Uses of BSP-MA / HA-NB hydrogel in the preparation of drugs for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage.

Abstract

This invention belongs to the biomedical field, specifically relating to the use of BSP-MA / HA-NB hydrogel in the preparation of drugs for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage. This invention introduces HA-NB (hyaluronic acid modified with o-nitrosobenzaldehyde) into BSP-MA (methacrylated Bletilla striata polysaccharide) to prepare BSP-MA / HA-NB hydrogel, which exhibits a significant synergistic effect in promoting cartilage and subchondral bone regeneration. Simultaneously, it can reduce inflammatory responses and promote tissue repair by inhibiting the activation of M1 macrophages and promoting the increase of M2 macrophages. It can be used to treat cartilage damage and / or degenerative diseases of articular cartilage, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of BSP-MA / HA-NB hydrogel in the preparation of drugs for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage. Background Technology

[0002] Articular cartilage is a specialized soft tissue composed of chondrocytes and extracellular matrix (ECM) located on the surface of bone joints. It plays a crucial role in maintaining lubrication and joint movement. Damage to articular cartilage due to trauma, congenital abnormalities, and disease often leads to joint pain and impaired motor function. Degenerative articular cartilage diseases refer to the gradual degeneration and damage of cartilage due to natural aging, chronic inflammation, or metabolic abnormalities; the most common type is osteoarthritis. Cartilage lesions and degenerative articular cartilage diseases are common skeletal system disorders, primarily characterized by cartilage damage and degeneration, accompanied by inflammatory responses and changes in bone tissue. Current treatment strategies for articular cartilage lesions mainly rely on surgical treatments such as microfractures, drilling, and autologous chondrocyte transplantation. However, these strategies often have limitations, including trauma, inefficiency, and postoperative fibrocartilage formation, which may impair joint function. Compared to the above strategies, biomedical hydrogels can provide an ECM-like environment similar to natural cartilage.

[0003] Hydrogels are three-dimensional hydrophilic polymer networks containing water in their matrix, capable of loading cells, drugs, and bioactive molecules while maintaining their activity; they have been widely used in cartilage injury repair. Hyaluronic acid (HA) is a polysaccharide composed of alternating d-galacturonic acid and N-acetylglucosamine, naturally present in cartilage and synovial fluid. Compared to other polysaccharides, HA influences the regulation of cartilage function and the repair of cartilage damage in multiple ways. Previous studies have demonstrated that HA can improve the lubricity of the cartilage interface, modulate inflammation at the site of cartilage injury, promote cell adhesion and proliferation, and improve cartilage ECM deposition and cartilage regeneration. Furthermore, functionalization of HA-based hydrogels can enhance the adhesion, proliferation, and cartilage differentiation of encapsulated stem cells and chondrocytes, modulate the inflammatory microenvironment at the site of cartilage injury, and promote cartilage ECM deposition.

[0004] Bletilla striata polysaccharide (BSP) is a glucomannan extracted and isolated from Bletilla striata. It has anti-inflammatory, antioxidant, and anti-tumor effects. Modern pharmacological experiments have shown that BSP has good biocompatibility and possesses various biological activities such as antioxidant, anti-tumor, anti-aging, immunomodulatory, and wound-healing promotion. It can also be used to treat osteoarthritis or degenerative changes in articular cartilage.

[0005] Due to the unique tissue structure of articular cartilage, its repair and treatment after injury present significant limitations and challenges. While existing treatments can alleviate symptoms to some extent, most drugs only relieve pain and inflammation, failing to repair cartilage, and often suffer from numerous side effects and low patient compliance. Therefore, there is an urgent need to develop a new drug that can promote cartilage repair and regeneration to meet the requirements of cartilage tissue repair. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides the use of BSP-MA / HA-NB hydrogel in the preparation of medicaments for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage.

[0007] This invention provides the use of BSP-MA / HA-NB hydrogel in the preparation of medicaments for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage; the BSP-MA / HA-NB hydrogel comprises the following raw materials in parts by weight:

[0008] 5-15 parts of methacrylated Bletilla striata polysaccharide and 2.5-10 parts of hyaluronic acid modified with o-nitrosobenzaldehyde.

[0009] Preferably, the BSP-MA / HA-NB hydrogel comprises the following raw materials in parts by weight:

[0010] 10 parts of methacrylated Bletilla striata polysaccharide and 5 parts of hyaluronic acid modified with o-nitrosobenzaldehyde.

[0011] Preferably, the preparation method of the BSP-MA / HA-NB hydrogel includes the following steps:

[0012] Step 1: Hyaluronic acid modified with o-nitrosobenzaldehyde and methacrylamide-modified Bletilla striata polysaccharide are added to a buffer solution to obtain a mixed solution;

[0013] Step 2: React the mixed solution with the photoinitiator and cure it to obtain the final product.

