Composite hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to composite hydrogel as well as a preparation method and application thereof. The composite hydrogel comprises a thermosensitive forward gel material, a thermosensitive reverse gel material, a biological material, an initiator and a hemostatic component, the thermosensitive positive gel material is prepared from at least one of hydroxybutyl chitosan and poly (N-isopropylacrylamide); the thermosensitive reverse gel material comprises at least one of methacrylated gelatin and double-bond sodium hyaluronate; the biological material comprises at least one of hydroxyapatite nanoparticles and bioglass. The composite hydrogel disclosed by the invention has double-temperature threshold thermal responsiveness, can be quickly gelated at the temperature of less than 18.6 DEG C or greater than 24.3 DEG C to construct a physical hemostasis barrier, can maintain a flowing state at the temperature of 18.6-24.3 DEG C, and has good injectability and controllability. Meanwhile, by combining biological materials and hemostatic components, good hemostatic and bone defect repairing effects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a composite hydrogel and a preparation method and application thereof. Background Art

[0002] Bone is a complex connective tissue with a rich distribution of blood vessels and filled with bone marrow. Severe impact, tumor resection or bone loss may lead to bone injury. After bone tissue damage, hemostasis and tissue repair are continuous processes that are closely intertwined and mutually reinforcing. In current clinical treatment, hemostasis and bone repair are usually performed separately, resulting in problems such as multiple operations, destruction of new tissue, and aggravation of the wound surface. Especially in harsh environments such as plateaus, extreme cold, and disasters, traditional materials are often unable to meet on-site emergency treatment needs due to their limited temperature response, complex operation, and single function.

[0003] Existing thermosensitive hydrogel materials mostly rely on a single critical temperature response, such as Pluronic F127 and poly (N-isopropylacrylamide) (PNIPAM), which can only gel when the body temperature is greater than 30°C. In low-temperature environments, they fail to gel and are prone to loss, making it impossible to achieve physical closure of the bleeding site and thus unable to adapt to low-temperature trauma treatment. Pure low-temperature gel systems often lack a controllable injection window, have poor operational flexibility, and are difficult to achieve precise positioning and treatment. This is because gelation at low temperatures means that in order to maintain injectability, the sol state must be maintained at a high temperature (close to or above room temperature). In addition, the high body temperature (>35°C) may cause the gel to partially or completely dissolve, which is particularly evident in deep tissues or high blood flow areas. The poor persistence of gelation seriously affects the positioning and support performance.

[0004] Therefore, it is of great significance to develop a composite hydrogel that can quickly achieve in situ gelation and wound closure in a wide temperature range, achieve good hemostasis and bone repair effects, have good flow state in the intermediate temperature zone, and have good injectability and controllability. Summary of the Invention

[0005] The present invention aims to address one or more technical issues existing in the aforementioned prior art and to provide at least a beneficial alternative. Specifically, the present invention provides a composite hydrogel with a dual temperature threshold response, enabling rapid in situ gelation and wound closure over a wide temperature range, demonstrating excellent hemostasis and bone repair. Furthermore, it maintains fluidity in the intermediate temperature range (18.6-24.3°C), demonstrating excellent injectability and manipulability.

[0006] The inventive concept of the present invention is as follows: The raw materials for preparing the composite hydrogel include a thermosensitive forward gelling material, a thermosensitive reverse gelling material, a biomaterial, an initiator, and a hemostatic component. The thermosensitive forward gelling material includes at least one of hydroxybutyl chitosan and poly(N-isopropylacrylamide); the thermosensitive reverse gelling material includes at least one of methacrylated gelatin and double-bonded sodium hyaluronate; and the biomaterial includes at least one of hydroxyapatite and bioglass. The thermosensitive forward gelling material of the present invention exhibits the characteristic of gradually gelling with increasing temperature, while the thermosensitive reverse gelling material exhibits the characteristic of gradually gelling with decreasing temperature. The combination of the two allows the composite hydrogel to be in a sol state within a specific temperature range and in a gel state below or above this temperature range, thus exhibiting dual-threshold thermal responsiveness. This means it can quickly gel at temperatures below 18.6°C or above 24.3°C, making it suitable for low- or body-temperature wound environments, physically blocking bleeding sites and creating a physical hemostatic barrier. While maintaining a fluid state between 18.6°C and 24.3°C facilitates intraoperative manipulation, injection, and positioning. Furthermore, the combined effect of biomaterials and hemostatic components gives the composite hydrogel excellent hemostatic and bone repair effects.

[0007] Therefore, a first aspect of the present invention provides a composite hydrogel.

[0008] Specifically, the raw materials for preparing the composite hydrogel include thermosensitive forward gel material, thermosensitive reverse gel material, biomaterial, initiator, and hemostatic component; The thermosensitive positive gel material includes at least one of hydroxybutyl chitosan and poly (N-isopropyl acrylamide); The thermosensitive reverse gel material comprises at least one of methacrylated gelatin and double-bonded sodium hyaluronate; The biomaterial includes at least one of hydroxyapatite and bioglass.

[0009] Specifically, conventional forward and reverse thermosensitive gel systems often exhibit a unidirectional temperature response after mixing due to physical mechanisms (interference between phase transitions), solution behavior (compression of the gelation window), shear failure, and changes in the solvent environment. The gelation mechanism of one material dominates the behavior of the mixed system, while the temperature response characteristics of the other material are lost, making it impossible to achieve bidirectional temperature response and functional synergy. This phenomenon is widespread in existing technologies and limits the development and application of multifunctional thermosensitive gel materials.

