Esterase response type composite hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to an esterase response type composite hydrogel microsphere and a preparation method and application thereof.The preparation method comprises the steps that KGN is connected with methacrylated gelatin GelMA, methacrylated hyaluronic acid HAMA, methacrylated sodium alginate AlgMA or methacrylated chondroitin sulfate ChsMA, and the composite hydrogel microsphere is obtained; the esterase response type composite hydrogel microspheres are prepared through a photo-crosslinking microfluidic technology. The esterase response type composite hydrogel microspheres constructed by the invention show good biocompatibility, minimally invasive injectability and sustained release characteristics, can improve the adaptability of NPCs to a severe IVDD microenvironment, and provides a promising synergistic method for cell therapy in IVDD regeneration.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, in particular to an esterase-responsive composite hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] Low back pain (LBP) is a major public health problem worldwide, affecting people of all ages. Authoritative surveys show that the global prevalence of LBP is about 9.4%, making it a major cause of disability, economic burden, and rehabilitation needs. Although the causes of LBP are diverse, intervertebral disc degeneration (IVDD) is the main factor. Conservative treatments, such as bed rest, medication, and physical therapy, can only provide limited relief and cannot restore the degenerated intervertebral disc (IVD) to a healthy state.

[0003] Surgical options include decompression and spinal fusion, which are common procedures. However, total disc replacement is challenged by technical difficulty, high cost, and comparable outcomes to spinal fusion. Decompression and fusion procedures are palliative in nature and may not restore the original biomechanical properties of the disc, especially in fusion procedures. These interventions may cause complications such as herniation of residual disc tissue, adjacent segment degeneration, and implant failure, necessitating revision surgery. Therefore, regenerative therapies are needed to slow or reverse the progression of IVDD.

[0004] The nucleus pulposus (NP), located in the center of the IVD, presents a gel-like structure composed of nucleus pulposus cells (NPCs) and extracellular matrix (ECM), which is synthesized and maintained by NPCs. ECM is essential for the mechanical properties of the NP and provides an essential microenvironment for cell homeostasis. The main components of ECM include water (70-90%), proteoglycans (50% of the water-free weight), and type II collagen (COL II, ~20% of the water-free weight).

[0005] The major proteoglycan in the nucleus pulposus is aggrecan (ACAN), which forms a complex with hyaluronic acid to promote water retention through osmotic pressure and cation attraction. The biomechanical function of the nucleus pulposus is crucial for IVD, which is surrounded by the annulus fibrosus (AF) and cartilaginous endplates and is able to withstand axial compression and torsional forces. IVDD involves the loss of nucleus pulposus cells and increased metabolic activity, resulting in proteoglycan depletion, reducing the ability of the nucleus pulposus to support compressive loads and indirectly increasing stress on the annulus fibrosus. Evidence suggests that degeneration originates primarily in the nucleus pulposus. Due to its avascularity and harsh microenvironment, the nucleus pulposus has limited regenerative capacity.

[0006] Recently, cell transplantation has attracted widespread attention as a potential therapeutic approach for regenerating neurons and restoring IVD function. However, the IVD microenvironment poses many challenges, including hypoxia, low pH, increased mechanical stress, hyperosmotic conditions, and nutrient depletion. IVDD exacerbates these conditions, leading to increased oxidative stress and inflammation, which reduces cell survival and engraftment rates after direct transplantation. To overcome these limitations, hydrogel microspheres have emerged as an effective cell delivery system.

[0007] Microspheres create a protective environment for cells, improve survival, and enable controlled release of bioactive molecules. Unlike traditional hydrogels, the interstices between microspheres and their high surface area to volume ratio promote cell proliferation and migration. Methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) are commonly used biocompatible polymers for the preparation of hydrogel microspheres.

[0008] GelMA is derived from natural gelatin and promotes cell adhesion through the RGD (arginine-glycine-aspartic acid) sequence; HAMA, as a derivative of hyaluronic acid (HA), enhances the viscoelastic properties of hydrogels; AlgMA is modified from seaweed Alg and has good biocompatibility. It can provide cells with a good microenvironment similar to that in the body; ChsMA is a modified chondroitin sulfate formed by introducing methacrylic acid groups into chondroitin sulfate molecules, which can regulate the differentiation direction of cells and promote the formation and regeneration support of specific tissues. The composite material formed by mixing any two or more of these materials not only mimics the extracellular matrix of NPs, but also provides enhanced mechanical strength. This composite material shows potential in applications such as bone regeneration, cartilage formation, and tendon-bone healing. However, its potential in NP regeneration still needs to be further explored. Summary of the invention

[0009] In view of the limitations of current IVDD clinical treatment, the present invention provides an esterase-responsive composite hydrogel microsphere and a preparation method and application thereof, which can enhance the adaptability of NPCs to the harsh IVDD microenvironment and provide a promising synergistic method for cell therapy in IVDD regeneration.

