A time sequence immune metabolism regulation hydrogel system for diabetic bone defect and a preparation method thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a time sequence immunometabolic regulation hydrogel system for diabetic bone defects and a preparation method thereof, which is used for repairing diabetic bone defects. The system takes oxidized sodium alginate-gelatin-chitosan as a self-crosslinking base, integrates immunoregulation, metformin / alpha-ketoglutaric acid metabolic regulation, BMP-2 osteogenic induction and glutathione antioxidant modules, realizes three-stage time sequence release through active oxygen targeted response, corrects the imbalance of immunity-metabolism-oxidative stress, and promotes bone regeneration. The preparation process parameters are clear, the biocompatibility is excellent, the clinical applicability is strong, and the system can be expanded to diabetic related soft tissue defect repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a time-series immune metabolic regulation hydrogel system for diabetic bone defects and its preparation method. Background Technology

[0002] Diabetic patients suffer from metabolic disorders, immune dysfunction, and oxidative stress damage caused by long-term hyperglycemia, resulting in a significant decrease in bone tissue regeneration capacity. This makes it a difficult type of bone defect repair to treat clinically. The postoperative healing rate of bone defects in diabetic patients is lower than that in non-diabetic patients, and the incidence of complications is significantly higher, which places a heavy burden on the medical system and seriously affects the quality of life of patients.

[0003] The pathological mechanisms of diabetic bone defects are significantly unique, mainly manifested as: 1) Imbalance of the immune microenvironment: Hyperglycemia induces macrophages to continuously polarize towards the pro-inflammatory M1 phenotype, releasing large amounts of pro-inflammatory factors such as TNF-α and IL-1β, inhibiting the transformation to the anti-inflammatory M2 phenotype, forming a chronic inflammatory microenvironment, and hindering stem cell recruitment and the initiation of bone regeneration; 2) Disorders of the metabolic microenvironment: Insulin resistance leads to abnormal metabolic reprogramming of bone tissue, reduced osteoblast activity, and hyperfunction of osteoclasts. At the same time, hyperglycemia induces the deposition of advanced glycation end products (AGEs), further damaging the bone matrix. 3) Decreased osteogenic potential: The osteogenic differentiation capacity of endogenous mesenchymal stem cells (MSCs) decreases, and insufficient angiogenesis leads to a lack of nutrient supply to bone tissue. In addition, the large amount of reactive oxygen species (ROS) generated by oxidative stress directly damages the survival and function of stem cells; 4) Amplified damage by oxidative stress: The ROS concentration in diabetic bone defects is significantly higher than that in normal bone defects. ROS not only exacerbates the inflammatory response, but also forms a vicious cycle of "inflammation-oxidative stress-metabolic disorder" by damaging biological macromolecules such as DNA and proteins, further deteriorating the bone regeneration microenvironment. Although existing clinical treatments (such as autologous bone transplantation, allogeneic bone transplantation, and traditional biomaterial implantation) have problems such as donor shortage, immune rejection, and poor repair effect, they are difficult to adapt to the complex pathological characteristics of diabetic bone defects.

[0004] Currently, to address the challenges of bone defect repair, various injectable hydrogel systems have emerged in the field of biomaterials in recent years. Their core design concept focuses on "in-situ molding and signal modulation," promoting bone regeneration by loading active ingredients (such as growth factors, stem cells, and immunomodulators). Among these, related technologies (such as the GelSSO / PDA@SDF fluid biomimetic hydrogel disclosed in CN121313938A) have achieved breakthroughs. Through photocrosslinking and in-situ molding, utilizing PDA nanoparticles to mediate immune regulation and SSO to mediate osteogenic transcriptional regulation, they achieve a dual-stage signal amplification of "immunity-osteoogenesis," providing an effective solution for the repair of common bone defects.

