Polyethylene glycol derivative-collagen injectable cartilage repair gel as well as preparation method and application thereof

Abstract

The invention provides a polyethylene glycol derivative-collagen cartilage repair gel which is characterized by being prepared from the following components in parts by weight: 8 to 12 parts of collagen (namely COL) solution, 0.5 to 2 parts of polydeoxyribonucleotide (namely PDRN), 4 to 8 parts of polyethylene glycol disuccinimide succinate (namely SS-PEG-SS) solution and 0.1 to 0.4 part of tannic acid (namely TA), and particularly discloses a preparation method and application of the polyethylene glycol derivative-collagen cartilage repair gel. In a word, the composite gel and the collagen are subjected to efficient amidation reaction through SS-PEG-SS to form a stable three-dimensional network, so that the structural support of the type I collagen, the tissue repair of PDRN and the anti-inflammatory and anti-oxidation characteristics of tannic acid are successfully integrated; therefore, the composite material has the comprehensive performance of good structural stability, high mechanical strength, good biocompatibility, controllable degradation period, excellent needle passing performance and the like, and shows the wide application prospects of clear repair effect, remarkable component synergistic effect, mature production process, outstanding cost effectiveness and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polyethylene glycol derivative-collagen injectable cartilage repair gel, its preparation method, and its application. Background Technology

[0002] Osteoarthritis is a common chronic joint disease that currently affects a large number of people worldwide. Its core manifestations are the degeneration, necrosis, and defects of articular cartilage. Current treatment options for this problem mainly include microfracture surgery, autologous cartilage transplantation, allogeneic cartilage transplantation, autologous chondrocyte implantation, and tissue engineering techniques. However, most of these methods are still in the clinical trial stage and have not yet been fully matured for clinical application. Furthermore, cartilage tissue itself is avascular tissue with extremely low cell density. These two characteristics directly lead to a significant deficiency in the migration and proliferation activity of chondrocytes, ultimately resulting in extremely poor self-repair ability of cartilage tissue.

[0003] Polyethylene glycol disuccinimide succinate (SS-PEG-SS) is a bifunctional PEG derivative with NHS active esters at both ends, and is mostly a solid or viscous powder at room temperature. The two succinimide succinate (SS) groups in its structure can rapidly react with primary amine groups of proteins, peptides, and lipids under pH 7–8.5 conditions to form stable amide bonds, thereby simultaneously crosslinking two PEG chains to the target molecule, achieving multi-point coupling of proteins / drugs or the construction of hydrogel networks. Its hydrophilic backbone endows the hydrogel with good biocompatibility and swelling properties, matching the moist physiological environment of cartilage tissue; at the same time, the mechanical strength of the hydrogel can be optimized by adjusting the crosslinking density, avoiding repair failure due to mechanical mismatch. It can also slowly degrade in cellular metabolic or reducing microenvironments, making room for cartilage regeneration, and the degradation products have no toxic side effects. Therefore, introducing SS-PEG-SS into the gel structure can be considered to promote crosslinking.

[0004] Collagen (COL) is a key structural protein that makes up human connective tissue. Among them, type I collagen, as a major component of the natural extracellular matrix (ECM), is widely used in the field of cartilage repair due to its excellent biocompatibility and ability to form three-dimensional scaffolds, providing a good environment for cell attachment and growth.

[0005] Polydeoxyribonucleotide (PDRN) is the "active signaling molecule" for cartilage repair, with its core role in regulating cell function and promoting tissue regeneration. As an endogenous adenosine receptor agonist, it can specifically bind to the A2A receptor on the surface of chondrocytes, activating downstream signaling pathways and significantly promoting chondrocyte proliferation and the synthesis of cartilage-specific matrices such as type II collagen and proteoglycans. Simultaneously, it inhibits the expression of matrix metalloproteinases (MMPs), reducing cartilage matrix degradation. Furthermore, PDRN can promote vascular endothelial cell migration and angiogenesis, improving blood supply to the cartilage repair area (especially addressing the natural avascularity of cartilage), providing sufficient nutrition for cell proliferation and matrix synthesis, and accelerating the repair process.