[0010] A method for preparing esterase-responsive composite hydrogel microspheres, comprising: KGN was connected with methacryloyl gelatin GelMA, methacryloyl hyaluronic acid HAMA, methacryloyl sodium alginate AlgMA or methacryloyl chondroitin sulfate ChsMA, and esterase-responsive composite hydrogel microspheres were prepared by photo-crosslinking microfluidic technology.

[0011] As a preference, the method comprises the following steps: Step S1, preparing methacrylylated gelatin GelMA, methacrylylated hyaluronic acid HAMA, methacrylylated sodium alginate AlgMA, and methacrylylated chondroitin sulfate ChSMA; Step S2, grafting katogenetin KGN onto methacryloyl gelatin GelMA, methacryloyl hyaluronic acid HAMA, methacryloyl sodium alginate AlgMA or methacryloyl chondroitin sulfate ChSMA to obtain GelMA-KGN or HAMA-KGN or AlgMA-KGN or ChSMA-KGN, respectively; Step S3, blending methacrylylated gelatin GelMA with GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN, preparing composite hydrogel microspheres by microfluidics, and then recovering the microspheres, washing them, and freeze-drying them.

[0012] Preferably, step S1 comprises: Step S11, weighing a certain amount of gelatin, hyaluronic acid, sodium alginate or chondroitin sulfate, and adding deionized water to the gelatin, hyaluronic acid, sodium alginate or chondroitin sulfate to prepare a gelatin solution, a hyaluronic acid solution, a sodium alginate solution or a chondroitin sulfate solution; Step S12, using a microinjection pump to drop methacrylic anhydride MA into the gelatin solution, hyaluronic acid solution, sodium alginate solution or chondroitin sulfate solution at a rate of 0.1-0.3 mL / min to react; Step S13, the reaction was terminated after 2 hours of reaction, the solution was dialyzed for 72 hours, and then freeze-dried; Step S14, after freeze-drying, methacrylylated gelatin GelMA, methacrylylated hyaluronic acid HAMA, methacrylylated sodium alginate AlgMA, and methacrylylated chondroitin sulfate ChSMA are obtained.

[0013] Preferably, step S2 comprises: Step S21, adding deionized water to the freeze-dried methacryloyl gelatin GelMA, methacryloyl hyaluronic acid HAMA, methacryloyl sodium alginate AlgMA or methacryloyl chondroitin sulfate ChSMA to dissolve, to obtain a methacryloyl gelatin solution, a methacryloyl hyaluronic acid solution, a methacryloyl sodium alginate solution or a methacryloyl chondroitin sulfate solution; wherein the dissolution temperature of GelMA is 37-60°C, more preferably 37°C, and the solution concentration is 2.5%-10% (w / v), more preferably 10% (w / v); the dissolution temperature of HAMA is room temperature, and the solution concentration is 0.5%-2% (w / v), more preferably 1% (w / v); the dissolution temperature of AlgMA is room temperature, and the solution concentration is 1%-4% (w / v), more preferably 2%; the dissolution temperature of ChSMA is room temperature, and the solution concentration is 5%-10% (w / v), more preferably 10% (w / v).

