Injectable sodium grass ammonia functionalized hydrogel microspheres loaded with TGFβ3 and application thereof

Abstract

The present application relates to the technical field of intervertebral disc degeneration treatment and drug delivery, and discloses an injectable oxynate sodium functionalized hydrogel microsphere loaded with TGFbeta3 and application, by loading glycolysis inhibitor SO in the GelMA microsphere matrix and post-adsorbing TGFbeta3, on the one hand, SO is used to inhibit the excessive active glycolysis in the degenerative microenvironment, reduce lactic acid accumulation and relieve inflammation, on the other hand, TGFbeta3 is used to effectively promote the synthesis of collagen and proteoglycan, both of which synergistically act from the two aspects of metabolic regulation and matrix synthesis, and jointly promote the recovery of intervertebral disc homeostasis, therefore, the oxynate sodium functionalized microsphere loaded with TGFbeta3 constructed by the present application can realize the synergistic treatment of multiple targets and multiple pathways by synchronously delivering metabolic regulators and synthesis promoters, so as to more effectively delay or reverse the intervertebral disc degeneration process, and finally achieve the beneficial effect of the double repair effect of simultaneously regulating abnormal glycolysis metabolism of degenerative nucleus pulposus cells and promoting extracellular matrix synthesis.

Description

Technical Field

[0001] This invention relates to the field of intervertebral disc degeneration treatment and drug delivery technology, specifically to an injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3 and its application. Background Technology

[0002] Intervertebral disc degeneration is one of the main causes of chronic low back pain. Its pathological features include metabolic disorders of nucleus pulposus cells, activation of local inflammatory responses, and degradation of the extracellular matrix. Current clinical treatments are mostly limited to symptom relief and cannot achieve tissue regeneration. The intervertebral disc is located in a unique microenvironment of ischemia and hypoxia, and nucleus pulposus cells mainly rely on glycolysis for energy. Studies have shown that the inflammatory microenvironment during degeneration leads to abnormally enhanced glycolysis, lactic acid accumulation, and further exacerbates extracellular matrix degradation and tissue structure damage. Therefore, regulating glycolysis and promoting matrix synthesis has become a new strategy for treating intervertebral disc degeneration. In existing technologies, studies have used hydrogels loaded with growth factors or anti-inflammatory drugs for intradiscal delivery, but these often target single pathological processes and suffer from problems such as drug burst release, short retention time, and low bioavailability. Sodium oxalate, as a specific inhibitor of lactate dehydrogenase, can inhibit excessive glycolysis and reduce lactic acid production; TGFβ3 can effectively promote extracellular matrix synthesis. However, there is still a lack of effective carrier systems for the synergistic, long-term, and targeted delivery of these two substances into the intervertebral disc. To address the aforementioned problems in the prior art, this invention provides an injectable, biocompatible, and synergistically controlled release of sodium oxalate and TGFβ3 functionalized hydrogel microspheres for the repair and treatment of intervertebral disc degeneration. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 and their applications. These microspheres possess advantages such as strong injectability, good tissue compatibility, uniform particle size, stable structure, controllable drug loading, and adjustable release behavior. Furthermore, they enable synergistic controlled release and dual repair of sodium oxalate and TGFβ3. This invention solves the problems of existing hydrogels that load only a single drug, target only a single pathological stage, and suffer from sudden drug release, short residence time at the lesion site, and low bioavailability, thus failing to effectively achieve efficient repair of intervertebral disc degeneration.

[0004] To achieve the above objectives, the present invention provides the following technical solution: an injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3, wherein the hydrogel microsphere is composed of methacrylamide gelatin, sodium oxalate, and transforming growth factor β3, wherein sodium oxalate is used to correct pathological metabolic states and provide a favorable microenvironment for cell function recovery; transforming growth factor β3 maximizes its pro-synthetic repair efficacy in the microenvironment; the methacrylamide gelatin matrix not only serves as a co-carrier for sodium oxalate and transforming growth factor β3, enabling controlled release, but also provides a biomimetic adhesion and growth scaffold for cells.