[0006] Tannic acid (TA), a natural polyphenol compound, possesses multiple functions including enhanced cross-linking, anti-inflammatory and antioxidant properties, and antibacterial activity. Its multiple phenolic hydroxyl groups can form hydrogen bonds and hydrophobic interactions with the amino and hydroxyl groups of type I collagen. Simultaneously, it synergistically enhances the cross-linking stability and mechanical properties of hydrogels with SS-PEG-SS, slowing down the hydrogel degradation rate. More importantly, tannic acid exhibits significant anti-inflammatory activity, inhibiting the release of pro-inflammatory factors such as TNF-α and IL-6, reducing inflammatory responses during cartilage repair, and preventing inflammation-mediated chondrocyte apoptosis and matrix destruction. Furthermore, its antioxidant properties can scavenge reactive oxygen species (ROS) in the body, reducing oxidative stress damage to chondrocytes and protecting chondrocyte activity. In addition, the antibacterial effect of tannic acid can reduce the risk of postoperative infection, creating a stable sterile microenvironment for cartilage repair.

[0007] In summary, how to obtain a cartilage repair gel that is simple to prepare, structurally stable, has a high elastic modulus, facilitates cell migration, and promotes cartilage regeneration is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a polyethylene glycol derivative-collagen cartilage repair gel, its preparation method, and its applications. This invention employs the following technical solution:

[0009] The first aspect of this invention provides a polyethylene glycol derivative-collagen cartilage repair gel, which, by weight, is composed of 8-12 parts of collagen (i.e., COL) solution, 0.5-2 parts of polydeoxyribonucleotide (i.e., PDRN), 4-8 parts of polyethylene glycol disuccinimide succinate (i.e., SS-PEG-SS) solution, and 0.1-0.4 parts of tannic acid (i.e., TA);

[0010] Furthermore, the polyethylene glycol derivative-collagen cartilage repair gel, by weight, is composed of 10 parts COL solution, 1 part PDRN, 5 parts SS-PEG-SS solution and 0.2 parts TA;

[0011] Furthermore, in step 1), the mass concentration of COL is 20~150 mg / mL, preferably 100 mg / mL;

[0012] Furthermore, in step 2), the mass concentration of SS-PEG-SS is 10~75 mg / mL, preferably 50 mg / mL;

[0013] A second aspect of the present invention provides a method for preparing any of the above-mentioned polyethylene glycol derivative-collagen cartilage repair gels, comprising the following preparation methods:

[0014] 1) Add COL and PDRN to PBS according to the above weight proportions, stir until fully dissolved, and obtain COL / PDRN solution;

[0015] 2) Add SS-PEG-SS and TA to PBS according to the above weight proportions, and sonicate to dissolve completely to obtain SS-PEG-SS / TA solution;

[0016] 3) Using parallel syringes, COL / PDRN solution and SS-PEG-SS / TA solution are mixed evenly and allowed to stand for cross-linking to obtain COL / PDRN-PEG / TA gel;

[0017] Preferably, in step 1), COL is selected from COL-I;

[0018] Preferably, in step 1), the mass concentration of COL is 20~150 mg / mL, more preferably 100 mg / mL;

[0019] Preferably, in step 1), the mass concentration of PDRN is 5~15 mg / mL, more preferably 10 mg / mL.

[0020] Preferably, in step 1), the COL and PDRN are sterilized by irradiation beforehand;

[0021] Preferably, in step 2), the mass concentration of SS-PEG-SS is 10~75 mg / mL, more preferably 50 mg / mL.

[0022] Preferably, in step 2), the mass concentration of TA is 1~3 mg / mL, more preferably 2 mg / mL;

[0023] Preferably, step 2) further includes filtration sterilization of the SS-PEG-SS / TA solution, preferably at a depth of 0.22 μm;

[0024] Preferably, in step 3), the mixing volume ratio of the COL / PDRN solution to the SS-PEG-SS / TA solution is 1:1~2; preferably, in step 3), the mixing volume ratio of the COL / PDRN solution to the SS-PEG-SS / TA solution is 1:1.

[0025] Preferably, in step 3), the crosslinking reaction temperature is 25~37℃, more preferably 37℃;

[0026] A third aspect of the present invention provides the use of any of the above-mentioned polyethylene glycol derivative-collagen cartilage repair gels in the preparation of tissue filler materials;

[0027] Furthermore, the tissue filling material has a cartilage repair function;

[0028] The present invention has the following beneficial effects:

[0029] This invention prepares a polyethylene glycol derivative-collagen gel formulation loaded with polydeoxyribonucleotides and tannic acid by crosslinking polyethylene glycol disuccinimide succinate (SS-PEG-SS), collagen (COL), polydeoxyribonucleotides (PDRN), and tannic acid (TA). In this cartilage repair hydrogel, type I collagen serves as the structural building block, providing a biomimetic microenvironment for chondrocytes, promoting cell adhesion and proliferation, maintaining cell phenotype, and providing a scaffold for matrix regeneration. SS-PEG-SS acts as a key crosslinking agent in this gel system, undergoing a highly efficient amidation reaction with the amino groups of collagen, thereby constructing a stable three-dimensional structure for the entire gel network. PDRN, as an active signaling molecule, promotes chondrocyte proliferation and specific matrix synthesis, inhibits matrix degradation, and improves blood supply to the repair area. Tannic acid synergistically enhances the stability and mechanical properties of the hydrogel, and also has anti-inflammatory and antioxidant properties, reducing the risk of infection. All four components work together to promote cartilage repair. This gel possesses excellent biocompatibility and accelerates cell proliferation, which is beneficial for cartilage regeneration at defect sites. It is an innovative tissue engineering and regenerative medicine material. Furthermore, the injectable cartilage repair gel prepared from the two solutions using a parallel syringe offers advantages such as simple preparation, green cross-linking process, and excellent mechanical properties when used as a tissue engineering and regenerative medicine material. In summary, this composite gel forms a stable three-dimensional network through an efficient amidation reaction between SS-PEG-SS and collagen, successfully integrating the structural support of type I collagen, the tissue repair properties of PDRN, and the anti-inflammatory and antioxidant properties of tannins. This results in a comprehensive performance characterized by good structural stability, high mechanical strength, excellent biocompatibility, controllable degradation cycle, and excellent needle penetration, demonstrating broad application prospects with clear repair effects, significant synergistic effects of components, mature production process, and outstanding cost-effectiveness. Attached Figure Description

[0030] Figure 1 A schematic flowchart illustrating the preparation method of the COL / PDRN-PEG / TA cartilage repair gel of Example 9 of the present invention;

[0031] Figure 2 This diagram shows the two-component ratio screening of COL-PEG gels prepared in Examples 1-3 of this invention.

[0032] Figure 3 Images showing the COL concentration gradient of the COL-PEG gels prepared in Examples 1 and 4-7 of this invention;

[0033] Figure 4 Images showing the mechanical strength of COL-PEG gels with different COL concentrations prepared in Examples 1 and 4-7 of this invention;

[0034] Figure 5 Visual representations of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention;

[0035] Figure 6 This shows scanning electron microscope images of the COL-PEG, COL / PDRN-PEG and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention after lyophilization.

[0036] Figure 7 This shows the elemental mapping diagram of the COL / PDRN-PEG / TA cartilage repair gel prepared in Example 9 of this invention after lyophilization.

[0037] Figure 8 Cell viability diagrams of COL-PEG, COL / PDRN-PEG and COL / PDRN-PEG / TA cartilage repair gels prepared according to Examples 7-9 of the present invention are shown.

[0038] Figure 9 Cell viability staining of COL-PEG, COL / PDRN-PEG and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention;

[0039] Figure 10 This represents the cell proliferation data of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention;

[0040] Figure 11The images show cell migration experiments of COL-PEG, COL / PDRN-PEG and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention.

[0041] Figure 12 The images show the cartilage repair gels prepared in Examples 7-9 of this invention during the collection of materials for treating articular cartilage defects in rats.

[0042] Figure 13 The images show micro-CT scan reconstructions at week 4 and week 8 of rats treated with COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention to treat articular cartilage bone defects.

[0043] Figure 14 The images show H&E staining images of cartilage tissue in rats with articular cartilage defects treated with COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 of this invention at weeks 4 and 8. Detailed Implementation

[0044] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0045] (I) Implementation Examples

[0046] Example 1

[0047] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0048] Step S1: Add 30 mg COL to 0.5 mL PBS and stir until fully dissolved; add 15 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0049] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0050] Step S3: The mixed solution was allowed to stand at 37 °C for 3 min for crosslinking to obtain COL-PEG gel.

[0051] Example 2

[0052] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0053] Step S1: Add 30 mg COL to 0.5 mL PBS and stir until fully dissolved; add 30 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0054] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0055] Step S3: The mixed solution was allowed to stand at 37 °C for 6 min for crosslinking to obtain COL-PEG gel.

[0056] Example 3

[0057] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0058] Step S1: Add 30 mg COL to 0.5 mL PBS and stir until fully dissolved; add 60 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0059] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0060] Step S3: The mixed solution was allowed to stand at 37 °C for 10 min for crosslinking to obtain COL-PEG gel.