[0014] Step S22, adding a strong acid cation exchange resin to the methacrylated gelatin solution, the methacrylated hyaluronic acid solution, the methacrylated sodium alginate solution or the methacrylated chondroitin sulfate solution, and reacting overnight to perform ion exchange; Step S23, adding tetrabutylammonium hydroxide TBA-OH, adjusting the pH to 7.0, to obtain solution A; Step S24, recovering the solution A in step S23 and freeze-drying it for 48 hours to obtain GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA; Step S25, dissolving catogenein KGN with anhydrous dimethyl sulfoxide (DMSO) under nitrogen protection to obtain a catogenein solution, wherein the final concentration of the catogenein solution is 5-10 mg / mL, more preferably 8 mg / mL; Step S26, adding 4-dimethylaminopyridine DMAP to the catogynine solution of step S25 until it is completely dissolved to obtain solution B, wherein the final concentration of solution B is 1.2-4 mg / mL, more preferably 2 mg / mL; Step S27, adding di-tert-butyl carbonate BOC2O melted at 37° C. to the solution B obtained in step S26 to obtain a mixture A, wherein the final concentration of the mixture A is 0.6%-0.8% (v / v), more preferably 0.75% (v / v); Step S28, stirring the mixture A in step S27 at 25° C. for 30 min to obtain an activated KGN solution; Step S29, dissolving GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBAHAMA-TBA in DMSO to a concentration of 10-30 mg / mL, more preferably 20 mg / mL; Step S210, slowly adding the activated KGN solution to the GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA solution, stirring continuously, and reacting overnight; Step S211, dialyzing in deionized water for 2 days, and freeze-drying after dialysis; Step S212: After freeze-drying for 2 days, dry GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN is obtained.

[0015] Preferably, the concentration of the GelMA solution in step S3 is 10-20%, more preferably 10% (w / v); The concentration of GelMA-KGN solution is 10-20%, more preferably 10% (w / v); The concentration of HAMA-KGN solution is 1-2%, more preferably 1% (w / v); The concentration of AlglMA-KGN solution is 2-5%, more preferably 2% (w / v); The concentration of ChSMA-KGN solution is 2-10%, more preferably 5% (w / v); The volume ratio of GelMA to GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN is 1:1-3:1, more preferably 1:1.

[0016] Preferably, in step S11, the gelatin dissolution temperature is 37-60°C, more preferably 37°C, and the gelatin solution concentration is 5%-10% (w / v), more preferably 5% (w / v); The dissolution temperature of hyaluronic acid is room temperature, and the concentration of hyaluronic acid solution is 0.5%-4% (w / v), more preferably 1% (w / v); The dissolution temperature of sodium alginate is room temperature, and the concentration of the sodium alginate solution is 1%-4% (w / v), more preferably 2% (w / v); The dissolution temperature of chondroitin sulfate is room temperature, and the concentration of the chondroitin sulfate solution is 2.5%-10% (w / v), more preferably 5% (w / v); In step S12, the final concentration of methacrylic anhydride MA added to gelatin is 0.1-0.8 mL / g gelatin, that is, 0.1-0.8 mL methacrylic anhydride MA is added to each gram of gelatin, preferably 0.5 mL / g gelatin; The final concentration of methacrylic anhydride MA added to hyaluronic acid is 2-5 mL / g hyaluronic acid, that is, 2-5 mL methacrylic anhydride MA is contained in each gram of hyaluronic acid, preferably 2 mL / g hyaluronic acid; The final concentration of methacrylic anhydride MA added to sodium alginate is 1-3 mL / g sodium alginate, that is, 1-3 mL methacrylic anhydride MA is contained in each gram of sodium alginate, preferably 2 mL / g sodium alginate; The final concentration of methacrylic anhydride MA added to chondroitin sulfate is 0.5-1 mL / g chondroitin sulfate, that is, 0.5-1 mL methacrylic anhydride MA is contained in each gram of chondroitin sulfate, preferably 0.5 mL / g chondroitin sulfate.

[0017] Preferably, the mass of the strongly acidic cation exchange resin in step S22 is 3-5 times the dry mass of GelMA, HAMA, AlgMA or ChSMA.

[0018] Preferably, the volume ratio of the activated KGN solution to the GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA solution in step S210 is 2:5-1:1, more preferably 2:5; the reaction temperature is 16-37°C, GelMA-TBA is more preferably 37°C, and the others are more preferably 20°C; the reaction time is 12-16h, more preferably 12h.

[0019] The invention discloses an esterase-responsive composite hydrogel microsphere, which is prepared by adopting a method for preparing the esterase-responsive composite hydrogel microsphere.

[0020] Application of esterase-responsive composite hydrogel microspheres in intervertebral disc degeneration products.

[0021] The present invention has significant technical effects due to the adoption of the above technical solution: The present invention successfully constructed an esterase-responsive composite hydrogel microsphere, which exhibited good biocompatibility, minimally invasive injectability, and sustained release properties. It not only provided a growth environment and mechanical support for neural progenitor cells, but also improved the adaptability of NPCs to the harsh IVDD microenvironment through esterase-responsive release. Overall, this esterase-responsive KGN-releasing composite hydrogel microsphere provides a promising synergistic approach for cell therapy in IVDD regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The scanning electron microscope images are of the esterase-responsive composite hydrogel microspheres GHKM, GCKM, GGKM and GAKM of Examples 1-4 of the present invention.

