogel scaffold with in-situ repair function for the anterior cruciate ligament (ACL) and its preparation method. The prepared hydrogel scaffold forms a multi-network structure through Schiff base reaction, metal chelation, electrostatic interaction, and self-polymerization, exhibiting self-healing properties and effectively improving the mechanical properties of the hydrogel. The prepared hydrogel scaffold also possesses good wet adhesion properties. Furthermore, the added magnesium ions promote the regeneration and repair of the integrated ligament and bone. The prepared hydrogel scaffold also exhibits good anti-degradation ability and certain antibacterial properties, and can undergo in-situ gelation, making it suitable for irregular wounds. In addition, the hydrogel scaffold repairs the ACL rapidly and can reduce osteoarthritis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing an injectable multi-crosslinked composite hydrogel scaffold with in-situ repair function of the anterior cruciate ligament, comprising the following steps:

[0009] (1) Chondroitin sulfate was oxidized with sodium periodate to obtain chondroitin sulfate oxide; under nitrogen protection, dopamine was grafted onto chondroitin sulfate oxide using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain dopamine-grafted chondroitin sulfate oxide; magnesium chloride solution was mixed with dopamine-grafted chondroitin sulfate oxide solution, reacted at 25°C for 1-5 h, and freeze-dried to obtain dopamine-grafted chondroitin sulfate oxide-magnesium;

[0010] (2) Mix ε-polylysine solution with dopamine-grafted chondroitin sulfate-magnesium solution and ferric chloride solution, add bioactive substances, adjust the pH value to 8.0, let stand to form a gel, and obtain an injectable multi-crosslinked composite hydrogel scaffold with in situ repair function of anterior cruciate ligament.

[0011] In step (1), the mass ratio of chondroitin sulfate to sodium periodate is 1:0.5-1.

[0012] In step (1), the mass ratio of dopamine to chondroitin sulfate is 1:0.5-1.

[0013] In step (1), the volume ratio of magnesium chloride solution to dopamine-grafted chondroitin sulfate solution is 0.01 to 0.05:1, the concentration of magnesium chloride solution is 20 wt% to 30 wt%, and the concentration of dopamine-grafted chondroitin sulfate solution is 1 wt% to 3 wt%.

[0014] In step (2), the volume ratio of ε-polylysine solution to dopamine-grafted chondroitin sulfate-magnesium solution and ferric chloride solution is 1-1.5:1-1.5:0.1-0.5, the concentration of ε-polylysine solution is 5wt%-30wt%, the concentration of dopamine-grafted chondroitin sulfate-magnesium solution is 5wt%-20wt%, and the concentration of ferric chloride solution is 1wt%-10wt%.

[0015] In step (2), the temperature for standing and gelling is 25-30℃ and the time is 10-40s.

[0016] In step (2), the bioactive substance is selected from any one or more of the following: epidermal growth factor, fibroblast growth factor, growth differentiation factor, insulin-like growth factor, platelet-derived growth factor, or transforming growth factor-β, mechanical growth factor E peptide, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, neural stem cells, tranexamic acid, celecoxib, glucosamine, and antibiotics.

[0017] The concentrations of the growth factors in the solution are between 0.001 and 1 mg / L, the concentrations of the stem cell solution are between 50 and 200 w / mL, the platelet enrichment concentrations of platelet-rich plasma are between 4 and 8 times, and the concentrations of the antibiotic solution are between 0.5 and 1 mmol / L.

[0018] An injectable multi-crosslinked composite hydrogel scaffold prepared by the above preparation method.

[0019] The above-mentioned application of an injectable multi-crosslinked composite hydrogel in the preparation of products for anterior cruciate ligament repair.

[0020] The significant advantages of this invention are:

[0021] (1) In this invention, chondroitin sulfate is used to prepare hydrogel scaffolds. Chondroitin sulfate can reduce pain in patients with osteoarthritis, improve joint function, and reduce joint swelling.

[0022] (2) The ε-polylysine material selected in this invention can remove hydrated cations on wet tissues, promote the tight binding between catechol groups and tissues through hydrogen bonds, π-π interactions and electrostatic attraction, provide excellent wet adhesion, and has certain antibacterial properties.

[0023] (3) The hydrogel scaffold formed by the present invention has a multi-network structure, which improves the mechanical properties of the hydrogel, and has self-healing ability and slow degradation.