[0005] However, existing hydrogel technologies still have significant limitations in addressing the specific pathological needs of diabetic bone defects, mainly in the following aspects: (1) They only focus on the dual-dimensional regulation of "immune regulation and osteogenic induction". The regulation dimension is singular, does not cover metabolic disorders, and does not design a dedicated regulation module for the metabolic microenvironment disorder unique to diabetes. As a result, the repair effect is constrained by metabolic abnormalities, making it difficult to reverse bone regeneration disorders from the root cause; (2) Existing hydrogels mostly rely on matrix hydrolysis or enzymatic hydrolysis to achieve passive release. The response mechanism lacks targeting, and the drug release efficiency is low. They do not design an active response mechanism in combination with the pathological characteristics of high ROS in diabetic defect sites, resulting in non-specific release of active ingredients in healthy tissues, which not only wastes drugs but may also cause off-target effects. (3) Most existing hydrogels are cross-linked and molded using 405nm visible light, which requires matching light irradiation equipment. Furthermore, photoinitiators may have potential biotoxicity. At the same time, it is difficult to achieve uniform light irradiation in complex defect sites, which limits the clinical translation of minimally invasive injection repair. (4) Existing technologies do not add antioxidant components in a targeted manner, which cannot effectively remove excessive ROS in diabetic defect sites, leading to a continuous amplification of inflammatory response, impaired stem cell survival and function, and difficulty in improving the bone regeneration microenvironment. (5) Existing technologies mostly adopt a "two-stage release" approach of early immune regulation and later osteogenic induction, without considering the three-stage pathological process of diabetic bone defects: "inflammatory period - metabolic repair period - bone regeneration period," which leads to a disconnect between signal regulation and tissue repair timing, affecting the repair effect.

[0006] Therefore, developing a time-series immune metabolic regulation hydrogel system for diabetic bone defects that is targeted, multidimensionally regulated, easily moldable, and time-matched has important practical significance. Summary of the Invention

[0007] In view of this, the present invention proposes a time-series immune metabolic regulation hydrogel system for diabetic bone defects and its preparation method, aiming to solve the problems of current technology, such as failure to specifically address the pathological metabolic disorders of diabetic bone defects, lack of targeted response mechanism of pathological microenvironment, low drug release efficiency and off-target risk, dependence on exogenous stimulation, reliance on the inherent biocompatibility of materials to passively resist oxidative stress, easy occurrence of nanoparticle aggregation, denaturation of bioactive components, and fluctuation of gel performance.

[0008] This invention proposes a time-series immunometabolic regulation hydrogel system for diabetic bone defects, comprising a self-linked substrate material, a ROS-responsive immunomodulatory module, a metabolic regulation module, an osteogenic induction module, and an antioxidant module. The hydrogel system is formed by temperature / pH dual-sensitive self-crosslinking. The self-linked substrate material is a sodium alginate-gelatin-chitosan composite system. The ROS-responsive immunomodulatory module consists of IL-10-loaded PLGA-PEG-TK core-shell nanoparticles, wherein the PLGA-PEG-TK contains ROS-sensitive ketthiols. The metabolic regulation module, a complex system of metformin and α-ketoglutarate. The osteogenic induction module is loaded with mesoporous silica nanoparticles containing BMP-2. The antioxidant module is grafted onto the gel backbone with glutathione.

[0009] Furthermore, the mass ratio of each component in the self-crosslinking substrate material is gelatin: sodium alginate: chitosan = 100:30:15, and the amount of glutathione added is 5% of the gel solid content.

[0010] Furthermore, in the ROS-responsive immunomodulatory module, the PLGA-PEG-TK core-shell nanoparticles have a particle size of 150-200 nm.

[0011] Furthermore, in the metabolic regulation module, the mass ratio of metformin to α-ketoglutarate is 1:1~3, and the pore size of the mesoporous silica nanoparticles in the osteogenic induction module is 20-50 nm, with a chitosan layer coating the surface having a thickness of 30-50 nm.

[0012] A method for preparing a time-series immunometabolic regulation hydrogel system for diabetic bone defects includes the following steps: (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, IL-10 aqueous solution was added, and ultrasonic emulsification was performed to prepare a primary emulsion. Then, PVA aqueous solution was added and stirred at high speed to form a secondary emulsion. After evaporation of organic solvent at room temperature, the nanoparticles were collected by centrifugation, washed, and freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then chitosan solution containing metformin and α-ketoglutarate was added to encapsulate the core-shell structure. The nanoparticles were then freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin was dissolved in deionized water under heating to prepare a mixed solution, sodium oxidized alginate was added and stirred, chitosan was added to adjust the pH, glutathione was added and reacted at room temperature to obtain hydrogel substrate precursor solution; (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (3), disperse by ultrasonication, add glycerol, and filter under sterile conditions to obtain an injectable hydrogel.

[0013] Furthermore, the ultrasonic emulsification parameters in step (1) are: power 50~150W, time 1~10min, frequency 20~80kHz; the high-speed stirring parameters are: rotation speed 8000~18000rpm, time 5~20min, temperature 0~10℃; the centrifugation parameters are: rotation speed 8000~15000rpm, time 5~15min, temperature 0~10℃; and the washing is 3~5 times.