[0023] Figure 2 This is a comparison of live-dead staining after co-culture of GHKM, GHM, GM and NPCs cells in Example 1 of the present invention and Comparative Examples 1-2.

[0024] Figure 3The figure is a graph showing the degradation of GHKM, GHM and GM in Example 1 of the present invention and Comparative Examples 1-2 in a similar physiological environment PBS.

[0025] Figure 4 This is a diagram showing the ROS fluorescence effect after GHKM, GHM, GM and NPCs cells were co-cultured and stimulated by 1L-1β in Example 1 of the present invention and Comparative Examples 1-2.

[0026] Figure 5 This is a HE staining effect diagram of GHKM, GHM, and GM microspheres used in Example 1 of the present invention and Comparative Examples 1-2 for treating rat tail bone marrow nuclear resection.

[0027] Figure 6 The safranin staining effect diagram of GHKM, GHM and GM microspheres used in Example 1 of the present invention and Comparative Examples 1-2 for treating rat tail bone marrow nuclear resection is shown. DETAILED DESCRIPTION

[0028] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the embodiments are only for explaining the present invention and are not intended to limit it. Example 1

[0029] A method for preparing esterase-responsive composite hydrogel microspheres comprises the following steps: Step S1, weigh 10 g of gelatin, add 200 mL of deionized water, and stir in a 37°C water bath to completely dissolve; add MA at a rate of 0.25 mL / min using a syringe pump, and stir to react for 2 h; dialyze the solution for 3 days and then freeze-dry to obtain GelMA; Step S2, weigh 5 g of sodium hyaluronate, add 500 mL of deionized water, and dissolve at room temperature; add MA by syringe pump at the speed of step S2, and react in an ice bath for 4 hours; dialyze the solution for 3 days and then freeze-dry to obtain HAMA; Step S3, dissolving 1 g of HAMA in 100 mL of deionized water, and adding 3 g of strongly acidic cation exchange resin; Step S4, after overnight reaction, add TBA-OH to adjust the pH to 7.0; Step S5, recovering and freeze-drying the solution to obtain HAMA-TBA; Step S6, dissolve 16 mg KGN in 2 mL DMSO, then add 4 mg DMAP and stir until completely dissolved; Step S7, add 12 μL of BOC2O melted at 37°C to the system, and stir at 25°C for 30 min to obtain an activated KGN solution; Step S8, taking 100 mg of HAMA-TBA and dissolving it thoroughly with 5 mL of DMSO; Step S9: slowly add the activated KGN solution in step S11 into the HAMA-TBA solution, continue stirring, and react at 20° C. for 12 h.

[0030] Step S10, dialyzing the solution in an acidified sodium chloride solution for 1 day and in deionized water for 2 days; Step S11, freeze-drying for 2 days to obtain dry HAMA-KGN; Step S12, weigh 5 g of GelMA, add 50 mL of deionized water containing photoinitiator LAP, and dissolve it fully at 37°C; Step S13, weigh 500 mg HAMA-KGN, add 50 mL deionized water containing photoinitiator LAP, and dissolve evenly at room temperature; Step S14, mixing the two solutions of step S12 and step S13 in equal volumes; Step S15, preparing GelMA / HAMA-KGN composite hydrogel microspheres by microfluidics; Step S16, recovering the microspheres and cleaning them; Step S17, freeze-drying to obtain esterase-responsive composite hydrogel microspheres (GelMA / HAMA-KGN composite hydrogel microspheres, GHKM). Example 2