[0010] In the present invention, the thermosensitive forward gelling material forms a solution at low temperatures and a gel at high temperatures; the thermosensitive reverse gelling material forms a gel at low temperatures and a solution at high temperatures. Thermosensitive forward gelling materials, such as hydroxybutyl chitosan, rely on a hydrophobic interaction-driven thermogelation mechanism, while thermosensitive reverse gelling materials, such as methacrylated gelatin, are based on hydrogen bond disruption and chain disentanglement. These two mechanisms operate in different temperature ranges without mutual exclusion or competition, fundamentally avoiding the problem of phase transition behavior interfering with each other. Therefore, the present invention utilizes the superposition of the opposing phase transition properties of two thermosensitive gelling materials to create a composite system with staggered temperature-sensitive windows, or dual temperature thresholds. The material can gel at temperatures below 18.6°C or above 24.3°C, while remaining fluid in the intermediate temperature window (18.6-24.3°C), providing a good operating window and environmental adaptability.

[0011] The following uses hydroxybutyl chitosan and methacrylated gelatin as examples to illustrate the principles of gelation at high and low temperatures, as well as the principle of fluidity in the intermediate temperature window: The principle of low-temperature gelation of the composite hydrogel system: methacryloylated gelatin forms a physical gel (triple helical association of gelatin chains) after cooling. At this time, hydroxybutyl chitosan is in a solution state with low participation. The gelation mechanism is dominated by the physical cross-linking of methacryloylated gelatin, which is in a gel state.

[0012] The intermediate temperature window shows the flow state principle: in this temperature range, methacryloyl gelatin begins to dissociate (triple helix → linear) and gradually turns into solution. Hydroxybutyl chitosan has not fully reached the gelation temperature and the system is in a transition state.

[0013] The principle of high-temperature gelation of the composite hydrogel system: When the temperature of hydroxybutyl chitosan exceeds its critical solution temperature (LCST) of "sol-gel transition temperature", the hydrophobic interaction is enhanced, the chains aggregate to form a three-dimensional network, the methacrylated gelatin is completely dissolved, and basically does not participate in cross-linking. The gelation mechanism is that hydroxybutyl chitosan dominates the thermosensitive physical gel and is in a gel state.

[0014] As for the hemostatic mechanism, the gel quickly forms and seals the wound, building a physical hemostatic barrier; at the same time, thermosensitive positive gel materials, such as hydroxybutyl chitosan, have a positive charge and interact with the negatively charged red blood cells and platelet surfaces on the wound, inducing their rapid aggregation, and combining with hemostatic components to work together to achieve a good hemostatic effect.

[0015] Regarding the bone repair mechanism, thermosensitive reverse gel materials, such as methacrylated gelatin, can provide a three-dimensional scaffold to promote osteoblast adhesion and growth; double-bonded sodium hyaluronate can quickly form a stable three-dimensional hydrogel network through photocrosslinking, providing a three-dimensional scaffold with cell adhesion and migration support functions; biomaterials, such as hydroxyapatite, can mimic the natural bone mineralization structure and induce osteogenic differentiation; bioactive glass can sustainably release silicon, calcium and phosphorus ions in a physiological environment, inducing the formation of a hydroxyapatite layer similar to the natural bone mineralization structure on the surface, and at the same time promote osteoblast differentiation and bone tissue regeneration by regulating the extracellular microenvironment; thermosensitive positive gel materials, such as hydroxybutyl chitosan, have osteogenic activity; the three work together to make the composite hydrogel have good bone repair ability.

[0016] Preferably, the hemostatic component includes at least one of thrombin and fibrin.

[0017] Preferably, the initiator comprises a photoinitiator.

[0018] Preferably, the photoinitiator comprises lithium acylphenyl-2,4,6-trimethylbenzenesulfonate.

[0019] Preferably, among the raw materials for preparing the composite hydrogel, the thermosensitive forward gel material accounts for 2-10wt%, the thermosensitive reverse gel material accounts for 2-15wt%, the biomaterial accounts for 1-10wt%, the initiator accounts for 0.05-0.5wt%, and the concentration of the hemostatic component is 10-500U / mL.

[0020] Preferably, the raw materials for preparing the composite hydrogel further include a solvent.

[0021] Preferably, among the raw materials for preparing the composite hydrogel, the thermosensitive forward gel material accounts for 2-10wt%, the thermosensitive reverse gel material accounts for 2-15wt%, the biomaterial accounts for 1-10wt%, the initiator accounts for 0.05-0.5wt%, the concentration of the hemostatic component is 10-500U / mL, and the remainder is solvent.

[0022] Preferably, the solvent comprises water.

[0023] Preferably, the gel temperature of the composite hydrogel is less than 18.6°C, or the gel temperature of the composite hydrogel is greater than 24.3°C.

[0024] Preferably, the gel temperature of the composite hydrogel is less than 18.56°C, or the gel temperature of the composite hydrogel is greater than 24.31°C.

[0025] Further preferably, the gel temperature of the composite hydrogel is greater than or equal to 4°C and less than 18.56°C, or the gel temperature of the composite hydrogel is greater than 24.31°C and less than or equal to 45°C.

[0026] More preferably, the gelling temperature of the composite hydrogel is 10-17°C, or the gelling temperature of the composite hydrogel is 25-40°C.

[0027] Preferably, the gel time of the composite hydrogel is 18-45 s; further preferably, the gel time of the composite hydrogel is 20-40 s.