[0014] Preferably, the mass fraction of methacrylamide-modified Bletilla striata polysaccharide in the BSP-MA / HA-NB hydrogel is 5-15%, and the mass fraction of hyaluronic acid modified with o-nitrosobenzaldehyde is 2.5-10%.

[0015] And / or, in step 2, the photoinitiator is selected from at least one of phenyl-2,4,6-trimethylbenzoylphosphine lithium, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, 1-hydroxycyclohexylphenyl ketone, 2-hydroxy-2-methyl-1-phenylpropanone, 2-methyl-1-[4-methylthiophenyl]-2-morpholino-1-propanone, and 2-isopropylthioxanthraphenone.

[0016] And / or, in step 2, the curing is ultraviolet curing, and the curing conditions are: the wavelength of the ultraviolet lamp is 360-405nm, and the light intensity of the ultraviolet light is 0.5-2W / cm². 2 The irradiation time is 8-30 seconds.

[0017] Preferably, the methacrylated Bletilla striata polysaccharide is prepared by reacting Bletilla striata polysaccharide with methacrylic anhydride; the mass ratio of Bletilla striata polysaccharide to methacrylic anhydride is 1.5-2.5:1.5-2.5, and the molecular weight of the methacrylated Bletilla striata polysaccharide is ≥3500 Da;

[0018] And / or, the o-nitrosobenzaldehyde-modified hyaluronic acid is prepared by reacting hyaluronic acid with an o-nitrosobenzaldehyde oxidizing agent; the molar ratio of hyaluronic acid to o-nitrosobenzaldehyde oxidizing agent is 1:(0.5-1).

[0019] Preferably, the pH of the reaction between the Bletilla striata polysaccharide and methacrylic anhydride is 8-9, and the temperature is 20-30℃.

[0020] And / or, the reaction of the Bletilla striata polysaccharide with methacrylic anhydride is carried out under light protection and the stirring frequency is 500±50 rpm;

[0021] The molar ratio of the disaccharide units of the hyaluronic acid to the o-nitrosobenzaldehyde oxidizing agent is 1:0.5-1;

[0022] And / or, the structural formula of the o-nitrosobenzaldehyde oxidizing agent is shown in Formula I:

[0023]

[0024] Among them, R1 and R2 are independently selected from hydrogen and C1-C, respectively. 10 Alkyl group, n is selected from 1-5;

[0025] And / or, the reaction of the hyaluronic acid with the o-nitrosobenzaldehyde reagent is carried out under the action of an activator and a coupling agent. The activator is selected from 1-(3-dimethylaminopropyl)-3-ethylcarboxylic acid diamine hydrochloride, dicyclohexylcarbodiimide, and diisopropylcarbodiimide. The coupling agent is selected from N-hydroxybenzotriazole, benzotriazole-1-oxytripyrrolylurea, and benzotriazole-1-oxytriphenylmethylurea. The reaction temperature is 20-30℃, the reaction time is 36-72h, and the reaction pH is 4-5.

[0026] Preferably, in Formula I, R1 is selected from hydrogen and C1 alkyl; R2 is selected from C1 alkyl, and n is 3.

[0027] Preferably, R1 is selected from hydrogen.

[0028] Preferably, the articular cartilage degenerative disease is at least one of osteoarthritis, cartilage damage, rheumatoid arthritis, and bone hyperplasia.

[0029] Preferably, the drug promotes cartilage formation.

[0030] Preferably, the drug can inhibit the inflammatory response;

[0031] And / or, the drug can reduce the degradation of cartilage matrix.

[0032] This invention also provides a pharmaceutical composition for treating bone defects, cartilage damage, or degenerative diseases of articular cartilage. It is a formulation prepared using BSP-MA / HA-NB hydrogel as the active ingredient and pharmaceutically acceptable excipients. The BSP-MA / HA-NB hydrogel comprises the following raw materials in parts by weight:

[0033] 5-15 parts of methacrylated Bletilla striata polysaccharide and 2.5-10 parts of hyaluronic acid modified with o-nitrosobenzaldehyde.

[0034] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0035] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0036] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～b Alkyl indicates any alkyl group containing one to two carbon atoms ("a" to "b"). Therefore, for example, "C..." 1～4 "Alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0037] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 member atoms, such as 1 to 4 member atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may optionally be substituted by one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

[0038] In this invention, "o-nitrosobenzaldehyde reagent" refers to a reagent containing at least one A compound containing a group and at least one amino group.

[0039] A "disaccharide unit" refers to the smallest repeating sugar unit in a polysaccharide molecule. Hyaluronic acid (HA) is a linear polysaccharide composed of alternating N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA). These two monosaccharide units together constitute the disaccharide unit of hyaluronic acid.

