Preparation and application of composite hydrogel for promoting diabetic bone regeneration

Abstract

The invention provides a composite hydrogel for promoting diabetic bone regeneration, the composite hydrogel is composed of L-histidine modified chitosan, oxidized konjac glucomannan, Zn and nano clay, and in-situ gelation is realized through coordination regulation, interlayer exchange and dynamic covalent bond synergistic effect. Through combined application of coordination regulation, interlayer exchange and dynamic covalent bonds, on-demand staged delivery of various ions is realized. According to the innovative design, the adhesive property and mechanical integrity of the hydrogel at a defect part are maintained, intelligent response can be realized according to the change of a bone microenvironment, and the defect that a single release mechanism in the prior art is difficult to meet the complicated demand of diabetic bone regeneration is overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to the preparation and application of a composite hydrogel that promotes bone regeneration in diabetic patients. Background Technology

[0002] Diabetic bone defects refer to fractures or bone defects that occur in a state of chronic hyperglycemia, characterized by intractable poor healing of local bone tissue. The number of people with diabetes worldwide is enormous and is projected to continue to grow. Fracture healing time is significantly prolonged in diabetic patients, with increased rates of delayed healing, nonunion, and infection.

[0003] The core obstacle to the repair of diabetic bone defects lies in the chronically adverse microenvironment at the lesion site. Long-term high glucose and advanced glycation end products (AGEs) activate NADPH oxidase and disrupt mitochondrial function through the action of RAGE receptors, leading to persistently high levels of reactive oxygen species (ROS), causing oxidative DNA damage, reducing mitochondrial membrane potential, disrupting cellular energy metabolism, impairing the migration, survival, and osteogenic differentiation of bone marrow mesenchymal stem cells, and inhibiting the expression of key osteogenic genes, thus delaying collagen deposition and the formation of mineralized nodules.

[0004] In terms of angiogenesis, endothelial cells are sensitive to oxidative stress. Reactive oxygen species (ROS) damage the cytoskeleton and adhesion junctions, and thickening of the basement membrane and loss of pericytes hinder vascular maturation, leading to a decrease in the quantity and quality of H-type vessels and interruption of vessel-bone coupling. Regarding the bone immune environment, the hyperglycemic-AGEs-RAGE axis, in conjunction with ROS, causes macrophages to remain in a pro-inflammatory M1 state. Activation of the NLRP3 inflammasome maintains high levels of pro-inflammatory mediators, hindering the transition to a pro-repair M2 state, inhibiting osteogenic gene expression in bone marrow mesenchymal stem cells, and promoting osteoclast formation. Oxidative stress, endothelial dysfunction, and abnormal macrophage polarization amplify each other, forming a self-sustaining cycle of inflammation-bone resorption-re-inflammation.

[0005] Traditional internal fixation and bone grafting methods fail to correct the wound environment characterized by high reactive oxygen species, acidic exudates, and chronic inflammation. Bone grafts often lack viability, and the risks of displacement and heterotopic ossification remain. Many early strategies addressed only one aspect of the problem, leading to bone scaffolds and carriers that focused on osteogenic activity but were incompatible with natural bone tissue, affecting normal biological functions such as inflammation regulation and biomineralization. Antioxidants can reduce reactive oxygen species levels, but in the diabetic environment, bioactive materials degrade rapidly, leading to drug or ion bursts and toxicity. Macrophages remain M1-dominated, resulting in poor durability of single-pathway treatment. In recent years, scaffolds combining immunomodulation and osteogenic activity have represented a promising strategy for bone repair, but platforms that can integrate immune repair, vascular support, and osteogenic activity and are clinically applicable to diabetic bone defects remain scarce.

[0006] Existing strategies generally suffer from fundamental problems such as single target, spatiotemporal mismatch, poor durability, insufficient integration, and limited clinical translation. These strategies only target one aspect of oxidative stress, inflammation, or osteogenic processes, failing to address multiple pathological mechanisms simultaneously. They lack precise response and temporal regulation to the needs of different repair stages. In the high reactive oxygen species and acidic environment of diabetes, materials degrade rapidly and release out of control. They also fail to effectively integrate antioxidant, immune remodeling, vascular reconstruction, and osteogenic promotion, and lack practical platforms that are injectable, can be solidified in situ, and are adaptable to irregular defects.

