Injectable bioactive matrix hydrogel with anti-angiogenesis and anti-neurogenesis microenvironment and application thereof

Abstract

The invention discloses injectable bioactive matrix hydrogel with an anti-angiogenesis and anti-neurogenesis microenvironment and application of the injectable bioactive matrix hydrogel. The injectable bioactive matrix hydrogel is prepared from bevacizumab, a concentrated growth factor and methacrylic acid chondroitin sulfate. The hydrogel prepared by the invention not only shows excellent injectability and biocompatibility, but also can inhibit pathological vascularization and neurogenesis, polarize macrophages to promote regeneration of M2 phenotype so as to relieve inflammation, and directly enhance deposition of cartilage specific ECM components (such as type II collagen and aggregation proteoglycan). By utilizing the coordinated multi-mechanism strategy, the comprehensive structure and function repair of the degenerated intervertebral disc is realized.

Description

Technical Field

[0001] This invention relates to the field of bone regeneration technology, specifically to an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment and its applications. Background Technology

[0002] Low back pain (LBP) is a significant global health challenge, consistently ranking as the leading cause of disability worldwide for many years. It places a heavy burden on individuals and healthcare systems. Over the past three decades, the number of people disabled by LBP has increased by 54%, and this trend is projected to continue. This upward trend is accompanied by declining quality of life for patients and increased healthcare expenditures, collectively placing immense pressure on societal resources. Intervertebral disc degeneration (IDD) is a major pathological basis. The pathogenesis of IDD is multifactorial and complex, characterized by a harmful cascade involving nucleus pulposus (NP) cell apoptosis, impaired extracellular matrix (ECM) metabolism, and a chronic inflammatory microenvironment.

[0003] Therefore, in addition to palliative symptom management, there is an urgent need to develop treatment strategies that can effectively stop intervertebral disc degeneration and promote the recovery of intervertebral disc function. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment and its applications.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides an injectable bioactive matrix hydrogel having an anti-angiogenic and anti-neurogenic microenvironment, wherein the injectable bioactive matrix hydrogel comprises bevacizumab, concentrated growth factors (CGF), and methacrylated chondroitin sulfate (CSMA).

[0006] As a preferred embodiment, the concentration of bevacizumab in the injectable bioactive matrix hydrogel is 10 μg / mL, and the concentration of concentrated growth factor is 10 mg / mL.

[0007] As a preferred embodiment, the preparation method of chondroitin sulfate methacrylate includes the following steps: dissolving chondroitin sulfate in deionized water to form a chondroitin sulfate solution; then adding methacrylic anhydride dropwise to the chondroitin sulfate solution, stirring and reacting at low temperature, and purifying the resulting product before freeze-drying to obtain chondroitin sulfate methacrylate.

[0008] As a preferred embodiment, the method for preparing the concentrated growth factor includes the following steps: centrifuging the collected blood sequentially at speeds of 2700 rpm, 2400 rpm, 2700 rpm and 3000 rpm for 2 minutes, 4 minutes, 4 minutes and 3 minutes respectively; after standing and separating into layers, extracting the intermediate layer; and then pre-cooling, freeze-drying and pulverizing the extracted intermediate layer to obtain the concentrated growth factor.

[0009] Secondly, the present invention provides a method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment, comprising the following steps: S1. Dissolve chondroitin sulfate methacrylated in a solvent containing a photoinitiator to obtain precursor solution a; S2. Add bevacizumab and concentrated growth factor to precursor solution a, stir well to obtain precursor solution b; S3. Induce photocrosslinking of precursor solution b under UV light irradiation to obtain injectable bioactive matrix hydrogel.

[0010] As a preferred embodiment, in step A, the concentration of chondroitin sulfate methacrylate in the precursor solution a is 3-7%.

[0011] As a preferred embodiment, the concentration of chondroitin sulfate methacrylate in the precursor solution a is 5%.

[0012] As a preferred embodiment, in step A, the photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the solvent is selected from PBS solution.

[0013] As a preferred embodiment, the concentration of the photoinitiator in the solvent containing the photoinitiator is 0.2-0.5%.

[0014] As a preferred embodiment, in step C, the UV irradiation conditions are: UV light wavelength of 320-400nm, light intensity of 8-12 mW / cm², and irradiation time of 3-8 seconds.