[0024] (4) Mg released from the hydrogel 2+ It can promote osteogenic differentiation mediated by calcitonin gene-associated peptide-α (CGRP) and bone marrow stem cells (BMSCs), thereby promoting the expression of OCN, COL, and ALP genes, and Mg 2+ It also has a positive effect on promoting the formation of mineralized bone matrix, and can promote ligament repair in an integrated manner;

[0025] (5) The hydrogel of the present invention can be injected in situ to form a gel for different degrees of anterior cruciate ligament tear, which has the advantages of convenient operation, targeting different populations and different degrees of anterior cruciate ligament rupture, thereby effectively promoting wound healing.

[0026] (6) The hydrogel scaffold of the present invention protects ligaments from the invasion of synovial fluid and enzymes in the joint cavity. The composite bioactive substances further enhance cell migration and adhesion and angiogenesis, promote ligament tissue regeneration, and restore the patient's original limb function to the maximum extent, thereby improving the clinical efficacy of ligament repair, which has great clinical demand. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of an injured anterior cruciate ligament (left image shows partial damage, right image shows complete rupture).

[0028] Figure 2 This is an injection diagram for a partial injury to the anterior cruciate ligament.

[0029] Figure 3 This is an enlarged view of an injection site for a partial injury to the anterior cruciate ligament.

[0030] Figure 4 This is an injection diagram for a complete anterior cruciate ligament injury.

[0031] Figure 5 This is an enlarged view of an injection site for a complete anterior cruciate ligament injury.

[0032] Figure 6 This is a picture of the injectable hydrogel scaffold product prepared in Example 1.

[0033] Figure 7 Fluorescence images of the injectable hydrogel scaffold prepared in Example 3 at 72h 3T3.

[0034] Figure 8 This image shows the repair of the anterior cruciate ligament in rabbits after 4 weeks using the injectable hydrogel scaffold prepared in Example 3.

[0035] Figure 9 The image shows the MRI of the injectable hydrogel scaffold prepared in Example 3 on a rabbit knee at 12 weeks. Detailed Implementation

[0036] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0037] In the following examples, the chondroitin sulfate had an average molecular weight of 20-30 kDa and was purchased from Aladdin Reagent (Shanghai) Co., Ltd., catalog number C832332.

[0038] In the following examples, the dopamine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number A902400.

[0039] In the following examples, the ε-polylysine has an average molecular weight of 70-150 kDa and was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S20058.

[0040] In the following examples, the glucosamine is D-glucosamine acid, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number D838500.

[0041] Example 1

[0042] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0043] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0044] S1-1: Preparation of chondroitin sulfate oxidized:

[0045] Chondroitin sulfate was dissolved in deionized water to prepare a 2 wt% solution, and sodium periodate was dissolved in deionized water to prepare an 18 wt% solution. Under stirring, 10 mL of the 18 wt% sodium periodate solution was added dropwise to 100 mL of the 2 wt% chondroitin sulfate solution. The reaction was carried out at 25°C in the dark for 2 hours, and then 2 mL of ethylene glycol was added and stirred for 1 hour to terminate the reaction. The resulting solution was poured into a dialysis bag with a molecular weight cutoff of 3500 MW and dialyzed with deionized water at room temperature for 3 days, with the water changed every 8 hours. The entire solution in the dialysis bag was poured into a polytetrafluoroethylene plate, pre-frozen at -80°C for 12 hours, and then freeze-dried at -55°C and a vacuum of 0.01 Pa for 48 hours to obtain oxidized chondroitin sulfate.

[0046] S1-2: Preparation of dopamine-grafted chondroitin sulfate-magnesium:

[0047] 1.0 g of chondroitin sulfate was dissolved in 100 mL of distilled water. 0.96 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.288 g of N-hydroxythiosuccinimide (NHS) were added. The pH of the system was adjusted to 4.8 with HCl. After stirring at room temperature for 1 hour, 1.0 g of dopamine was added. The mixture was stirred overnight at room temperature under nitrogen protection. The resulting solution was poured into a dialysis bag with a molecular weight cutoff of 3000 MW and dialyzed with deionized water at room temperature for 3 days, changing the water every 8 hours. The entire solution in the dialysis bag was poured into a polytetrafluoroethylene plate and pre-frozen at -80°C for 12 hours. Then, it was freeze-dried at -55°C and a vacuum of 0.01 Pa for 48 hours to obtain dopamine-grafted chondroitin sulfate.