[0007] In summary, the treatment of diabetic bone defects urgently requires the development of a multimodal repair platform that can simultaneously achieve redox correction, immune remodeling, vascular support, and osteogenic enhancement. This platform should be injectable, have in situ adhesion, pH responsiveness, and multi-ion time-gated release capability to restore the balance of the immune-vascular-osteogenic axis and provide a well-defined and clinically feasible solution for diabetic bone regeneration. Summary of the Invention

[0008] To address the shortcomings of existing technologies in the repair of diabetic bone defects, this invention aims to solve the following technical problems: a) Temporal control issues in multi-ion delivery: Existing bone repair materials have difficulty in achieving on-demand release and sequential delivery of multiple therapeutic ions, and cannot maintain adhesion and mechanical integrity at the defect site for a long time, resulting in limited treatment effects.

[0009] b) Insufficient microenvironment responsiveness: Diabetic bone defect wounds are weakly acidic and have abnormally high levels of reactive oxygen species (ROS). Existing materials lack the ability to intelligently respond to this pathological microenvironment and cannot form a closed-loop regulatory system of "stimulus perception - controlled release - gradual microenvironment remodeling".

[0010] c) Poor synergy of multiple biological functions: Traditional materials often focus only on a single therapeutic target, making it difficult to achieve synergistic effects of multiple functions such as oxidative stress clearance, antioxidant network reconstruction, macrophage M1 to M2 polarization, angiogenesis promotion, and bone marrow stromal cell osteogenic differentiation enhancement on a unified platform.

[0011] d) Lack of regulation of the immune-vascular-osteogenic axis: Current technologies have failed to systematically restore the dynamic balance of the three major axes of immunity, vascularity and osteogenicity in diabetic bone defects, and lack a holistic treatment strategy based on pathological mechanisms.

[0012] This invention constructs a composite hydrogel system based on coordination regulation, interlayer exchange, and dynamic covalent bond synergy, and establishes a mechanism-driven material design strategy to provide a clinically promising solution for diabetic bone regeneration. Overcoming the aforementioned deficiencies of existing technologies, this invention proposes a composite hydrogel for promoting diabetic bone regeneration and its application.

[0013] To solve the aforementioned technical problems, the technical solution adopted by the present invention is as follows: A composite hydrogel that promotes bone regeneration in diabetic patients, the composite hydrogel being composed of L-histidine-modified chitosan, oxidized konjac glucomannan, Zn²⁺ and nanoclay, achieving in-situ gelation through coordination regulation, interlayer exchange and dynamic covalent bond synergy.

[0014] Preferably, the method for preparing the L-histidine-modified chitosan is as follows: Chitosan was weighed and dissolved in acetate buffer. L-histidine was dissolved in deionized water. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred to activate the mixture. The mixture was then added dropwise to the chitosan solution. After the reaction, the pH was adjusted, dialyzed, and freeze-dried to obtain the final product.

[0015] Preferably, the preparation method of the oxidized konjac glucomannan is as follows: Weigh out konjac glucomannan, dissolve it in deionized water, add sodium periodate and stir to react in the dark, then add ethylene glycol to quench the residual oxidant, dialyze, and freeze dry to obtain the product.

[0016] Preferably, the composite hydrogel is prepared by a two-component in-situ mixing method: Component A is L-histidine modified chitosan dissolved in HEPES buffer, then dispersed with ZnCl2 stock solution and nano-clay and brought to volume. Component B is oxidized konjac glucomannan dissolved in PBS buffer and brought to volume. Before use, mix equal volumes of component A and component B, then inject the mixture evenly back and forth into the bone defect area.

[0017] Preferably, the composite hydrogel achieves in-situ gelation through dynamic Schiff base crosslinking, His-Zn²⁺ coordination crosslinking, and interlayer ion exchange of nanoclay.

[0018] Preferably, the composite hydrogel can intelligently respond to changes in the bone microenvironment, forming a closed-loop regulatory system of "stimulus perception - controllable release - gradual microenvironment remodeling".