[0015] Thirdly, the present invention provides the use of the aforementioned injectable bioactive matrix hydrogel or the injectable bioactive matrix hydrogel prepared according to the aforementioned method in the preparation of products for intervertebral disc degeneration repair and / or promoting cartilage regeneration.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a "three-in-one" strategy for intervertebral disc degeneration by constructing a novel injectable bioactive matrix hydrogel with anti-angiogenic, anti-neurogenic, and immunomodulatory microenvironment to promote intervertebral disc matrix remodeling. This strategy has not been previously reported. This invention synthesizes an injectable bioactive matrix (cbCSMA) hydrogel by combining the anti-angiogenic and anti-neurogenic agent bevacizumab, along with concentrated growth factor (CGF), into a photocrosslinked chondroitin sulfate methacrylate (CSMA) network. This cbCSMA hydrogel not only exhibits excellent injectability and biocompatibility but also inhibits pathological angiogenesis and neurogenesis, polarizes macrophages to a pro-regenerative M2 phenotype to reduce inflammation, and directly enhances the deposition of cartilage-specific ECM components (such as type II collagen and proteoglycans). Utilizing this coordinated multi-mechanism strategy, comprehensive structural and functional repair of intervertebral disc degeneration is achieved.

[0017] The cbCSMA hydrogel prepared in this invention has significant clinical translational potential for IDD repair, reducing angiogenesis and neurogenesis, and reconstructing the intervertebral disc matrix and resolving LBP through immunomodulation. Attached Figure Description

