Cartilage tissue repair hydrogel and method of making same

Abstract

The application relates to the field of biological medicines, in particular to a cartilage tissue repair hydrogel and a preparation method thereof. The preparation components of the cartilage tissue repair hydrogel are doped with 10-15 g of mulberry silk, 0.1-0.5 g of glycidyl methacrylate, 0-1 g of lithium magnesium silicate, 0-1 g of tannic acid and 0.1 g of a photo initiator per 100 ml of the hydrogel. The preparation method of the cartilage tissue repair hydrogel is as follows: degumming and dissolving treatment are conducted on the mulberry silk to obtain a silk fibroin solution, glycidyl methacrylate is added into the silk fibroin solution and stirring is conducted to obtain a methacrylated silk fibroin solution, lithium magnesium silicate solution and tannic acid solution are added into the methacrylated silk fibroin solution to obtain a silk fibroin mixed solution, and then the photo initiator is added into the silk fibroin mixed solution to prepare the cartilage tissue repair hydrogel. The tissue repair hydrogel has a hydrophilic three-dimensional network structure, can provide mechanical support and biological induction activity for a bone cartilage defect site, and is suitable for bone injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a cartilage tissue repair hydrogel and a preparation method thereof. Background Art

[0002] Musculoskeletal and joint diseases have garnered significant attention from the World Health Organization over the past decade, impacting the health of tens of millions of people. However, effective treatment options for osteochondral damage are currently lacking. Osteochondral damage involves two tissues (cartilage and subchondral bone) with distinct characteristics and regenerative capacities. During repair, a lack of tight connection and asynchronous repair rates between the two tissues can alter joint biomechanics, impacting the long-term performance of the cartilage. Therefore, repairing osteochondral defects presents significant challenges.

[0003] Currently, common clinical treatments for osteochondral injuries, such as joint cavity lavage and debridement, microfracture, and other procedures, have poor therapeutic effects. While these procedures aim to alleviate joint pain and symptoms, they are ineffective in promoting the repair and regeneration of osteochondral tissue and are limited by factors such as donor availability and immune rejection. Therefore, developing effective methods to promote the repair of osteochondral injuries is of great clinical significance and application value.

[0004] In recent years, advances in tissue engineering and regenerative medicine have offered new hope for the repair and regeneration of osteochondral defects. Finding suitable tissue engineering scaffolds is crucial for the repair of osteochondral defects. Gelatin methacrylate (GelMA) hydrogels, composed of photocrosslinked methacrylic anhydride (MA) and skin-derived gelatin, have been widely used in tissue engineering due to their cell adhesion, excellent biocompatibility, and highly tunable mechanical properties. However, there is a lack of GelMA hydrogels with multifunctional properties that can provide a supportive microenvironment for osteochondral regeneration.

[0005] Tannic acid (TA), a US Food and Drug Administration (FDA)-approved drug, has recently been widely used in the biomedical field for its antioxidant, antimicrobial, and anti-inflammatory properties. TA is a polyphenolic compound originating from various natural plants. Its abundant phenolic hydroxyl groups impart a redox potential to TA, promoting its binding to substrates through oxidation-induced oligomerization, forming a composite hydrogel. Within the hydrogel, TA can scavenge reactive oxygen species and enhance the survival of bone marrow mesenchymal stem cells in response to oxidative stress.