[0048] Dopamine-grafted chondroitin sulfate was dissolved in deionized water to prepare a 2 wt% solution, and magnesium chloride was dissolved in deionized water to prepare a 20 wt% solution. 100 μL of the 20 wt% magnesium chloride solution was added to 10 mL of the 2 wt% dopamine-grafted chondroitin sulfate solution, and the reaction was carried out at 25 °C for 3 hours. After the reaction was completed, the solution was pre-frozen at -80 °C for 12 hours, and then freeze-dried at -55 °C and a vacuum of 0.01 Pa for 48 hours to obtain dopamine-grafted chondroitin sulfate-magnesium.

[0049] S2: Multi-network cross-linking reaction:

[0050] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed, and 3 mL of the 10 wt% ferric chloride solution was added dropwise while stirring. The pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0051] Example 2

[0052] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0053] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0054] The same as step S1 in Example 1.

[0055] S2: Multi-network cross-linking reaction:

[0056] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed, and 4 mL of the 10 wt% ferric chloride solution was added dropwise while stirring. The pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0057] Example 3

[0058] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0059] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0060] The same as step S1 in Example 1.

[0061] S2: Multi-network cross-linking reaction:

[0062] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 15 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed, and 4 mL of the 10 wt% ferric chloride solution was added dropwise while stirring. The pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0063] Example 4

[0064] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0065] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0066] The same as step S1 in Example 1.

[0067] S2: Multi-network cross-linking reaction:

[0068] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 15 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed. While stirring, 4 mL of the 10 wt% ferric chloride solution and 5 mL of platelet-rich plasma were added dropwise. The pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0069] The method for preparing platelet-rich plasma is as follows: 5 mL of fresh rabbit whole blood is collected and centrifuged for the first time (4℃, 350g, 10 min). The centrifuged blood should separate into three layers: the top layer is the plasma layer, accounting for 40% of the centrifuged whole blood volume, containing a large number of platelets; the middle layer is a thin white membrane layer containing a large number of white blood cells, platelets, and a small number of red blood cells; the bottom layer is the red blood cell layer, containing a large number of red blood cells. The top layer and most of the middle layer are extracted and mixed evenly, and then centrifuged a second time (4℃, 2000g, 10 min). After centrifugation, platelet precipitate and plasma supernatant are formed. The excess plasma supernatant is discarded, and 500 μL of plasma supernatant is retained to resuspend the platelet precipitate. The mixture is then blown and stirred to obtain platelet-rich plasma. Calculations show that the platelet enrichment factor of the prepared platelet-rich plasma is 5 times.

[0070] Example 5

[0071] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0072] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0073] The same as step S1 in Example 1.

[0074] S2: Multi-network cross-linking reaction:

[0075] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed. While stirring, 5 mL of the 10 wt% ferric chloride solution and 2 mL of 0.01 mg / L epidermal growth factor (EGF) were added dropwise. The pH was adjusted to 8.0 with NaOH to obtain the hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament.

[0076] Example 6

[0077] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0078] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0079] The same as step S1 in Example 1.

[0080] S2: Multi-network cross-linking reaction:

[0081] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, dopamine-grafted chondroitin sulfate-magnesium solution was dissolved in deionized water to prepare a 15 wt% solution, and ferric chloride solution was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed. While stirring, 4 mL of the 10 wt% ferric chloride solution and 2 mL of 1 mmol / L glucosamine were added dropwise. The pH was adjusted to 8.0 with NaOH to obtain the hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0082] Comparative Example 1

[0083] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0084] S1: Preparation of chondroitin sulfate oxidized and dopamine-grafted chondroitin sulfate oxidized and magnesium-containing compounds:

[0085] The same as step S1 in Example 1.

[0086] S2: Crosslinking reaction:

[0087] ε-polylysine was dissolved in deionized water to prepare a 20 wt% solution, and dopamine-grafted chondroitin sulfate-magnesium was dissolved in deionized water to prepare a 15 wt% solution. 10 mL of the 20 wt% ε-polylysine solution and 10 mL of the 15 wt% dopamine-grafted chondroitin sulfate-magnesium solution were rapidly mixed, and the pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0088] Comparative Example 2

[0089] A method for preparing an injectable hydrogel scaffold with in-situ repair function of the anterior cruciate ligament includes the following steps:

[0090] S1: Preparation of chondroitin sulfate oxidized:

[0091] The same as step S1 in Example 1.

