A composite hydrogel for regulating energy metabolism and a preparation method and application thereof

Abstract

The present application belongs to the technical field of hydrogel materials, and particularly relates to a composite hydrogel for regulating energy metabolism and a preparation method and application thereof. The preparation raw material of the composite hydrogel for regulating energy metabolism comprises creatine modified double bond hydroxybutyl chitosan, hydroxyapatite, curcumin, an initiator and a solvent. CHBC provides energy metabolism support at the cell level, is conducive to the proliferation and differentiation of osteogenesis-related cells; HA provides an inorganic environment similar to bone mineralization, and plays a bone conduction and bone induction role; Cur regulates inflammatory response and improves oxidative stress state, and optimizes the repair microenvironment. Through synergy in different aspects such as energy metabolism support, structure mineralization and microenvironment regulation, the regulation of the energy metabolism state of cells in the bone repair area is realized, which is conducive to meeting the high energy requirement of osteoblasts in the repair process, and can promote new bone formation and improve the inflammatory and oxidative stress microenvironment of the injury area, and provides more favorable repair conditions for bone tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel materials technology, and specifically relates to a composite hydrogel that regulates energy metabolism, its preparation method, and its application. Background Technology

[0002] Bone injuries, including bone defects, fractures, and bone tissue damage, have a high incidence in trauma, orthopedic surgery, and the treatment of bone diseases. Due to the complex structure of bone tissue and the long repair cycle, how to construct a stable repair microenvironment conducive to bone regeneration in the defect area has always been a key research focus in the field of bone repair.

[0003] Existing bone repair materials and related technologies primarily focus on providing structural support and the material basis for osteogenic formation at the injury site. For example, by introducing hydroxyapatite, bioceramics, or polymeric scaffold materials, they provide inorganic mineral sources and osteogenic conduction conditions for new bone growth. These materials can promote bone tissue regeneration to some extent, and their design typically focuses on the material's mechanical properties, structural stability, and the supply of osteogenic substances. However, in actual bone repair, osteoblast proliferation, differentiation, and new bone formation are all highly energy-dependent biological processes. Insufficient local energy supply or restricted cellular energy metabolism in the injury area often affects the continuation of the osteogenic process, thereby reducing bone repair efficiency. Most existing bone repair material systems do not involve the regulation of cellular energy metabolism in the repair area, making it difficult to meet the vigorous energy demands of cells during bone repair. Furthermore, early bone injury is often accompanied by adverse microenvironmental factors such as inflammatory responses and oxidative stress, which may inhibit osteoblast function and interfere with new bone formation. Current technologies still have limited ability to synergistically regulate the improvement of the inflammatory microenvironment and the promotion of bone regeneration.

[0004] Therefore, it is of great significance to provide a composite hydrogel that can meet the high energy requirements of osteoblasts during proliferation and differentiation, improve the inflammatory and oxidative stress microenvironment in the damaged area, and provide favorable conditions for bone tissue regeneration. Summary of the Invention

[0005] The present invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial alternative. Specifically, the present invention provides a composite hydrogel that can regulate the energy metabolism state of cells in the bone repair area, which is beneficial to meeting the high energy demand of osteoblasts during the repair process, and can promote new bone formation and improve the inflammatory and oxidative stress microenvironment of the damaged area, providing more favorable repair conditions for bone tissue regeneration and achieving good repair effects.

[0006] The inventive concept of this invention: The raw materials for preparing the composite hydrogel of this invention include creatine-modified double-bonded hydroxybutyl chitosan, hydroxyapatite, curcumin, initiator, and solvent.

[0007] This invention introduces creatine into a chitosan material system to modify chitosan with creatine, constructing creatine-modified double-bonded hydroxybutyl chitosan with potential for energy metabolism regulation. This allows the bone repair material to provide material support while participating in the regulation of local cellular energy metabolism. Simultaneously, this invention combines creatine-modified double-bonded hydroxybutyl chitosan (CHBC) with hydroxyapatite (HA) and curcumin (Cur) to construct a CHBC / HA / Cur multifunctional composite hydrogel system. This system is not a simple superposition of multiple functional materials, but rather achieves synergistic effects at different key regulatory levels during bone injury repair: CHBC provides energy metabolism support at the cellular level, promoting the proliferation and differentiation of osteoblast-related cells; HA provides an inorganic environment for osteomineralization, exerting osteoconduction and osteoinduction effects; and Cur regulates inflammatory responses and improves oxidative stress, optimizing the repair microenvironment. By working synergistically at different levels, such as energy metabolism support, structural mineralization, and microenvironment regulation, this composite hydrogel achieves multi-level synergistic regulation of the bone repair process at different levels, such as cell metabolism, tissue structure, and local microenvironment, which is significantly different from existing bone repair materials that only have a single function or simple superposition of multiple functions.

[0008] Therefore, a first aspect of the present invention provides a composite hydrogel for regulating energy metabolism.

[0009] Specifically, the raw materials for preparing the composite hydrogel that regulates energy metabolism include creatine-modified double-bonded hydroxybutyl chitosan, hydroxyapatite, curcumin, initiator, and solvent.