[0018] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The ¹H NMR results for the CSMA prepared in Example 1; Figure 2 The curing effect of CSMA hydrogels of different concentrations prepared in Example 1 is shown in the following images from left to right: before light exposure, inverted image before light exposure, image during light exposure, image after light exposure, and inverted image after light exposure. Figure 3 The results of the injectability evaluation of CSMA hydrogels of different concentrations prepared in Example 1; Figure 4 The CCK8 assay results are for bCSMA hydrogels containing different concentrations of bevacizumab and cCSMA hydrogels containing different concentrations of CGF prepared in Example 2; wherein, Figure 4 A is a bCSMA hydrogel; Figure 4 B is cCSMA hydrogel; Figure 5 This is a schematic diagram of the preparation of cbCSMA hydrogel and the performance test results in Example 2; wherein, Figure 5 A is a schematic diagram of the preparation process; Figure 5 B represents the final result of G'; Figure 5C represents the viscosity result; Figure 5 D represents the compression modulus result; Figure 5 E represents the swelling result; Figure 5 F represents the degradation result; Figure 5 G represents the release results of CGF and bevacizumab in the hydrogel; Figure 6 The morphology and elemental analysis results of the CSMA hydrogel, bCSMA hydrogel, and cbCSMA hydrogel prepared in Example 2 are shown below; Figure 6 A shows the SEM images of each hydrogel (images at different magnifications, from left to right). Figure 6 B shows the elemental images of each hydrogel; Figure 6 C represents the XRD analysis of each hydrogel; Figure 6 D represents the FTIR analysis of each hydrogel; Figure 6 E represents the quantitative analysis of carbon, nitrogen, oxygen, and sulfur in each hydrogel; Figure 7 Example 3 presents the results of biocompatibility assessment and biological function analysis of CSMA hydrogel, bCSMA hydrogel, and cbCSMA hydrogel; wherein, Figure 7 A is a schematic diagram of chondrocytes loaded on cbCSMA hydrogel; Figure 7 B shows the results of live and dead cell staining of chondrocytes on various hydrogel samples; Figure 7 C is a schematic diagram of macrophages loaded on cbCSMA hydrogel; Figure 7 D represents the staining results of live and dead cells of macrophages on various hydrogel samples; Figure 7 E represents the quantitative analysis results of live cells in chondrocytes; Figure 7 F represents the quantitative analysis results of dead cells in chondrocytes; Figure 7 G represents the quantitative analysis results of live cells in macrophages; Figure 7 H represents the quantitative analysis results of dead cells in macrophages; Figure 7 I represents the relative mRNA expression of Col2 in chondrocytes; Figure 7 J represents the relative mRNA expression of SOX9 in chondrocytes; Figure 7 K represents the relative mRNA expression of TNFα in macrophages; Figure 7 L represents the relative mRNA expression of IL6 in macrophages; Figure 8 The results are the histopathological analysis of cbCSMA hydrogel treatment for IDD in Example 4; wherein, Figure 8 A is a schematic diagram of the implantation of cbCSMA hydrogel; Figure 8B shows the HE, SO, and Sirius red staining results of the nucleus pulposus (NP) and annulus fibrosus (AF) in the Sham, Ctrl, and cbCSMA groups; Figure 8 C represents the HE staining results of the heart, liver, spleen, lungs, and kidneys in the Sham, Ctrl, and cbCSMA groups; Figure 8 D represents the relative mRNA expression result of SOX9; Figure 8 E represents the relative mRNA expression result of Col2; Figure 8 F represents the relative mRNA expression of TNFα; Figure 8 G represents the relative mRNA expression of IL-6; Figure 8 H represents the relative mRNA expression of CD31; Figure 8 I represents the relative mRNA expression result of CALCA; Figure 9 The results of cartilage-specific biomarker analysis for cbCSMA hydrogel treatment of IDD in Example 4 are shown; wherein, Figure 9 A is a schematic diagram of cartilage-specific markers in IDD repair; Figure 9 B shows immunofluorescence staining images of ACAN and Col2 in the Sham, Ctrl, and cbCSMA groups; Figure 9 C represents the quantitative analysis of ACAN in NP; Figure 9 D represents the quantitative analysis result of Col2 in NP; Figure 9 E represents the quantitative analysis result of Col2 in AF; Figure 9 F represents the quantitative analysis results of ACAN in AF; Figure 10 The results of the analysis of M1-specific and M2-specific biomarkers for cbCSMA hydrogel treatment of IDD in Example 4 are shown; among them, Figure 10 A is a schematic diagram of M1 and M2 specific biomarkers for immune regulation in IDD repair; Figure 10 B shows immunofluorescence staining images of Arg1 and iNOS in the Sham, Ctrl, and cbCSMA groups; Figure 10 C represents the quantitative analysis results of Arg1 in NP; Figure 10 D represents the quantitative analysis results of Arg1 in AF; Figure 10 E represents the quantitative analysis results of iNOS in NP; Figure 10 F represents the quantitative analysis results of iNOS in AF; Figure 11 The results of the analysis of neurospecific and vascular specific biomarkers in cbCSMA hydrogel treatment of IDD in Example 4 are shown; among them, Figure 11A is a schematic diagram illustrating the anti-angiogenic and anti-neurogenic functions in IDD repair; Figure 11 B shows immunofluorescence staining images of CGRP and CD31 in the Sham, Ctrl, and cbCSMA groups; Figure 11 C represents the quantitative analysis results of CGRP in NP; Figure 11 D represents the quantitative analysis results of CGRP in AF; Figure 11 E represents the quantitative analysis results of CD31 in NP; Figure 11 F represents the quantitative analysis results of CD31 in AF. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0020] Existing research indicates that angiogenesis and macrophage infiltration are known key drivers of exacerbating inflammation and matrix catabolism, creating a vicious cycle that promotes intervertebral disc degeneration (IDD). Furthermore, neurovascularization of cartilage can promote a sustained supply of inflammatory mediators (interleukins and TNFα) to surrounding cartilage tissue, thereby accelerating matrix degradation and exacerbating pain, leading to further cartilage degeneration. Therefore, inhibiting angiogenesis and innervation while simultaneously reconstructing the intervertebral disc matrix structure may effectively alleviate or reverse the symptoms of IDD and chronic pain.

[0021] Meanwhile, biomaterial-based systems, particularly injectable hydrogels, offer the potential for comprehensive and integrated therapeutic approaches as a multifunctional modality for minimally invasive delivery, mechanical support, and controlled release of therapeutic agents.

[0022] Therefore, this invention has developed a "three birds with one stone" strategy for intervertebral disc degeneration. By constructing a novel injectable bioactive matrix hydrogel with anti-angiogenesis, anti-neurogenesis and immune-regulating microenvironment, it promotes intervertebral disc matrix reconstruction, thereby achieving the effect of treating intervertebral disc degeneration and thus treating and relieving lower back pain.

[0023] This invention is implemented through the following specific embodiments: In the following specific embodiments, an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment is provided. The injectable bioactive matrix hydrogel comprises 10 μg / mL bevacizumab, 10 mg / mL concentrated growth factor and 5% (w / v) chondroitin sulfate methacrylate.

[0024] In the following specific embodiments, all raw materials and reagents used are commercially available products or prepared by conventional methods. This invention does not impose any particular limitations.