[0005] Preferably, the methacrylamide gelatin has structural formula I, abbreviated as GelMA; sodium oxalate has structural formula II, abbreviated as SO; transforming growth factor β3 is abbreviated as TGFβ3, and structural formula I is expressed as:

[0006] In the formula, R represents the gelatin backbone; n represents the number of lysine residues substituted with methacryloyl groups; Structural formula II is expressed as:

[0007] In the formula, Na⁺ represents sodium ions, which form ionic bonds with oxalate ions.

[0008] Preferably, an injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3 comprises the following preparation steps: Step 1: Preparation of reagents and materials: Methacrylamide gelatin, photoinitiator LAP, sodium oxalate and TGFβ3 are used as functional components, and sterile PBS, mineral oil, surfactant Span 80, isopropanol and ethanol are used as auxiliary reagents. Step 2, GelMA-SO prepolymer solution preparation: Under light-protected conditions, dissolve methacrylamide gelatin and photoinitiator LAP in sterile PBS, heat and stir until completely dissolved, then add sodium oxalate and mix well to obtain the prepolymer solution; Step 3: Preparation of W / O type microdroplets by microfluidic method: Using a coaxial microfluidic device, the prepolymer obtained in step 2 is injected as the dispersed phase into the inner diameter capillary, and a mineral oil solution containing surfactant is injected as the continuous phase into the outer diameter sleeve. The flow rate of the two phases is controlled by a precision injection pump, and uniform water-in-oil microdroplets are formed at the capillary outlet and collected in low temperature mineral oil. Step 4, Photocrosslinking Curing and Washing and Drying: The microdroplets in the receiving dish are immediately placed under a 405nm ultraviolet light source to irradiate the methacrylamide gelatin to undergo a photocrosslinking reaction and solidify into spheres. The microspheres are then collected by centrifugation, washed and freeze-dried to obtain white SO@GelMA microsphere powder. Step 5, TGFβ3 post-loading: Place the lyophilized SO@GelMA microspheres from Step 4 into a sterile container, add freshly prepared TGFβ3 PBS solution, and gently shake and incubate at 3-4℃ to allow TGFβ3 to be loaded into the porous network of the microspheres through physical adsorption. After loading is complete, remove the supernatant, rinse with sterile PBS, and obtain the final sodium oxalate functionalized hydrogel microspheres loaded with TGFβ3.

[0009] Preferably, in step one, the degree of methacrylyl substitution of the methacrylated gelatin is 60%; the mass concentration of the methacrylated gelatin in the final cross-linked microsphere network is 7% (w / v); the concentration of sodium oxalate added to the methacrylated gelatin prepolymer solution is 10 mM; and the loading concentration of TGFβ3 is 100 ng / mL.

[0010] Preferably, the preparation conditions of the GelMA-SO prepolymer solution in step two are as follows: under light-protected conditions, sterile PBS is used as a solvent, and the dissolution conditions are water bath stirring at 44-45℃; LAP is used as a photoinitiator; after the sodium oxalate is added, the mixture is thoroughly mixed in a vortex mixer for 30-45 seconds to obtain a homogeneous and clear prepolymer solution.

[0011] Preferably, the preparation of W / O type microdroplets in step three includes: (1) The continuous phase is a mineral oil solution containing 1% to 10% (v / v) of surfactant Span 80; (2) When the prepolymer is injected as the dispersed phase, the flow rate is controlled between 0.5-2 mL / h, and when the mineral oil solution containing surfactant is injected, the flow rate is controlled between 50-200 mL / h, with the preferred flow rate ratio being 1:100. (3) The water-in-oil micro-liquid formed by shearing at the capillary outlet drips into a receiving dish containing low-temperature mineral oil after dropping.

[0012] Preferably, the photocrosslinking curing conditions in step four are: UV irradiation time of 5-15 minutes; centrifugation conditions of 2500-3000 rpm for 5-8 minutes.