[0010] Creatine is a small molecule naturally occurring in the body that plays an important physiological role in energy metabolism. Through the creatine / phosphocreatine system, it participates in the rapid regeneration and energy buffering of intracellular adenine triphosphate (ATP), playing a key regulatory role in high-energy-demand tissues such as skeletal muscle and nerve tissue. Cellular energy metabolism is closely related to cell proliferation, differentiation, and functional maintenance. However, in the design of existing bone repair materials, the role of energy metabolism-related molecules such as creatine has not been fully emphasized, and energy metabolism regulation is generally not incorporated into bone repair strategies. This invention introduces creatine into a chitosan material system, modifies chitosan with creatine, and constructs creatine-modified double-bonded hydroxybutyl chitosan with potential for energy metabolism regulation. This allows the bone repair material to participate in regulating local cellular energy metabolism while providing material support. Simultaneously, it is compounded with hydroxyapatite and curcumin to construct a CHBC / HA / Cur multifunctional composite hydrogel system, achieving excellent bone repair effects.

[0011] Preferably, in the creatine-modified double-bonded hydroxybutyl chitosan, the double-bonded hydroxybutyl chitosan is hydroxybutyl chitosan grafted with double bonds.

[0012] Preferably, the proportion of creatine-modified double-bonded hydroxybutyl chitosan in the raw materials for preparing the composite hydrogel is 1-10 wt%.

[0013] Preferably, the proportion of the initiator in the raw materials for preparing the composite hydrogel is 0.01-1 wt%.

[0014] Preferably, the solvent accounts for 85.95-98.889 wt% of the raw materials used to prepare the composite hydrogel.

[0015] Preferably, the proportion of hydroxyapatite in the raw materials for preparing the composite hydrogel is 0.1-3 wt%.

[0016] Preferably, the proportion of curcumin in the raw materials for preparing the composite hydrogel is 0.001-0.05 wt%.

[0017] Preferably, in the raw materials for preparing the composite hydrogel, the proportion of creatine-modified double-bonded hydroxybutyl chitosan is 5 wt%, the proportion of initiator is 0.1 wt%, the proportion of solvent is 93.885 wt%, the concentration of hydroxyapatite is 10 mg / mL (1 wt%), and the concentration of curcumin is 150 μg / mL (0.015 wt%).

[0018] A second aspect of the present invention provides a method for preparing the composite hydrogel for regulating energy metabolism described in the first aspect of the present invention.

[0019] Specifically, the preparation method of the composite hydrogel that regulates energy metabolism includes the following steps: (1) Mix creatine-modified double-bonded hydroxybutyl chitosan solution and initiator to obtain precursor solution; (2) The precursor solution, hydroxyapatite solution and curcumin solution are mixed and photocured to obtain the product.

[0020] Preferably, the creatine-modified double-bond hydroxybutyl chitosan solution contains 1-10 wt% creatine-modified double-bond hydroxybutyl chitosan; more preferably, the creatine-modified double-bond hydroxybutyl chitosan solution contains 3-7 wt% creatine-modified double-bond hydroxybutyl chitosan; for example, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, etc.

[0021] Preferably, the solvent in the creatine-modified double-bond hydroxybutyl chitosan solution includes water.

[0022] Preferably, the initiator in the precursor solution has a mass fraction of 0.01-1 wt%; more preferably, the initiator in the precursor solution has a mass fraction of 0.05-0.2 wt%, such as 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, etc.

[0023] Preferably, the initiator includes a photoinitiator.

[0024] Preferably, the photoinitiator includes 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (Irgacure 2959) and lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP).

[0025] Preferably, the concentration of the hydroxyapatite solution is 1-30 mg / mL; more preferably, the concentration of the hydroxyapatite solution is 5-20 mg / mL, such as 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, etc.

[0026] Preferably, the solvent in the hydroxyapatite solution includes water.

[0027] Preferably, the concentration of the curcumin solution is 10-500 μg / mL; more preferably, the concentration of the curcumin solution is 50-300 μg / mL, such as 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, etc.

[0028] Preferably, the solvent in the curcumin solution includes water.

[0029] Preferably, the volume ratio of the precursor solution, hydroxyapatite solution, and curcumin solution is 1:(0.8-1.2):(0.8-1.2); more preferably, the volume ratio of the precursor solution, hydroxyapatite solution, and curcumin solution is 1:(0.9-1.1):(0.9-1.1); even more preferably, the volume ratio of the precursor solution, hydroxyapatite solution, and curcumin solution is 1:1:1.

[0030] Specifically, the precursor solution, hydroxyapatite solution, and curcumin solution are uniformly dispersed by stirring to obtain a homogeneous hydrogel precursor solution. Under light irradiation, the precursor solution can undergo in-situ photocrosslinking reaction and solidify to form a stable hydrogel structure.

[0031] Preferably, the method for preparing creatine-modified double-bond hydroxybutyl chitosan in the creatine-modified double-bond hydroxybutyl chitosan solution includes the following steps: mixing double-bond hydroxybutyl chitosan and a solvent; then adding creatine, reacting, and obtaining the solution.

[0032] Preferably, after the double-bonded hydroxybutyl chitosan and solvent are mixed, an activator is added for activation.