[0006] To promote the regeneration of OC defects, osteogenic and chondrogenic factors are often introduced to enhance the bilineage differentiation of bone marrow mesenchymal stem cells. Synthetic lithium magnesium silicate (laponite) is a two-dimensional disc-shaped silicate nanoclay with anisotropic charge distribution. It can be used as a crosslinker and rheology modifier for hydrogels and has drug delivery capabilities. Notably, previous studies have confirmed that lithium magnesium silicate has bilineage osteochondral bioactivity both in vitro and in vivo, and hydrogels prepared from it can enhance the integrated osteochondral regeneration of patients with full-thickness osteochondral defects. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention proposes a cartilage tissue repair hydrogel and a preparation method thereof. The cartilage tissue repair hydrogel is prepared by the cross-linking reaction of lithium magnesium silicate and tannic acid with silk fibroin. The prepared cartilage tissue repair hydrogel can promote the bidirectional differentiation of endogenous bone marrow mesenchymal stem cells into osteoblasts and chondrocytes, effectively promoting the repair and regeneration of osteochondral defects.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A cartilage tissue repair hydrogel is prepared by doping 100 ml of the hydrogel with the following ingredients: 10-15 g of mulberry silk, 0.1-0.5 g of glycidyl methacrylate, 0-1 g of lithium magnesium silicate, 0-1 g of tannic acid, and 0.1 g of a photoinitiator.

[0010] Preferably, the concentration of glycidyl methacrylate is 100-1000 mM.

[0011] Preferably, the concentration of the lithium magnesium silicate solution is 0-1% (w / v).

[0012] Preferably, the concentration of the tannic acid solution is 0-1% (w / v).

[0013] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and the concentration of the photoinitiator is 0.1% (w / v).

[0014] Preferably, the preparation method of the cartilage tissue repair hydrogel is:

[0015] First, mulberry silk is degummed and dissolved to obtain a silk fibroin solution. Glycidyl methacrylate is added to the silk fibroin solution and stirred to obtain a methacrylated silk fibroin solution. Then, a lithium magnesium silicate solution and a tannic acid solution are added to the methacrylated silk fibroin solution to obtain a silk fibroin mixed solution. Then, a photoinitiator is added to the silk fibroin mixed solution and a photocrosslinking reaction is performed to solidify the silk fibroin mixed solution, thereby finally obtaining a cartilage tissue repair hydrogel.

[0016] The photo-crosslinking reaction is carried out by irradiating ultraviolet light, and the wavelength of the ultraviolet light is 200-400nm.

[0017] Preferably, the specific method of the degumming treatment is: placing the mulberry silk in a sodium carbonate aqueous solution, water bathing at 100°C for 30 minutes, and then using pure water for oscillation washing to remove the sericin in the mulberry silk, leaving the fibroin in the mulberry silk, and then drying the fibroin for later use.

[0018] Preferably, the specific method of the dissolution treatment is to dissolve the dried silk fibroin in a lithium bromide aqueous solution, heat it at 60°C for 1 hour until the silk fibroin is dissolved, and obtain a mixed solution containing silk fibroin and a small amount of insoluble particles, and then dialyze the mixed solution to remove the insoluble particles and lithium bromide ions in the mixed solution.

[0019] Preferably, the cartilage tissue repair hydrogel is prepared by the preparation method of the cartilage tissue repair hydrogel.

[0020] Preferably, the cartilage tissue repair hydrogel or the cartilage tissue repair hydrogel prepared by the cartilage tissue repair hydrogel preparation method can be used to prepare osteochondral loss repair material.

[0021] Beneficial effects of the present invention:

[0022] 1. The tissue repair hydrogel prepared by the cross-linking reaction of lithium magnesium silicate, tannic acid and silk fibroin of the present invention is a hydrophilic three-dimensional network structure gel that can provide mechanical support and biological induction activity for the osteochondral defect site and is suitable for the repair of osteochondral damage.