[0019] Preferably, the composite hydrogel simultaneously achieves multiple functions on a unified platform, including oxidative stress clearance, antioxidant network reconstruction, macrophage M1 to M2 polarization, angiogenesis promotion, and enhanced osteogenic differentiation of bone marrow stromal cells.

[0020] Preferably, the composite hydrogel systematically restores the dynamic balance of the three major axes of immunity, vascularity and osteogenicity in diabetic bone defects.

[0021] Application of a composite hydrogel that promotes bone regeneration in diabetes in the preparation of drugs for repairing diabetic bone defects.

[0022] Preferably, the composite hydrogel is applied to diabetic bone defects via injection to promote bone regeneration.

[0023] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves on-demand, staged delivery of multiple ions through the combined application of coordination regulation, interlayer exchange, and dynamic covalent bonds. This innovative design maintains the adhesion and mechanical integrity of the hydrogel at defect sites while intelligently responding to changes in the bone microenvironment, overcoming the limitations of existing technologies where a single release mechanism cannot meet the complex needs of bone regeneration in diabetes.

[0024] The composite hydrogel of this invention can form a closed-loop regulatory system of "stimulus sensing - controlled release - gradual microenvironment remodeling" in the weakly acidic and highly reactive oxygen environment unique to diabetic wounds. This intelligent response mechanism enables the hydrogel to actively adapt to the pathological environment, realizing a leap from passive material to active treatment platform, and solving the technical problem that traditional biomaterials cannot dynamically respond to the complex pathological environment of diabetic bone defects.

[0025] This invention addresses the root pathological mechanism of diabetic bone defects by restoring the balance of the three major axes of immunity, vascularity, and osteogenicity, thus achieving a systematic intervention in diabetic bone regeneration disorders. Compared to existing treatments that target only a single pathological aspect, this invention provides a mechanism-based solution for diabetic bone regeneration with greater clinical application prospects, significantly improving the repair efficiency and clinical treatment outcomes of diabetic bone defects.

[0026] The material strategy adopted in this invention is rationally designed and has a high degree of functional integration, providing a practical and feasible treatment method for the clinical challenge of diabetic bone defects. It has important clinical application value and broad market prospects. Attached Figure Description

[0027] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts. Wherein: Figure 1 This is a schematic diagram illustrating the preparation of the composite hydrogel of the present invention; Figure 2 This is a schematic diagram of the oscillatory rheological testing of the hydrogel of the present invention; Figure 3 This is a scanning electron microscope image of the composite hydrogel of the present invention; Figure 4 This is a schematic diagram of the energy spectrum distribution of the hydrogel of the present invention; Figure 5 This is a schematic diagram showing the porosity of the composite hydrogel of the present invention; Figure 6 This is a schematic diagram of the pore size distribution of the composite hydrogel of the present invention; Figure 7 This is a schematic diagram of XPS analysis of the hydrogel of the present invention; Figure 8 This is a schematic diagram of the mechanical compression experiment of the composite hydrogel of the present invention; Figure 9 This is a schematic diagram of the Zn²⁺ release curves of the hydrogel of the present invention under different pH conditions; Figure 10 This is a schematic diagram of the degradation curve of the composite hydrogel of the present invention; Figure 11 This is a schematic diagram illustrating the biocompatibility of the composite hydrogels of the present invention; wherein, a) BMSCs were co-cultured with different composite hydrogels for 3 days and then subjected to live / dead staining; b) Quantitative analysis of live cell staining (n=5); c) CCK8 analysis was performed on BMSCs after co-culturing with different composite hydrogels for 1, 3, and 5 days. Figure 12 This diagram illustrates the effect of the composite hydrogels of this invention on macrophage polarization. Bone marrow-derived macrophages (BMDMs) were co-cultured with different composite hydrogels, followed by immunofluorescence staining and quantitative analysis of CD86 (a, b) and CD206 (c, d) cells. *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the HSCO group; #P<0.05, ##P<0.01 and ###P<0.001 indicate statistically significant differences compared to the HCSO@ZL group. Figure 13 This diagram illustrates the effect of the composite hydrogel of the present invention on angiogenesis; wherein, a) conditioned medium extracted from BMDMs treated with the composite hydrogel was used to evaluate its angiogenesis capacity in endothelial cells (HUVECs). b) Transwell crystal violet staining experiment. c) Quantitative analysis of migrating cells (n=5). d) Tube formation experiment of HUVECs. ef) Quantitative analysis of tube length and junction points using ImageJ (n=5). g) Immunofluorescence staining of CD31, a marker of HUVECs: green (CD31) and blue (DAPI). h) Quantitative analysis of CD31 fluorescence intensity (n=5). *P<0.05,**P<0.01 and***P<0.001 indicate statistical difference compared with the HSCO group; #P<0.05,##P<0.01 and###P<0.001 indicate statistical difference compared with the HCSO@ZL group; Figure 14This diagram illustrates the effect of the composite hydrogel of the present invention on intracellular reactive oxygen species (ROS) scavenging and osteogenic differentiation. The analysis includes: a) intracellular ROS fluorescence staining; b) ROS fluorescence intensity analysis; c) flow cytometry analysis of intracellular ROS levels in BMSCs; d) ALP and ARS staining; e) quantitative analysis of ALP activity; and f) quantitative analysis of calcium nodules. *P<0.05, **P<0.01 and ***P<0.001 indicate statistical differences compared to the HSCO group; #P<0.05, ##P<0.01 and ###P<0.001 indicate statistical differences compared to the HCSO@ZL group. Figure 15 This diagram illustrates the in vivo bone regeneration assessment of the HCSO@ZL hydrogel in a rat femoral defect model. A) Representative 3D reconstructed images of the rat femoral defect obtained by Micro-CT scanning at 4 and 8 weeks post-surgery. B) Quantitative analysis of bone volume / total bone area (BV / TV), bone thickness (Tb.Th), and bone mineral density (BMD) in the bone injury area (n=4). C) Representative images stained with H&E. Detailed Implementation