[0013] Preferably, in step four, the washing and drying process involves sequentially washing with isopropanol, 75% ethanol, and sterile PBS; the freeze-dried microspheres are then sealed and stored at -20°C.

[0014] Preferably, in step five, the concentration of the freshly prepared TGFβ3 PBS solution is 100 ng / mL; the incubation time is 12-24 hours, and the particle size distribution of the TGFβ3-loaded sodium oxalate functionalized hydrogel microspheres is in the range of 100-300 μm.

[0015] Application of injectable sodium oxalate functionalized hydrogel microspheres loaded with TGFβ3: The sodium oxalate functionalized hydrogel microspheres prepared according to the above method are used for the repair treatment of intervertebral disc degeneration. They are injected through a 21G-27G injection needle and are suitable for percutaneous minimally invasive interventional treatment.

[0016] Compared with the prior art, the present invention provides injectable sodium oxalate functionalized hydrogel microspheres loaded with TGFβ3 and their applications, which have the following beneficial effects: 1. This invention loads the glycolysis inhibitor SO onto a GelMA microsphere matrix and then adsorbs TGFβ3. On one hand, SO inhibits excessive glycolysis in the degenerative microenvironment, reducing lactic acid accumulation and alleviating inflammation. On the other hand, TGFβ3 effectively promotes collagen and proteoglycan synthesis. These two mechanisms work synergistically at both the metabolic regulation and matrix synthesis levels to jointly promote the restoration of intervertebral disc homeostasis. Therefore, the TGFβ3-loaded sodium oxalate functionalized microspheres constructed in this invention achieve multi-target, multi-pathway synergistic therapy by simultaneously delivering metabolic regulators and synthesis-promoting factors. This more effectively delays or reverses the degenerative process of the intervertebral disc, achieving the beneficial effect of simultaneously regulating abnormal glycolytic metabolism in degenerated nucleus pulposus cells and promoting extracellular matrix synthesis.

[0017] 2. This invention utilizes microfluidic technology combined with photocrosslinking curing process to achieve uniform particle size and good monodispersity of SO@GelMA microspheres, while also meeting the requirements of simple process, good repeatability, and suitability for large-scale production. Furthermore, by optimizing the microsphere particle size range (100-300 μm) and preparation process, ST@GM microspheres can be successfully delivered to the intervertebral disc nucleus pulposus region through 21G-27G injection needles, achieving long-term sustained release and tissue retention in vivo. This makes them more suitable for percutaneous minimally invasive interventional therapy, achieving the beneficial effects of convenient use and long-lasting therapeutic effects.

[0018] 3. This invention was verified by an in vivo rat caudal degeneration model. ST@GM microsphere injection can effectively maintain intervertebral disc height and nucleus pulposus water content, promote COL II expression and inhibit COX2 expression, thereby significantly inhibiting the progression of intervertebral disc degeneration and promoting tissue repair. Attached Figure Description

[0019] Figure 1 This is a scanning electron microscope image of the microspheres in an embodiment of the present invention (showing their surface morphology and pore structure). Figure 2 The infrared spectrum of the microspheres in this embodiment of the invention (used to prove the successful introduction of SO); Figure 3 This is a diagram illustrating the effect of microspheres on the regulation of lactate production in nucleus pulposus cells in vitro, according to an embodiment of the present invention. Figure 4 This is an imaging result of SD rats maintaining intervertebral disc height and water content after 4 weeks of using hydrogel microspheres, as described in this embodiment of the invention. Figure 5 This is a histological staining diagram showing the effect of the hydrogel microspheres on collagen II accumulation and inhibition of inflammatory factor expression after 4 weeks of treatment, as described in this embodiment of the invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figures 1-5 An injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3 is disclosed. The hydrogel microsphere is composed of methacrylamide gelatin, sodium oxalate, and transforming growth factor β3. Sodium oxalate is used to correct pathological metabolic states and provide a favorable microenvironment for cell function recovery. Transforming growth factor β3 maximizes its pro-synthetic repair efficacy in the microenvironment. The methacrylamide gelatin matrix not only serves as a co-carrier for sodium oxalate and transforming growth factor β3, enabling controlled release, but also provides a biomimetic adhesion and growth scaffold for cells. The three components work synergistically in the hydrogel microsphere.