[0014] Furthermore, in step (2), the mass ratio of metformin to α-ketoglutarate in the chitosan solution is 1:1~3.

[0015] Furthermore, the heating in step (3) is 40~60℃; the solid-liquid ratio of the gelatin and deionized water is 1:8~15g / mL; the stirring parameters are: rotation speed 100~500rpm, time 10~60min, temperature 45~55℃; and the pH is adjusted to 6.0-6.5.

[0016] Furthermore, the ultrasonic dispersion parameters in step (4) are: power 20~80W and time 1~5min.

[0017] This invention also provides the application of the hydrogel system described above in the preparation of drugs for treating diabetic bone defects, including skull defects and femoral defects. The hydrogel system can be self-crosslinked and formed in situ at the defect site through minimally invasive injection, and can also be extended to the repair of soft tissue defects related to diabetic complications such as diabetic foot ulcers.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention constructs a closed-loop regulatory system of immune regulation, metabolic improvement, osteogenic induction, and antioxidation. Compared with existing technologies that can only solve the problems of immunity and osteogenicity, this invention reverses the vicious cycle of the bone regeneration microenvironment in diabetes from the root and significantly improves the repair success rate.

[0019] In the metabolic regulation module of this invention, metformin and α-ketoglutarate work synergistically to improve insulin resistance, reduce the deposition of advanced glycation end products, and regulate stem cell metabolic reprogramming, enabling mesenchymal stem cells to maintain high osteogenic activity in a high-glucose environment. This solves the technical shortcomings of existing technologies in that they do not have targeted regulation of metabolic disorders.

[0020] The antioxidant module of this invention triggers glutathione desorption and activation through ROS, precisely removes excess ROS from defective sites, inhibits the activation of pro-inflammatory signaling pathways, and creates a stable microenvironment for immune balance and osteogenic differentiation, filling the gap in the lack of active antioxidant design in existing technologies.

[0021] In this invention, metabolic regulators and osteogenic factors are released in stages through encapsulation with mesoporous silica nanoparticles and chitosan, precisely matching the degradation rate of the hydrogel substrate. This ensures that the drugs remain effective in the corresponding repair stages. Compared with the signal disorder problem of passive degradation release in existing technologies, the drug release efficiency is improved and the effect is more specific in this invention.

[0022] The hydrogel system described in this invention has a simple preparation process, controllable cost, requires no special equipment, can be mass-produced, and its minimally invasive injection method conforms to clinical practice, has good patient tolerance, and has extremely high clinical translation potential. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0024] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention proposes a time-series immunometabolic regulation hydrogel system for diabetic bone defects, comprising a self-linked substrate material, a ROS-responsive immunomodulatory module, a metabolic regulation module, an osteogenic induction module, and an antioxidant module. The hydrogel system is formed by temperature / pH dual-sensitive self-crosslinking. The self-linked substrate material is a sodium alginate-gelatin-chitosan composite system. The ROS-responsive immunomodulatory module consists of IL-10-loaded PLGA-PEG-TK core-shell nanoparticles, wherein the PLGA-PEG-TK contains ROS-sensitive ketthiols. The metabolic regulation module, a complex system of metformin and α-ketoglutarate. The osteogenic induction module is loaded with mesoporous silica nanoparticles containing BMP-2. The antioxidant module is grafted onto the gel backbone with glutathione.

[0029] In this invention, the mass ratio of each component in the self-crosslinking substrate material is gelatin: sodium alginate: chitosan = 100:30:15, and the amount of glutathione added is 5% of the gel solid content.

[0030] In this invention, the particle size of the PLGA-PEG-TK core-shell nanoparticles in the ROS-responsive immunomodulatory module is preferably 150~200nm, and more preferably 160~200nm.

[0031] In this invention, in the metabolic regulation module, the mass ratio of metformin to α-ketoglutarate is preferably 1:1~3, more preferably 1:2~3; the pore size of the mesoporous silica nanoparticles in the osteogenic induction module is preferably 20-50 nm, more preferably 30-50 nm; and the thickness of the chitosan layer coating the surface is preferably 30-50 nm, more preferably 40-50 nm.