[0040] This invention introduces HA-NB (hyaluronic acid modified with o-nitrosobenzaldehyde) into BSP-MA (methacrylated Bletilla striata polysaccharide) to prepare BSP-MA / HA-NB hydrogel. It exhibits a significant synergistic effect in promoting the regeneration of cartilage and subchondral bone. At the same time, it can help reduce the inflammatory response and promote tissue repair by inhibiting the activation of M1 macrophages and promoting the increase of M2 macrophages. It can be used to treat osteochondral injuries and / or degenerative diseases of articular cartilage and has broad application prospects.

[0041] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0042] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0043] Figure 1 (a) Fluorescence detection of intracellular reactive oxygen species levels in cells treated with BSP-MA / HA-NB hydrogels; (b) TUNEL staining of oxidative stress bone marrow mesenchymal stem cells showed differences in the degree of DNA breakage (scale bar: 100 μm).

[0044] Figure 2 (c) The migration capacity of bone marrow mesenchymal stem cells was assessed at 0, 12 and 24 hours after treatment (scale bar: 100 μm); (d) The proliferation capacity of bone marrow mesenchymal stem cells in different treatment groups was assessed on days 0, 1, 3, 5 and 7 after intervention; (e) The migration area of ​​bone marrow mesenchymal stem cells in different treatment groups.

[0045] Figure 3 The expression of target genes (Col2A1, SOX9, ACAN, IL-1β, TNF-α, MMP13) in each group was detected by real-time quantitative PCR.

[0046] Figure 4 (a) Micro-CT three-dimensional reconstructed images, transverse and sagittal two-dimensional projection images of each group at 12 weeks postoperatively; (be) Quantitative analysis of Micro-CT parameters of the defect area at 6 and 12 weeks postoperatively (bone volume fraction BV / TV, bone mineral density BMD, trabecular thickness Tb.Th, trabecular spacing Tb.Sp) (n=6); one-way ANOVA was used to determine statistical significance.

[0047] Figure 5 (a) H&E staining of osteochondral repair tissue at week 6 post-operation (scale bar: 400 μm); (b) SO staining of osteochondral repair tissue at week 6 post-operation (scale bar: 400 μm); (c) TB staining of osteochondral repair tissue at week 6 post-operation (scale bar: 400 μm); (d) Distribution of M2 macrophages in regenerated osteochondral tissue as shown by co-localization of CD206 (green) and CD68 (red) fluorescence at week 6 post-implantation (scale bar: 400 μm for 4X images, 50 μm for others); (e) and (f) Immunohistochemical staining of ADAMTS5 and MMP13 in regenerated osteochondral tissue at week 6 post-implantation (scale bar: 400 μm). Statistical significance was determined by one-way ANOVA, P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001. Detailed Implementation

[0048] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0049] Example 1: Preparation of BSP-MA / HA-NB hydrogel

[0050] I. Synthesis of Methacrylated Bletilla striata Polysaccharide (BSP-MA)

[0051] 1. Place 200 mL of 1×PBS buffer solution into a 500 mL Spherical flask, start the magnetic stirrer and set the stirring speed to 500±50 rpm. Then, add 2 g of BSP (Bletilla striata polysaccharide, S27914, Yuanye Biotechnology, 70% purity) into the Spherical flask and continue stirring at room temperature until the BSP is completely dissolved.

[0052] 2. To avoid light interference with the reaction, except for the material addition area, the spherical bottle should be completely wrapped with aluminum foil.

[0053] 3. Turn on the stirrer at a frequency of 500±50 rpm. Using a 1 mL pipette, accurately measure 2 mL of methacrylic anhydride (Aladdin, M102519, ≥94%, containing 0.2% Topanol as a stabilizer) and add it dropwise to the 500 mL spherical flask. The dropping rate should be controlled at 1 mL / min. After the addition is complete, begin adding a 3 mol / L NaOH solution to adjust the pH of the reaction system to the range of 8-9. The dropping rate should be controlled at 1 mL / min. The specific dropping process is as follows: the first drop is 3 mL, and the reaction is stirred for 15 min after the first drop is completed; then the second drop is 2 mL, and the reaction is stirred for 15 min after the second drop is completed; finally, the third drop is approximately 1 mL.

[0054] 4. Transfer the reacted solution to a dialysis bag with a molecular weight cutoff of 3500 Da. Add 15 times the volume of pure water based on the volume of the reaction solution and dialyze for 15 days. Keep the dialysis process away from light.

[0055] 5. To avoid light interference, completely block out light from the freeze dryer with black plastic bags. Start the freeze dryer and wait until the internal temperature drops to -40℃. Place the pre-frozen product into the freeze dryer, turn on the vacuum system, and freeze dry under vacuum conditions for 48 hours. After the freeze drying is complete, remove the product.