[0022] Specifically, methacrylamide gelatin is structural formula I, abbreviated as GelMA; sodium oxalate is structural formula II, abbreviated as SO; transforming growth factor β3 is abbreviated as TGFβ3, and structural formula I is expressed as:

[0023] In the formula, R represents the gelatin backbone; n represents the number of lysine residues substituted with methacryloyl (i.e., the degree of methacrylation). Structural formula II is expressed as:

[0024] In the formula, Na⁺ represents sodium ions; it forms an ionic bond with oxalate ions (-OOC-CO-NH2).

[0025] Specifically, an injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3 includes the following preparation steps: Step 1: Preparation of reagents and materials: Methacrylamide gelatin (GelMA), photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphinate), sodium oxalate (SO) and TGFβ3 (transforming growth factor β3) are used as core functional components, and sterile PBS, mineral oil, surfactant Span 80, isopropanol and ethanol are used as auxiliary reagents. Step 2, GelMA-SO prepolymer solution preparation: Under light-protected conditions, dissolve methacrylamide gelatin (GelMA) and photoinitiator LAP in sterile PBS, heat and stir until completely dissolved, then add sodium oxalate (SO) and mix well to obtain a homogeneous and clear prepolymer solution. Step 3: Preparation of W / O type microdroplets by microfluidic method: Using a coaxial microfluidic device, the prepolymer obtained in step 2 is injected as the dispersed phase into the inner diameter capillary, and a mineral oil solution containing surfactant is injected as the continuous phase into the outer diameter sleeve. The flow rate of the two phases is controlled by a precision injection pump, and uniform water-in-oil microdroplets are formed at the capillary outlet and collected in low temperature mineral oil. Step 4, Photocrosslinking Curing and Washing & Drying: The microdroplets in the receiving dish were immediately placed under a 405nm ultraviolet light source to irradiate the methacrylamide gelatin (GelMA) to undergo a photocrosslinking reaction and solidify into spheres. The microspheres were then collected by centrifugation and washed sequentially with isopropanol, 75% ethanol and sterile PBS to remove residual oil phase and surfactants. The washed microspheres were then freeze-dried to obtain white SO@GelMA microsphere powder. Step 5, TGFβ3 post-loading: Place the lyophilized SO@GelMA microspheres from Step 4 into a sterile container, add freshly prepared TGFβ3 PBS solution, ensuring the microspheres are completely submerged, and gently incubate with shaking at 3-4℃ to allow TGFβ3 to be loaded into the porous network of the microspheres through physical adsorption. After loading is complete, remove the supernatant and gently rinse 1-2 times with an equal volume of sterile PBS to obtain the final TGFβ3-loaded sodium oxalate functionalized hydrogel microspheres (ST@GM microspheres).

[0026] Specifically, in step one, the degree of methacrylyl substitution of the methacrylated gelatin (GelMA) is preferably 60% to ensure photocrosslinking efficiency and retain the bioactivity of the gelatin; the mass concentration of methacrylated gelatin (GelMA) in the final crosslinked microsphere network is in the range of 5%-10% (w / v), preferably 7% (w / v); the concentration of sodium oxalate (SO) added to the methacrylated gelatin (GelMA) prepolymer solution is 5mM-10mM, preferably 10mM; and the loading concentration of TGFβ3 is 50ng / mL-200ng / mL, preferably 100ng / mL.

[0027] By setting the component and parameter ranges in step one above, the synergistic compatibility of the GelMA matrix, SO and TGFβ3 is ensured, thus providing a stable material basis for the subsequent microsphere preparation.

[0028] Specifically, the preparation conditions for the GelMA-SO prepolymer solution in step two are as follows: under light-protected conditions, sterile PBS is used as the solvent, and the dissolution conditions are water bath stirring at 44-45℃; LAP is used as the photoinitiator; after the sodium oxalate (SO) is added, it is thoroughly mixed in a vortex mixer for 30-45 seconds to obtain a homogeneous and clear prepolymer solution, which is used for subsequent microfluidic droplet preparation.