[0092] S2: Crosslinking reaction:

[0093] ε-polylysine was dissolved in deionized water to prepare a 10 wt% solution, and chondroitin sulfate was dissolved in deionized water to prepare a 10 wt% solution. 10 mL of the 10 wt% ε-polylysine solution and 10 mL of the 10 wt% chondroitin sulfate solution were rapidly mixed, and the pH was adjusted to 8.0 with NaOH to obtain a hydrogel precursor solution. The solution was then allowed to stand at 25°C for a period of time to form a gel, thus obtaining an injectable hydrogel scaffold with in-situ repair function for the anterior cruciate ligament.

[0094] The in-situ repair of the anterior cruciate ligament injectable hydrogel scaffold was subjected to rheological testing using a rheometer. The test parameters were as follows: at 25°C, the rheological study was conducted on a cylinder (5 mm high × 10 mm diameter) with a gap distance of 0.8 mm using a rheometer (MCR 302, Austria). The frequency scan was performed at 1% strain and oscillation frequencies from 0.1 to 100 rad / s.

[0095] The degradation capacity of the prepared injectable hydrogel scaffold for in situ repair of the anterior cruciate ligament was tested. The specific steps were as follows: the hydrogel scaffold was immersed in PBS buffer containing 0.4 U / ml collagenase and 0.4 U / ml lysozyme, and cultured at 37℃ with constant temperature shaking at 120 rpm. The medium was changed once a day. After 30 days, the hydrogel scaffold was taken out, lyophilized, weighed, and the degradation rate of the hydrogel scaffold was calculated.

[0096] The tensile mechanical properties of the in-situ repair anterior cruciate ligament injectable hydrogel scaffold were tested using a universal testing machine (LLOYD LR100K, CN). After the clamps at both ends were fixed, the scaffold was stretched at a speed of 15 mm / min to obtain the maximum breaking load and tensile displacement of the hydrogel scaffold. The experiment was repeated 5 times, and the results are expressed as (mean ± standard deviation).

[0097] Compression tests were performed on the in-situ repaired anterior cruciate ligament injectable hydrogel scaffold using a Stable Micro Systems texture analyzer (TA-XT plus, UK). The measurement mode of the shear texture analyzer was set to compression, and the test was conducted at a speed of 20 mm / min under 80% strain.

[0098] The adhesion strength of the in-situ repair of the anterior cruciate ligament injectable hydrogel scaffold was tested using a porcine skin overlap shear test. A fresh piece of porcine skin was degreased and shaved, then cut into 10 mm × 30 mm rectangles. The skin was rinsed with water, removed from the water, and used directly without drying. A hydrogel precursor solution was injected between two 10 mm × 15 mm pieces of porcine skin. The adhesive area was then clamped and left at room temperature for 2 hours. Tensile testing was performed using a general-purpose testing machine (Instron 5567) at a speed of 10 mm / min. The adhesion strength (Pa = N / m) was measured. 2 The calculation method is to divide the maximum force (N) by the adhesive area (m²). 2 ).

[0099] Burst pressure tests were performed on the prepared in-situ repair injectable hydrogel scaffold for anterior cruciate ligament. Fresh pigskin was cut into rectangles 20 mm wide and 50 mm long and adhered to a plastic tubing coated with cyanoacrylate adhesive. A 3 mm diameter circular notch was then made in the pigskin and tubing using a needle. A hydrogel precursor solution (300 μL) was injected into the notch area and left to stand for 2 hours. One end of the tubing was connected to an injection pump, and the other end to a digital pressure gauge. PBS was then filled into the syringe and pumped at a rate of 150 mL / h. The burst pressure of the multi-network hydrogel was recorded.

[0100] The antibacterial test was performed on the prepared in-situ injectable hydrogel scaffold for repairing the anterior cruciate ligament. Specifically, the activated 10 7 CFU bacterial culture medium (E. coil and MRSA) was co-cultured with a hydrogel scaffold for 12 hours. Then, the bacterial culture medium was used to measure the absorbance at OD600nm using an ELISA reader and spread on plates to calculate the sterilization rate.