[0025] In the following specific embodiments, the methods used for characterization, detection and quantitative analysis are all conventional methods used in the field, and the present invention does not impose any particular limitations.

[0026] Example 1 This embodiment provides a method for preparing chondroitin sulfate methacrylate (CSMA), the specific steps of which are as follows: 1 g of chondroitin sulfate (CS, Aladdin) was dissolved in 150 mL of deionized water at room temperature until completely dissolved. Then, 1 mL of methacrylic anhydride (Aladdin) was added dropwise to the CS solution over 30 minutes, with the resulting reaction mixture kept in an ice bath. The reaction mixture was placed in the dark and reacted with continuous stirring at 4°C for 24 hours. The resulting product was purified by dialysis (3.5 kDa) against deionized water for 3 days, with water changed every 4 hours to remove unreacted reagents and methacrylic anhydride. Finally, the purified CSMA solution was lyophilized to obtain a white porous sponge (CSMA powder) and stored at -20°C for later use. To further verify the successful synthesis of CSMA, ¹H NMR (Bruker AVANCE III 500 MHz) was performed on CS and CSMA. The results are shown below. Figure 1 As shown, by Figure 1 The results confirmed the successful synthesis of CSMA.

[0027] Hydrogels of different concentrations exhibit varying mechanical properties, pore size, and rheological characteristics, all of which significantly affect their manufacturability, cell adhesion, and proliferation. Therefore, selecting an appropriate CSMA concentration is crucial. Based on this, a CSMA concentration screening experiment was further conducted, with the specific steps as follows: 1. The CSMA powder synthesized by the above method was dissolved in a PBS solution containing 0.2% (w / v) lithium acetylacetonate (photoinitiator) to obtain precursor solutions with CSMA concentrations of 0%, 1%, 3%, 5%, and 7% (w / v). 2. The precursor solution was exposed to UV light (wavelength 365 nm, light intensity 10 mW / cm²) for 5 seconds to induce photocrosslinking, resulting in CSMA hydrogels of different concentrations. Figure 2 The curing results show that 0% and 1% CSMA cannot be effectively cured, while 3%, 5%, and 7% CSMA can be effectively cured.

[0028] 3. Considering that injection therapy is the most ideal method for repairing IDD, the injectability of CSMA hydrogels was further investigated. CSMA hydrogels at concentrations of 3%, 5%, and 7% (w / v) were prepared in syringes using the methods described in steps 1) and 2) above. The injectability of each prepared CSMA hydrogel was then evaluated. The results are as follows: Figure 3 As shown, 3% and 5% CSMA hydrogels are easy to extrude and exhibit good injectability; while 7% CSMA hydrogel is difficult to extrude.

[0029] 4. Chondrocytes (prepared in Example 3 below) were loaded into 3% and 5% CSMA hydrogels at a density of 1 × 10^6 / ml and cultured for 24 hours. The effect of CSMA hydrogels on chondrocyte proliferation was then evaluated using a live-dead cell staining method. The results showed that 5% CSMA had a greater ability to promote chondrocyte proliferation than 3% CSMA.

[0030] Example 2 This embodiment provides a method for preparing an injectable bioactive matrix (cbCSMA) hydrogel with an anti-angiogenic and anti-neurogenic microenvironment (see schematic diagram of preparation). Figure 5 As shown in A), the following steps are included: 1. Preparation of Autologous Concentrated Growth Factor (CGF): Rat blood was collected using non-anticoagulation blood collection tubes, with a volume ranging from 3 to 5 ml. The blood was then centrifuged sequentially at 2700 rpm, 2400 rpm, 2700 rpm, and 3000 rpm for 2 minutes, 4 minutes, 4 minutes, and 3 minutes, respectively. After standing for 15 minutes, the CGF in the intermediate layer was extracted. The extracted CGF was pre-cooled, lyophilized, and powdered (ground) before use. CGF is rich in various growth factors, such as TGFβ, FGF, PDGF, and IGF, highlighting its significant potential for application in regenerative repair.