[0029] By optimizing the light-shielding dissolution conditions, water bath temperature, and vortex oscillation method in step two above, a uniform and clear GelMA-SO prepolymer solution is obtained, thereby ensuring the uniform distribution of SO in the microspheres and the stability of subsequent photocrosslinking.

[0030] Specifically, step three, the preparation of W / O type microdroplets, includes: (1) The continuous phase is a mineral oil solution containing 1% to 10% (v / v) of surfactant Span 80; (2) When the prepolymer is injected as the dispersed phase, the flow rate is controlled between 0.5-2 mL / h, and when the mineral oil solution containing surfactant is injected, the flow rate is controlled between 50-200 mL / h, with the preferred flow rate ratio being 1:100. (3) The water-in-oil micro-liquid formed by shearing at the capillary outlet drips into a receiving dish containing low-temperature mineral oil after dropping.

[0031] By controlling the flow rates, flow rate ratio, and surfactant concentration of the dispersed and continuous phases in step three above, uniform shearing and stable shaping of microdroplets can be achieved, thus laying the technological foundation for the preparation of microspheres with controllable particle size and good monodispersity.

[0032] Specifically, in step four, the photocrosslinking curing conditions are as follows: the UV irradiation time is set to 5-15 minutes; the centrifugation conditions are 2500-3000 rpm for 5-8 minutes.

[0033] By setting the UV irradiation time, centrifugation conditions, and multi-stage washing process in step four above, we can ensure that the photocrosslinking and curing of the microspheres are complete and that the oil phase and surfactants are thoroughly removed, thereby obtaining SO@GelMA microsphere powder with a complete structure and pure and dry powder.

[0034] Specifically, in step four, washing and drying: the microspheres are washed sequentially with isopropanol, 75% ethanol, and sterile PBS to ensure their purity; the freeze-dried microspheres are then sealed and stored at -20°C.

[0035] Through the above process, the porous structure of the microspheres and the bioactivity of SO can be effectively maintained, thus providing an ideal carrier state for the subsequent efficient loading of TGFβ3.

[0036] Specifically, in step five, the concentration of the freshly prepared TGFβ3 PBS solution is in the range of 50-200 ng / mL, preferably 100 ng / mL; the incubation time is 12-24 hours, and the resulting TGFβ3-loaded sodium oxalate functionalized hydrogel microspheres have uniform particle size, distributed in the range of 100-300 μm, and have good monodispersity. When used, they can be redispersed in sterile PBS or physiological saline to form a suspension that can be injected.

[0037] By using the TGFβ3 post-loading conditions (concentration, temperature, incubation time) and gentle rinsing method adopted in step five above, efficient physical adsorption and uniform distribution of TGFβ3 in the porous network of SO@GelMA microspheres are achieved. The resulting ST@GM microspheres have uniform particle size (100-300μm), good monodispersity, and can be redispersed in sterile PBS or physiological saline to form a stable suspension suitable for minimally invasive injection.

[0038] An application of injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3: The sodium oxalate-functionalized hydrogel microspheres prepared according to the above method are used for the repair treatment of intervertebral disc degeneration. During use, they are successfully injected through a 21G-27G injection needle to ensure that the ST@GM microspheres have good injectability and intradiscal delivery capability, thus making them suitable for percutaneous minimally invasive interventional treatment.

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments: Example

[0040] Preparation of SO@GelMA microspheres: At room temperature, 0.35 g of GelMA (60% substitution) and 0.25 mg of LAP were weighed and added to 5 mL of PBS. The mixture was dissolved at 45 °C in the dark. SO was added to a final concentration of 10 mM and mixed to obtain the precursor solution. A coaxial microfluidic device was used with an aqueous phase flow rate of 0.5 mL / h and an oil phase (containing 5% Span 80 mineral oil) flow rate of 50 mL / h. The droplets were collected in an ice bath and crosslinked under 405 nm UV light for 10 min. Finally, the microspheres were washed sequentially with isopropanol, 75% ethanol, and PBS to obtain lyophilized microspheres. Example

[0041] Loading with TGFβ3: Take 10 mg of lyophilized microspheres, immerse them in 1 mL of PBS containing 100 ng / mL TGFβ3, incubate at 37 °C for 12 h, and wash with PBS to obtain ST@GM microspheres (sodium oxalate functionalized hydrogel microspheres loaded with TGFβ3 of this invention).