[0028] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0029] Specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0030] This embodiment presents the preparation and application of a composite hydrogel that promotes bone regeneration in diabetic patients.

[0031] 1. Preparation of injectable composite hydrogel (HCSO@ZL) Figure 1 ) (1) Preparation of L-histidine modified chitosan (CS-HIS) Weigh 1.00 g of chitosan and dissolve it in 100 mL of acetate buffer (0.1 mol / L acetic acid / sodium acetate, pH 5.5), stirring magnetically until completely dissolved. Separately, dissolve 0.123 g (0.000792 mol) of L-histidine in 50 mL of deionized water, then add 0.205 g (0.001069 mol, approximately 1.35 equivalents to histidine) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.091 g (0.000792 mol) of N-hydroxysuccinimide (NHS), stirring at room temperature for 1 hour. Add the activated mixture dropwise to the chitosan solution while continuously stirring. After reacting at room temperature for 24 hours, the pH of the reaction system was adjusted to 7.5 with 1 mol / L NaOH solution, and the mixture was transferred to a dialysis bag (molecular weight cutoff 3.5 kDa). Dialysis was performed with excess deionized water for 72 hours, with the water changed three times daily. The retentate after dialysis was freeze-dried to obtain a white powder of L-histidine-modified chitosan (CS-HIS), which was stored in a dry environment for later use.

[0032] (2) Preparation of oxidized konjac glucomannan (OKGM) Weigh 1.00 g of konjac glucomannan and dissolve it in 100 mL of deionized water, stirring until the solution is clear and transparent. Dissolve 1.00 g of sodium periodate (NaIO4) in 50 mL of deionized water and slowly add it to the above konjac glucomannan solution under light-protected conditions, stirring and reacting for 12 hours in the dark. After the reaction is complete, add 15 mL of ethylene glycol to quench any remaining oxidizing agent and continue stirring for 30 minutes. Transfer the reaction mixture to a dialysis bag (molecular weight cutoff 3.5 kDa) and dialyze with deionized water for 72 hours, changing the water three times a day. After dialysis, freeze-dry to obtain oxidized konjac glucomannan (OKGM) powder.