[0031] The same as Example 1, except that the present example prepares GelMA / ChSMA-KGN composite hydrogel microspheres, and the specific steps are as follows: Step S1, weigh 10 g of gelatin, add 200 mL of deionized water, and stir in a 37°C water bath to completely dissolve; add MA at a rate of 0.25 mL / min using a syringe pump, and stir to react for 2 h; dialyze the solution for 3 days and then freeze-dry to obtain GelMA; Step S2, weigh 5 g of chondroitin sulfate, add 500 mL of deionized water, and dissolve at room temperature; add MA at the speed of step S2 by a syringe pump, and react at room temperature for 4 hours; dialyze the solution for 3 days and then freeze-dry to obtain ChSMA; Step S3, dissolving 1 g of ChSMA in 100 mL of deionized water, and adding 3 g of strongly acidic cation exchange resin; Step S4, after overnight reaction, add TBA-OH to adjust the pH to 7.0; Step S5, recovering and freeze-drying the solution to obtain ChSMA-TBA; Step S6: Dissolve 16 mg KGN in 2 mL DMSO, then add 4 mg DMAP and stir until completely dissolved; Step S7, add 12 μL of BOC2O melted at 37°C to the system, and stir at 25°C for 30 min to obtain an activated KGN solution; Step S8, take 100 mg of ChSMA-TBA and dissolve it thoroughly with 5 mL of DMSO; Step S9: slowly add the activated KGN solution in step S11 into the ChSMA-TBA solution, continue stirring, and react at 20° C. for 12 h.

[0032] Step S10, dialyzing the solution in an acidified sodium chloride solution for 1 day and in deionized water for 2 days; Step S11, freeze-drying for 2 days to obtain dry ChSMA-KGN; Step S12, weigh 5 g of GelMA, add 50 mL of deionized water containing photoinitiator LAP, and dissolve it fully at 37°C; Step S13, weigh 500 mg ChSMA-KGN, add 50 mL deionized water containing photoinitiator LAP, and dissolve evenly at room temperature; Step S14, mixing the two solutions of step S12 and step S13 in equal volumes; Step S15, preparing GelMA / ChSMA-KGN composite hydrogel microspheres by microfluidics; Step S16, recovering the microspheres and cleaning them; Step S17, freeze-drying to obtain esterase-responsive composite hydrogel microspheres (GelMA / ChSMA-KGN composite hydrogel microspheres, GCKM). Example 3

[0033] The same as Example 1, except that the final product prepared in this example is GelMA / GelMA-KGN composite hydrogel microspheres GGKM. Example 4

[0034] The same as Example 1, except that the final product prepared in this example is GelMA / AlgMA-KGN composite hydrogel microspheres GAKM. Comparative Example 1

[0035] The same as Example 1, except that this embodiment specifically includes the following steps: Step S1: using LAP solution containing photoinitiator to prepare 1% HAMA and 10% GelMA hydrogel solutions respectively, Step S2, mixing the two solutions in equal volumes; Step S3, preparing GelMA / HAMA composite hydrogel microspheres by microfluidic technology; Step S4, recovering the microspheres and cleaning them; Step S5: freeze-drying to obtain GelMA / HAMA microspheres GHM. Comparative Example 2

[0036] The same as Example 1, except that this embodiment specifically includes the following steps: Step S1: Prepare 10% GelMA hydrogel solution using LAP solution containing photoinitiator. Step S2, preparing GelMA hydrogel microspheres by microfluidic technology; Step S3, recovering the microspheres and cleaning them; Step S4: freeze-drying to obtain GelMA microspheres GM. Comparative Example 3 Microsphere Degradation Process

[0037] To simulate physiological conditions, 10 mg of GM, GHM, and GHKM were placed in 1 mL of PBS containing 0.5 U / mL type II collagenase (Sigma-Aldrich). The conditions were maintained at 37°C and pH 7.4, with continuous shaking at 100 rpm. At the same time, 10 mg of each type of microspheres were placed in 1 mL of PBS without type II collagenase as a control. At predetermined time intervals (weeks 1, 4, and 8), the degradation status of each group of microspheres was observed by optical microscopy. After observation, the microspheres were carefully rinsed twice with deionized water. The samples were then freeze-dried and weighed to measure the remaining mass, after which the microspheres were placed back in fresh 1 mL of PBS containing 0.5 U / mL type II collagenase to continue degradation.