[0101] Biocompatibility tests were performed on the prepared in situ repair of the anterior cruciate ligament injectable hydrogel scaffold: (1) Cell viability staining: Cylindrical hydrogel scaffolds with a diameter of 10 mm were prepared for use. Before the experiment, the hydrogel scaffolds were soaked in PBS buffer and irradiated under ultraviolet light for 24 h. Then, the hydrogel was placed in a 24-well plate and 50 μL of 4×10⁻⁶ cells were added. 5 3T3 cell suspensions at a concentration of 3 cells / mL were seeded onto hydrogel scaffolds and co-cultured for 72 hours. The cells were then stained with live / dead cell staining solution, and cell growth was observed under a fluorescence microscope. Figure 7 (2) Cytotoxicity assay: After 72 h of culture in the well plate, the culture medium and hydrogel scaffold were removed, and 200 μL of 0.05 mg / mL MTT solution was added to each well. The plates were then incubated at 37 °C for 4 h in a 5% CO2 incubator. The MTT solution was removed, and 200 μL of LDMSO was added. The plates were incubated at 37 °C with shaking for 15 min. The incubator was then aliquoted into 96-well plates, and the absorbance was measured at 490 nm using a microplate reader. The relative cell proliferation rate was calculated.

[0102] As shown in Table 1, compared with Comparative Examples 1-2, the injectable hydrogel scaffolds for in-situ repair of the anterior cruciate ligament prepared in Examples 1-6 have good wet adhesion properties, superior mechanical properties and biocompatibility, and slow degradation. Therefore, they are more likely to maintain a relatively intact shape during the long-term healing of the ligament, thus preventing synovial fluid from eroding the anterior cruciate ligament stump. At the same time, the injectable hydrogel scaffolds for in-situ repair of the anterior cruciate ligament prepared in Examples 1-6 have certain antibacterial properties. By combining bioactive substances, they can further promote cell proliferation and promote the healing of the anterior cruciate ligament.

[0103] Table 1 Performance test results of injectable hydrogel scaffolds for in situ repair of anterior cruciate ligament

[0104]

[0105] Animal experiments were conducted using the injectable hydrogel scaffolds for in-situ repair of the anterior cruciate ligament (ACL) prepared in Examples 1-6 and Comparative Examples 1-2. The ACL of the right knee joint of male New Zealand white rabbits weighing 2.5 kg to 3 kg was partially and completely torn. (The scaffold used in Example 1 was used for the partial tear model (Example 1-1) and for the complete tear model (Example 1-2); the scaffold used in Example 2 was used for the partial tear model (Example 2-1) and for the complete tear model (Example 2-2); the scaffold used in Example 3 was used for the partial tear model (Example 3-1) and for the complete tear model (Example 3-2); the scaffold used in Example 4 was used for the partial tear model (Example 4-1) and for the complete tear model (Example 4-2); the scaffold used in Example 5 was used for the partial tear model (Example 5-1) and for the complete tear model (Example 5-2). The scaffolds used in Example 6 were employed for the partial fracture model (Example 6-1) and for the complete fracture model (Example 6-2); the scaffolds used in Comparative Example 1 were employed for the partial fracture model (Comparative Example 1-1) and for the complete fracture model (Comparative Example 1-2); the scaffolds used in Comparative Example 2 were employed for the partial fracture model (Comparative Example 2-1) and for the complete fracture model (Comparative Example 2-2). After surgically suturing the fracture ends, a hydrogel precursor solution was injected into the suture site and allowed to settle into a gel. Control groups 1-1 (partial fracture with only ligament sutured and no scaffold) and 1-2 (complete fracture with only ligament sutured and no scaffold), as well as control groups 2-1 (partial fracture with untreated ligament) and 2-2 (complete fracture with untreated ligament) were established. After wound treatment, the rabbit's right knee joint was immobilized with a brace, which was removed after two weeks. Ligament repair was observed at 4, 8, and 12 weeks of brace immobilization, and the ligament and surrounding cartilage tissue were observed through pathological staining. Markin scoring was used, and the scoring criteria are shown in Table 2. The ligament repair results are shown in Tables 3-1 and 3-2.