[0033] (3) Preparation of injectable composite hydrogel (HCSO@ZL) This invention employs a two-component on-site mixing method to prepare injectable composite hydrogels. The final target composition is: CS-HIS 2% (w / v), OKGM 5% (w / v), Zn²⁺ 100μm, and nano-clay (Lp) 10mg / mL. Taking a total preparation volume of 10mL as an example, the specific steps are as follows: Preparation of Component A: Weigh 0.200 g of CS-HIS and dissolve it in approximately 4.70–4.80 mL of 50 mmol / L HEPES buffer (pH 7.4); add 0.10 mL of 10 mmol / L ZnCl2 stock solution (to make the Zn²⁺ concentration in Component A 200 μm, achieving 100 after 1:1 mixing); add 0.10 g of nano-clay (Lp) and disperse it thoroughly by vortex mixing and short-term sonication; bring the volume to 5.00 mL with HEPES buffer. The final concentration of Component A is: CS-HIS 4% (w / v), Lp 20 mg / mL.

[0034] Preparation of component B: Weigh 0.500g of OKGM, dissolve it in PBS buffer (pH 7.4), and bring the volume to 5.00mL, with a concentration of 10% (w / v).

[0035] In-situ formation of the hydrogel: Before use, equal volumes of component A and component B are mixed using a dual-syringe connector or a static mixer, and injected back and forth 20–30 times to ensure uniform mixing, resulting in an HCSO@ZL composite hydrogel, which is then injected into the bone defect site. After 1:1 mixing, the final concentrations of each component in 10 mL of the formulation are: CS-HIS 0.200 g / 10 mL (2% w / v), OKGM 0.500 g / 10 mL (5% w / v), Zn²⁺ 100 μm, and Lp 10 mg / mL.

[0036] 2. Physicochemical characterization of injectable composite hydrogel (HCSO@ZL) (1) The oscillatory rheological test of HCSO@ZL hydrogel showed that after mixing the precursor, the storage modulus G' steadily increased and exceeded the loss modulus G'' at about 170 seconds. Figure 2 ).

[0037] (2) Scanning electron microscopy revealed that all hydrogels possessed a highly porous microstructure, which facilitates cell invasion, proliferation, and migration. The addition of Zn²⁺ or nanoclay produced granular surface features, with nanoclay exhibiting more pronounced granular characteristics, such as… Figure 3 As shown.

[0038] (3) Energy dispersive spectroscopy imaging and local analysis confirmed that zinc, silicon, and magnesium are uniformly distributed in HCSO@ZL, such as Figure 4 As shown.

[0039] (4) The porosity measurement results show that the cumulative porosity of HCSO is 92.2576%, that of HCSO@Z is 92.1642%, that of HCSO@L is 89.4481%, and that of HCSO@ZL is 89.9896% (e.g. Figure 5 (As shown).

[0040] (5) Pore size distribution shows that 31.92% of the pores in HCSO are in the 104-105 nm range, and 68.06% are in the 105-106 nm range. HCSO@Z accounts for 33.67% and 33.32% in the same range, respectively, reflecting a decrease in overall pore size, which is likely due to the enhanced cross-linking caused by the coordination of histidine and zinc. However, when used with nanoclay, the pore size increases. The proportions of HCSO@L in these two ranges are 20.86% and 79.13%, respectively, and the proportions of HCSO@ZL in these ranges are 12.55% and 87.44%, respectively, indicating that the pore size regulated by the clay is appropriate. Figure 6 As shown.

[0041] (6) XPS analysis confirmed the inclusion of the components. Decomposition of the C1s spectrum revealed the C–C, C=O, and C=N components in the HCSO@ZL fraction, such as... Figure 7 As shown.

[0042] (7) Mechanical compression tests show that HCSO@ZL can withstand a pressure of about 150 kPa at 50% strain, which is about 2.5 times that of HCSO. This indicates the synergistic reinforcing effect of zinc and bentonite (e.g., Figure 8 (As shown).

[0043] (8) The zinc ion release experiment showed that the zinc release rate was faster at pH 5.5 than at pH 7.4, but the chelating effect of histidine on zinc allowed the release to continue for about 21 days, reaching a steady state at about 14 days, with the final cumulative release approaching 80%. Figure 9 As shown.

[0044] (9) Degradation curves show that all hydrogels can last up to 21 days (e.g. Figure 10 As shown in the figure, this coincides with the mineralization stage of bone regeneration.