[0038] Figure 1-Figure 6 As shown, the GHKM, GHM and GM microspheres prepared by Example 1 and Comparative Examples 1-2 were co-cultured with NPCs cells, and 1L-1β was added to stimulate the co-culture system, and the groups were set as GM-group, GM+group, GHM+group and GHKM+group (- / + represents the presence or absence of 1L-1β stimulation). The co-culture system was exposed to 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C in the dark for 20 min, and the fluorescence of ROS was photographed by confocal microscopy after the end. Comparative Example 4 ROS Measurement

[0039] Figure 1-Figure 6 As shown, the GHKM, GHM and GM microspheres prepared by Example 1 and Comparative Examples 1-2 were co-cultured with NPCs cells, and 1L-1β was added to stimulate the co-culture system, and the groups were set as GM-group, GM+group, GHM+group and GHKM+group (- / + represents the presence or absence of 1L-1β stimulation). The co-culture system was exposed to 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C in the dark for 20 min, and the fluorescence of ROS was photographed by confocal microscopy after the end. Comparative Example 5 Animal Experiment

[0040] Forty male Sprague-Dawley rats, each weighing 300 to 350 g, were selected and randomly assigned to five different groups: sham operation group (Sham), nuclear resection + PBS group (PBS), nuclear resection + NPCs group (NPCs), nuclear resection + NPCs@GHM group (NPCs@GHM), and nuclear resection + NPCs@GHKM group (NPCs@GHKM).

[0041] Rats were anesthetized by intraperitoneal injection of 10% (weight ratio) chloral hydrate. After successful anesthesia, the surgical area was disinfected with povidone-iodine and 75% ethanol. A dorsal longitudinal incision of approximately 1 cm in length was made at the center of the Co5-6 intervertebral disc, followed by stepwise dissection to expose the dorsal AF of the Co5-6 IVD. The AF was longitudinally incised using a No. 11 scalpel (1.5 mm), and the NP was completely removed using a 1 mm microscratcher. For the sham group, only the skin incision was made without puncturing the intervertebral disc. The PBS group received 10 μL PBS injected into the intervertebral disc, while the NPCs group received 10 μL NPCs suspension. NPCs@GHM and NPCs@GHKM received 10 μL of the corresponding co-cultured microspheres, respectively. After implantation, the AF and skin were sutured using 5-0 nylon sutures, and the incision was covered with a sterile dressing. The surgery was performed under sterile and warm conditions, and the rats were placed in a temperature-controlled, well-ventilated area after surgery.

[0042] Coccyx specimens of rats were collected after euthanasia at 4 and 8 weeks after surgery. They were first fixed with 4% paraformaldehyde and then decalcified in 10% EDTA for 8 weeks. After paraffin embedding, the specimens were cut into 6 μm thick sections, dewaxed and hydrated. Afterwards, the tissue sections were stained with hematoxylin-eosin and safranin and photographed under a microscope to obtain the histological staining results.

[0043] The GHKM, GCKM, GGKM and GAKM hydrogel microspheres prepared in Examples 1-4 were photographed by scanning electron microscope, which proved that the hydrogel microspheres obtained in Examples 1-4 had uniform particle sizes and had good three-dimensional porous structures, such as Figure 1 As shown; the four microspheres were co-cultured with NPCs cells, and the live-dead staining results of the cells after 3 days indicated that none of the four hydrogel microspheres had obvious biological toxicity, such as Figure 2 shown.

[0044] Through the microsphere degradation experiments of Example 1 and Comparative Examples 1, 2, and 3, it was found that the GHKM microspheres took longer to completely degrade and could play a more sustained role in the body.

[0045] The ROS measurement results of Example 1 and Comparative Examples 1, 2, and 4 show that ROS significantly increases under inflammatory conditions, and the esterase-responsive composite hydrogel microspheres GHKM can significantly inhibit the generation of ROS. Figure 3 shown.

[0046] Through the experiments of Example 1 and Comparative Examples 1, 2, and 5, it was found that GHKM can promote structural recovery and prevent further degeneration of the NP removal site. H&E and safranin staining showed that NP was missing in the PBS group and the AF laminar structure was gradually destroyed over time. The cartilage endplates became discontinuous and irregular, resulting in severe damage and significant collapse of the intervertebral disc space. In the NPCs group, although the continuity of the cartilage endplates was maintained, the intervertebral disc degeneration was still severe. Both the NPCs@GHM and NPCs@GHKM groups showed significant deposition of sulfated glycosaminoglycans in the nuclear fissure area, while the AF, bone, and cartilage structures were intact, especially in the NPCs@GHKM group, as shown in Figure 2. Figure 4 shown. Example 5

[0047] An esterase-responsive composite hydrogel microsphere is obtained by the preparation method of the hydrogel microsphere in any one of Examples 1-4, and can achieve uniform particle size and good dispersibility. The prepared microsphere can simulate the natural nucleus pulposus extracellular matrix, has good biocompatibility, and while ensuring good biocompatibility, can also provide a growth environment and mechanical support for neural precursor cells, and improve the adaptability of NPCs to the harsh IVDD microenvironment through the esterase-responsive release of KGN, can promote or protect nucleus pulposus cells, can promote the regeneration of neural precursor cells after rat tail nuclear resection, and provides a promising synergistic method for cell therapy in IVDD regeneration. Example 6

[0048] An application of the esterase-responsive composite hydrogel microspheres in Example 5 in intervertebral disc degeneration products.