[0032] A method for preparing a time-series immunometabolic regulation hydrogel system for diabetic bone defects includes the following steps: (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, IL-10 aqueous solution was added, and ultrasonic emulsification was performed to prepare a primary emulsion. Then, PVA aqueous solution was added and stirred at high speed to form a secondary emulsion. After evaporation of organic solvent at room temperature, the nanoparticles were collected by centrifugation, washed, and freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then chitosan solution containing metformin and α-ketoglutarate was added to encapsulate the core-shell structure. The nanoparticles were then freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin was dissolved in deionized water under heating to prepare a mixed solution, sodium oxidized alginate was added and stirred, chitosan was added to adjust the pH, glutathione was added and reacted at room temperature to obtain hydrogel substrate precursor solution; (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (3), disperse by ultrasonication, add glycerol, and filter under sterile conditions to obtain an injectable hydrogel.

[0033] In this invention, the ultrasonic emulsification parameters in step (1) are preferably: power 50~150W, time 1~10min, frequency 20~80kHz, and more preferably power 80~150W, time 3~10min, and frequency 30~80kHz; the high-speed stirring parameters are preferably: rotation speed 8000~18000rpm, time 5~20min, and temperature 0~10℃, and more preferably rotation speed 10000~18000rpm, time 10~20min, and temperature 3~10℃; the centrifugation parameters are preferably: rotation speed 8000~15000rpm, centrifugation time 5~15min, and temperature 0~10℃, and more preferably 8000~12000rpm, centrifugation time 8~15min, and temperature 5~10℃; the washing is preferably 3~5 times, and more preferably 3~4 times.

[0034] In this invention, the ultrasonic emulsification process requires maintaining a low system temperature to avoid the denaturation of IL-10 and the degradation of PLGA-PEG-TK caused by ultrasonic heat generation. Furthermore, magnetic stirring can be used in conjunction with ultrasonication to promote mass transfer between the oil and water phases and improve emulsification uniformity.

[0035] In this invention, the entire process is centrifuged at low temperatures (avoiding room temperature or high temperature) to prevent the heat generated by centrifugation from causing denaturation of IL-10 and hydrolysis of PLGA-PEG-TK nanoparticles. Excessive temperature will significantly reduce protein activity and nanoparticle stability, and reduce the risk of nanoparticle aggregation.

[0036] In this invention, the mass ratio of metformin to α-ketoglutarate in the chitosan solution in step (2) is preferably 1:1~3, and more preferably 1:2~3.

[0037] In this invention, the chitosan solution contains metformin and α-ketoglutarate. Metformin improves insulin resistance through the AMPK pathway, while α-ketoglutarate promotes stem cell metabolic reprogramming by regulating TET enzyme activity. The ratio of the two must meet the synergistic logic of first correcting the overall metabolic imbalance and then activating local stem cell function. When the α-ketoglutarate content is insufficient, the stem cell metabolic reprogramming effect is weak, and the osteogenic induction efficiency decreases. When the metformin content is too low, the improvement of insulin resistance is insufficient, the metabolic microenvironment is difficult to correct, and excessive α-ketoglutarate will reduce the viscosity of the chitosan solution, affecting the encapsulation stability of mesoporous silica nanoparticles (MSNs).

[0038] In this invention, the heating in step (3) is preferably 40~60℃, more preferably 45~60℃; the solid-liquid ratio of the gelatin and deionized water is preferably 1:8~15g / mL, more preferably 1:10~15g / mL; the stirring parameters are preferably: rotation speed 100~500rpm, time 10~60min, temperature 45~55℃, more preferably rotation speed 200~500rpm, time 10~50min, temperature 48~55℃; the pH adjustment is preferably adjusted to 6.0-6.5, more preferably adjusted to 6.2-6.5.

[0039] In this invention, the gelatin is added to deionized water and self-crosslinked through Schiff base reaction. The resulting hydrogel has a high compressive modulus, which matches the mechanical environment of cancellous bone. This not only avoids stress concentration at the interface caused by excessive stiffness, but also provides stable support for stem cell adhesion and proliferation. Furthermore, it meets the needs of nutrient exchange and cell infiltration, and is precisely matched with the bone regeneration cycle.

[0040] In this invention, the ultrasonic dispersion parameters in step (4) are preferably: power 20~80W and time 1~5min, and more preferably power 20~60W and time 2~5min.

[0041] This invention also provides the application of the hydrogel system described above in the preparation of drugs for treating diabetic bone defects, including skull defects and femoral defects. The hydrogel system can be self-crosslinked and formed in situ at the defect site through minimally invasive injection, and can also be extended to the repair of soft tissue defects related to diabetic complications such as diabetic foot ulcers.