[0056] II. Synthesis of hyaluronic acid o-nitrosobenzaldehyde (HA-NB)

[0057] 1. Step 1: Synthesis of methyl 4-(4-formyl-2-methoxyphenoxy)butyrate:

[0058] 4-Hydroxy-3-methoxybenzaldehyde (vanillin) (8.90 g, 58.5 mmol, 1.06 eq.), methyl 4-bromobutyrate (9.89 g, 55.0 mmol, 1.0 eq.), and potassium carbonate (10.2 g, 73.8 mmol, 1.34 eq.) were dissolved in N,N-dimethylformamide (DMF) (40 mL). The mixture was stirred at room temperature for 16 h, and the resulting solution was poured into chilled water (200 mL) and precipitated at 0 °C for 15 min. The solid was filtered off, washed with water, dissolved in dichloromethane, and dried over magnesium sulfate. The solvent was removed under reduced pressure to give a white solid, methyl 4-(4-formyl-2-methoxyphenoxy)butyrate.

[0059] 2. Second step: Synthesis of methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butyrate

[0060] Methyl 4-(4-formyl-2-methoxyphenoxy)butyrate (9.4 g, 37.3 mmol) was slowly added to a pre-cooled (-2 °C) nitric acid (70%, 140 mL) solution and stirred at -2 °C for 3 h. The resulting solution was poured into 500 mL of chilled water and precipitated at 0 °C for 15 min (Note: the precipitation time should be short because the product will saponify under these conditions). The product was filtered, washed with water, and dissolved in dichloromethane. The organic layer was dried on magnesium sulfate and the solvent was removed under reduced pressure to obtain a pale yellow powder, methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butyrate.

[0061] 3. Third step: Synthesizing mNB

[0062] Sodium borohydride (1.50 g, 39.7 mmol) was slowly added at 0 °C to a solution of methyl methyl-(4-formyl-2-methoxy-5-nitrophenoxy)butyrate (7.7 g, 25.9 mmol) in ethoxyacetic acid / tetrahydrofuran (1:1 v / v, 100 mL). After 3 h, all solvents were removed under vacuum, and the residue was suspended in water (50 mL) and dichloromethane (50 mL). The aqueous layer was extracted twice with dichloromethane (50 mL), and the combined organic layer was dried on magnesium sulfate. (To improve the overall yield and remove some saponification products, methanol (100 mL) and formyl acid (50 mg) were added to the residue and stirred overnight at room temperature.) The solvent was removed under reduced pressure to obtain a yellow solid. Using the above yellow solid as raw material, the residue was purified by silica gel column chromatography with hexane / ethyl acetate = 1:1 (Rf = 0.6) to finally obtain a pale yellow powder mNB.

[0063] 4. Fourth step: Synthesis of NB

[0064] 0.5 g (1.8 mmol) of mNB and 1.1 mL (2 mmol, Sigma-Aldrich) of ethylenediamine were dissolved in methanol. The mixture was refluxed overnight until the initial single component could not be detected by thin-layer chromatography. After the reaction was complete, the solvent was evaporated under vacuum. The crude precipitate was dissolved in methanol and precipitated three more times with ethyl acetate. The filter cake was dried under vacuum at 30 °C for 12 h until NB was a pale yellow powder (0.4 g, 1.2 mmol, 66.7%). The structural formula of NB is [insert structural formula here].

[0065] 5. Fifth step: Synthesis of HA-NB

[0066] HA (408 mg, 1 mmol, disaccharide units, molecular weight 20 w-40 w Da) was dissolved in 50 mL of room temperature deionized water, NB (224 mg, 0.69 mmol) was added, followed by HOBt (153 mg, 1 mmol, Sigma-Aldrich). The pH of the mixture was adjusted to pH 4.5, and 1-(3-dimethylaminopropyl)-3-ethylcarboxylic acid diamine hydrochloride (200 mg, 1.04 mmol, Sigma-Aldrich) was added. The mixture was stirred at room temperature for 48 h. The solution was then loaded into a dialysis tube (molecular weight: 3500 Da) and dialyzed for 2 days in diluted HCl (pH 3.5) containing 0.1 M NaCl, followed by dialysis for 2 days in deionized water. The solution was then lyophilized to obtain powdered HA-NB.

[0067] III. Preparation of BSP-MA / HA-NB hydrogel

[0068] Weigh out BSP-MA and HA-NB powders to the target concentration, add them to 180 μL of 1×PBS buffer, and dissolve by magnetic stirring at 200 rpm. Then add 20 μL of 4% photoinitiator LAP solution and apply the solution using a handheld UV lamp (365 nm, 1 W / cm²). 2 Irradiation for 10 seconds induced gelation, forming a stable three-dimensional network structure, and a 10% BSP-MA + 5% HA-NB hydrogel was prepared, wherein the mass fraction of BSP-MA was 10% and the mass fraction of HA-NB was 5%.