[0049] Inflammation and oxidative stress are key drivers of IVDD pathogenesis. Inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) have been implicated in key pathophysiological processes in IVDD. Interleukin-1β (IL-1β) stimulation increases the levels of IL-6, IL-8, and interleukin-17 (IL-17) in IVD cells, triggering an inflammatory cascade. These cytokines promote extracellular matrix degradation, induce cell senescence and apoptosis, and enhance oxidative stress by producing excessive reactive oxygen species (ROS), leading to oxidative damage. Excessive ROS further disrupt extracellular matrix homeostasis, impair extracellular matrix synthesis, and promote its degradation. ROS also activate inflammatory pathways through nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, forming a vicious cycle of inflammation and oxidative stress. Therefore, combining anti-inflammatory and antioxidant strategies is promising for nanoparticle regeneration.

[0050] Catogenetin (KGN) promotes chondrogenesis, has anti-inflammatory effects, and acts as an antioxidant, making it a promising approach for the treatment of IVDD. Our previous studies have shown that KGN enhances matrix synthesis and inhibits degradation in NPCs, possibly through the activation of nuclear factor erythroid 2-related factor 2 (N RF 2) and its downstream antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (GPX1), which are able to scavenge reactive oxygen species and protect cells from oxidative damage.

[0051] In order to achieve sustained release in the NP area, the microspheres used in this example connected KGN with GelMA, HAMA, AlgMA or ChsMA, and the esterase activity of the implanted cells was enhanced under the acidic conditions of IVDD; composite hydrogel microspheres were prepared by photocrosslinking microfluidic technology, and the microstructure, biocompatibility and degradability of the microspheres were evaluated, and their effect in promoting NPCs regeneration of nanoparticles was further discovered; finally, the in vivo regenerative potential of NPCs-loaded composite hydrogel microspheres on nanoparticle defects was evaluated by the rat caudate nucleotomy model, which provides a promising synergistic approach for cell therapy in IVDD regeneration, enabling its better application in intervertebral disc degeneration products.

[0052] It is easy to understand that those skilled in the art can combine, split, reorganize, etc. the embodiments of the present application to obtain other embodiments based on one or several embodiments provided in the present application, and these embodiments do not exceed the protection scope of the present application.

[0053] The present invention and its implementation methods are described schematically above, and the description is not restrictive. The embodiments shown in the embodiments are only part of the implementation methods of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by the embodiments and designs a structure and an implementation method similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing esterase-responsive composite hydrogel microspheres, characterized in that: include: KGN was connected with methacryloyl gelatin GelMA, methacryloyl hyaluronic acid HAMA, methacryloyl sodium alginate AlgMA or methacryloyl chondroitin sulfate ChsMA, and esterase-responsive composite hydrogel microspheres were prepared by photo-crosslinking microfluidic technology.

2. The method for preparing an esterase-responsive composite hydrogel microsphere according to claim 1, comprising the following steps: Step S1, preparing methacrylylated gelatin GelMA, methacrylylated hyaluronic acid HAMA, methacrylylated sodium alginate AlgMA, and methacrylylated chondroitin sulfate ChSMA; Step S2, grafting katogenetin KGN onto methacryloyl gelatin GelMA, methacryloyl hyaluronic acid HAMA, methacryloyl sodium alginate AlgMA or methacryloyl chondroitin sulfate ChSMA to obtain GelMA-KGN or HAMA-KGN or AlgMA-KGN or ChSMA-KGN, respectively; Step S3, blending methacrylylated gelatin GelMA with GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN to prepare composite hydrogel microspheres by microfluidics.