[0023] 2. The preparation process of the present invention is simple, and the biological activity of lithium magnesium silicate and tannic acid is guaranteed to the greatest extent. Lithium magnesium silicate and tannic acid have good biocompatibility and can effectively promote the bidirectional differentiation of bone marrow mesenchymal stem cells into osteoblasts and chondrocytes. Silk fibroin hydrogel can effectively carry lithium magnesium silicate and tannic acid and provide mechanical support for the defect site, thereby promoting the repair and regeneration of osteochondral damage sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 is a general morphological diagram of the tissue repair hydrogel having osteochondrogenic induction activity in Example 1 of the present invention;

[0027] Figure 2 This is an electron microscopic photograph of the tissue repair hydrogel with osteochondrogenic induction activity in Example 1 of the present invention;

[0028] Figure 3 is a graph showing the rheological properties of the tissue repair hydrogel having osteochondrogenic induction activity in Example 1 of the present invention;

[0029] Figure 4 This is a picture of the gross morphology of rat osteochondral damage repaired 8 weeks after Example 2 of the present invention;

[0030] Figure 5 This is a SO staining picture of rat osteochondral damage repair 8 weeks after Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1

[0033] A tissue repair hydrogel of pure silk fibroin is prepared by photocrosslinking of silk fibroin, and the preparation method thereof comprises the following steps:

[0034] (1) Preparation of methacrylated silk fibroin solution:

[0035] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0036] b) Dissolution: The dried silk fibroin was dissolved in 9.3M lithium bromide aqueous solution at 20% (w / v) and heated at 60°C for 1 hour until the silk fibroin was dissolved to obtain a mixture containing silk fibroin and a small amount of insoluble particles.

[0037] c) Methacrylate esterification: 424 mM glycidyl methacrylate was added to the above mixture, and the mixture was heated and stirred at 60°C and 300 rpm for 3 hours until the mixture was uniformly mixed;

[0038] d) Dialysis: The mixed solution was dialyzed with 5, and then dialyzed 6 times in 3 days with 10 times the volume of sterile pure water to remove lithium bromide ions in the solution and obtain a retentate;

[0039] (2) Preparation of a tissue repair hydrogel of pure silk fibroin: The high-concentration methacrylated silk fibroin solution prepared by the method (1) was diluted with sterile pure water to a solution with a final concentration of 10% (w / v). 1000 μl of the above silk fibroin solution was placed in a 1.5 ml EP tube, and a photoinitiator LAP was added to the solution with a final concentration of 0.2% (w / v). Mix well and irradiate under a 365 nm ultraviolet lamp for 10 seconds. Place in an oven and heat at 37°C for 60 minutes to obtain a tissue repair hydrogel of pure silk fibroin.

[0040] Example 2

[0041] A tissue repair hydrogel with antioxidant stress activation is prepared by photocrosslinking of silk fibroin and tannic acid. The preparation method comprises the following steps:

[0042] (1) Preparation of methacrylated silk fibroin solution:

[0043] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0044] b) Dissolution: The dried silk fibroin was dissolved in 9.3M lithium bromide aqueous solution at 20% (w / v) and heated at 60°C for 1 hour until the silk fibroin was dissolved to obtain a mixture containing silk fibroin and a small amount of insoluble particles.

[0045] c) Methacrylate esterification: 424 mM glycidyl methacrylate was added to the above mixture, and the mixture was heated and stirred at 60°C and 300 rpm for 3 hours until the mixture was uniformly mixed;

[0046] d) Dialysis: The mixed solution was dialyzed with 5, and then dialyzed 6 times in 3 days with 10 times the volume of sterile pure water to remove lithium bromide ions in the solution and obtain a retentate;

[0047] e) freeze-drying the retentate at -45°C and 20 Pa for 48 hours and storing at -80°C for later use;

[0048] (2) Tannic acid was diluted with sterile water to a storage solution with a concentration of 10 mg / ml and stored at 4°C.

[0049] (3) A tissue repair hydrogel with antioxidant stress activation: The high-concentration methacrylated silk fibroin solution prepared by method (1) is diluted with sterile pure water to a solution with a final concentration of 10% (w / v). 900 μl of the above silk fibroin solution is placed in a 1.5 ml EP tube, and 100 μl of the tannic acid solution prepared by method (2) is added to obtain a silk fibroin mixed solution with a final tannic acid concentration of 1% (w / v). A photoinitiator LAP is added to the solution with a final concentration of 0.2% (w / v). Mix well and irradiate under a UV lamp with a wavelength of 365 nm for 10 seconds. Place in an oven and heat at 37°C for 60 minutes to obtain a silk fibroin hydrogel with a composite tannic acid, i.e., a tissue repair hydrogel with antioxidant stress activation.