[0042] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, and IL-10 aqueous solution was added. The mixture was ultrasonically emulsified for 10 min at a power of 80 W and a frequency of 30 kHz to prepare a primary emulsion. PVA aqueous solution was then added and the mixture was stirred at high speed at 10000 rpm and 3℃ for 20 min to form a secondary emulsion. After evaporating the organic solvent at room temperature, the mixture was centrifuged at 8000 rpm and 5℃ for 15 min to collect the emulsion. After washing three times, the nanoparticles were freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then a chitosan solution of metformin and α-ketoglutarate mixed at a mass ratio of 1:2 was added to encapsulate the core-shell structure, and the nanoparticles were freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin and deionized water were mixed at a solid-liquid ratio of 1:10 at 45°C. Sodium oxidized alginate was added and stirred at 200 rpm and 48°C for 10 min. Chitosan was added to adjust the pH to 6.2. Glutathione was added and reacted at room temperature to obtain the hydrogel substrate precursor solution. (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (4), ultrasonically disperse for 5 min under a power of 20W, add glycerol, and sterile filter to obtain an injectable hydrogel.

[0045] Example 2 (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, and IL-10 aqueous solution was added. The mixture was ultrasonically emulsified for 5 min at a power of 100W and a frequency of 50kHz to prepare a primary emulsion. PVA aqueous solution was then added and the mixture was stirred at high speed at 14000rpm and 6℃ for 15 min to form a secondary emulsion. After evaporating the organic solvent at room temperature, the mixture was centrifuged at 10000rpm and 8℃ for 10 min to collect the emulsion. After washing three times, the nanoparticles were freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then a chitosan solution of metformin and α-ketoglutarate mixed at a mass ratio of 1:2 was added to encapsulate the core-shell structure, and the nanoparticles were freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin and deionized water were mixed at a solid-liquid ratio of 1:12 at 50°C to prepare a mixed solution. Sodium oxidized alginate was added and stirred at 300 rpm and 50°C for 30 min. Chitosan was added to adjust the pH to 6.3. Glutathione was added and reacted at room temperature to obtain the hydrogel substrate precursor solution. (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (4), disperse them by ultrasonication for 3 min under a power of 40W, add glycerol, and filter aseptically to obtain an injectable hydrogel.

[0046] Example 3 (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, and IL-10 aqueous solution was added. The mixture was ultrasonically emulsified for 3 min at a power of 150W and a frequency of 80kHz to prepare a primary emulsion. PVA aqueous solution was then added and the mixture was stirred at high speed at 18000rpm and 10℃ for 10 min to form a secondary emulsion. After evaporating the organic solvent at room temperature, the mixture was centrifuged at 12000rpm and 10℃ for 8 min to collect the emulsion. After washing 4 times, the nanoparticles were freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then a chitosan solution of metformin and α-ketoglutarate mixed in a mass ratio of 1:3 was added to encapsulate the core-shell structure, and the nanoparticles were freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin and deionized water were mixed at a solid-liquid ratio of 1:15 at 55℃ to form a mixed solution. Sodium oxidized alginate was added and stirred at 500 rpm and 55℃ for 10 min. Chitosan was added to adjust the pH to 6.5. Glutathione was added and reacted at room temperature to obtain the hydrogel substrate precursor solution. (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (4), ultrasonically disperse for 2 min under a power of 60W, add glycerol, and sterile filter to obtain an injectable hydrogel.

[0047] Comparative Example 1 (1) Preparation of PDA@SDF-1α nanoparticles: Dopamine hydrochloride was dissolved in Tris-HCl buffer (pH 8.5), SDF-1α aqueous solution was added, and the mixture was ultrasonically dispersed for 5 min at a power of 100 W and a frequency of 40 kHz. PDA nanoparticles were formed by stirring at room temperature for 24 h. The nanoparticles were collected by centrifugation at a speed of 10000 rpm and a temperature of 4 °C for 10 min, washed 3 times, and then freeze-dried to obtain PDA@SDF-1α nanoparticles. (2) Preparation of SSO loading system: Dissolve SSO (sequence-specific oligodeoxynucleotides) in sterile water, add 1% sodium alginate solution, and stir magnetically for 30 min to form a uniform dispersion for later use; (3) Preparation of GelMA hydrogel substrate: GelMA and deionized water were mixed at a solid-liquid ratio of 1:10 at 50℃ to prepare a mixed solution. 0.5% photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) was added and stirred at 250 rpm and 50℃ for 20 min to obtain hydrogel substrate precursor solution. (4) Assemble the hydrogel system: Add the PDA@SDF-1α nanoparticles obtained in step (1) and the SSO dispersion obtained in step (2) to the hydrogel substrate precursor solution in step (3), and ultrasonically disperse for 3 min under a power of 40W. After sterile filtration, crosslink the hydrogel by irradiation with 405nm visible light for 30s to obtain the photocrosslinked hydrogel.