[0061] Example 4

[0062] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0063] Step S1: Add 10 mg COL to 0.5 mL PBS and stir until fully dissolved; add 5 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0064] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0065] Step S3: The mixed solution was allowed to stand at 37 °C for cross-linking, but no gel system was formed and no COL-PEG gel was obtained.

[0066] Example 5

[0067] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0068] Step S1: Add 20 mg COL to 0.5 mL PBS and stir until fully dissolved; add 10 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0069] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0070] Step S3: The mixed solution is allowed to stand at 37 °C for crosslinking to obtain COL-PEG gel, which has poor mechanical properties.

[0071] Example 6

[0072] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0073] Step S1: Add 40 mg COL to 0.5 mL PBS and stir until fully dissolved; add 20 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0074] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0075] Step S3: The mixed solution was allowed to stand at 37 °C for 10 min for crosslinking to obtain COL-PEG gel, which had poor mechanical properties.

[0076] Example 7

[0077] A method for preparing COL-PEG cartilage repair gel, the method comprising the following steps:

[0078] Step S1: Add 50 mg COL to 0.5 mL PBS and stir until fully dissolved; add 25 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0079] Step S2: Mix the above 0.5 mL COL solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0080] Step S3: The mixed solution was allowed to stand at 37 °C for 10 min for crosslinking to obtain COL-PEG gel.

[0081] Example 8

[0082] A method for preparing a COL / PDRN-PEG cartilage repair gel, the method comprising the following steps:

[0083] Step S1: Add 50 mg COL and 5 mg PDRN to 0.5 mL PBS and stir until fully dissolved; add 25 mg SS-PEG-SS to 0.5 mL PBS and sonicate until fully dissolved;

[0084] Step S2: Mix the above 0.5 mL COL / PDRN solution and 0.5 mL SS-PEG-SS solution at a volume ratio of 1:1 using a parallel syringe;

[0085] Step S3: The mixed solution was allowed to stand at 37 °C for 10 min for crosslinking to obtain COL / PDRN-PEG gel.

[0086] Example 9

[0087] A method for preparing a COL / PDRN-PEG / TA cartilage repair gel, the method comprising the following steps:

[0088] Step S1: Add 50 mg COL and 5 mg PDRN to 0.5 mL PBS and stir until fully dissolved; add 25 mg SS-PEG-SS and 1 mg TA to 0.5 mL PBS and sonicate until fully dissolved;

[0089] Step S2: Mix the above 0.5 mL COL / PDRN solution and 0.5 mL SS-PEG-SS / TA solution at a volume ratio of 1:1 using a parallel syringe;

[0090] Step S3: The mixed solution was allowed to stand at 37 °C for 10 min for crosslinking to obtain COL / PDRN-PEG / TA gel.

[0091] (II) Experimental Examples

[0092] 1. Performance analysis of COL / PDRN-PEG / TA cartilage repair gel:

[0093] Figure 2 A two-component ratio screening diagram of COL-PEG gel prepared according to Examples 1-3 of the present invention is shown. It can be seen from the diagram that when COL and SS-PEG-SS are mixed in a ratio of 2:1, the gel formation time is the shortest.

[0094] Figure 3 The images show the COL concentration gradient of the COL-PEG gels prepared according to Examples 1, 4-7 of the present invention. It can be seen from the images that the gel formation time is shortest when the COL concentration is 10%.

[0095] Figure 4 Images of the mechanical strength of COL-PEG gels with different COL concentrations prepared according to Examples 1, 4-7 of the present invention are shown. It can be seen from the images that when the COL concentration is 10%, the resulting gel has the highest mechanical strength, which meets the strength required after filling.

[0096] Figure 5 Visual illustrations of COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA injectable gels prepared according to Examples 7-9 of the present invention are shown. As can be seen from the figures, the gel is transparent without the addition of TA, and is white with the addition of TA.

[0097] Figure 6 Scanning electron microscope images of COL / PDRN-PEG / TA gels prepared according to Examples 7-9 of the present invention after lyophilization are shown. As can be seen from the images, the COL / PDRN-PEG / TA gels have a porous network structure with pore sizes of about 100-300 μm.

[0098] Figure 7 An elemental mapping diagram of the COL / PDRN-PEG / TA gel prepared according to Example 9 of the present invention is shown. It can be seen from the figure that it has a uniform distribution of P and S elements, which proves that the gel contains PDRN and SS-PEG-SS components.