[0069] Example 2: Preparation of BSP-MA / HA-NB hydrogel

[0070] BSP-MA / HA-NB hydrogels were prepared according to the preparation method of Example 1, with the difference being:

[0071] The prepared BSP-MA / HA-NB colloid contained 15% BSP-MA and 2.5% HA-NB by mass.

[0072] Example 3: Preparation of BSP-MA / HA-NB hydrogel

[0073] BSP-MA / HA-NB hydrogels were prepared according to the preparation method of Example 1, with the difference being:

[0074] The prepared BSP-MA / HA-NB colloid contained 5% BSP-MA and 10% HA-NB by mass.

[0075] Example 4: Effect of BSP-MA / HA-NB hydrogel on H2O2-induced oxidative damage cell model

[0076] I. Experimental Methods

[0077] 1. Cell Culture

[0078] The rat-derived BMSCs of P3 were used at 5 × 10 5 Cells were seeded at a concentration of 100 cells / well in 12-well plates. After cell adhesion, except for the control group, BMSCs were induced by H2O2-induced oxidative damage in L-DMEM complete medium with 400 μM H2O2 for 30 minutes. Afterward, except for the control group, each group was rinsed three times with sterile medium for 3 minutes each time. According to the experimental groups, L-DMEM complete medium, 5% HA-NB gel extract complete medium, and 10% BSP-MA + 5% HA-NB gel extract complete medium were added for culture.

[0079] The preparation method of 5% HA-NB gel extract is as follows: HA-NB powder is dissolved in 1XPBS, stirred until dissolved, and then 10% (v / v) of 4% photoinitiator LAP is added. The mixture is then cured under UV light to obtain a 5% (w / w) HA-NB gel. The obtained 5% HA-NB gel is added to 1 mL of L-DMEM complete culture medium and soaked at 37°C for 24 hours to obtain the extract. The extract is then sterilized using a 0.22 μm filter. The preparation method of 10% BSP-MA + 5% HA-NB gel extract is as follows: The obtained 10% BSP-MA + 5% HA-NB hydrogel is added to 1 mL of L-DMEM complete culture medium and soaked at 37°C for 24 hours to obtain the extract. The extract is then sterilized using a 0.22 μm filter.

[0080] Experimental Groups:

[0081] (1) Control group

[0082] (2) H2O2 group (oxidative damage cell model group)

[0083] (3) 5% HA-NB group (5% HANB): Oxidative damage cell model + 5% HA-NB gel extract complete culture medium, cultured for 12 hours.

[0084] (4) 10% BSP-MA+5% HA-NB group (10% BSPMA+5% HANB): The oxidative damage cell model was added to the complete culture medium containing 10% BSP-MA and 5% HA-NB gel extract and cultured for 12 hours.

[0085] 2. Fluorescence detection: Fluorescence detection of intracellular reactive oxygen species levels; the specific operation steps are as follows: after the above cells are treated for 12 hours, the old culture medium is removed, and the cells are rinsed 3 times with sterile PBS; after staining with DCFH-DA kit (Beyotime, S0033M) for 30 minutes, excess dye is removed by rinsing with PBS, and the cells are observed by fluorescence microscope.

[0086] 3. TUNEL: After 24 hours of treatment, the old culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 minutes; washed twice with PBS for 10 minutes each time; 0.5% Triton X-100 in PBS was added, and the cells were incubated at room temperature for 5 minutes; the cells were stained with a one-step TUNEL apoptosis detection kit (Beyotime, C1088) for 30 minutes, rinsed with PBS to remove excess dye, and the cell nuclei were stained with DAPI staining solution; washed twice with PBS for 10 minutes each time, and observed under a fluorescence microscope.

[0087] 4. Scratch test: P3 rat-derived BMSCs were subjected to a scratch test at 5 × 10⁻⁶ mm. 5 Cells at a concentration of 1 cell / well are seeded in 6-well plates. Once the cell density reaches more than 80%, a 200 μL pipette tip is used to make incisions perpendicular to the horizontal line on the back of the plate, using a ruler as a guide. The pipette tip must be vertical and not tilted. After scratching, cells were washed three times with PBS to remove detached cells. Except for the control group, BMSCs in each group were induced to undergo H2O2-induced oxidative damage by adding 400 μM H2O2 to L-DMEM complete medium for 30 minutes. Afterward, except for the control group, each group was rinsed three times with sterile medium for 3 minutes each time. According to the experimental groups, L-DMEM complete medium, 5% HA-NB gel extract complete medium, and 10% BSP-MA + 5% HA-NB gel extract complete medium were added for culture. The culture plates were placed in a 37°C, 5% CO2 incubator. Cells were removed at 0, 12, and 24 hours, and the scratch width was observed and photographed under a microscope. The images were opened with ImageJ software, 6-8 horizontal lines were randomly drawn, and the mean distance between cells was calculated.