[0028] The second aspect of the present invention provides a method for preparing the composite hydrogel according to the first aspect of the present invention.

[0029] Specifically, the preparation method of the composite hydrogel comprises the following steps: The raw materials for preparing the composite hydrogel are mixed, incubated at a temperature lower than 18.6° C. or higher than 24.3° C., and then photocured to obtain the composite hydrogel.

[0030] Specifically, the composite hydrogel is first incubated at a temperature lower than 18.6° C. or higher than 24.3° C. to form a preliminary gel, and then further cross-linked and cured by light curing to obtain the composite hydrogel.

[0031] Preferably, the light curing method includes ultraviolet light irradiation.

[0032] Specifically, after the initial gel is formed, further cross-linking and curing is performed by photocuring, which can enhance the mechanical strength of the composite hydrogel and stabilize the three-dimensional network structure.

[0033] Preferably, the preparation method of hydroxybutyl chitosan comprises the following steps: After chitosan is fully alkalized, it is fully stirred in a dispersion medium, and an alkylene oxide reactant is slowly added dropwise under heating conditions to react, and the pH value is adjusted to neutral, followed by dialysis and freeze-drying to obtain the hydroxybutyl chitosan.

[0034] Preferably, the alkalization process is completed by mixing alkali solution and chitosan and reacting them.

[0035] Preferably, the alkali solution comprises sodium hydroxide solution.

[0036] Preferably, the mass concentration of the alkali solution is 9-33%; further preferably, the mass concentration of the alkali solution is 10-30%.

[0037] Preferably, during the alkalization process, the reaction temperature is 25-55° C., and the reaction time is 1.8-6.5 h; further preferably, during the alkalization process, the reaction temperature is 25-50° C., and the reaction time is 2-6 h.

[0038] Preferably, the dispersion medium comprises isopropyl alcohol.

[0039] Specifically, isopropyl alcohol as a dispersion medium can not only provide a solvent environment, but also effectively regulate the reaction rate and inhibit the occurrence of side reactions.

[0040] Preferably, the alkylene oxide reactant includes at least one of 1,2-epoxyhexane, 1,2-epoxybutane, 1,2-epoxypentane and propylene oxide.

[0041] Specifically, the alkylene oxide reactants include but are not limited to the aforementioned types, such as other alkylene oxides that can undergo ring-opening addition reaction with amino groups or hydroxyl groups of chitosan.

[0042] Preferably, the stirring time is 22-52 hours; further preferably, the stirring time is 24-48 hours.

[0043] Preferably, the heating temperature is 45-65°C; more preferably, the heating temperature is 50-60°C.

[0044] Preferably, during the reaction process after the dropwise addition of the alkylene oxide reactant, the reaction time is 11-26 hours; more preferably, the reaction time is 12-24 hours.

[0045] Preferably, the method for preparing the methacrylated gelatin comprises the following steps: Gelatin is dissolved in PBS buffer, methacrylic anhydride is added dropwise, reacted, PBS buffer is added to terminate the reaction, and then dialyzed and freeze-dried to obtain the methacrylated gelatin.

[0046] Preferably, during the preparation of the methacrylated gelatin, the reaction temperature is 35-55°C, and the reaction time is 2-4 hours; further preferably, the reaction temperature is 40-50°C, and the reaction time is 2.5-3.5 hours; further preferably, the reaction temperature is 45°C, and the reaction time is 3 hours.

[0047] A third aspect of the present invention provides a biomedical material.

[0048] Specifically, the biomedical material includes the composite hydrogel described in the first aspect of the present invention.

[0049] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: (1) The thermosensitive forward gel material and the thermosensitive reverse gel material of the present invention are used in combination to make the composite hydrogel have dual-temperature threshold thermal responsiveness, breaking through the traditional single temperature point limitation. It can quickly form a gel under conditions below 18.6°C or above 24.3°C, achieve physical blockage of the bleeding site, and build a physical hemostatic barrier. It is suitable for in situ rapid hemostasis and repair at temperatures below 18.6°C or above 24.3°C. Maintaining a flow state between 18.6-24.3°C is an operating window, which is convenient for intraoperative operation, injection, and positioning, and has good injectability and controllability. At the same time, combined with biomaterials and hemostatic components, they work together to make the composite hydrogel have good hemostasis and bone repair effects.

[0050] (2) The gel of the present invention quickly forms and seals the wound surface, constructing a physical hemostatic barrier. At the same time, the thermosensitive positive gel material, such as hydroxybutyl chitosan, can also synergize with the hemostatic component to promote blood coagulation, quickly form a gel barrier, and effectively stop bleeding.

[0051] (3) The thermosensitive reverse gel material of the present invention, such as methacrylated gelatin, can provide a cell attachment environment; the thermosensitive forward gel material, such as hydroxybutyl chitosan, has osteogenic activity; and the biomaterial, such as hydroxyapatite, can promote bone induction. The three work together to make the composite hydrogel have good bone repair ability, which is suitable for repairing bone defects in the skull, limbs and other parts.

[0052] (4) The present invention integrates hemostasis and repair functions into a single material system, reducing the number of operation steps and avoiding secondary damage; and the composite hydrogel of the present invention can provide a molding support matrix in minimally invasive or precision injection surgery.

[0053] (5) The present invention can still be applied in scenarios without constant temperature or equipment support, such as extreme cold in plateaus, high-temperature fire scenes, battlefields, remote field medical emergency, and other complex and harsh application scenarios.