[0031] 2. Preparation of CSMA hydrogels, bCSMA hydrogels, and cCSMA hydrogels: 2.1 The CSMA powder synthesized by the method in Example 1 was dissolved in a PBS solution containing 0.25% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) to obtain a precursor solution a with a CSMA concentration of 5% (w / v); 2.2 Bevacizumab at concentrations of 1 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL was added to precursor solution a, and the mixture was stirred until homogeneous to prepare bCSMA precursor solutions with different bevacizumab concentrations. 2.3 Add CGF prepared in step 1 at concentrations of 1 μg / mL, 10 μg / mL, 100 μg / mL, 1 mg / mL and 10 mg / mL respectively to precursor solution a, stir well and prepare cCSMA precursor solutions with different CGF concentrations; 2.4 Precursor solution a, each of the bCSMA precursor solutions and cCSMA precursor solutions were exposed to UV light (wavelength 365 nm, light intensity 10 mW / cm²) for 5 seconds to induce photocrosslinking, and the corresponding CSMA hydrogels, bCSMA hydrogels and cCSMA hydrogels were prepared.

[0032] 2.5 Cell proliferation was assessed using the CCK8 Kit (Dojindo, Japan) according to the manufacturer's instructions. 20 μL of CSMA hydrogel, bCSMA hydrogel, and cCSMA hydrogel were placed at the bottom of 96-well plates. After swelling equilibrium was reached, chondrocytes were directly seeded onto each hydrogel at a density of 5 × 10³ cells / well, and 100 μL of fresh culture medium was added for 24 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well containing 100 μL of fresh culture medium, and the plates were incubated at 37°C for 2 hours. The absorbance of the formazan product was measured at 450 nm using a microplate reader. The results are as follows: Figure 4 A and Figure 4 As shown in Figure B, the results indicated that 10 μg / mL bevacizumab had no significant effect on cell proliferation, while 10 mg / mL CGF significantly enhanced cell proliferation. Therefore, 10 μg / mL bevacizumab and 10 mg / mL CGF were selected for subsequent experiments.

[0033] 3. Preparation of cbCSMA hydrogel: 3.1 In the precursor solution a with a CSMA concentration of 5% (w / v) prepared in step 2.1, add 10 μg / ml bevacizumab (Merck) and 10 mg / ml CGF prepared in step 1, and stir well to prepare cbCSMA precursor solution; 3.2 The cbCSMA precursor solution was exposed to UV light (365 nm, 10 mW / cm²) for 5 seconds to induce photocrosslinking and prepare cbCSMA hydrogel.

[0034] 4. Characterization of the rheological properties, mechanical properties, swelling properties, degradation properties, and drug release properties of each prepared hydrogel: 4.1 Using custom molds, standard-sized cylindrical CSMA hydrogel samples (5% CSMA), bCSMA hydrogel samples (bevacizumab concentration of 10 μg / mL, 5% CSMA), and cbCSMA hydrogel samples (bevacizumab concentration of 10 μg / mL, CGF concentration of 10 mg / mL, 5% CSMA) with a diameter of 10 mm and a thickness of 3 mm were prepared according to the methods in steps 2 and 3 above.

[0035] 4.2 The final G' and viscosity of each sample were tested using a rheometer (HAAKE MARS60). The results are as follows: Figure 5 B and Figure 5 As shown in Figure C, the addition of CGF and bevacizumab does not affect the rheological properties of the CSMA hydrogel.

[0036] 4.3 The compressive mechanical properties of the hydrogel were evaluated using a universal testing machine (Instron 5943, Norwood, MA, USA) via uniaxial compression testing. Each sample was compressed between two parallel plates at a constant strain rate of 1 mm / min until 60% of the maximum strain was reached. The compressive modulus was determined by the slope of the initial linear elastic region of the obtained stress-strain curve. The results are as follows: Figure 5 As shown in Figure D, there was no significant difference in the compressive modulus of the three samples, indicating that the addition of CGF and bevacizumab does not affect the mechanical properties of the CSMA hydrogel.

[0037] 4.4 Each sample was placed in PBS, and its volume was measured at 0, 2, 4, 8, 12, and 24 hours. The rate of volume change was used as the swelling data. Results are as follows: Figure 5 As shown in E, the swelling ratios of the three samples showed similar trends over time, indicating that the addition of CGF and bevacizumab did not affect the swelling properties of the CSMA hydrogel.

[0038] 4.5 Each sample was placed in PBS containing 0.1% (w / v) collagenase (Solarbio Science) and incubated at 37 °C for 1, 3, and 5 hours. The remaining mass ratio was calculated based on the weight of the remaining hydrogel to assess degradation performance. Results are as follows: Figure 5 As shown in F, the remaining mass ratios of the three samples showed similar trends over time, indicating that the addition of CGF and bevacizumab did not affect the degradation performance of the CSMA hydrogel.