[0088] II. Experimental Results

[0089] This invention uses BMSCs as an in vitro cell model to evaluate the therapeutic effect of BSP-MA / HA-NB hydrogel on oxidatively damaged cells. Figure 1 As shown in Figure a, DCFH-DA detection revealed that the H2O2 group exhibited more green fluorescence, indicating that H2O2 can induce oxidative stress damage in cells, significantly increasing intracellular ROS levels. Compared to the H2O2 group, the 10% BSP-MA + 5% HA-NB group showed significantly reduced green fluorescence, indicating a decrease in intracellular ROS levels and thus inhibiting apoptosis. Figure 1 b), and significantly restored the migration of oxidatively damaged BMSCs ( Figure 2 c, e) and proliferation capacity ( Figure 2 d). The results show that the BSP-MA / HA-NB hydrogel prepared in this invention can significantly remove intracellular ROS from oxidatively damaged BMSCs, inhibit apoptosis, and restore migration and proliferation capabilities, thereby treating oxidatively damaged BMSCs.

[0090] Example 5: Effect of BSP-MA / HA-NB hydrogel on the chondrogenic capacity of BMSCs

[0091] I. Experimental Methods

[0092] 1. Cell Culture

[0093] The rat-derived BMSCs of P3 were used at a rate of 1×10 6 Cells were concentrated at a concentration of 100 cells / tube and centrifuged to the bottom of 15 mL centrifuge tubes. Except for the control group, each group was subjected to H2O2-induced oxidative damage to BMSCs by adding 400 μM H2O2 in L-DMEM complete medium for 30 minutes. Afterward, each group was washed three times with PBS for 5 minutes each time. According to the experimental groups, chondrogenic induction medium (CIA), 5% HA-NB gel extract chondrogenic induction medium, and 10% BSP-MA + 5% HA-NB gel extract chondrogenic induction medium were added respectively. The centrifuge tubes were loosely capped and incubated at 37°C for 14 days, with fresh induction medium changed every 2 days. The composition of the chondrogenic induction medium is shown in Table 1. Taking the preparation of 50 mL of chondrogenic induction medium as an example, it includes 40 mL of basal medium + 10 mL of supplemental medium.

[0094] Table 1

[0095]

[0096] Experimental Groups:

[0097] (1) Control group: Add cartilage induction solution and induce for 14 days.

[0098] (2) H2O2 group: Oxidative damage cell model group + cartilage induction solution, induced for 14 days.

[0099] (3) 5% HA-NB group (HANB group): Oxidative damage cell model + 5% HA-NB gel extract chondrogenic solution, induced for 14 days.

[0100] (4) 10% BSP-MA+5% HA-NB group (BSPMA+HANB group): Oxidative damage cell model + 10% BSP-MA+5% HA-NB gel extract chondrogenic solution, induced for 14 days.

[0101] 2. Real-time quantitative PCR: After 14 days of induction, cells were ground into pellets using a grinding tube, and RNA was extracted using the Trizon (Takara) method. The extracted RNA was transcribed into cDNA using a reverse transcription kit (Novizan, R333-01). The expression of the target genes (Col2A1, SOX9, ACAN, IL-1β, TNF-α, MMP13) was then detected.

[0102] II. Experimental Results

[0103] like Figure 3 As shown, qPCR results revealed that the BSP-MA+HA-NB group significantly upregulated the expression of cartilage-related genes in bone marrow mesenchymal stem cells (BMSCs), including type II collagen (Col2A1), Sox9, and acanthose (ACAN), while downregulating the expression of inflammation-related genes such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Furthermore, the BSP-MA+HA-NB group also reduced the expression level of matrix metalloproteinase 13 (MMP13). These results indicate that the BSPMA / HANB hydrogel not only alleviates oxidative stress but also promotes the differentiation of BMSCs into cartilage, providing a novel strategy for enhancing osteochondral repair under inflammatory conditions.

[0104] Example 6: The role of BSP-MA / HA-NB hydrogel in an osteochondral defect model

[0105] I. Experimental Methods

[0106] Animal model establishment: Male Sprague-Dawley (SD) rats aged 9-11 weeks were used for the experiment. They were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (50 mg / kg body weight). After exposing the knee joint through a medial parapatellar incision, a critical-sized defect (3 mm in diameter, 2 mm in depth) was prepared using a surgical drill to establish an osteochondral defect model. Subsequently, 15 μL each of 10% BSPMA, 5% HANB, and a 10% BSPMA+5% HANB composite hydrogel were precisely injected into the defect area. The incision was sutured and disinfected with povidone-iodine solution. Tissue samples were harvested at 6 and 12 weeks postoperatively for histological, biochemical, and biomechanical analysis.