[0054] (6) The preparation process of the present invention is simple and convenient for large-scale promotion and application, and the photocrosslinking and curing in the preparation process can further enhance the mechanical properties of the gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the thermal responsive rheological diagram of the composite hydrogel in Example 1 of the present invention; Figure 2 This is the thermal responsive rheological diagram of the composite hydrogel of Comparative Example 3 of the present invention; Figure 3 This is the thermal responsive rheological diagram of the composite hydrogel of Comparative Example 4 of the present invention; Figure 4This is a live-dead staining image of cell culture of the extracts of the composite hydrogels of Example 1 and Comparative Examples 1-2 of the present invention; Figure 5 This is a graph showing the cytotoxicity test results of the composite hydrogels of Example 1 and Comparative Examples 1-2 of the present invention; Figure 6 This is a graph showing the effects of the composite hydrogels of Example 1 and Comparative Examples 1-2 on the expression of bone-related genes; Figure 7 This is a micro-computed tomography image of a rat skull defect model 12 weeks after creation; Figure 8 The results of bone repair volume and bone volume fraction in the modeling area 12 weeks after rat skull defect modeling; Figure 9 This is the result graph of mouse liver hemostasis time and total blood loss. DETAILED DESCRIPTION

[0056] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0057] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0058] Example 1 A method for preparing a composite hydrogel comprises the following steps: (1) Preparation of hydroxybutyl chitosan (HBC): Chitosan was mixed with a sodium hydroxide solution with a mass concentration of 50%, and the mixture was reacted at 25°C for 6 h to fully alkalize the chitosan. The mixture was then stirred in isopropanol for 24 h. 1,2-epoxyhexane was slowly added dropwise at 60°C for 12 h. The pH value was adjusted to pH 7, the mixture was fully dialyzed, and the mixture was freeze-dried to obtain HBC. Preparation of methacryloyl gelatin (GelMA): Dissolve 5 g of gelatin in 75 mL of PBS buffer, add methacrylic anhydride dropwise, react at 45°C for 3 h, terminate the reaction by adding 375 mL of PBS, dialyze thoroughly, and lyophilize to obtain GelMA. (2) Preparation of composite hydrogel: 0.5 g HBC was dissolved in 10 mL ultrapure water, 0.5 g GelMA was added, and the mixture was stirred until completely dissolved. 0.01 g photoinitiator lithium acylphenyl-2,4,6-trimethylbenzenesulfonate (LAP) was added, and after mixing, 0.3 g nano-hydroxyapatite (nHA) was added, and 1000 U thrombin (concentration of 100 U / mL) was added. After mixing, the mixture was incubated at 37 °C to form a preliminary gel, and then irradiated with 365 nm ultraviolet light for 10 s to form a stable gel, which is the composite hydrogel.

[0059] Example 2 A method for preparing a composite hydrogel comprises the following steps: (1) Preparation of HBC: Chitosan was mixed with a sodium hydroxide solution with a mass concentration of 50%, and the mixture was reacted at 25°C for 6 h to fully alkalize the chitosan. The mixture was then stirred in isopropanol for 24 h. 1,2-epoxyhexane was slowly added dropwise at 60°C for 12 h. The pH value was adjusted to pH 7, and the mixture was fully dialyzed and freeze-dried to obtain HBC. Preparation of GelMA: Dissolve 5 g of gelatin in 75 mL of PBS buffer, add methacrylic anhydride dropwise, react at 45°C for 3 h, then add 375 mL of PBS to terminate the reaction. After thorough dialysis, freeze-dry to obtain GelMA. (2) Preparation of composite hydrogel: Dissolve 0.3 g HBC in 10 mL ultrapure water, add 0.3 g GelMA, stir until completely dissolved, add 0.005 g photoinitiator LAP, add 0.2 g nHA after mixing, add 500 U thrombin (concentration of 50 U / mL), mix well and incubate at 37 °C to form a preliminary gel, then irradiate with 365 nm ultraviolet light for 10 s to form a stable gel, which is the composite hydrogel.

[0060] Example 3 A method for preparing a composite hydrogel comprises the following steps: (1) Preparation of HBC: Chitosan was mixed with a sodium hydroxide solution with a mass concentration of 50%, and the mixture was reacted at 25°C for 6 h to fully alkalize the chitosan. The mixture was then stirred in isopropanol for 24 h. 1,2-epoxyhexane was slowly added dropwise at 60°C for 12 h. The pH value was adjusted to pH 7, and the mixture was fully dialyzed and freeze-dried to obtain HBC. Preparation of GelMA: 5 g of gelatin was dissolved in 75 mL of PBS buffer, methacrylic anhydride was added dropwise, and the mixture was reacted at 45°C for 3 h. Then, 375 mL of PBS was added to terminate the reaction. The mixture was dialyzed and freeze-dried to obtain GelMA. (2) Preparation of composite hydrogel: Dissolve 0.6 g HBC in 10 mL ultrapure water, add 0.5 g GelMA, stir until completely dissolved, add 0.02 g photoinitiator LAP, mix, add 0.5 g nHA, add 2000 U thrombin (concentration of 200 U / mL), mix well, incubate at 37 °C to form a preliminary gel, and then irradiate with 365 nm ultraviolet light for 10 s to form a stable gel, which is the composite hydrogel.

[0061] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain nHA and thrombin, and the mass of the missing components is supplemented by an equal mass of deionized water to maintain the total mass of the formula consistent. Others are the same as Example 1.