[0039] 4.6 bCSMA and cbCSMA hydrogel samples were placed in PBS for 1, 7, 14, 21, and 28 days. The supernatant was then collected, and the levels of bevacizumab and CGF (a TGF-β substitute) were analyzed using ELISA kits (ab237642, ab215715, Abcam). The cumulative release pattern was calculated based on the initial concentrations. Results are shown below. Figure 5 As shown in G, the results indicate that the release pattern of CGF and bevacizumab in the CSMA hydrogel is continuous, and the release lasts for more than 4 weeks.

[0040] 4.7 The morphology of the hydrogels was observed using a scanning electron microscope (SEM, Hitachi SU8010). Elemental distribution was analyzed using energy-dispersive X-ray spectroscopy (EDS). Chemical composition and crystallinity were examined using Fourier transform infrared spectroscopy (FTIR, Nicoleti S50) and X-ray diffraction analysis (XRD, Ultima IV). Results are as follows: Figure 6 As shown, structural analysis of the hydrogel revealed an interconnected porous structure, which is beneficial for cell colonization and adhesion. Figure 6 A). X-ray diffraction (XRD) analysis results indicate that its structure is a low-crystallinity scaffold, which is beneficial for tissue regeneration and reconstruction. Figure 6 C). Additional elemental analysis showed that the scaffold was mainly composed of carbon, nitrogen, oxygen, and sulfur, with slight differences in content between groups, indicating that CGF and bevacizumab had successfully bound to the CSMA. Figure 6 B and 6E). Similarly, Fourier transform infrared (FTIR) spectroscopy results further confirmed the successful addition of CGF and bevacizumab (B and 6E). Figure 6 D). Therefore, the successful preparation of cbCSMA hydrogel provides IDD with a microenvironment that is anti-neurogenesis, anti-angiogenesis, and promotes tissue regeneration.

[0041] Example 3 Biocompatibility of cbCSMA hydrogel is a fundamental requirement for its application in IDD treatment. Therefore, this embodiment evaluates the biocompatibility and performs biological function analysis on the CSMA hydrogel samples, bCSMA hydrogel samples, and cbCSMA hydrogel samples prepared in Example 2. The specific steps are as follows: 1. Isolation and Culture of Chondrocytes: Chondrocytes were isolated from rat cartilage tissue. Harvested rat cartilage was minced into fragments of approximately 1 mm³ and then digested in DMEM at 37 °C with 0.2% (w / v) type II collagenase solution for 4–8 hours with continuous stirring. The isolated primary chondrocytes were then resuspended in complete culture medium (composed of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin). The resuspended primary chondrocytes were then seeded into culture flasks and maintained in a humidified incubator at 37 °C and 5% CO2. The culture medium was changed every 2–3 days until passage P2, approximately 10 days later, at which point sufficient chondrocytes were obtained.

[0042] 2. Chondrocytes or RAW 264.7 macrophages (purchased from Cyagen Bioscience Inc.) obtained in step 1 were loaded into each hydrogel sample at a density of 1 × 10^6 / ml. After culturing for 7 days, live and dead cell staining was performed using a relevant kit (Dojindo Laboratories). Schematic diagrams of chondrocytes and macrophages loaded onto cbCSMA hydrogels are shown below. Figure 7 A and Figure 7 As shown in Figure C, the staining results of live and dead cells for chondrocytes and macrophages on various hydrogel samples are as follows: Figure 7 B and Figure 7 As shown in Figure D, the quantitative analysis results of live and dead cells in chondrocytes are as follows: Figure 7 E and Figure 7 As shown in Figure F, the quantitative analysis results of live and dead cells in macrophages are as follows: Figure 7 G and Figure 7 Figure 7H shows the results. The results indicate that a large number of chondrocytes and macrophages survived in CSMA, bCSMA, and cbCSMA hydrogel samples, with only a very small number of dead cells, and there was no significant difference in the number of dead cells among the samples (Figures 7B, 7D, 7F, and 7H). Furthermore, there was no difference in the number of live cells between CSMA and bCSMA hydrogel samples, indicating that the addition of bevacizumab did not significantly affect cell viability (Figures 7E and 7G). In contrast, the cbCSMA hydrogel sample significantly enhanced cell survival, indicating that the introduction of CGF can improve the activity of chondrocytes and macrophages (Figures 7E and 7G). These findings suggest that cbCSMA hydrogel has good biocompatibility, and that the introduction of CGF can enhance cell survival and proliferation.