[0048] Comparative Example 2 (1) Preparation of ROS-responsive core-shell nanoparticles: completely consistent with step (1) of Example 2 (ultrasonic emulsification at 100W power and 50kHz frequency for 5min, high-speed stirring at 14000rpm and 6℃ for 15min, and centrifugation at 10000rpm and 8℃ for 10min). (2) Preparation of BMP-2 loaded nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, etched with hydrochloric acid, and then coated with pure chitosan solution without metformin and α-ketoglutarate, loading only BMP-2, freeze-drying for later use, to obtain BMP-2@MSNs; (3) Preparation of self-crosslinking hydrogel substrate: completely consistent with step (3) of Example 2 (50°C, solid-liquid ratio 1:12, stirring at 300 rpm for 30 min, pH adjusted to 6.3, glutathione added and reacted at room temperature). (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the BMP-2@MSNs obtained in step (2) to the hydrogel substrate precursor solution in step (3), sonicate for 3 min under a power of 40W, add glycerol, and filter aseptically to obtain the injectable hydrogel.

[0049] Comparative Example 3 (1) Preparation of ordinary PLGA-PEG nanoparticles: Ordinary PLGA-PEG (without TK sensitive bond) was dissolved in dichloromethane, and IL-10 aqueous solution was added. The primary emulsion was prepared by ultrasonic emulsification for 5 min at a power of 100W and a frequency of 50kHz. Then, PVA aqueous solution was added and the mixture was stirred at high speed at 14000rpm and 6℃ for 15 min to form a secondary emulsion. After evaporating the organic solvent at room temperature, the mixture was collected by centrifugation at 10000rpm and 8℃ for 10 min. After washing 3 times, the mixture was freeze-dried to obtain ordinary IL-10 nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: completely consistent with step (2) of Example 2 (metformin to α-ketoglutarate mass ratio 1:2, encapsulated in chitosan solution). (3) Preparation of self-crosslinking hydrogel substrate: Same as step (3) in Example 2, but without adding glutathione, only gelatin-OSA-chitosan mixed reaction to obtain hydrogel substrate precursor solution; (4) Assemble the hydrogel system: Add the ordinary nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (3), ultrasonically disperse for 3 min under a power of 40W, add glycerol, and sterile filter to obtain an injectable hydrogel.

[0050] Comparative Example 4 (1) Preparation of ROS-responsive core-shell nanoparticles: completely consistent with step (1) of Example 2; (2) Preparation of metabolic-osteogenic composite nanoparticles: completely consistent with step (2) of Example 2; (3) Preparation of GelMA photocrosslinking substrate: GelMA and deionized water were mixed at a solid-liquid ratio of 1:12 at 50°C to prepare a mixed solution. Sodium oxidized alginate was added and stirred at 300 rpm and 50°C for 30 min. Chitosan was added to adjust the pH to 6.3. Glutathione was added and reacted at room temperature for 30 min. Finally, 0.5% photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) was added and stirred evenly to obtain the photocrosslinking substrate precursor solution. (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the substrate precursor solution in step (3), disperse them by ultrasonication for 3 min under a power of 40W, add glycerol, filter under sterile conditions, and crosslink them by irradiation with 405nm visible light for 30s to obtain a photocrosslinked injectable hydrogel.

[0051] Performance testing The in vitro simulated environment simulated the pathological microenvironment of diabetes (high glucose 25 mmol / L, ROS 10 μmol / L), with a temperature of 37℃ and a pH of 6.0-6.5 (matching the environment of bone defect sites); the in vitro simulated environment was based on GB / T 16886.1-2011 "Biological evaluation of medical devices - Part 1: Evaluation and testing in the risk management process".

[0052] In vivo environment: A type II diabetes model (fasting blood glucose ≥16.7 mmol / L) was established in SD rats, and a skull defect model with a diameter of 5 mm was established. Postoperative monitoring was performed regularly. The design of the in vivo environment was based on GB / T 16886.16-2021 "Biological evaluation of medical devices - Part 16: In vivo degradation test".