[0062] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain thrombin, and the mass of the missing component is supplemented by an equal mass of deionized water to keep the total mass of the formula consistent. Others are the same as Example 1.

[0063] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain GelMA, and the mass of the missing component is supplemented by an equal mass of deionized water to keep the total mass of the formula consistent. Others are the same as Example 1.

[0064] Since Comparative Example 3 does not contain a photocrosslinkable component, no further crosslinking occurs after UV irradiation, and the stability of the gel depends on the temperature-responsive physical gel structure.

[0065] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain HBC, and the mass of the missing component is supplemented by an equal mass of deionized water to maintain the total mass of the formula consistent. Other details are the same as in Example 1.

[0066] Since Comparative Example 4 does not contain HBC with positive temperature responsiveness, no preliminary gel is formed during incubation at 37°C. By irradiating with 365nm ultraviolet light for 10s, photocrosslinking of GelMA is achieved to form a stable gel, namely a composite hydrogel.

[0067] Performance Testing 1. Thermal responsive rheological behavior test To evaluate the thermoresponsive rheological behavior of the composite hydrogel, rheological tests under temperature gradient were carried out on an Anton Paar MCR 302 rheometer (Anton Paar, Austria).

[0068] The test used a C25 concentric cylinder measurement system with a shear frequency set to 1 Hz. During the test, the pre-configured composite hydrogel precursor solutions of Example 1 and Comparative Examples 3-4 (solutions that were not gelled after mixing the various prepared raw materials) were placed in a measuring fixture, and a layer of anhydrous mineral oil was covered on the surface of the sample to prevent water evaporation due to temperature changes during the test, which would interfere with the data. The temperature was set from 10°C to 40°C at a heating rate of 1°C / min. Within this temperature gradient range, the storage modulus (G') and loss modulus (G'') of the sample were continuously monitored in oscillation mode to determine its elastic-viscosity transition behavior and gelation characteristics. The test was repeated at least three times for each sample, and the average value was taken to plot the modulus-temperature response curve.

[0069] The thermal responsive rheological diagrams of the composite hydrogels of Example 1 and Comparative Examples 3-4 are shown as follows: Figure 1-3 shown.

[0070] Depend on Figure 1-3 It can be seen from the rheological test results that the composite hydrogel system of the present invention has significant dual-temperature response characteristics. In the two temperature zones of less than 18.56°C and greater than 24.31°C, the storage modulus (G') is higher than the loss modulus (G''), and the system is in a gel state dominated by elasticity, which is conducive to physical blocking of bleeding sites in low temperature or body temperature environments and building a physical hemostatic barrier; between 18.56°C and 24.31°C, the storage modulus is lower than the loss modulus, and the material exhibits a sol state with good fluidity, which is suitable for injection operations.

[0071] In contrast, the HBC-nHA-thrombin system of Comparative Example 3 exhibits a storage modulus exceeding loss modulus only at high temperatures (>23.7°C), forming a gel structure. At lower temperatures, it remains in a solution state and struggles to maintain a stable gel state. Furthermore, the GelMA-nHA-thrombin system of Comparative Example 4 exhibits a storage modulus exceeding loss modulus only at lower temperatures (<11.98°C), entering a gel state. Upon increasing the temperature, it transforms into a fluid, lacking structural support. Neither Comparative Examples 3 nor 4 achieve a dual-temperature threshold response.

[0072] It can be seen that the present invention achieves the composite hydrogel material maintaining good fluidity within the intraoperative temperature range (18.56°C-24.31°C) through the synergistic design of the cold gel properties of GelMA (gel at low temperature and solution at high temperature) and the thermosensitive gel properties of HBC (solution at low temperature and gel at high temperature), and can quickly gel under low-temperature storage before surgery or body temperature conditions after surgery, showing a typical dual-temperature threshold response behavior, which is a responsiveness that single-component GelMA or HBC systems do not have, and has higher application adaptability and clinical operability.

[0073] 2. Cytocompatibility Test The composite hydrogels of Example 1 and Comparative Examples 1-2 were sterilized and disinfected with 75% (volume fraction) ethanol, and fully minced. The mixture was added to GibcoDMEM culture medium containing dual antibiotics (100 U / mL of penicillin and 100 μg / mL of streptomycin) at a mass-to-volume ratio of 0.1 g / mL, and incubated at 37°C and 60 rpm for 24 hours on a shaking table. The resulting solution was then sterile-filtered through a 0.22 μm pore size filter membrane, and 10% (v / v) fetal bovine serum (FBS) was added to prepare a cell culture extract for later use. The resulting extract can be used for subsequent cell proliferation activity observation, morphological assessment, cytotoxicity detection, and osteogenesis-related gene expression analysis.

[0074] (a) Assessment of cell viability of bone marrow mesenchymal stem cells: Live / Dead double staining experiment The live / dead cell double staining method was used to evaluate the effects of the composite hydrogels of Example 1 and Comparative Examples 1-2 on the cell viability of bone marrow mesenchymal stem cells (BMSCs). 5 1 mL of a BMSC cell suspension of cells / mL was evenly inoculated into a 24-well plate, and the extracts of the composite hydrogels of Example 1 and Comparative Example 1-2 and the complete DMEM medium of the control group were added respectively, and then incubated in a constant temperature incubator with 5% (volume fraction) carbon dioxide and 37°C for 1, 3, and 5 days; after the incubation, the plates were gently washed twice with pre-cooled PBS, and live cell / dead cell double staining working solution (Calcein-AM / PI staining working solution) was added, that is, live cell green fluorescence (Calcein-AM) and dead cell red fluorescence (PI) double staining was performed, and the plates were incubated in the dark for 20 minutes; after staining, a fluorescence microscope (FITC / TRITC channel) was used to observe and photograph the cells to analyze cell survival and morphological distribution.