[0033] Preferably, the solvent used in the preparation process of creatine-modified double-bond hydroxybutyl chitosan includes water.

[0034] Preferably, after the double-bonded hydroxybutyl chitosan and the solvent are mixed, the mass-volume percentage concentration (w / v) of the double-bonded hydroxybutyl chitosan is 1-2% (w / v); for example, 1% (w / v), 1.5% (w / v), 2% (w / v), etc.

[0035] Specifically, 1-2% (w / v) means that 100 mL of solvent contains 1-2 g of double-bonded hydroxybutyl chitosan.

[0036] Preferably, the activator comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS).

[0037] Preferably, the molar ratio of EDC to NHS is 1:(1-2); for example, 1:1, 1:1.5, 1:2, etc.

[0038] Preferably, the mass ratio of the double-bonded hydroxybutyl chitosan to creatine is 1:(0.05-0.5); more preferably, the mass ratio of the double-bonded hydroxybutyl chitosan to creatine is 1:(0.1-0.33), such as 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.33, etc.

[0039] Preferably, in the preparation process of creatine-modified double-bonded hydroxybutyl chitosan, the reaction temperature is room temperature and the reaction time is 12-48 hours.

[0040] Preferably, the preparation process of the creatine-modified double-bonded hydroxybutyl chitosan further includes dialysis and lyophilization after the reaction.

[0041] Preferably, the method for preparing the double-bonded hydroxybutyl chitosan includes the following steps: mixing hydroxybutyl chitosan and a solvent, then adding a methacrylamide reagent, reacting, and obtaining the product.

[0042] Preferably, in the preparation process of the double-bonded hydroxybutyl chitosan, the mass ratio of the hydroxybutyl chitosan to the methacrylating agent is 1:(1-10); for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.

[0043] Preferably, after the hydroxybutyl chitosan and solvent are mixed, the concentration of hydroxybutyl chitosan is 0.5-20 mg / mL; for example, 0.5 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, etc.

[0044] Preferably, the methacrylating agent includes at least one of methacrylic anhydride, methacryloyl chloride, and active ester compounds containing a methacryloyl group.

[0045] More preferably, the methacrylating agent comprises methacrylic anhydride.

[0046] Preferably, in the preparation of the double-bonded hydroxybutyl chitosan, the reaction temperature is room temperature to 70°C, and the reaction time is 12 to 48 hours; more preferably, the reaction temperature is 40 to 60°C.

[0047] Preferably, the preparation process of the double-bonded hydroxybutyl chitosan further includes dialysis and freeze-drying after the reaction.

[0048] Preferably, the method for preparing the hydroxybutyl chitosan includes the following steps: Chitosan was activated under alkaline conditions, and then a solvent was added to obtain a mixture. An epoxide compound was added to the mixture, and the mixture was reacted to obtain the final product.

[0049] Preferably, the chitosan undergoes a purification process before activation.

[0050] Preferably, the purification process is as follows: chitosan and acid solution are mixed, swollen, and then the pH of the solution system is adjusted to neutral, filtered, and dried to obtain the product.

[0051] Preferably, the acid solution comprises an aqueous solution of acetic acid.

[0052] Preferably, in the preparation process of the hydroxybutyl chitosan, the reagent that provides alkaline conditions includes an alkaline solution.

[0053] Preferably, the alkaline solution includes at least one of sodium hydroxide solution and potassium hydroxide solution.

[0054] Preferably, the concentration of the alkaline solution is 1-20M; for example, concentrations of 1M, 5M, 10M, 15M, 19.1M, 20M, etc.

[0055] Preferably, in the preparation process of the hydroxybutyl chitosan, the solvent includes any one of organic solvents and water / organic solvent mixtures.

[0056] Preferably, the organic solvent includes at least one of isopropanol and ethanol.

[0057] Preferably, in the water / organic solvent mixture system, the volume ratio of organic solvent to water is 1:9-9:1.

[0058] Preferably, the mass ratio of the epoxy compound to chitosan is (1-20):1.

[0059] Preferably, the epoxy compound includes 1,2-epoxybutane, through which hydroxybutyl groups are introduced.

[0060] Preferably, in the preparation of the hydroxybutyl chitosan, the reaction temperature is 50-70℃ and the reaction time is 12-48h.

[0061] Preferably, the preparation process of the hydroxybutyl chitosan further includes dialysis and freeze-drying after the reaction.

[0062] A third aspect of the present invention provides the application of the composite hydrogel for regulating energy metabolism described in the first aspect of the present invention in the preparation of drugs or devices for bone injury repair.

[0063] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) Based on the physiological function of creatine as a key substance of endogenous energy metabolism, this invention introduces creatine into the chitosan material system, modifies chitosan with creatine, and constructs creatine-modified double-bonded hydroxybutyl chitosan with potential for energy metabolism regulation. This enables bone repair materials to participate in regulating the energy metabolism state of local cells while providing material support, thereby achieving regulation of the energy metabolism state of cells in the bone repair area, which is beneficial to meeting the high energy demand of osteoblasts during the repair process.