[0050] Example 3

[0051] A tissue repair hydrogel with osteochondrogenic activity is prepared by photocrosslinking of silk fibroin, lithium magnesium silicate and tannic acid. The preparation method comprises the following steps:

[0052] (1) Preparation of methacrylated silk fibroin solution:

[0053] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0054] b) Dissolution: The dried silk fibroin was dissolved in 9.3M lithium bromide aqueous solution at 20% (w / v) and heated at 60°C for 1 hour until the silk fibroin was dissolved to obtain a mixture containing silk fibroin and a small amount of insoluble particles.

[0055] c) Methacrylate esterification: 424 mM glycidyl methacrylate was added to the above mixture, and the mixture was heated and stirred at 60°C and 300 rpm for 3 hours until the mixture was uniformly mixed;

[0056] d) Dialysis: The mixed solution was dialyzed with 5, and then dialyzed 6 times in 3 days with 10 times the volume of sterile pure water to remove lithium bromide ions in the solution and obtain a retentate;

[0057] e) freeze-drying the retentate at -45°C and 20 Pa for 48 hours and storing at -80°C for later use;

[0058] (2) Lithium magnesium silicate was diluted with sterile pure water to a storage solution with a concentration of 40 mg / ml and stored at 4°C.

[0059] (3) Tannic acid was diluted with sterile pure water to a storage solution with a concentration of 10 mg / ml and stored at 4°C.

[0060] (4) Preparation of a tissue repair hydrogel with osteochondrogenic activity: The high-concentration methacrylated silk protein solution prepared by method (1) was diluted with sterile pure water to a solution with a final concentration of 10% (w / v). 875 μl of the above silk protein solution was placed in a 1.5 ml EP tube, and 25 μl of lithium magnesium silicate prepared by method (2) and 100 μl of tannic acid solution prepared by method (3) were added respectively to obtain a silk protein mixed solution with a final concentration of lithium magnesium silicate and tannic acid of 1% (w / v). A photoinitiator LAP was added to the solution with a final concentration of 0.2% (w / v). Mix well and irradiate under a UV lamp with a wavelength of 365 nm for 10 seconds. Place in an oven and heat at 37°C for 60 minutes to obtain a silk protein hydrogel with a composite of lithium magnesium silicate and tannic acid, i.e., a tissue repair hydrogel with osteochondrogenic activity ( Figure 1 The freeze-dried silk fibroin hydrogel showed a porous three-dimensional network structure under an electron microscope ( Figure 2 Compared with the traditional photo-crosslinked silk fibroin hydrogel, the tissue repair hydrogel with osteochondrogenic activity prepared by the present invention has significantly improved rheological properties after photo-crosslinking reaction ( Figure 3 ), which can better meet the mechanical performance requirements of osteochondral damage repair.

[0061] Example 4

[0062] A tissue repair hydrogel with osteochondrogenic activity is prepared by cross-linking of 0-dimensional carbon nanomaterials lithium magnesium silicate and tannic acid with silk fibroin. The application method thereof comprises the following steps:

[0063] (1) Osteochondral injury modeling: 6-8 week old male SD rats (weighing 200-220 g) were selected. After general anesthesia, a longitudinal incision of about 1 cm was made on the right side of the patella. The joint capsule was cut from the medial side of the patella and pushed outward to dislocate the patella. The femoral condyle trochlea was exposed, and a sternal puncture needle (1 mm in diameter) was used to create an osteochondral defect of 1 mm in diameter and 1 mm in depth in the center of the trochlea.