[0107] Experimental groups (N=6):

[0108] (1) Damage group;

[0109] (2) 10% BSP-MA group (BSPMA group);

[0110] (3) 5% HA-NB group (HANB group);

[0111] (4) 10% BSP-MA + 5% HA-NB group (BSPMA + HANB group).

[0112] 2. Histological and imaging evaluation: After 6 weeks of treatment, the rats were euthanized by injecting an overdose of anesthetic, and the femoral tissue was collected. The collected tissue was fixed with 4% paraformaldehyde for 48 hours. After fixation, it was rinsed with water overnight to remove excess paraformaldehyde. The subchondral bone CT condition was evaluated using a small animal in vivo tomography imaging system (Milabs, Netherlands, U-CT-XUHR), and the subchondral bone repair was quantitatively evaluated.

[0113] The indicators are:

[0114] (1) Volumetric bone mineral density (vBMD);

[0115] (2) Bone volume fraction (BV / TV): This represents the ratio of bone tissue volume to tissue volume, which can directly reflect changes in bone mass. This indicator is the most important in microct bone tissue analysis.

[0116] (3) Trabecular thickness (Tb.Th): The average thickness of the trabecular bone;

[0117] (4) Trabecular separation (Tb.Sp): Represents the average width of the medullary cavity between trabeculae. It reflects the distance between trabeculae in porous bone tissue.

[0118] 3. Immunofluorescence staining

[0119] II. Experimental Results

[0120] Figure 4 a shows Micro-CT scan images of subchondral bone repair at 12 weeks in each group. Statistical analysis results based on cylindrical regions of interest (ROIs) with a diameter of 3 mm and a height of 2 mm are provided below. Figure 4At 6 weeks post-surgery, the 10% BSPMA / 5% HANB group showed significantly improved bone mineral density (BMD) and bone volume fraction (BV / TV) compared to other groups, indicating its advantage in early bone repair. By 12 weeks, BMD and BV / TV in this group further increased, demonstrating that the BSP-MA / HA-NB hydrogel, combining a triple strategy of immunomodulation, biomechanical adaptation, and tissue adhesion, has significant subchondral bone repair capacity. The significant increase in trabecular thickness (Tb.Th) further confirms the enhanced subchondral bone repair capacity of this group, reflecting improved integrity and functionality of the newly formed bone structure. Simultaneously, the trabecular spacing (Tb.Sp) significantly decreased at 12 weeks, indicating the formation of a denser and more interconnected trabecular network, resulting in better integration of the new tissue with surrounding tissues. Although Tb.Th increased in the 10% BSPMA group, Tb.Sp increased at 12 weeks, suggesting that the lack of tissue adhesion and biomechanical adaptation may impair the integration of new tissue, thereby increasing the risk of osteoporotic features in the newly formed subchondral bone.

[0121] The repair of osteochondral defects at 6 and 12 weeks post-implantation was systematically evaluated using histological staining results. At 6 weeks post-operation, H&E staining (…) Figure 5 a), SO staining ( Figure 5 b) with TB staining ( Figure 5 c) Combined analysis showed that the Damage group exhibited fibrous tissue hyperplasia accompanied by significant hematoma formation, and delayed repair of the cartilage-subchondral bone interface. While the 10% BSPMA group significantly reduced hematoma in the bone defect area through BSP release from BSPMA, secondary fibrosis occurred due to the material's lack of tissue adhesion and mechanical support. Although the HANB group achieved cartilage layer reconstruction, fibrous tissue remained in the subchondral bone region, confirming the crucial role of interfacial integration in osteochondral repair. In contrast, the 10% BSPMA / 5% HANB hydrogel, through a triple strategy of immunomodulation, mechanical adaptation, and tissue adhesion, achieved rapid subchondral bone repair in the early stages of osteochondral defect repair.

[0122] To assess macrophage polarization at bone defect sites, immunofluorescence co-staining with CD68 and either CD206 or CD80 was used. Results showed ( Figure 5 d): At six weeks, the Damage group was dominated by M1 macrophages. Although BSPMA could promote the polarization of M2 macrophages in the defect area, the lack of tissue adhesion and mechanical support led to the continued activation of M1 macrophages. Although HANB provided tissue adhesion, its limited immunomodulatory capacity resulted in weak M2 macrophage polarization, failing to construct a favorable immune microenvironment for osteochondral repair. In contrast, the combination of BSPMA and HANB effectively inhibited the polarization of M1 macrophages in the bone defect site, thereby inhibiting ADAMTS5 (… Figure 5 e) and MMP13 ( Figure 5f) secretion, while promoting the polarization of M2 macrophages, establishing an immune microenvironment conducive to the repair of osteochondral defects, and achieving optimal tissue remodeling.