[0064] (2) The present invention promotes new bone formation and improves the inflammatory and oxidative stress microenvironment of the damaged area through the synergistic effect of hydroxyapatite and curcumin, providing more favorable repair conditions for bone tissue regeneration.

[0065] (3) The composite hydrogel material system of the present invention has injectability and in-situ molding capability, which can adapt to irregular bone defects and highly match the defect morphology. It overcomes the problem that traditional molding scaffolds are difficult to adapt to complex defect shapes, improves the flexibility and applicability of the material in clinical applications, and thus improves the application effect of the material in the repair of complex bone injuries.

[0066] (4) This invention constructs a CHBC / HA / Cur multifunctional composite hydrogel system by combining creatine-modified double-bonded hydroxybutyl chitosan, hydroxyapatite and curcumin. The multi-component synergistic design avoids the problem of limited repair efficiency of single functional materials and realizes multi-dimensional comprehensive regulation of the bone repair process. Attached Figure Description

[0067] Figure 1 This is a schematic diagram illustrating the composition and usage of the composite hydrogel in Example 1 of the present invention; Figure 2 These are scanning electron microscope (SEM) images of the hydrogel samples from Example 1 and Comparative Examples 1-2 of this invention. Figure 3 Representative images of surviving bacterial colonies on agar plates from different treatment groups; Figure 4 The graph shows the quantitative analysis results of surviving bacterial colonies on agar plates of different treatment groups; Figure 5 Figure showing the effect of different creatine concentrations on ATP levels in bone marrow mesenchymal stem cells; Figure 6 A comparison of ATP content in bone marrow mesenchymal stem cells after different hydrogel treatments. Figure 7 Figure showing the results of quantitative analysis of mitochondrial membrane potential in bone marrow mesenchymal stem cells after different hydrogel treatments; Figure 8 A graph showing the changes in mitochondrial membrane potential of bone marrow mesenchymal stem cells after different hydrogel treatments; Figure 9 Three-dimensional reconstructed images of skull defects in animals from different treatment groups after 12 weeks of repair using micro-computed tomography. Figure 10 The figure shows the quantitative analysis results of the new bone volume fraction in the skull defect area of ​​animals in different treatment groups. Detailed Implementation

[0068] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0069] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0070] Example 1 This embodiment provides a method for preparing a composite hydrogel that regulates energy metabolism. The specific process is as follows: Creatine-modified double-bonded hydroxybutyl chitosan was dissolved in deionized water to prepare a CHBC solution with a mass fraction of 3 wt%. LAP photoinitiator was added to the CHBC solution to make its mass fraction in the system 0.05 wt%. The mixture was stirred at room temperature until the solution was homogeneous to obtain the CHBC basic precursor solution. Hydroxyapatite was dispersed in deionized water to prepare an HA dispersion with a mass concentration of 5 mg / mL. Curcumin was added to deionized water and stirred to obtain a uniformly dispersed Cur dispersion system with a mass concentration of 50 μg / mL. The CHBC basic precursor solution, HA dispersion and Cur dispersion system were mixed in equal volume ratios and stirred to ensure uniform distribution of each component, thus obtaining a CHBC / HA / Cur hydrogel precursor solution. The CHBC / HA / Cur hydrogel precursor solution was then irradiated under 365nm ultraviolet light for 30s to carry out an in-situ photocrosslinking reaction and solidified to obtain a CHBC / HA / Cur composite hydrogel.

[0071] The preparation process of creatine-modified double-bond hydroxybutyl chitosan is as follows: (1) Preparation of hydroxybutyl chitosan (HBC) Chitosan was dissolved in an acidic aqueous system (0.1M acetic acid aqueous solution) and stirred at 26℃ for 12h to induce swelling. The solution was then adjusted to neutral with 1M NaOH aqueous solution, filtered to remove impurities, and dried at 50℃ to obtain refined chitosan. 4g of refined chitosan was dispersed in a 19.1M NaOH aqueous solution for 24h activation. After activation, the system was transferred to an 80mL isopropanol and water mixture (volume ratio 1:1). 80mL (66g) of 1,2-epoxybutane was added dropwise to the reaction system. The reaction was carried out at 60℃ with magnetic stirring for 24h. After the reaction, the mixture was dialyzed for 5 days at room temperature using a 14 kDa molecular weight cutoff dialysis bag to remove unreacted substances. Hydroxybutyl chitosan (HBC) was obtained by lyophilization. (2) Preparation of double bond modified hydroxybutyl chitosan (HBC_m) HBC was dissolved in deionized water at a concentration of 10 mg / mL. Methacrylic anhydride was added to the solution to introduce a photocrosslinkable double bond structure, with a mass ratio of methacrylic anhydride to HBC of 3.2. The reaction was carried out at 40 °C for 24 h. After the reaction was completed, unreacted reagents were removed by dialysis, and the mixture was lyophilized to obtain modified hydroxybutyl chitosan with a double bond structure, namely double bond modified hydroxybutyl chitosan (double bond hydroxybutyl chitosan, HBC_m). (3) Preparation of creatine-modified double-bonded hydroxybutyl chitosan (CHBC) Dissolve 2g of HBC_m in 100mL of ultrapure water to obtain a homogeneous and transparent HBC_m solution; 0.66 g of creatine was dissolved in 100 mL of ultrapure water. Then, 1.92 g of EDC·HCl and 1.42 g of NHS were added to the solution, and the mixture was stirred at room temperature for 20 min to activate the carboxyl group in the creatine molecule. The activated creatine solution was then added to the HBC_m solution, and the reaction was continued at room temperature for 24 h to allow the activated creatine to undergo an amidation reaction with the residual amino groups on the HBC_m molecular chain. After the reaction was completed, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed in deionized water for 3 days to remove unreacted small molecules and byproducts. The solution was then freeze-dried to obtain creatine-modified double-bonded hydroxybutyl chitosan HBC_m_Cre, abbreviated as CHBC.