[0053] The gelation time was detected using the inverted test tube method: the time it took for the solution to lose its fluidity at 37°C was recorded. Based on the principle of "instant formation upon injection" in clinical minimally invasive injection, the detection was conducted in accordance with the "General Technical Requirements for Medical Gels" YBB00142004-2015.

[0054] The compressive modulus was tested using a universal testing machine at a compression rate of 1 mm / min. The gel compression stress-strain curve was plotted, and the slope of the elastic stage was used for calculation. The test was conducted in accordance with GB / T 22849-2014, "Mechanical Properties Test Method for Biomedical Materials - Compression Test".

[0055] The pore size (μm) of the gel was detected by scanning electron microscopy (SEM), freeze-drying and sputtering with gold, observing the cross-section and counting the diameter of 50 pores. The detection was based on GB / T16594-2008 "Determination of Specific Surface Area and Particle Size Distribution of Metal Powders by Air Permeation Method".

[0056] The degradation cycle (weeks) was determined by immersion in an in vitro simulated body fluid (SBF) at 37°C and pH 7.4, and the time it took for the mass loss rate to reach 90% was calculated. The determination was based on GB / T 16886.15-2022, "Biological Evaluation of Medical Devices Part 15: Degradation Products of Metals and Alloys".

[0057] The biocompatibility was tested using the CCK-8 method: the extract was co-cultured for 7 days, and the absorbance was measured to calculate the survival rate. The test was based on GB / T 16886.5-2017, "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests".

[0058] The ROS scavenging rate was detected using the DCFH-DA fluorescent probe method: changes in intracellular fluorescence intensity were detected, and the detection was based on the "General Rules for Detection of Reactive Oxygen Species" YY / T 1544-2017.

[0059] The in vivo bone repair effect was detected by Micro-CT scanning analysis and HE / Masson staining observation, and the detection was based on GB / T 16886.16-2021 "Biological Evaluation of Medical Devices Part 16: In Vivo Degradation Test" and GB / T 33889-2017 "Methods for Morphometry of Bone Tissue".

[0060] The performance test results of the products of Examples 1-3 and Comparative Examples 1-4 in in vitro simulated environment and in vivo environment are shown in Table 1 below; Table 1. Performance test results of the products from Examples 1-3 and Comparative Examples 1-4 In summary, the in vivo bone repair effect of Examples 1-3 of the present invention is 28%~35%, which is an improvement compared to 20%~22% of Comparative Example 1, thus solving the root cause problems of metabolic disorders and oxidative stress in diabetic bone defects. The in vivo bone repair effect of the time-series immunometabolic regulation hydrogel system for diabetic bone defects described in Comparative Example 2 (Met / α-KG deficiency) was 25%~27%, significantly lower than the 32%~35% in Example 2. This confirms that the synergistic use of metformin and α-ketoglutarate can effectively correct the metabolic imbalance in diabetes and lay the microenvironmental foundation for osteogenic regeneration. Comparative Example 3 (no ROS response, no GSH) showed a ROS clearance rate of 55%~60%, compared to 78%~80% in Example 2, resulting in a significant decline in osteogenic activity and repair effect. This indicates that the PLGA-PEG-TK... The synergistic effect of the ROS-sensitive design of nanoparticles and the GSH antioxidant module can achieve closed-loop regulation of pathological signal triggering, precise drug release and oxidative stress clearance, thereby improving lesion targeting and regulatory efficiency. Comparative Example 4 (photocrosslinking molding) has a 6% to 8% decrease in cell survival rate due to photoinitiator toxicity, and its mechanical strength is slightly lower than that of Example 2, with a potential risk of uneven crosslinking in deep defects. Moreover, Examples 1-3 of this application can be adapted to different clinical scenarios of patients with insulin resistance-dominated defects, osteogenic potential decline defects or most diabetic bone defects according to their parameter design, and the process is highly selective.

[0061] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A time-series immunometabolic regulation hydrogel system for diabetic bone defects, characterized in that, The system includes a self-linked substrate material, a ROS-responsive immunomodulatory module, a metabolic regulation module, an osteogenic induction module, and an antioxidant module. The hydrogel system is formed through temperature / pH dual-sensitive self-crosslinking. The self-linked substrate material is a sodium alginate-gelatin-chitosan composite system. The ROS-responsive immunomodulatory module consists of IL-10-loaded PLGA-PEG-TK core-shell nanoparticles, wherein the PLGA-PEG-TK contains ROS-sensitive ketethiolates. The metabolic regulation module, a complex system of metformin and α-ketoglutarate. The osteogenic induction module is loaded with mesoporous silica nanoparticles containing BMP-2. The antioxidant module is grafted onto the gel backbone with glutathione.