[0042] Example 3 (In vitro lactate detection) To evaluate the regulatory effect of functionalized hydrogel microspheres on glycolytic metabolism in degenerated nucleus pulposus cells, lactate production was detected using an in vitro cell model. The specific experimental methods are as follows: T1. Primary nucleus pulposus cells from P2 generation rats were collected, at a density of 1 × 10⁻⁶ cells per well. ^4 Cells were seeded at a density of 10% in 96-well plates and cultured in DMEM / F12 complete medium containing 10% fetal bovine serum until the cells adhered. T2, after starvation treatment with 2% low serum culture medium for 6 hours, was divided into groups for intervention: (1) Normal control group (Control group): Cells were cultured in complete culture medium; (2) Degeneration model group (TNF-α group): Cells were cultured in complete medium containing 10 ng / mL TNF-α; (3) SO@GM intervention group: Cells were co-cultured with GelMA microspheres loaded with SO (5mM or 10mM) and the culture medium contained 10ng / mL TNF-α; (4) ST@GM intervention group: Cells were co-cultured with GelMA microspheres loaded with SO (10mM) and TGFβ3 (100ng / mL), and the culture medium contained 10ng / mL TNF-α. Five replicates were set up for each group. T3. After 72 hours of intervention, cell culture supernatant was collected, and the lactate detection kit (WST-8 method) was used according to the instructions. The absorbance of each well was measured at 450 nm using an ELISA reader, and the lactate concentration was calculated based on the standard curve. The experimental results showed that compared with the Control group (5.38±0.28 μmol / L), the lactate production in the TNF-α group was significantly increased (14.52±1.09 μmol / L, p<0.01), while the 5 mM SO@GM group (9.78±0.89 μmol / L) and the 10 mM SO@GM group (6.40±0.24 μmol / L) significantly reduced the lactate level (p<0.05). Among them, there was no statistically significant difference in lactate concentration between the 10 mM SO@GM group and the Control group (p>0.05). These results indicate that SO-functionalized microspheres can effectively inhibit TNF-α-induced abnormal glycolysis in nucleus pulposus cells and restore their lactate metabolic homeostasis.

[0043] Example 4 (In vivo treatment of intervertebral disc degeneration) To evaluate the therapeutic effects of functionalized hydrogel microspheres in vivo, a rat model of caudal vertebral degeneration was established using percutaneous acupuncture, and different microsphere interventions were performed. The specific experimental methods are as follows: S1. Healthy male SD rats aged 6-8 weeks were selected and randomly divided into 5 groups: (1) Sham surgery group; (2) Degeneration model + PBS injection group (Defect group); (3) Degeneration model + SO@GM microsphere injection group (S@GM group); (4) Degeneration model + T@GM microsphere injection group (T@GM group); (5) Degeneration model + ST@GM microsphere injection group (ST@GM group); Six animals were included in each group. After anesthesia, punctures were performed at the Co7-8 and Co9-10 segments of the caudal vertebrae. A 20G needle was used to percutaneously puncture the nucleus pulposus to the middle, rotated 360° and held for 30 seconds to establish a degeneration model. After the model was established, 10 μL of the corresponding preparation (PBS or PBS suspension containing microspheres) was injected into the nucleus pulposus of the injured segment in each group. The animals were fed routinely after the operation, and radiographic and histological evaluations were performed at 4 and 8 weeks after the intervention. S2. Imaging Evaluation: After euthanizing the animals, the tails were separated, and lateral X-ray radiography and 1.5T T2-weighted magnetic resonance imaging were performed. The Disc Height Index (DHI) and nucleus pulposus signal intensity were measured using ImageJ software. The results showed that compared with the Sham group, the DHI and nucleus pulposus signal in the Defect group decreased significantly over time (p<0.05). The ST@GM group effectively maintained disc height and nucleus pulposus water content at both time points, with no significant difference from the Sham group (p>0.05), and the effect was better than other intervention groups.