[0072] Example 1: A schematic diagram illustrating the composition and usage of the composite hydrogel is shown below. Figure 1 As shown.

[0073] Example 2 This embodiment provides a method for preparing a composite hydrogel that regulates energy metabolism. The specific process is as follows: Creatine-modified double-bonded hydroxybutyl chitosan was dissolved in deionized water to prepare a CHBC solution with a mass fraction of 5 wt%. LAP photoinitiator was added to the CHBC solution to make its mass fraction in the system 0.1 wt%. The mixture was stirred at room temperature until the solution was homogeneous to obtain the CHBC basic precursor solution. Hydroxyapatite was dispersed in deionized water to prepare an HA dispersion with a mass concentration of 10 mg / mL. Curcumin was added to deionized water and stirred to obtain a uniformly dispersed Cur dispersion system with a mass concentration of 150 μg / mL. The CHBC basic precursor solution, HA dispersion and Cur dispersion system were mixed in equal volume ratios and stirred to ensure uniform distribution of each component, thus obtaining a CHBC / HA / Cur hydrogel precursor solution. The CHBC / HA / Cur hydrogel precursor solution was then irradiated under 365nm ultraviolet light for 30s to carry out an in-situ photocrosslinking reaction and solidified to obtain a CHBC / HA / Cur composite hydrogel.

[0074] The preparation process of creatine-modified double-bonded hydroxybutyl chitosan is the same as in Example 1.

[0075] Example 3 This embodiment provides a method for preparing a composite hydrogel that regulates energy metabolism. The specific process is as follows: Creatine-modified double-bonded hydroxybutyl chitosan was dissolved in deionized water to prepare a CHBC solution with a mass fraction of 7 wt%. LAP photoinitiator was added to the CHBC solution to make its mass fraction in the system 0.2 wt%. The mixture was stirred at room temperature until the solution was homogeneous to obtain the CHBC basic precursor solution. Hydroxyapatite was dispersed in deionized water to prepare an HA dispersion with a mass concentration of 20 mg / mL. Curcumin was added to deionized water and stirred to obtain a uniformly dispersed Cur dispersion system with a mass concentration of 300 μg / mL. The CHBC basic precursor solution, HA dispersion and Cur dispersion system were mixed in equal volume ratios and stirred to ensure uniform distribution of each component, thus obtaining a CHBC / HA / Cur hydrogel precursor solution. The CHBC / HA / Cur hydrogel precursor solution was then irradiated under 365nm ultraviolet light for 30s to carry out an in-situ photocrosslinking reaction and solidified to obtain a CHBC / HA / Cur composite hydrogel.

[0076] The preparation process of creatine-modified double-bonded hydroxybutyl chitosan is the same as in Example 1.

[0077] Comparative Example 1 Comparative Example 1 provides a CHBC hydrogel, the preparation process of which is as follows: First, the photoinitiator LAP was weighed and dissolved in ultrapure water to prepare a 0.2 wt% LAP aqueous solution. Then, CHBC material was weighed at a mass ratio of 5 wt% and added to the above LAP aqueous solution at room temperature. The solution was stirred continuously until completely dissolved to obtain a homogeneous and transparent CHBC hydrogel precursor solution. The precursor solution was then irradiated under 365 nm ultraviolet light for 30 s to induce an in-situ photocrosslinking reaction. After curing, the solution was sealed and stored for subsequent experimental use.

[0078] The preparation process of CHBC material is the same as in Example 1.

[0079] Comparative Example 2 Comparative Example 2 provides a CHBC / HA composite hydrogel, the preparation process of which is as follows: The CHBC basic precursor solution and HA dispersion were mixed in equal volume ratio and stirred to ensure uniform distribution of each component, thus obtaining the CHBC / HA hydrogel precursor solution. The CHBC / HA hydrogel precursor solution was then irradiated under 365nm ultraviolet light for 30s to carry out an in-situ photocrosslinking reaction and solidified to obtain the CHBC / HA composite hydrogel.

[0080] The preparation processes of the CHBC basic precursor solution, HA dispersion, and creatine-modified double-bonded hydroxybutyl chitosan are the same as in Example 1.