[0064] (2) Application of a silk fibroin hydrogel composited with lithium magnesium silicate and tannic acid: A tissue repair hydrogel with osteochondrogenic activity from Example 1 was filled into the osteochondral lesion created in (1), with 50 μl per wound. After modeling, the patella was repositioned and sutured layer by layer. Postoperatively, the rats were fed a standard animal feed and allowed to move freely.

[0065] (3) Evaluation of repair effect: The animals were killed 8 weeks after surgery and samples were collected for gross and histological evaluation. Figure 4 a) and pure silk fibroin hydrogel group ( Figure 4 b) Compared with the silk fibroin hydrogel group ( Figure 4 c) It can significantly promote the repair of osteochondral defects, and the surface cartilage of the defect site is well repaired; the results of histological sections show that the silk fibroin hydrogel composited with lithium magnesium silicate and tannic acid can promote the repair and regeneration of the surface cartilage and subchondral bone at the site of osteochondral injury, and the cell morphology, matrix secretion, and osteochondral composition are close to those of natural osteochondral tissue ( Figure 5 ).

[0066] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0067] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A cartilage tissue repair hydrogel, characterized in that: The cartilage tissue repair hydrogel is prepared by doping 100 ml of the hydrogel with the following ingredients: 10-15 g of mulberry silk, 0.1-0.5 g of glycidyl methacrylate, 0-1 g of lithium magnesium silicate, 0-1 g of tannic acid, and 0.1 g of a photoinitiator, wherein the lithium magnesium silicate is not 0 g, and the tannic acid is not 0 g; The preparation method of the cartilage tissue repair hydrogel is as follows: First, mulberry silk is degummed and dissolved to obtain a silk fibroin solution. Glycidyl methacrylate is added to the silk fibroin solution and stirred to obtain a methacrylated silk fibroin solution. Then, a lithium magnesium silicate solution and a tannic acid solution are added to the methacrylated silk fibroin solution to obtain a silk fibroin mixed solution. Then, a photoinitiator is added to the silk fibroin mixed solution and a photocrosslinking reaction is performed to solidify the silk fibroin mixed solution, thereby finally obtaining a cartilage tissue repair hydrogel. The photo-crosslinking reaction is carried out by irradiating ultraviolet light, and the wavelength of the ultraviolet light is 200-400nm.

2. The cartilage tissue repair hydrogel according to claim 1, characterized in that: The concentration of glycidyl methacrylate is 100-1000 mM.

3. The cartilage tissue repair hydrogel according to claim 1, characterized in that: The concentration of the lithium magnesium silicate solution is 0-1% (w / v), and the concentration of the lithium magnesium silicate solution is not 0.

4. The cartilage tissue repair hydrogel according to claim 1, characterized in that: The concentration of the tannic acid solution is 0-1% (w / v), and the concentration of the tannic acid solution is not zero.

5. The cartilage tissue repair hydrogel according to claim 1, characterized in that: The photoinitiator is 2,4,6-trimethylbenzoylphenylphosphonic acid lithium salt, and the concentration of the photoinitiator is 0.1% (w / v).

6. The cartilage tissue repair hydrogel according to claim 1, characterized in that: The specific method of the degumming treatment is: putting the mulberry silk into a sodium carbonate aqueous solution, water bathing at 100°C for 30 minutes, and then using pure water to shake and wash to remove the sericin in the mulberry silk, leaving the fibroin in the mulberry silk, and then drying the fibroin for later use.

7. The cartilage tissue repair hydrogel according to claim 1, characterized in that The specific method of the dissolution treatment is to dissolve the dried silk fibroin in a lithium bromide aqueous solution, heat it at 60°C for 1 hour until the silk fibroin is dissolved, and obtain a mixed solution containing silk fibroin and a small amount of insoluble particles, and then dialyze the mixed solution to remove the insoluble particles and lithium bromide ions in the mixed solution.

8. Use of the cartilage tissue repair hydrogel according to any one of claims 1 to 7 for preparing a material for repairing osteochondral loss.

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