[0106] Table 2 Scoring Criteria

[0107] Scoring Indicators Score I. Cartilage Structure normal 0 Surface irregularity 1 Pannus formation and surface irregularities 2 Cracks enter the transition layer 3 Cracks penetrate the radiation layer 4 Cracks penetrate the calcified layer 5 The structure was completely destroyed. 6 II chondrocytes normal 0 Diffuse cell increase 1 Local cell increase 2 The number of cells was significantly reduced. 3 III. Cartilage matrix staining (Safranin O) normal 0 Slight reduction 1 Moderate reduction 2 Severe reduction 3 Uncolored 4 IV. Tidal Line Integrity whole 0 Damaged 1

[0108] As can be seen from the experimental data in Tables 3-1 and 3-2, the in situ repair of the anterior cruciate ligament injectable hydrogel scaffolds in Examples 1 to 6 of the present invention can better promote the rapid repair of the anterior cruciate ligament, promote the formation of new blood vessels, accelerate the repair speed and quality of the anterior cruciate ligament, and reduce the occurrence of osteoarthritis.

[0109] Table 3-1 Partial Fracture Model Test Data

[0110]

[0111] Table 3-2 Test data of the complete fracture model

[0112]

[0113] In addition, ligament observations were taken from the complete injury model in week four of Example 3 ( Figure 8 Significant new ligament-like tissue was observed at the anterior cruciate ligament, indicating good ligament connection and suggesting that the scaffold effectively promoted the repair of the injured ligament. Simultaneously, MRI was performed on the knee of the completely injured ligament model from Week 12 of Example 3. Figure 9 The results showed that the knee area had a continuous low signal morphology, less joint effusion, and the repaired anterior cruciate ligament was relatively intact. This indicates that the stents can promote the repair of the anterior cruciate ligament and reduce the production of intra-articular inflammation, which is conducive to the recovery of motor function.

[0114] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing an injectable multi-crosslinked composite hydrogel scaffold with in-situ repair function of the anterior cruciate ligament, characterized in that: Includes the following steps: (1) Chondroitin sulfate was oxidized with sodium periodate to obtain chondroitin sulfate oxidized; under nitrogen protection, dopamine was grafted onto chondroitin sulfate oxidized using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain dopamine-grafted chondroitin sulfate oxidized; magnesium chloride solution was mixed with dopamine-grafted chondroitin sulfate oxidized solution, reacted at 25°C for 1-5 h, and freeze-dried to obtain dopamine-grafted chondroitin sulfate oxidized magnesium; (2) Mix ε-polylysine solution with dopamine-grafted chondroitin sulfate-magnesium solution and ferric chloride solution, add bioactive substances, adjust the pH value to 8.0, let stand to form a gel, and obtain an injectable multi-crosslinked composite hydrogel scaffold with in situ repair function of anterior cruciate ligament.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of chondroitin sulfate to sodium periodate is 1:0.5~1.

3. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of dopamine to chondroitin sulfate is 1:0.5~1.

4. The preparation method according to claim 1, characterized in that: In step (1), the volume ratio of magnesium chloride solution to dopamine-grafted chondroitin sulfate solution is 0.01~0.05:1, the mass concentration of magnesium chloride solution is 20%~30%, and the mass concentration of dopamine-grafted chondroitin sulfate solution is 1%~3%.

5. The preparation method according to claim 1, characterized in that: In step (2), the volume ratio of ε-polylysine solution to dopamine-grafted chondroitin sulfate-magnesium solution and ferric chloride solution is 1~1.5:1~1.5:0.1~0.5, the mass concentration of ε-polylysine solution is 5%~30%, the mass concentration of dopamine-grafted chondroitin sulfate-magnesium solution is 5%~20%, and the mass concentration of ferric chloride solution is 1%~10%.

6. The preparation method according to claim 1, characterized in that: In step (2), the temperature for settling into gel is 25~30℃ and the time is 10~40s.

7. The preparation method according to claim 1, characterized in that: In step (2), the bioactive substance is selected from any one or more of the following: epidermal growth factor, fibroblast growth factor, growth differentiation factor, insulin-like growth factor, platelet-derived growth factor, or transforming growth factor-β, mechanical growth factor E peptide, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, neural stem cells, tranexamic acid, celecoxib, glucosamine, and antibiotics.

8. An injectable multi-crosslinked composite hydrogel scaffold prepared by the preparation method of claim 1.

9. The use of the injectable multi-crosslinked composite hydrogel as described in claim 8 in the preparation of products for anterior cruciate ligament repair.

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