[0081] Performance testing 1. Scanning electron microscopy observation To characterize the microstructure of the composite hydrogel material of this invention, the morphology of hydrogel samples from different groups in Example 1 and Comparative Examples 1-2 was observed. In the experiment, the prepared hydrogel samples were freeze-dried to maintain their internal structure, and then the sample surface was sputter-coated with gold for conductivity. The microstructure was observed using a scanning electron microscope, and the pore structure distribution and overall morphological characteristics of the hydrogel were recorded. The scanning electron microscope images of the hydrogel samples from Example 1 and Comparative Examples 1-2 are shown below. Figure 2 As shown, where, Figure 2 Figure (a) in the figure is a scanning electron microscope image of the CHBC hydrogel in Comparative Example 1. Figure 2 Figure (b) in the figure is a scanning electron microscope image of the CHBC / HA composite hydrogel in Comparative Example 2. Figure 2 Figure (c) is a scanning electron microscope image of the CHBC / HA / Cur composite hydrogel of Example 1.

[0082] Depend on Figure 2 It can be seen that all groups of hydrogels exhibit a porous network structure. Among them, the CHBC hydrogel of Comparative Example 1 has a continuous three-dimensional pore morphology. After the introduction of hydroxyapatite, the inorganic phase can be observed to be uniformly distributed in the polymer network in the CHBC / HA composite hydrogel, and the pore wall structure is obvious. After further introducing curcumin, the overall pore structure of the CHBC / HA / Cur composite hydrogel remains intact, and no obvious collapse or phase separation phenomenon is observed. This indicates that the multi-component composite does not destroy the basic microstructure of the hydrogel, providing a good structural basis for subsequent biological properties and bone repair applications.

[0083] 2. Antibacterial test To verify the antibacterial properties of the composite hydrogel material of the present invention, methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) and Escherichia coli (E. coli, ATCC 25922) were selected as representative Gram-positive and Gram-negative bacteria, respectively, for the experiment.

[0084] The above-mentioned bacteria were inoculated into LB broth bacterial culture medium and cultured at 37°C until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶ by centrifugation and resuspending in sterile PBS. 7 CFU / mL was used as the standard bacterial solution. The standard bacterial solution was co-cultured with different groups of hydrogel materials. The experiment was divided into a control group (containing only bacteria, without hydrogel material) and experimental groups, each containing 0.1 mL of Comparative Example 1 CHBC hydrogel, Comparative Example 2 CHBC / HA composite hydrogel, and Example 1 CHBC / HA / Cur composite hydrogel, respectively. The cultures were incubated at 37°C for 24 h. After incubation, samples were taken from each system, and the bacterial solutions were subjected to 10 μL sterile PBS. 4 The bacterial culture was serially diluted and evenly spread on the surface of a solid culture medium (agar plate). After incubation at 37°C until colonies formed, the number of colonies formed in each culture dish was counted, and the bacterial survival rate of each experimental group was calculated with the control group as a reference.

[0085] Bacterial survival rate (%) = Number of colonies in the experimental group / Number of colonies in the control group (CFU) 实验组 / CFU 对照组 ) × 100%.

[0086] Representative images of surviving bacterial colonies on agar plates from different treatment groups are shown below. Figure 3 As shown.

[0087] Quantitative analysis results of surviving bacterial colonies on agar plates of different treatment groups are as follows: Figure 4 As shown.

[0088] Depend on Figure 3 , 4As can be seen, bacteria grew vigorously in the control group, forming a large number of colonies on the surface of the culture dish. In contrast, the number of colonies was significantly reduced after the addition of CHBC hydrogel and CHBC / HA composite hydrogel, indicating that the above materials have a certain inhibitory effect on bacterial growth. The addition of CHBC / HA / Cur composite hydrogel further significantly reduced bacterial survival rates, with the survival rates of Staphylococcus aureus and Escherichia coli decreasing to approximately 15.12±4.1% and 13.51±3.1%, respectively, showing a more prominent antibacterial effect. This demonstrates that the CHBC / HA / Cur composite hydrogel of this invention can effectively inhibit the growth of common pathogenic bacteria, and its antibacterial performance is superior to the control hydrogel material without curcumin, which is beneficial for reducing the potential infection risk during bone injury repair and improving the local repair microenvironment.

[0089] 3. Verification of the effect of energy metabolism regulation To verify the regulatory effect of the composite hydrogel material of this invention on energy metabolism of bone repair-related cells, bone marrow mesenchymal stem cells were extracted from the femur of 2-week-old mice. Fourth-generation bone marrow mesenchymal stem cells were selected as the research object and cultured routinely at 37°C and 5% (volume fraction) CO2. After the cells were stably adhered, the following treatment was performed. First, to verify the direct effect of creatine on cellular energy metabolism, different concentrations of creatine monomer were added to the cell culture system and co-cultured with the cells for 24 h. The creatine monomer concentrations were 0 mM, 0.1 mM, 1 mM, 5 mM and 10 mM, respectively. After the culture was completed, the intracellular ATP content of each group of cells was measured to analyze the effect of changes in creatine concentration on the cellular ATP production level. Secondly, to evaluate the regulatory effects of different material systems on cellular energy metabolism, three hydrogels—CHBC, CHBC / HA, and CHBC / HA / Cur—were freeze-dried, sealed, and then sterilized with ethylene oxide. Subsequently, in a cell culture clean bench, different masses of sterilized hydrogel samples were accurately weighed and immersed in DMEM complete culture medium. After sealing, the system was transferred to a shaker (37℃, 120 rpm) and extracted continuously for one week to obtain the hydrogel material extract. The obtained material extract was added to the cell culture system at a concentration of 10 mg / mL of the freeze-dried hydrogel, and co-cultured with the cells for 24 h. Cells without any added material extract served as a blank control group. After culture, the intracellular ATP level was analyzed using an ATP content detection method. Specifically: Cells were lysed and centrifuged at 12000g for 5 min at 4℃. The cell lysate supernatant was collected, and 100 μL of the supernatant was mixed with an equal volume of ATP detection working solution (Yeasen, 40210ES80). The luminescence signal intensity was detected immediately, and the ATP content was calculated using the ATP standard curve (concentration range 0.1~10 μM). The ATP content was normalized to the protein content.