[0075] The preparation process of BMSC cell suspension is as follows: Bone marrow mesenchymal stem cells at passages 3-5 were placed in Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's medium) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin, cultured until approximately 80% confluence, and digested with 0.25% (v / v) trypsin-ethylenediaminetetraacetic acid solution. After termination of digestion, the cells were collected by centrifugation (1000 rpm, 5 min); resuspended in PBS buffer and counted, and the cell concentration was adjusted to 1×10 5 cells / mL to obtain BMSC cell suspension.

[0076] Example 1, Comparative Example 1-2 composite hydrogel extract cell culture live-dead staining images as shown Figure 4Group A represents the composite hydrogel of Comparative Example 1, Group B represents the composite hydrogel of Comparative Example 2, and Group C represents the composite hydrogel of Example 1.

[0077] Depend on Figure 4 It can be seen that the composite hydrogels in different groups can all support cell adhesion and survival well, the living cells are evenly distributed, the green fluorescence signal is clear, and the red fluorescence signal is less, indicating that the material has no obvious toxic effect on cells, has good biocompatibility, and meets the basic standards of biomaterials in application fields such as tissue repair and biomedicine.

[0078] (b) BMSC cell proliferation activity detection: Cell counting kit-8 (CCK-8) experiment The density is 2×10 4 200 μL of a BMSC cell suspension of 10 cells / mL was inoculated into a 96-well plate and allowed to stand overnight to allow the cells to adhere to the wall. The culture medium was then replaced with the extract of the composite hydrogel of Example 1, Comparative Example 1-2, and the blank control group DMEM complete medium for culture. The culture medium was placed in an incubator with 5% (volume fraction) carbon dioxide and 37°C, and the culture medium was replaced every 24 hours. Samples were taken out on the 1st, 3rd, and 5th days, and 10 μL of CCK-8 reagent was added to each well. After incubation for another 2 hours, the absorbance value was measured at a wavelength of 450 nm using a microplate reader to evaluate the cell activity level and obtain the cytotoxicity test results. The cytotoxicity test results are shown in FIG. Figure 5 shown.

[0079] The preparation process of BMSC cell suspension is the same as above, except that the cell concentration is different.

[0080] Figure 5 In the figure, hydroxybutyl chitosan + methacryloyl gelatin, hydroxybutyl chitosan + methacryloyl gelatin + nano-hydroxyapatite, and hydroxybutyl chitosan + methacryloyl gelatin + nano-hydroxyapatite + thrombin represent the composite hydrogels of Comparative Example 1, Comparative Example 2, and Example 1, respectively. ns indicates no significant difference, * indicates significant difference (P<0.05), and ** indicates very significant difference (P<0.01).

[0081] Depend on Figure 5 As can be seen, on days 1, 3, and 5, the absorbance values ​​of Example 1 and Comparative Examples 1-2 remained at similar levels to those of the blank control group, with no statistically significant differences. This indicates that the composite hydrogels of Example 1 and Comparative Examples 1-2 had no significant inhibitory effect on the growth activity of BMSC cells and exhibited no detectable cytotoxicity. The absorbance of the composite hydrogel of Example 1 was stable throughout the culture period, and cell viability was good, further verifying the biocompatibility of this material at the cellular level and meeting the basic requirements for tissue engineering and biomedical material applications.

[0082] 3. Analysis of Osteogenesis-related Gene Expression The density is 5×10 5 1 mL of a BMSC cell suspension with a cell density of 10 cells / mL was inoculated into a 6-well plate and induced cultured using the extracts of the composite hydrogels of Example 1 and Comparative Examples 1-2, respectively. The blank control group used a standard osteogenic induction medium (DMEM high glucose medium, 10% (v / v) fetal bovine serum, 1% (v / v) penicillin / streptomycin, 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone). The cells were collected after 7 and 14 days of culture, respectively, and total RNA was extracted using the Trizol method. The RNA concentration and purity were detected by a Nanodrop 2000 spectrophotometer.

[0083] A reverse transcription kit from TransGen Biotech was used according to the instructions. 1.0 μg of RNA was used for complementary DNA (cDNA) synthesis. The synthesized cDNA was used as a template to prepare a 20 μL quantitative polymerase chain reaction solution (qPCR reaction solution), which contained SYBR Green real-time fluorescence quantitative polymerase chain reaction (SYBR Green PCR) premix, specific primers (forward + reverse), nuclease-free water, and cDNA template. The expression of target genes, such as bone morphogenetic protein (BMP) and alkaline phosphatase (ALP), was detected. Fluorescence signal acquisition and amplification curve analysis were performed using a real-time fluorescence quantitative polymerase chain reaction instrument to evaluate the effects of the composite hydrogels of Example 1 and Comparative Examples 1-2 on the osteogenic differentiation potential of BMSCs. The expression results of bone-related genes BMP and ALP are shown in Figure 2. Figure 6 shown.