[0123] The above results indicate that the BSP-MA / HA-NB hydrogel prepared in this invention has significant subchondral bone repair capabilities and exhibits a clear synergistic effect. It can help reduce inflammatory responses and promote tissue repair by inhibiting the activation of M1 macrophages and promoting the increase of M2 macrophages. It can be used to treat osteochondral injuries or degenerative diseases of articular cartilage.

[0124] In summary, this invention introduces HA-NB (hyaluronic acid modified with o-nitrosobenzaldehyde) into BSP-MA (methacrylated Bletilla striata polysaccharide) to prepare BSP-MA / HA-NB hydrogel. This hydrogel exhibits a significant synergistic effect in promoting cartilage and subchondral bone regeneration. Furthermore, it can help reduce inflammatory responses and promote tissue repair by inhibiting the activation of M1 macrophages and promoting the increase of M2 macrophages. Therefore, it has broad application prospects for treating osteochondral injuries or degenerative articular cartilage diseases.

Claims

1. The use of BSP-MA / HA-NB hydrogel in the preparation of drugs for treating bone defects; the BSP-MA / HA-NB hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylated Bletilla striata polysaccharide and 5 parts of hyaluronic acid modified with o-nitrosobenzaldehyde; The BSP-MA / HA-NB hydrogel contains 10% by mass of methacrylamide Bletilla striata polysaccharide and 5% by mass of o-nitrosobenzaldehyde-modified hyaluronic acid. The methacrylated Bletilla striata polysaccharide was prepared by reacting Bletilla striata polysaccharide with methacrylic anhydride; the o-nitrosobenzaldehyde-modified hyaluronic acid was prepared by reacting hyaluronic acid with an o-nitrosobenzaldehyde oxidizing agent. The o-nitrosobenzaldehyde reagent refers to one containing at least one A compound containing a group and at least one amino group.

2. The use according to claim 1, characterized in that, The preparation method of the BSP-MA / HA-NB hydrogel includes the following steps: Step 1: Hyaluronic acid modified with o-nitrosobenzaldehyde and methacrylamide-modified Bletilla striata polysaccharide are added to a buffer solution to obtain a mixed solution; Step 2: React the mixed solution with the photoinitiator and cure it to obtain the final product.

3. The use according to claim 2, characterized in that, In step 2, the photoinitiator is selected from at least one of phenyl-2,4,6-trimethylbenzoylphosphine lithium, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, 1-hydroxycyclohexylphenyl ketone, 2-hydroxy-2-methyl-1-phenylpropanone, 2-methyl-1-(4-methylthiophenyl)-2-morpholino-1-propanone, and 2-isopropylthioxanthraphenone; And / or, in step 2, the curing is ultraviolet curing, and the curing conditions are: the wavelength of the ultraviolet lamp is 360-405nm, the light intensity of the ultraviolet light is 0.5-2W / cm², and the irradiation time is 8-30 seconds.

4. The use according to claim 3, characterized in that, The mass ratio of Bletilla striata polysaccharide to methacrylic anhydride is 1.5-2.5:1.5-2.5; The molar ratio of hyaluronic acid to o-nitrosobenzaldehyde reagent is 1:(0.5-1).

5. The use according to claim 4, characterized in that, The reaction of Bletilla striata polysaccharide with methacrylic anhydride is carried out at a pH of 8-9 and a temperature of 20-30℃. And / or, the reaction of the Bletilla striata polysaccharide with methacrylic anhydride is carried out under light protection and the stirring frequency is 500±50 rpm; The molar ratio of the disaccharide units of the hyaluronic acid to the o-nitrosobenzaldehyde oxidizing agent is 1:0.5-1; And / or, the reaction of the hyaluronic acid with the o-nitrosobenzaldehyde reagent is carried out under the action of an activator and a coupling agent, wherein the activator is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide, and the coupling agent is selected from N-hydroxybenzotriazole; the reaction temperature is 20-30℃, the reaction time is 36-72h, and the reaction pH is 4-5.

6. A pharmaceutical composition for treating bone defects, characterized in that: It is a formulation prepared using BSP-MA / HA-NB hydrogel as the active ingredient and pharmaceutically acceptable excipients; the BSP-MA / HA-NB hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylated Bletilla striata polysaccharide and 5 parts of hyaluronic acid modified with o-nitrosobenzaldehyde; The BSP-MA / HA-NB hydrogel contains 10% by mass of methacrylamide Bletilla striata polysaccharide and 5% by mass of o-nitrosobenzaldehyde-modified hyaluronic acid. The methacrylated Bletilla striata polysaccharide was prepared by reacting Bletilla striata polysaccharide with methacrylic anhydride; the o-nitrosobenzaldehyde-modified hyaluronic acid was prepared by reacting hyaluronic acid with an o-nitrosobenzaldehyde oxidizing agent. The o-nitrosobenzaldehyde reagent refers to one containing at least one A compound containing a group and at least one amino group.

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