[0090] In addition, the mitochondrial membrane potential detection method was used to analyze the mitochondrial functional status of each group of cells. Specifically, cells treated in the same way as those in the ATP content detection were incubated with JC-1 fluorescent probe working solution (Beyon C2006, final concentration 5 μg / mL) at 37°C in the dark for 20 min. Then, the cells were washed three times with preheated PBS, and the red and green fluorescence signals of the cells were collected. The ratio of red to green fluorescence intensity was quantitatively analyzed.

[0091] The effects of different creatine concentrations on ATP levels in bone marrow mesenchymal stem cells are as follows: Figure 5 As shown; ns indicates no statistically significant difference, ** indicates p<0.01, and *** indicates p<0.001.

[0092] The comparison results of ATP content in bone marrow mesenchymal stem cells after different hydrogel treatments are as follows: Figure 6 As shown; ns indicates no statistically significant difference, and ** indicates p<0.01.

[0093] Quantitative analysis results of mitochondrial membrane potential of bone marrow mesenchymal stem cells after different hydrogel treatments are as follows: Figure 7 As shown; ns indicates no statistically significant difference, and *** indicates p<0.001.

[0094] The changes in mitochondrial membrane potential of bone marrow mesenchymal stem cells after treatment with different hydrogels are shown in the figure below. Figure 8 As shown.

[0095] Depend on Figure 5 It can be seen that in the creatine monomer treatment experiment, the intracellular ATP level gradually increased with the increase of exogenous creatine concentration. When the creatine concentration reached 5 mM and 10 mM, the cellular ATP content was significantly higher than that of the untreated group and the low-concentration creatine treatment group, indicating that creatine itself can directly affect the cellular ATP production level.

[0096] Depend on Figure 6 It can be seen that, in the material extract treatment experiment, compared with the blank control group, the intracellular ATP level increased after adding CHBC extract, CHBC / HA extract and CHBC / HA / Cur extract respectively, indicating that the material system containing creatine-modified components can continue the supporting role of creatine in cellular energy metabolism at the material level.

[0097] Furthermore, by Figure 7 and 8 It can be seen that, compared with the blank control group, the red-green fluorescence intensity ratio of cells in the material extract treatment group was significantly increased, indicating that the mitochondrial membrane potential level was enhanced and the mitochondrial functional state of the cells was improved. Among them, the cell groups with increased ATP content were usually accompanied by increased mitochondrial membrane potential, and the two showed a certain correlation, which can mutually confirm the effect of the material from two aspects: energy metabolism level and mitochondrial functional state.

[0098] In summary, the composite hydrogel material of this invention, by introducing creatine-modified components, can increase the level of cellular ATP production and enhance mitochondrial function under the action of the material extract, thereby providing favorable support for the energy metabolism needs of cells during bone repair.

[0099] 4. Verification of the effect of animal bone defect repair To further verify the application effect of the composite hydrogel material of the present invention in in vivo bone defect repair, an animal skull defect model was established for experimentation.

[0100] Healthy SD rats were selected as experimental animals. Standardized circular bone defect areas were prepared at specific locations on the skull under anesthesia. The animals were randomly divided into a control group, a CHBC group, a CHBC / HA group, and a CHBC / HA / Cur group. In the control group, the defect area was not filled with repair material, while in the other groups, 50 μL of the corresponding component of hydrogel material was implanted into the defect area. After implantation, the surgical area was routinely sutured, and necessary postoperative care was provided to prevent infection. At the predetermined repair time point, tissue samples from the skull defect areas of each group were taken for imaging analysis. Micro-computed tomography (MICR-CT) was used to observe new bone formation in the defect area, and the obtained images were reconstructed in three dimensions to evaluate the impact of different materials on bone defect repair. Simultaneously, parameters such as the new bone volume fraction (the ratio of bone tissue volume to total volume (BV / TV)) within the defect area were quantitatively analyzed to compare the differences in repair effects among the groups.

[0101] Micro-CT 3D reconstruction images of skull defects in animals from different treatment groups 12 weeks after repair are shown below. Figure 9 As shown.