[0084] Figure 6 The left figure shows the effect of the composite hydrogels of Example 1 and Comparative Examples 1-2 on the expression of bone-related genes BMP; Figure 6 The right figure in the figure is the effect of the composite hydrogel of Example 1 and Comparative Example 1-2 on the expression of bone-related gene ALP; and hydroxybutyl chitosan + methacryloylated gelatin, hydroxybutyl chitosan + methacryloylated gelatin + nano-hydroxyapatite, and hydroxybutyl chitosan + methacryloylated gelatin + nano-hydroxyapatite + thrombin represent the composite hydrogels of Comparative Example 1-2 and Example 1, respectively, * indicates significant difference (P < 0.05), ** indicates very significant difference (P < 0.01), and *** indicates extremely significant difference (P < 0.001).

[0085] Depend on Figure 6It can be seen that compared with the blank control group, the expression levels of bone formation-related genes BMP and ALP in the composite hydrogel groups of Example 1 and Comparative Examples 1-2 were increased, indicating that the composite hydrogel material can promote the activation of BMSC osteogenesis-related phenotypes to a certain extent, and the relative expression levels of the composite hydrogel of Example 1 of the present invention in both BMP and ALP indicators are higher than those of Comparative Examples 1-2, indicating that the composite hydrogel of Example 1 has stronger activity in promoting BMSC osteogenesis differentiation.

[0086] By optimizing its material composition, the composite hydrogel of the present invention effectively induces the upregulation of bone-related genes, potentially enhancing the osteogenic differentiation of stem cells. This composite hydrogel, while maintaining biosafety, further demonstrates excellent osteoinductive properties, providing a strong basis for its application in bone tissue engineering and bone defect repair.

[0087] 4. Bone defect repair test A rat skull defect model was prepared, and the composite hydrogel materials of Example 1 and Comparative Examples 1-2 were injected into the defect area. Twelve weeks after surgery, the bone repair effect was evaluated by micro-computed tomography (Micro-CT). Specifically, Twenty adult male SD rats (190-210 g) were randomly divided into four groups. After anesthesia, a 6 mm diameter defect model was established in the skull of each rat. Subsequently, the composite hydrogel precursor solutions of Example 1 and Comparative Examples 1-2 (i.e., non-gelled solutions obtained by mixing the prepared raw materials) were injected into the defect sites of three of the groups of rats, respectively. The solutions were then UV-cured for 10 seconds. Rats that did not receive any treatment served as a blank control group. Finally, the skin of the rat heads was sutured and disinfected. Twelve weeks after surgery, the rat skulls were harvested for Micro-CT analysis, and the bone repair volume and volume fraction of the modeled area were calculated.

[0088] Micro-computed tomography images of rats after 12 weeks of skull defect modeling Figure 7 As shown. Among them, Figure 7 Figures (a), (b), (c) and (d) are micro-computed tomography images of the blank control group, comparative example 1, comparative example 2 and example 1 composite hydrogel after 12 weeks of modeling rat skull defects.

[0089] Depend on Figure 7As can be seen, the composite hydrogels in Example 1, Comparative Example 1, and Comparative Example 2 all promoted the formation of new bone tissue to a certain extent, but there were significant differences in the size of the repaired area. After repair using the composite hydrogel in Example 1 of the present invention, the residual area of ​​the bone defect was significantly smaller than that in Comparative Examples 1 and 2, with more continuous bone bridge formation and higher bone density, indicating a more significant repair effect in promoting new bone formation in the skull defect area. However, the repaired areas in Comparative Examples 1 and 2 were smaller, and a large unhealed area remained at the edge of the bone defect. The blank control group showed almost no significant bone regeneration.

[0090] These results demonstrate that the composite hydrogel system of the present invention exhibits excellent efficacy in promoting bone defect repair. This system combines the physical gelation of methacrylated gelatin at low temperatures and the enhanced three-dimensional scaffold after photocrosslinking with the temperature-responsive physical support structure of hydroxybutyl chitosan at body temperature. Furthermore, it introduces osteogenic calcium and phosphate ions released by nanohydroxyapatite, supplemented by thrombin to promote hemostasis and cell migration, forming a functional platform that combines physical support, bioactivity, and an osteogenic microenvironment. Micro-CT evaluation results further validated the potential of this system for bone tissue regeneration.

[0091] The results of bone repair volume and bone volume fraction in the modeling area after 12 weeks of rat skull defect modeling are as follows Figure 8 As shown. Among them, Figure 8 Figure (a) shows the bone repair volume of the modeling area 12 weeks after the rat skull defect modeling. Figure 8 Panel (b) shows the bone volume fraction (bone volume to tissue volume ratio, BV / TV) of the modeled area 12 weeks after rat skull defect modeling. Hydroxybutyl chitosan + methacryloylated gelatin, hydroxybutyl chitosan + methacryloylated gelatin + nano-hydroxyapatite, and hydroxybutyl chitosan + methacryloylated gelatin + nano-hydroxyapatite + thrombin represent the composite hydrogels of Comparative Examples 1-2 and Example 1, respectively.

[0092] Depend on Figure 8 It can be seen that after the repair of the composite hydrogel of Example 1, the bone volume and bone volume fraction of the defect area were significantly higher than those of Comparative Example 1, Comparative Example 2 and the blank control group. Example 1 formed a larger volume of new bone tissue in the defect area, and the proportion of bone tissue in the entire region of interest was higher, suggesting that it has a significant promoting effect on new bone formation and bone mass filling. In contrast, Comparative Example 1 and the blank control group showed lower new bone volume and bone volume fraction, and the repair effect was limited; Comparative Example 2 contained nano-hydroxyapatite, which made its osteogenesis effect better than Comparative Example 1, but its bone formation amount and bone volume fraction were still significantly lower than Example 1.