[0043] 3. Chondrocytes and macrophages obtained after 7 days of culture in step 2 underwent total RNA extraction using standard methods. The expression of cartilage-specific and inflammation-specific genes was then further analyzed at the gene level to verify the regulatory function of cbCSMA hydrogel in cartilage regeneration and immune regulation. Primers were designed and synthesized by Sangon. The relative mRNA expression results of Col2 and SOX9 in chondrocytes from different samples are shown below. Figure 7 I, Figure 7 As shown in Figure J, the relative mRNA expression results of TNFα and IL6 in macrophages obtained from each sample are as follows: Figure 7 K, Figure 7 As shown in Figure 7I and 7J, the cbCSMA hydrogel sample significantly enhanced the expression levels of cartilage-specific genes Col2 and SOX9 compared to the CSMA and bCSMA hydrogel samples (Figure 7I and 7J), while decreasing the expression levels of inflammation-related genes TNFα and IL6. Figure 7 (K and 7L). Therefore, cbCSMA hydrogel can not only regulate cartilage regeneration, but also modulate local immune inflammation.

[0044] Example 4 This embodiment studies the efficacy of cbCSMA hydrogel in treating IDD through in vivo animal experiments. The specific steps are as follows: 1. Adult rats (200g) were purchased from Silaike Biotechnology Co., Ltd (Beijing, China). All animal procedures followed the Laboratory Animal Care and Use Guidelines of Tongliao People's Hospital and were approved by the Animal Ethics Committee of Tongliao People's Hospital. An IDD model was established in the intervertebral discs of adult rats using acupuncture, resulting in successfully modeled IDD rats.

[0045] 2. The cbCSMA hydrogel prepared according to the method of Example 2 was injected into the intervertebral discs of IDD model mice for 4 weeks as the experimental group (cbCSMA), the untreated IDD model mice were designated as the untreated group (Ctrl), and the sham-operated group was designated as the control group (Sham). A schematic diagram of the cbCSMA hydrogel implantation using a syringe is shown below. Figure 8 As shown in Figure A.

[0046] 3. Four weeks after treatment, the harvested spine and major organs (heart, liver, spleen, lung, and kidney) were fixed, decalcified, paraffin-embedded, and sectioned. The obtained tissue sections were stained with HE, Safranin O (SO), and Sirius Red (PSR) to assess general morphology and collagen deposition. Results are as follows: Figure 8 B and Figure 8Figure 8C shows the HE staining results of spinal sections. The results indicated that the nucleus pulposus (NP) and annulus fibrosus (AF) structures in the Ctrl group were disordered, failing to achieve effective reconstruction. In contrast, the cbCSMA group showed a significant degree of structural reconstruction, with a morphology similar to the Sham group (Figure 8B). SO and Sirius red staining results of spinal sections showed that cbCSMA hydrogel effectively promoted the deposition of cartilage-associated collagen, thus preliminarily confirming its potential application in the treatment of IDD (Figure 8B). HE staining results for organ toxicity verification showed that cbCSMA hydrogel was non-toxic to the heart, liver, spleen, lungs, and kidneys (Figure 8C).

[0047] 4. After removing excess spinal tissue, the intervertebral discs were carefully separated and placed in Trizol. Further gene analysis was performed at the gene level to identify the expression of cartilage-specific and inflammation-specific genes, as well as genes related to angiogenesis and neurogenesis. The relative mRNA expression results of SOX9, Col2, CD31, CALCA, TNFα, and IL-6 are shown below. Figure 8 D、 Figure 8 E, Figure 8 F, Figure 8 G, Figure 8 H, Figure 8 As shown in Figure I, cbCSMA hydrogel upregulated the expression levels of cartilage-specific genes Col2 and SOX9, while reducing the expression levels of inflammation-related genes TNF and IL6, and inhibiting the expression levels of angiogenesis and neurogenesis-related genes CALCA and CD31 (inhibition of angiogenesis and neurogenesis may contribute to local immune inflammation regulation, thereby leading to the treatment and structural reconstruction of IDD).