[0102] Quantitative analysis results of the new bone volume fraction in the skull defect area of ​​animals in different treatment groups are as follows: Figure 10 As shown in the figure. Here, ns indicates no statistically significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0103] Depend on Figure 9and 10 It can be seen that the control group showed less new bone formation and a clearer defect outline in the defect area; a certain degree of new bone tissue could be observed at the edge of the defect in the CHBC group, but the overall repair effect was limited, suggesting that although energy metabolism support alone helps bone repair-related cell activities, it is difficult to achieve sufficient new bone reconstruction on its own; after the introduction of hydroxyapatite, new bone formation in the defect area of ​​the CHBC / HA group increased significantly, bone tissue continuity was improved, and the new bone volume fraction increased, indicating that inorganic mineral support plays an important role in promoting new bone formation; after further compounding with curcumin, the new bone filling in the defect area of ​​the CHBC / HA / Cur group was more complete, the connection between the defect area and the surrounding normal bone tissue was more complete, and the new bone volume fraction in the defect area was significantly increased.

[0104] The above results indicate that, under conditions that simultaneously provide energy metabolism support, osteogenic basis, and inflammatory microenvironment regulation, new bone formation and structural reconstruction in bone defect areas can be promoted more effectively. The CHBC / HA / Cur composite hydrogel of this invention exhibits superior repair effects compared to the control group and materials containing only certain components in animal bone defect models.

[0105] Tests showed that the properties of the composite hydrogels in Examples 2 and 3 were comparable to those in Example 1.

[0106] In summary, this invention combines creatine-modified double-bonded hydroxybutyl chitosan, hydroxyapatite, and curcumin, which have energy metabolism regulating effects, to construct a CHBC / HA / Cur composite hydrogel system. Each material exerts a synergistic effect at different key regulatory levels during bone injury repair: CHBC provides energy metabolism support at the cellular level, promoting the proliferation and differentiation of osteoblast-related cells; HA provides an inorganic environment for osteo-like mineralization, exerting osteoconduction and osteoinduction effects; and Cur regulates the inflammatory response and improves oxidative stress, optimizing the repair microenvironment. Through synergistic effects and regulation at different levels, including energy metabolism support, structural mineralization, and microenvironment regulation, the energy metabolism state of cells in the bone repair area is controlled. This helps meet the high energy demands of osteoblasts during the repair process, promotes new bone formation, and improves the inflammatory and oxidative stress microenvironment of the damaged area, providing more favorable repair conditions for bone tissue regeneration.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composite hydrogel, characterized in that, The raw materials for preparing the composite hydrogel include creatine-modified double-bonded hydroxybutyl chitosan, hydroxyapatite, curcumin, initiator, and solvent.

2. The composite hydrogel according to claim 1, characterized in that, In the creatine-modified double-bonded hydroxybutyl chitosan, the double-bonded hydroxybutyl chitosan is hydroxybutyl chitosan grafted with double bonds.

3. The method for preparing the composite hydrogel according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: (1) Mix creatine-modified double-bonded hydroxybutyl chitosan solution and initiator to obtain precursor solution; (2) The precursor solution, hydroxyapatite solution and curcumin solution are mixed and photocured to obtain the product.

4. The preparation method according to claim 3, characterized in that, In the creatine-modified double-bond hydroxybutyl chitosan solution, the mass fraction of creatine-modified double-bond hydroxybutyl chitosan is 1-10 wt%; and / or, in the precursor solution, the mass fraction of the initiator is 0.01-1 wt%.

5. The preparation method according to claim 3, characterized in that, The concentration of the hydroxyapatite solution is 1-30 mg / mL; and / or the concentration of the curcumin solution is 10-500 μg / mL; And / or, the volume ratio of the precursor solution, hydroxyapatite solution, and curcumin solution is 1:(0.8-1.2):(0.8-1.2).

6. The preparation method according to claim 4, characterized in that, The method for preparing creatine-modified double-bond hydroxybutyl chitosan in the creatine-modified double-bond hydroxybutyl chitosan solution includes the following steps: Double-bonded hydroxybutyl chitosan and solvent were mixed; then creatine was added, and the mixture was reacted to obtain the product.

7. The preparation method according to claim 6, characterized in that, The mixture of the double-bonded hydroxybutyl chitosan and the solvent also includes the addition of an activator for activation. And / or, the mass ratio of the double-bonded hydroxybutyl chitosan to creatine is 1:(0.05-0.5). And / or, the reaction temperature is room temperature, and the reaction time is 12-48 hours.

8. The preparation method according to claim 6, characterized in that, The method for preparing the double-bonded hydroxybutyl chitosan The process includes the following steps: mixing hydroxybutyl chitosan and a solvent, then adding a methacrylating agent, reacting, and obtaining the product.

9. The preparation method according to claim 8, characterized in that, In the preparation of the double-bonded hydroxybutyl chitosan, the mass ratio of the hydroxybutyl chitosan to the methacrylating agent is 1:(1-10). And / or, after the hydroxybutyl chitosan and solvent are mixed, the concentration of hydroxybutyl chitosan is 0.5-20 mg / mL; And / or, in the preparation of the double-bonded hydroxybutyl chitosan, the reaction temperature is room temperature to 70°C, and the reaction time is 12 to 48 hours.

10. The use of the composite hydrogel according to any one of claims 1-2 in the preparation of drugs or devices for bone injury repair.

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