[0045] (10) Detection of the toxicity of HCSO@ZL hydrogel to cells and its effect on cell proliferation ( Figure 11 The results of live / dead cell staining after three days of co-culturing bone marrow mesenchymal stem cells (BMSCs) with hydrogel showed excellent cell compatibility. Fluorescence images showed that HCSO, HCSO@Z, HCSO@L, and HCSO@ZL contained mainly live cells with strong green signals and few dead cells. Figure 11 a). Quantitative analysis showed that cell viability remained above 90% in all groups, with no significant differences between groups, indicating that the incorporation of zinc²⁺ and nano-clay did not impair cell compatibility. Figure 11b). CCK-8 assays of BMSC proliferation over five days showed an increasing trend in metabolic activity across all groups, indicating that the controlled release of bioactive ions not only did not decrease but actually preserved proliferative capacity. Figure 11 c).

[0046] (11) To investigate the immunomodulatory effects of HCSO@ZL hydrogel, we detected surface markers in bone marrow-derived macrophages (BMDMs) co-cultured with the hydrogel. Figure 12 Compared to HCSO, the M1 marker CD86 was significantly reduced in all ion-loaded groups, with the largest reduction observed in the HCSO@ZL group. The fluorescence intensity of CD86 in the HCSO@ZL group was approximately 70% lower than that in the HCSO group, indicating a strong inhibition of pro-inflammatory activation. Figure 12 ab). In contrast, the M2 marker CD206 showed a stepwise increase across different treatments, reaching its highest level in HCSO@ZL, consistent with the best effect in promoting the tissue repair phenotype. Figure 12 cd).

[0047] (12) Next, we tested whether hydrogel-induced immunomodulation could promote angiogenesis. We cultured human umbilical vein endothelial cells (HUVECs) in media produced by BMDMs treated with each hydrogel ( Figure 13 a). Transwell migration assays revealed that the ion-loaded culture medium significantly increased the migration ability of HUVECs, much more so than HCSO. HCSO@Z and HCSO@L showed comparable effects, while HCSO@ZL exhibited the most significant effect, with its migration ability approximately three times that of HCSO (P<0.001). Figure 13 b–c). Tube formation experiments also reflected these results: HCSO@ZL-treated BMDMs induced a more extensive and complex tubular network in the culture medium. Figure 13 d). Quantitative analysis of tube length and number of connection points showed a gradually increasing trend among the groups, with HCSO@ZL exhibiting the most significant tube formation. Figure 13 e–f). CD31 immunofluorescence further confirmed the activation of angiogenesis. The CD31 intensity in HCSO@ZL was approximately six times that in HCSO (P<0.001). Figure 13 These results indicate that hydrogel-induced M2 polarization translates into robust pro-angiogenic AGE signaling. In HCSO@ZL, the synergistic effect of zinc (Zn²⁺) and lithium (Li⁺) ions appears to optimize the immune-angiogenic axis, likely through the synergistic release of multiple angiogenic factors from polarized macrophages, thereby creating a favorable microenvironment for angiogenesis in diabetic bone defects.

[0048] (13) Since oxidative stress impairs bone formation at diabetic bone defects, we evaluated the antioxidant capacity of these hydrogels. Fluorescence imaging showed that intracellular reactive oxygen species (ROS) levels gradually decreased from HCSO to HCSO@ZL hydrogels, with HCSO@ZL exhibiting the lowest ROS levels. Figure 14 ab). Flow cytometry confirmed a leftward shift of the reactive oxygen species peak, indicating a significant reduction in oxidative stress in HCSO@ZL. Figure 14 c). Decreased oxidative stress was associated with enhanced osteogenic differentiation of BMSCs. ALP staining gradually increased in all groups, with HCSO@ZL staining being the most intense, indicating a strong tendency for early osteogenic formation. Figure 14 d). The quantitative ALP activity of HCSO@ZL was significantly higher than that of other groups (P<0.001). Figure 14 e). Alizarin Red S staining showed a continuous increase in calcium deposition from HCSO to HCSO@ZL, with HCSO@ZL exhibiting extensive mineralized nodule formation typical of mature osteoblasts (e). Figure 14 d, f).