2. The time sequence immune-metabolic regulation hydrogel system for diabetic bone defect according to claim 1, characterized in that, The mass ratio of each component in the self-crosslinking substrate material is gelatin: sodium alginate: chitosan = 100:30:15, and the amount of glutathione added is 5% of the gel solid content.

3. The time sequence immune-metabolic regulation hydrogel system for diabetic bone defect according to claim 1, characterized in that, In the ROS-responsive immunomodulatory module, the PLGA-PEG-TK core-shell nanoparticles have a particle size of 150-200 nm.

4. The time sequence immune-metabolic regulation hydrogel system for diabetic bone defect according to claim 1, characterized in that, In the metabolic regulation module, the mass ratio of metformin to α-ketoglutarate is 1:1~3, and the pore size of the mesoporous silica nanoparticles in the osteogenic induction module is 20-50 nm, with a chitosan layer thickness of 30-50 nm covering the surface.

5. The method for preparing the time-series immunometabolic regulation hydrogel system for diabetic bone defects according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of ROS-responsive core-shell nanoparticles: PLGA-PEG-TK was dissolved in dichloromethane, IL-10 aqueous solution was added, and ultrasonic emulsification was performed to prepare a primary emulsion. Then, PVA aqueous solution was added and stirred at high speed to form a secondary emulsion. After evaporation of organic solvent at room temperature, the nanoparticles were collected by centrifugation, washed, and freeze-dried to obtain the nanoparticles. (2) Preparation of metabolic-osteogenic composite nanoparticles: Mesoporous silica nanoparticles were synthesized by sol-gel method, then hydrochloric acid was added for etching, and then chitosan solution containing metformin and α-ketoglutarate was added to encapsulate the core-shell structure. The nanoparticles were then freeze-dried for later use to obtain composite nanoparticles. (3) Preparation of self-crosslinking hydrogel substrate: Gelatin was dissolved in deionized water under heating to prepare a mixed solution, sodium oxidized alginate was added and stirred, chitosan was added to adjust the pH, glutathione was added and reacted at room temperature to obtain hydrogel substrate precursor solution; (4) Assemble the hydrogel system: Add the nanoparticles obtained in step (1) and the composite nanoparticles obtained in step (2) to the hydrogel substrate precursor solution in step (3), disperse by ultrasonication, add glycerol, and filter under sterile conditions to obtain an injectable hydrogel.

6. The method of claim 5, wherein the hydrogel system is prepared by the following steps: (a) mixing the first and second components to form a mixture; (b) incubating the mixture at a temperature of 37°C for 24 hours; and (c) removing the supernatant and washing the hydrogel system with a buffer solution. The ultrasonic emulsification parameters in step (1) are: power 50~150W, time 1~10min, frequency 20~80kHz; the high-speed stirring parameters are: rotation speed 8000~18000rpm, time 5~20min, temperature 0~10℃; the centrifugation parameters are: rotation speed 8000~15000rpm, time 5~15min, temperature 0~10℃; the washing is 3~5 times.

7. The method for preparing a time-series immunometabolic regulation hydrogel system for diabetic bone defects according to claim 5, characterized in that, In step (2), the mass ratio of metformin to α-ketoglutarate in the chitosan solution is 1:1~3.

8. The method for preparing a time-series immunometabolic regulation hydrogel system for diabetic bone defects according to claim 5, characterized in that, The heating in step (3) is 40~60℃; the solid-liquid ratio of the gelatin and deionized water is 1:8~15g / mL; the stirring parameters are: speed 100~500rpm, time 10~60min, temperature 45~55℃; the pH is adjusted to 6.0-6.

5.

9. The method for preparing a time-series immunometabolic regulation hydrogel system for diabetic bone defects according to claim 5, characterized in that, The ultrasonic dispersion parameters mentioned in step (4) are: power 20~80W, time 1~5min.

10. Use of the hydrogel system according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of a diabetic bone defect, characterized in that, The diabetic bone defects include skull defects and femoral defects. The hydrogel system can be injected in situ into the defect site via minimally invasive injection to form a self-crosslinking structure. It can also be extended to the repair of soft tissue defects related to diabetic complications such as diabetic foot ulcers.

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