[0048] 5. Spinal samples were fixed in paraformaldehyde, embedded, and sectioned. Immunofluorescence staining for Col2, ACAN, iNOS, Arg-1, CD31, and CGRP was then performed to assess cartilage regeneration. Fluorescence images were captured using a confocal microscope (Zeiss LSM 880) and quantified using ImageJ software. The immunofluorescence staining results for each group are shown below. Figure 9 , Figure 10 , Figure 11 As shown. ACAN and Col2 are cartilage-specific markers in IDD repair ( Figure 9 As shown in Figure A), immunofluorescence staining results indicated that cbCSMA hydrogel significantly enhanced the secretion of specific cartilage markers to repair local damage, while the Ctrl group secreted less specific proteins and polysaccharides (Figures 9B-9F). Arg1 and iNOS are M1-specific and M2-specific markers of immunomodulatory function in IDD repair, respectively. Figure 10 As shown in Figure A), immunofluorescence staining results indicate that cbCSMA hydrogel can regulate macrophage polarization towards M2, thereby achieving local inflammation regulation (Figures 10B-10F). CGRP and CD31 are neurospecific and vascular-specific markers, respectively, in IDD repair. A schematic diagram of anti-angiogenic and anti-neurogenic functions in IDD repair is shown below. Figure 11 As shown in Figure A, the immunofluorescence staining results of CGRP and CD31 indicate that cbCSMA hydrogel can reduce local angiogenesis and nerve formation (Figures 11B-11F).

[0049] The above in vivo repair results demonstrate that cbCSMA hydrogel exhibits good biocompatibility, biological function, and no organ toxicity. Furthermore, cbCSMA hydrogel can significantly inhibit angiogenesis and neurogenesis, thereby modulating local immune inflammatory responses. Ultimately, cbCSMA hydrogel can significantly enhance the secretion of cartilage-specific proteins and polysaccharides, thus achieving effective IDD repair and cartilage regeneration.

[0050] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. An injectable bioactive matrix hydrogel possessing an anti-angiogenic and anti-neurogenic microenvironment, characterized in that, The injectable bioactive matrix hydrogel comprises bevacizumab, concentrated growth factors, and chondroitin sulfate methacrylate.

2. The injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 1, characterized in that, In the injectable bioactive matrix hydrogel, the concentration of bevacizumab is 10 ug / mL, and the concentration of concentrated growth factor is 10 mg / mL.

3. The injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 1 or 2, characterized in that, The preparation method of the methylacrylic acid chondroitin sulfate includes the following steps: dissolving chondroitin sulfate in deionized water to form a chondroitin sulfate solution; then adding methylacrylic anhydride dropwise to the chondroitin sulfate solution, stirring and reacting at low temperature, and purifying the resulting product before freeze-drying to obtain methylacrylic acid chondroitin sulfate.

4. The injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 1 or 2, characterized in that, The method for preparing the concentrated growth factor includes the following steps: the collected blood is centrifuged at 2700 rpm, 2400 rpm, 2700 rpm and 3000 rpm for 2 minutes, 4 minutes, 4 minutes and 3 minutes respectively. After standing and separating into layers, the middle layer is extracted. The extracted middle layer is then pre-cooled, freeze-dried and powdered to obtain the concentrated growth factor.

5. A method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Dissolve chondroitin sulfate methacrylated in a solvent containing a photoinitiator to obtain precursor solution a; S2. Add bevacizumab and concentrated growth factor to precursor solution a, stir well to obtain precursor solution b; S3. Induce photocrosslinking of precursor solution b under UV light irradiation to obtain injectable bioactive matrix hydrogel.

6. The method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 5, characterized in that, In step A, the concentration of chondroitin sulfate methacrylate in the precursor solution a is 3-7%.

7. The method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 6, characterized in that, The concentration of chondroitin sulfate methacrylated in the precursor solution a is 5%.

8. The method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 5, characterized in that, In step A, the photoinitiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphonate; the solvent is selected from PBS solution; The concentration of the photoinitiator in the solvent containing the photoinitiator is 0.2-0.5%.

9. The method for preparing an injectable bioactive matrix hydrogel with an anti-angiogenic and anti-neurogenic microenvironment according to claim 5, characterized in that, In step C, the UV light irradiation conditions are: UV light wavelength of 320-400nm, light intensity of 8-12 mW / cm², and irradiation time of 3-8 seconds.

10. An injectable bioactive matrix according to any one of claims 1-4 The use of hydrogels or injectable bioactive matrix hydrogels prepared according to any one of claims 5-9 in the preparation of products for intervertebral disc degeneration repair and / or promoting cartilage regeneration.

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