[0044] S3. Histological evaluation: Intervertebral disc samples were fixed with 4% paraformaldehyde, decalcified with EDTA, and embedded in paraffin. 6μm sections were prepared and stained with H&E and Safranin O-Fast Green. Semi-quantitative analysis was performed according to the histological scoring system. The staining results showed that the nucleus pulposus structure in the Defect group was severely damaged, with loss of extracellular matrix and significant inflammatory cell infiltration; the nucleus pulposus tissue in the ST@GM group was better preserved, with deeper matrix staining and relatively intact structure, and the histological score was significantly better than that of the Defect group (p<0.01).

[0045] S4. Immunofluorescence analysis of tissues: Intervertebral disc sections were co-stained with COL II and COX2 immunofluorescence. The results showed that COL II expression was stronger and COX2 expression was weaker in the ST@GM group, suggesting that it can promote matrix synthesis and inhibit inflammatory response.

[0046] In summary, this invention successfully constructed injectable sodium oxalate-functionalized hydrogel microspheres (ST@GM microspheres) loaded with TGFβ3. Using methacrylamide gelatin (GelMA) as a matrix, the microfluidic technology combined with photocrosslinking and curing was employed to simultaneously load the glycolysis inhibitor sodium oxalate (SO) and subsequently adsorb the growth factor TGFβ3. These microspheres exhibit uniform particle size (100-300 μm), good monodispersity, and excellent injectability and tissue retention. In vitro experiments showed that SO@GelMA microspheres effectively inhibited TNF-α-induced abnormal glycolysis in nucleus pulposus cells and restored lactate metabolic homeostasis. ST@GM microspheres further synergistically enhanced TGFβ3, exerting a dual repair effect of metabolic regulation and matrix synthesis. In vivo experiments using a rat caudal degeneration model confirmed that ST@GM microspheres effectively maintained intervertebral disc height and nucleus pulposus water content at 4 and 8 weeks post-injection and promoted type II collagen (COL) synthesis. II) Synthesis to inhibit the expression of inflammatory factor (COX2), the histological score is significantly better than the single factor intervention group, and the preparation process of this invention is simple and reproducible, providing a novel carrier system with synergistic regulation of metabolic-synthetic dual pathways for minimally invasive treatment and tissue regeneration of intervertebral disc degeneration.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An injectable sodium oxalate-functionalized hydrogel microsphere loaded with TGFβ3, characterized in that, The hydrogel microspheres are composed of methacrylamide gelatin, sodium oxalate, and transforming growth factor β3. Sodium oxalate is used to correct pathological metabolic states and provide a favorable microenvironment for cell function recovery. Transforming growth factor β3 maximizes its pro-synthetic repair efficacy in the microenvironment. The methacrylamide gelatin matrix not only serves as a co-carrier for sodium oxalate and transforming growth factor β3, enabling controlled release, but also provides a biomimetic adhesion and growth scaffold for cells.

2. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 1, characterized in that, The methacrylamide gelatin has structural formula I, abbreviated as GelMA; sodium oxalate has structural formula II, abbreviated as SO; transforming growth factor β3 is abbreviated as TGFβ3, and structural formula I is expressed as: In the formula, R represents the gelatin backbone; n represents the number of lysine residues substituted with methacryloyl groups; Structural formula II is expressed as: In the formula, Na⁺ represents sodium ions, which form ionic bonds with oxalate ions.

3. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 1, characterized in that, The preparation steps include the following: Step 1: Preparation of reagents and materials: Methacrylamide gelatin, photoinitiator LAP, sodium oxalate and TGFβ3 are used as functional components, and sterile PBS, mineral oil, surfactant Span 80, isopropanol and ethanol are used as auxiliary reagents. Step 2, GelMA-SO prepolymer solution preparation: Under light-protected conditions, dissolve methacrylamide gelatin and photoinitiator LAP in sterile PBS, heat and stir until completely dissolved, then add sodium oxalate and mix well to obtain the prepolymer solution; Step 3: Preparation of W / O type microdroplets by microfluidic method: Using a coaxial microfluidic device, the prepolymer obtained in step 2 is injected as the dispersed phase into the inner diameter capillary, and a mineral oil solution containing surfactant is injected as the continuous phase into the outer diameter sleeve. The flow rate of the two phases is controlled by a precision injection pump, and uniform water-in-oil microdroplets are formed at the capillary outlet and collected in low temperature mineral oil. Step 4, Photocrosslinking Curing and Washing and Drying: The microdroplets in the receiving dish are immediately placed under a 405nm ultraviolet light source to irradiate the methacrylamide gelatin to undergo a photocrosslinking reaction and solidify into spheres. The microspheres are then collected by centrifugation, washed and freeze-dried to obtain white SO@GelMA microsphere powder. Step 5, TGFβ3 post-loading: Place the lyophilized SO@GelMA microspheres from Step 4 into a sterile container, add freshly prepared TGFβ3 PBS solution, and gently shake and incubate at 3-4℃ to allow TGFβ3 to be loaded into the porous network of the microspheres through physical adsorption. After loading is complete, remove the supernatant, rinse with sterile PBS, and obtain the final sodium oxalate functionalized hydrogel microspheres loaded with TGFβ3.

4. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, In step one, the degree of methacrylyl substitution of the methacrylamide gelatin is preferably 60%; the mass concentration of methacrylamide gelatin in the final cross-linked microsphere network is in the range of 5%-10% (w / v); the concentration of sodium oxalate added to the methacrylamide gelatin prepolymer solution is 5mM-10mM; and the loading concentration of TGFβ3 is 50ng / mL-200ng / mL.

5. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, In step two, the preparation conditions for the GelMA-SO prepolymer solution are as follows: under light-protected conditions, sterile PBS is used as the solvent, and the dissolution conditions are water bath stirring at 44-45℃; LAP is used as the photoinitiator; after the sodium oxalate is added, the mixture is thoroughly mixed in a vortex mixer for 30-45 seconds to obtain a homogeneous and clear prepolymer solution.

6. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, The preparation of W / O type microdroplets in step three includes: (1) The continuous phase is a mineral oil solution containing 1% to 10% (v / v) of surfactant Span 80; (2) When the prepolymer is injected as the dispersed phase, the flow rate is controlled between 0.5-2 mL / h, and when the mineral oil solution containing surfactant is injected, the flow rate is controlled between 50-200 mL / h. (3) The water-in-oil micro-liquid formed by shearing at the capillary outlet drips into a receiving dish containing low-temperature mineral oil after dropping.

7. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, In step four, the photocrosslinking curing conditions are as follows: the UV irradiation time is set to 5-15 minutes; the centrifugation conditions are 2500-3000 rpm for 5-8 minutes.

8. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, In step four, the microspheres are washed sequentially with isopropanol, 75% ethanol, and sterile PBS. After freeze-drying, the microspheres are sealed and stored at -20°C.

9. The injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3 according to claim 3, characterized in that, In step five, the concentration of the freshly prepared TGFβ3 PBS solution is in the range of 50-200 ng / mL; the incubation time is 12-24 hours, and the particle size distribution of the TGFβ3-loaded sodium oxalate functionalized hydrogel microspheres is in the range of 100-300 μm.

10. An application of injectable sodium oxalate-functionalized hydrogel microspheres loaded with TGFβ3, characterized in that, The sodium oxalate functionalized hydrogel microspheres prepared according to claim 3 are used for the repair and treatment of intervertebral disc degeneration. They are injected through a 21G-27G needle and are suitable for percutaneous minimally invasive interventional treatment.

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