[0049] (14) We tested the therapeutic effect in a diabetic rat femoral defect model and assessed bone regeneration at four and eight weeks post-implantation. Figure 15 Three-dimensional reconstruction showed that, compared with poor healing in the defect group, implantation of HCSO@Z and HCSO@L promoted new bone formation at the defect margin. Figure 15 a). HCSO@ZL exhibits superior regeneration capabilities ( Figure 15 a). Quantitative indicators show that HCSO@ZL achieves optimal improvement in new bone microstructure ( Figure 15 bd). Compared with the defect group, the HCSO@ZL group showed a significant increase in bone mineral density at four and eight weeks ( Figure 15 d). At eight weeks, the bone thickness (Tb.Th) and bone volume / total volume (BV / TV) of the HCSO@ZL group were 1.86 times and 2.73 times that of the defect group, respectively. Figure 15 bc). H&E staining showed a significantly enhanced regeneration effect ( Figure 15 e). At four weeks, callus formation in the defect group was limited and persistent gaps remained, while the hydrogel group showed accelerated healing and different types of new bone formation. HCSO@ZL exhibited the best regenerative effect, with orderly trabecular arrangement and abundant bone matrix. By eight weeks, HCSO@ZL had almost restored the continuity of the bone cortex and formed mature lamellar bone, while the defect group was still largely unhealed, consisting mainly of fibrous tissue. Figure 15 e).

[0050] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A composite hydrogel that promotes bone regeneration in diabetic patients, characterized in that, The composite hydrogel is composed of L-histidine-modified chitosan, oxidized konjac glucomannan, Zn²⁺ and nanoclay, and achieves in-situ gelation through coordination regulation, interlayer exchange and dynamic covalent bond synergy.

2. The composite hydrogel for promoting diabetic bone regeneration according to claim 1, characterized in that, The method for preparing the L-histidine-modified chitosan is as follows: Chitosan was weighed and dissolved in acetate buffer. L-histidine was dissolved in deionized water. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and stirred to activate the mixture. The mixture was then added dropwise to the chitosan solution. After the reaction, the pH was adjusted, dialyzed, and freeze-dried to obtain the final product.

3. The composite hydrogel for promoting diabetic bone regeneration according to claim 1, characterized in that, The preparation method of the oxidized konjac glucomannan is as follows: Weigh out konjac glucomannan, dissolve it in deionized water, add sodium periodate and stir to react in the dark, then add ethylene glycol to quench the residual oxidant, dialyze, and freeze dry to obtain the product.

4. The composite hydrogel for promoting diabetic bone regeneration according to claim 1, characterized in that, The composite hydrogel was prepared by a two-component in-situ mixing method. Component A is L-histidine modified chitosan dissolved in HEPES buffer, then dispersed with ZnCl2 stock solution and nano-clay and brought to volume. Component B is oxidized konjac glucomannan dissolved in PBS buffer and brought to volume. Before use, mix equal volumes of component A and component B, then inject the mixture evenly back and forth into the bone defect area.

5. The composite hydrogel for promoting diabetic bone regeneration according to claim 4, characterized in that, The composite hydrogel achieves in-situ gelation through dynamic Schiff base crosslinking, His-Zn²⁺ coordination crosslinking, and interlayer ion exchange of nanoclay.

6. The composite hydrogel for promoting diabetic bone regeneration according to claim 5, characterized in that, The composite hydrogel can intelligently respond to changes in the bone microenvironment, forming a closed-loop regulatory system of "stimulus perception - controlled release - gradual microenvironment remodeling".

7. The composite hydrogel for promoting diabetic bone regeneration according to claim 6, characterized in that, The composite hydrogel simultaneously achieves multiple functions on a unified platform, including oxidative stress clearance, antioxidant network reconstruction, macrophage M1 to M2 polarization, angiogenesis promotion, and enhanced osteogenic differentiation of bone marrow stromal cells.

8. The composite hydrogel for promoting diabetic bone regeneration according to claim 7, characterized in that, The composite hydrogel systematically restores the dynamic balance of the three major axes of immunity, vascularity, and osteogenicity in diabetic bone defects.

9. The use of the composite hydrogel according to any one of claims 1-8 in the preparation of a drug for repairing diabetic bone defects.

10. The application of the composite hydrogel according to claim 9 in the preparation of a drug for repairing diabetic bone defects, characterized in that, The composite hydrogel is applied to diabetic bone defects via injection to promote bone regeneration.

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