[0099] 2. Good cell compatibility

[0100] 2.1 Cell Count

[0101] Bone marrow mesenchymal stem cells were seeded in 96-well cell culture plates and cell culture dishes, with approximately 4000 cells per well. After 24 hours, the cells were fully adhered to the culture dish. After another 24 hours, CCK-8 assay reagent was added to the 96-well plate, and the cell absorbance was measured using an ELISA reader to calculate the cell viability.

[0102] Figure 8 The cytotoxicity data of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA injectable gels prepared in Examples 7-9 are shown. As can be seen from the figures, the cell viability of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA injectable gels prepared in Examples 7-9 is approximately 100%, indicating good cell compatibility. Furthermore, the COL / PDRN-PEG / TA cartilage repair gel group prepared in Example 9 achieved a cell viability of approximately 110%, demonstrating a stronger promotion of cell growth and greater benefit for tissue regeneration and repair.

[0103] 2.2 Cell staining

[0104] Bone marrow mesenchymal stem cells were seeded into cell culture dishes, approximately 100,000 cells per well. After 24 hours, the cells were fully adhered to the dish. Extracts of COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels were then added for incubation. After 24 hours, the cells in the cell culture dishes were stained using a live / dead cell assay kit, with live cells stained green and dead cells stained red, to assess cell viability.

[0105] Figure 9 Images of live / dead cells of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 are shown. As can be seen from the images, the number of live cells in the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gel groups prepared in Examples 7-9 is consistent with that in the control group, and almost no dead cells are observed, showing good cell compatibility.

[0106] 3. The effect of promoting cell proliferation is more significant.

[0107] Bone marrow mesenchymal stem cells were seeded into 24-well cell culture plates, approximately 20,000 cells per well. After 24 hours, the cells were fully adherent. COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA chondrocyte repair gels were then added to the wells for co-culture. After 1, 3, and 5 days of co-culture, CCK-8 assay reagent was added to the 24-well plates, and cell absorbance was measured using a microplate reader to calculate cell viability.

[0108] Figure 10 The cell proliferation data of the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 are shown. As can be seen from the figure, the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 can effectively promote the proliferation of fibroblasts in vitro. Among them, the COL / PDRN-PEG / TA cartilage repair gel prepared in Example 9 has a more significant effect on promoting cell proliferation.

[0109] 4. Significantly promotes cell migration

[0110] Well-morphologically intact BMSCs were digested with trypsin at a concentration of 2.5 × 10⁻⁶. 4Cells were diluted to a density of 1 cell / mL using serum-free medium to form a cell suspension. 200 μL of the cell suspension was added to the upper Transwell chamber of a 24-well plate, and 500 μL of serum-containing medium was added to the lower chamber. The plates were incubated for 24 h and then stained with crystal violet. The cells were photographed and their migration rate was analyzed.

[0111] Figure 11 The images show crystal violet staining of BMSC cell migration after 24 h using the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9. As shown in the figures, compared to other groups, the COL / PDRN-PEG / TA group exhibited a significantly increased number of migrating cells, demonstrating that the COL / PDRN-PEG / TA cartilage repair gel can significantly promote cell migration.

[0112] 5. Animal experiments have shown that it promotes bone repair.

[0113] Six-week-old male Sprague-Dawley rats were selected and, after a week of acclimatization feeding, weighed and anesthetized with sodium pentobarbital. The left leg of the rat was shaved and disinfected with povidone-iodine. The skin and muscle at the knee joint cartilage were incised, creating a circular defect (depth: 1.5 mm, diameter: 2 mm) at the distal femoral trochlea. During this process, cold saline was continuously dripped into the defect using a syringe to prevent overheating and necrosis caused by drill friction. After hemostasis with sterile gauze, the COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared in Examples 7-9 were filled into the defect, ensuring the material adhered closely to the defect. The incised muscle and skin were sutured with sterile surgical sutures. No material was implanted in the control group. Immediately after surgery, the rats' right leg was injected intramuscularly with penicillin once daily for three consecutive days to prevent wound infection. The rats' vital signs were closely monitored until stable before being placed in cages.

[0114] Rats were euthanized four and eight weeks after implantation, respectively. The femurs of the rats were collected, placed in 4% paraformaldehyde, fixed at room temperature for 24 h, and then stored at 4 ℃ for subsequent experiments. Figure 12Tissue photographs of rats with knee cartilage defects treated with COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared according to Examples 7-9 of the present invention are shown at weeks 4 and 8. Compared with the control group (no gel was added, but all other conditions were the same), the defect sites in the COL / PDRN-PEG / TA group showed almost complete regeneration of new cartilage tissue with a relatively intact surface, indicating that COL / PDRN-PEG / TA can promote the repair of knee cartilage defects and accelerate cartilage regeneration.