3. According to the method for preparing an esterase-responsive composite hydrogel microsphere according to claim 2, step S1 comprises: Step S11, adding deionized water to gelatin, hyaluronic acid, sodium alginate or chondroitin sulfate to prepare a gelatin solution, a hyaluronic acid solution, a sodium alginate solution or a chondroitin sulfate solution; Step S12, adding methacrylic anhydride MA to the gelatin solution, hyaluronic acid solution, sodium alginate solution or chondroitin sulfate solution to react Step S13, after the reaction, dialyzing and freeze-drying the solution; Step S14, after freeze-drying, methacrylylated gelatin GelMA, methacrylylated hyaluronic acid HAMA, methacrylylated sodium alginate AlgMA, and methacrylylated chondroitin sulfate ChSMA are obtained.

4. According to the method for preparing an esterase-responsive composite hydrogel microsphere according to claim 2, step S2 comprises: Step S21, adding deionized water to dissolve the freeze-dried methacrylylated gelatin GelMA, methacrylylated hyaluronic acid HAMA, methacrylylated sodium alginate AlgMA or methacrylylated chondroitin sulfate ChSMA to obtain a methacrylylated gelatin solution, a methacrylylated hyaluronic acid solution, a methacrylylated sodium alginate solution or a methacrylylated chondroitin sulfate solution; Step S22, adding a strong acid cation exchange resin to the methacrylated gelatin solution, the methacrylated hyaluronic acid solution, the methacrylated sodium alginate solution or the methacrylated chondroitin sulfate solution, and reacting overnight to perform ion exchange; Step S23, adding tetrabutylammonium hydroxide TBA-OH, adjusting the pH to 7.0, to obtain solution A; Step S24, recovering and freeze-drying the solution A in step S23 to obtain GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA; Step S25, dissolving catogeneine KGN with anhydrous dimethyl sulfoxide (DMSO) under nitrogen protection to obtain a catogeneine solution; Step S26, adding 4-dimethylaminopyridine DMAP to the catogynine solution of step S25 until it is completely dissolved to obtain solution B; Step S27, adding di-tert-butyl carbonate BOC2O melted at 37° C. to the solution B obtained in step S26 to obtain a mixture A; Step S28, stirring the mixture A in step S27 at 25° C. for 30 min to obtain an activated KGN solution; Step S29, dissolving GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBAHAMA-TBA in DMSO; Step S210, slowly adding the activated KGN solution to the GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA solution, stirring continuously, and reacting overnight; Step S211, dialysis in deionized water, and freeze-drying after dialysis; Step S212: freeze-drying to obtain dry GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN.

5. The method for preparing an esterase-responsive composite hydrogel microsphere according to claim 2, wherein in step S3, the concentration of GelMA solution is 10-20%, the concentration of GelMA-KGN solution is 10-20%, the concentration of HAMA-KGN solution is 1-2%, the concentration of AlglMA-KGN solution is 2-5%, the concentration of ChSMA-KGN solution is 2-10%, and the mixed volume ratio of GelMA to GelMA-KGN, HAMA-KGN, AlgMA-KGN or ChSMA-KGN is 1:1-3:

1.

6. The method for preparing an esterase-responsive composite hydrogel microsphere according to claim 2, characterized in that: In step S11, the concentration of the gelatin solution is 5%-10%, the concentration of the hyaluronic acid solution is 0.5%-4%, the concentration of the sodium alginate solution is 1%-4%, and the concentration of the chondroitin sulfate solution is 2.5%-10%; In step S12, the final concentration of methacrylic anhydride MA added to gelatin is 0.1-0.8 mL / g gelatin, the final concentration of methacrylic anhydride MA added to hyaluronic acid is 2-5 mL / g hyaluronic acid, the final concentration of methacrylic anhydride MA added to sodium alginate is 1-3 mL / g sodium alginate, and the final concentration of methacrylic anhydride MA added to chondroitin sulfate is 0.5-1 mL / g chondroitin sulfate.

7. According to the method for preparing an esterase-responsive composite hydrogel microsphere according to claim 4, the mass of the strongly acidic cation exchange resin in step S22 is 3-5 times the dry mass of GelMA, HAMA, AlgMA or ChSMA.

8. According to the method for preparing an esterase-responsive composite hydrogel microsphere according to claim 4, the volume ratio of the activated KGN solution to the GelMA-TBA, HAMA-TBA, AlgMA-TBA or ChSMA-TBA solution in step S210 is 2:5-1:

1.

9. An esterase-responsive composite hydrogel microsphere, characterized in that: The esterase-responsive composite hydrogel microspheres are prepared by the preparation method of any one of claims 1 to 8.

10. Use of the esterase-responsive composite hydrogel microspheres according to claim 9 in intervertebral disc degeneration products.

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