[0093] Micro-CT quantitative analysis results show that the composite hydrogel material of the present invention has good bone repair ability during bone tissue regeneration, and can effectively promote the formation and filling of new bone in the defect area, providing an important experimental basis for its application in skull defect repair and bone tissue engineering.

[0094] 5. Hemostatic effect test In a mouse liver puncture injury model, the composite hydrogel precursor solutions of Example 1, Comparative Example 1, and Comparative Example 2 (i.e., non-gelled solutions obtained by mixing the prepared raw materials) were injected onto the liver surface and photocured. The hemostasis time and total blood loss were recorded. Mice that did not receive any treatment after liver puncture injury served as a blank control group.

[0095] The hemostasis time and total blood loss of mouse liver Figure 9 As shown. Among them, Figure 9 (A) shows the results of mouse liver hemostasis time, *** indicates extremely significant differences (P<0.001); Figure 9 Figure (B) shows the results of total blood loss in mouse liver; hydroxybutyl chitosan + methacryloyl gelatin, hydroxybutyl chitosan + methacryloyl gelatin + nano-hydroxyapatite, and hydroxybutyl chitosan + methacryloyl gelatin + nano-hydroxyapatite + thrombin represent the composite hydrogels of Comparative Example 1, Comparative Example 2, and Example 1, respectively.

[0096] Depend on Figure 9 It can be seen that the composite hydrogel of Example 1 performed the most stably in terms of bleeding control, with its average hemostasis time and bleeding volume significantly lower than those of the blank control group and comparative examples 1 and 2, and the difference was statistically significant (p < 0.05). The composite hydrogel of Example 1 can achieve effective hemostasis within a short period of time after application, and the amount of bleeding is significantly reduced, indicating that the composite hydrogel has good hemostatic efficiency in in vivo applications. In contrast, although comparative examples 1 and 2 have a certain hemostatic effect, their hemostasis time and bleeding volume are both higher than that of Example 1, especially in the early stage of wound bleeding, the blood exudation control is worse than that of the present invention. The blank control group had the highest bleeding volume and the longest hemostasis time without any treatment. This shows that the choice of materials plays a key role in bleeding control.

[0097] The experimental results of the liver bleeding model show that the composite hydrogel of the present invention has good in vivo hemostatic ability under the synergistic action of various functional components such as thrombin, can quickly form a barrier and reduce bleeding in a short period of time, and has laid a solid foundation for its promotion in clinical application scenarios such as practical trauma treatment and intraoperative hemostasis.

[0098] In summary, the combined use of the thermosensitive forward gel material and the thermosensitive reverse gel material of the present invention enables the composite hydrogel to exhibit dual-temperature threshold thermal responsiveness, breaking through the traditional single-temperature limitation. It can rapidly gel at temperatures below 18.6°C or above 24.3°C, physically blocking the bleeding site and creating a physical hemostatic barrier. It is suitable for rapid in situ hemostasis and repair at low temperatures (<18.6°C) or body temperature (>24.3°C). Maintaining a fluid state in the 18.6-24.3°C range creates an operational window, facilitating intraoperative manipulation, injection, and positioning, and exhibiting excellent injectability and controllability. Furthermore, the combined action of biomaterials and hemostatic components ensures that the composite hydrogel exhibits excellent hemostatic and bone defect repair effects.

[0099] 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 the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents 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 thermosensitive forward gel material, thermosensitive reverse gel material, biomaterial, initiator, and hemostatic component; The thermosensitive positive gel material includes at least one of hydroxybutyl chitosan and poly (N-isopropyl acrylamide); The thermosensitive reverse gel material comprises at least one of methacrylated gelatin and double-bonded sodium hyaluronate; The biomaterial includes at least one of hydroxyapatite and bioglass.

2. The composite hydrogel according to claim 1, wherein: The hemostatic component includes at least one of thrombin and fibrin.

3. The composite hydrogel according to claim 1, wherein: The initiator includes a photoinitiator.

4. The composite hydrogel according to claim 1, wherein: Among the raw materials for preparing the composite hydrogel, the thermosensitive forward gel material accounts for 2-10wt%, the thermosensitive reverse gel material accounts for 2-15wt%, the biomaterial accounts for 1-10wt%, the initiator accounts for 0.05-0.5wt%, and the concentration of the hemostatic component is 10-500U / mL.

5. The composite hydrogel according to claim 1, wherein: The raw materials for preparing the composite hydrogel also include a solvent.

6. The composite hydrogel according to any one of claims 1 to 5, characterized in that: The gel temperature of the composite hydrogel is less than 18.6°C, or the gel temperature of the composite hydrogel is greater than 24.3°C.

7. The composite hydrogel according to any one of claims 1 to 5, characterized in that: The gel time of the composite hydrogel is 18-45s.

8. The method for preparing the composite hydrogel according to any one of claims 1 to 7, characterized in that: The following steps are involved: The raw materials for preparing the composite hydrogel are mixed, incubated at a temperature lower than 18.6° C. or higher than 24.3° C., and then photocured to obtain the composite hydrogel.

9. The preparation method according to claim 8, characterized in that: The light curing method includes ultraviolet light irradiation.

10. A biomedical material, characterized in that: The composite hydrogel comprises the composite hydrogel according to any one of claims 1 to 7.

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