[0115] Micro-CT analysis was performed on bone tissue at weeks 4 and 8. Figure 13 Three-dimensional micro-CT images of COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared according to Examples 7-9 of the present invention are shown. As can be seen from the figures, at week 8, all showed varying degrees of repair compared to week 4. The control group (without any gel added, but all other conditions were the same) showed slow repair, and defects were still visible. In contrast, the COL / PDRN-PEG / TA group showed intact cartilage tissue with uniform thickness and complete cortical bone morphology, indicating that COL / PDRN-PEG / TA has a significant therapeutic effect on knee cartilage defects and can accelerate cartilage regeneration.

[0116] Figure 14 The images show H&E staining of cartilage tissue from rats with knee cartilage defects treated with COL-PEG, COL / PDRN-PEG, and COL / PDRN-PEG / TA cartilage repair gels prepared according to Examples 7-9 of the present invention at weeks 4 and 8. Compared with the control group (no gel added, but all other conditions were the same), the COL / PDRN-PEG / TA group showed a certain tissue regeneration effect, with a significantly smaller defect area and the defect almost completely filled with newly formed cartilage tissue, indicating its excellent cartilage regeneration capacity. This demonstrates that COL / PDRN-PEG / TA has a significant therapeutic effect on knee cartilage defects and can promote cartilage tissue regeneration.

[0117] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various modifications and alterations without departing from the spirit and scope of the present invention, and all such modifications and alterations shall be within the protection scope of the present invention.

Claims

1. A polyethylene glycol derivative-collagen cartilage repair gel, characterized in that, By weight, it consists of 8-12 parts collagen (COL) solution, 0.5-2 parts polydeoxyribonucleotide (PDRN), 4-8 parts polyethylene glycol disuccinimide succinate (SS-PEG-SS) solution and 0.1-0.4 parts tannic acid (TA).

2. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 1, characterized in that, The polyethylene glycol derivative-collagen cartilage repair gel, by weight, consists of 10 parts COL solution, 1 part PDRN, 5 parts SS-PEG-SS solution and 0.2 parts TA.

3. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 1 or 2, characterized in that, In step 1), the mass concentration of COL is 20~150 mg / mL, preferably 100 mg / mL.

4. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 1, characterized in that, In step 2), the mass concentration of SS-PEG-SS is 10~75 mg / mL, preferably 50 mg / mL.

5. A method for preparing the polyethylene glycol derivative-collagen cartilage repair gel according to any one of claims 1 to 4, characterized in that, The preparation methods include the following: 1) Add COL and PDRN to PBS according to the above weight proportions, stir until fully dissolved, and obtain COL / PDRN solution; 2) Add SS-PEG-SS and TA to PBS according to the above weight proportions, and sonicate to dissolve completely to obtain SS-PEG-SS / TA solution; 3) Using parallel syringes, mix the COL / PDRN solution and SS-PEG-SS / TA solution evenly, allow to stand for cross-linking, and obtain COL / PDRN-PEG / TA gel.

6. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 1), COL is selected from COL-I.

7. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 1), the mass concentration of COL is 20~150 mg / mL, preferably 100 mg / mL.

8. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 1), the mass concentration of PDRN is 5~15 mg / mL, preferably 10 mg / mL.

9. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 1), the COL and PDRN are sterilized by irradiation beforehand.

10. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 2), the mass concentration of SS-PEG-SS is 10~75 mg / mL, preferably 50 mg / mL.

11. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 2), the mass concentration of TA is 1~3 mg / mL, preferably 2 mg / mL.

12. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, Step 2) also includes filtration sterilization of the SS-PEG-SS / TA solution, preferably at a depth of 0.22 μm.

13. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 3), the mixing volume ratio of the COL / PDRN solution to the SS-PEG-SS / TA solution is 1:1 to 2; preferably, in step 3), the mixing volume ratio of the COL / PDRN solution to the SS-PEG-SS / TA solution is 1:

1.

14. The polyethylene glycol derivative-collagen cartilage repair gel according to claim 5, characterized in that, In step 3), the crosslinking reaction temperature is 25~37℃, preferably 37℃.

15. The use of the polyethylene glycol derivative-collagen cartilage repair gel according to any one of claims 1 to 4 in the preparation of cartilage repair materials; further, the cartilage repair gel is a tissue regeneration medical material.

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