A composite hydrogel and a preparation method and application thereof

Abstract

The present invention belongs to the field of biomedical material technology, and particularly relates to a composite hydrogel, its preparation method, and application. The composite hydrogel of the present invention includes a thermosensitive forward gel material, a thermosensitive reverse gel material, a biomaterial, an initiator, and a hemostatic component; the thermosensitive forward gel material includes at least one of hydroxybutyl chitosan and poly (N-isopropyl acrylamide); the thermosensitive reverse gel material includes at least one of methacrylated gelatin and double-bonded sodium hyaluronate; the biomaterial includes at least one of hydroxyapatite nanoparticles and bioglass. The composite hydrogel of the present invention has dual-temperature threshold thermal responsiveness, and can quickly gel at temperatures below 18.6°C or above 24.3°C, constructing a physical hemostatic barrier, while maintaining a fluid state at 18.6-24.3°C, and has good injectability and controllability. At the same time, combined with biomaterials and hemostatic components, good hemostasis and bone defect repair effects are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a composite hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Bone is a complex connective tissue with rich blood vessels on its surface and filled with bone marrow. Severe impact, tumor resection or bone loss can all cause bone injury. After bone tissue injury, hemostasis and tissue repair are a continuous process closely intertwined and promoting each other. Currently, hemostasis and bone repair are usually performed separately in clinical treatment, which has problems such as multiple operations, destruction of new tissues and aggravation of wounds. Especially in harsh environments such as high altitude, extreme cold and disaster, traditional materials often cannot meet the on-site emergency treatment requirements due to limited temperature response, complex operation and single function.

[0003] Existing thermosensitive hydrogel materials mostly rely on single critical temperature response, such as Pluronic F127, poly (N-isopropylacrylamide) (PNIPAM) and the like, which only gel at a body temperature greater than 30℃. In low-temperature environments, they fail to gel and are easily lost, which cannot achieve physical plugging of bleeding sites, and thus cannot adapt to low-temperature wound treatment. A pure low-temperature gel system often lacks a controllable injection window, has poor operation flexibility, and is difficult to achieve precise positioning and treatment. This is because gelling at low temperature means that in order to maintain injectability, the sol state must be maintained at high temperature (close to or above room temperature). In addition, the high temperature in the body (>35℃) can cause partial or complete dissolution of the gel, which is particularly evident in deep tissues or high blood flow areas, and the gelling persistence is poor, which seriously affects the positioning and support performance.

[0004] Therefore, it is of great significance to develop a composite hydrogel that can quickly realize in-situ gelation and close the wound surface in a wide temperature range, achieve good hemostasis and bone repair effect, and have good flow state in the intermediate temperature zone, good injectability and operability. SUMMARY

[0005] The present application aims to solve one or more technical problems in the prior art described above, and at least provide a beneficial alternative. Specifically, the present application provides a composite hydrogel having a double temperature threshold response, which can quickly realize in-situ gelation and close the wound surface in a wide temperature range, has good hemostasis and bone repair effect, and can maintain a flow state in the intermediate temperature zone (18.6-24.3℃), has good injectability and operability.

[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;

[0009] The thermosensitive positive gel material includes at least one of hydroxybutyl chitosan and poly (N-isopropyl acrylamide);

[0010] The thermosensitive reverse gel material comprises at least one of methacrylated gelatin and double-bonded sodium hyaluronate;

[0011] The biomaterial includes at least one of hydroxyapatite and bioglass.

[0012] 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.

[0013] In the present application, the heat-sensitive positive gel material is a solution at low temperature and a gel at high temperature; the heat-sensitive reverse gel material is a gel at low temperature and a solution at high temperature. The heat-sensitive positive gel material, such as hydroxybutyl chitosan, is a heat gel mechanism driven by hydrophobic interaction, and the heat-sensitive reverse gel material, such as methacrylated gelatin, is based on hydrogen bond breaking / segment disentanglement. The two mechanisms act in different temperature zones and do not produce mutual repulsion or competition, fundamentally avoiding the problem of mutual interference of phase change behavior. Therefore, the present application utilizes the opposite phase change characteristics of the two heat-sensitive gel materials to superimpose, so that the composite system has an interlaced temperature-sensitive window, i.e., a double temperature threshold, which can be gelled at less than 18.6℃ or more than 24.3℃, and remains in a flowing state in the intermediate temperature window (18.6-24.3℃), with good operating window and environmental adaptability.

[0014] The following takes hydroxybutyl chitosan and methacrylated gelatin as an example to explain the high and low temperature gel forming principle, and the principle of the intermediate temperature window being in a flowing state:

[0015] The low temperature gel forming principle of the composite hydrogel system: methacrylated gelatin forms a physical gel after cooling (gelatin chain triple helix association), at this time the hydroxybutyl chitosan is in a solution state, the participation is low, and the gel forming mechanism is dominated by the physical crosslinking of methacrylated gelatin, showing a gel state.

[0016] The principle of the intermediate temperature window being in a flowing state: in this temperature range, methacrylated gelatin begins to disassociate (triple helix→linear), gradually turning into a solution, and the hydroxybutyl chitosan does not reach the gel temperature completely, and the system is in a transition state.

[0017] The high temperature gel forming principle of the composite hydrogel system: the temperature of hydroxybutyl chitosan exceeds the critical solution temperature (LCST) of the "sol-gel transition temperature", the hydrophobic interaction is enhanced, the chain aggregation forms a three-dimensional network, and the methacrylated gelatin is completely dissolved and basically does not participate in crosslinking, the gel forming mechanism is dominated by the heat-sensitive physical gel of hydroxybutyl chitosan, showing a gel state.

[0018] For the hemostatic mechanism, the gel quickly forms to seal the wound surface and construct a physical hemostatic barrier; at the same time, the heat-sensitive positive gel material, such as hydroxybutyl chitosan, has a positive charge, interacts with the negatively charged surface of red blood cells and platelets on the wound surface, induces rapid aggregation, and combines with hemostatic components, together to achieve good hemostatic effect.

[0019] For bone repair mechanism, thermosensitive reverse gel materials such as methacrylated gelatin can provide a three-dimensional scaffold to promote osteoblast adhesion and growth; double bond hyaluronic acid sodium can quickly form a stable three-dimensional hydrogel network through photo-crosslinking to provide a three-dimensional scaffold with cell adhesion and migration support function; biomaterials such as hydroxyapatite can mimic the natural bone mineralization structure to induce osteogenic differentiation; bioactive glass can continuously release silicon, calcium and phosphorus ions in a physiological environment to induce the formation of a hydroxyapatite layer similar to the natural bone mineralization structure, while regulating the extracellular microenvironment to promote the differentiation of osteoblasts and bone tissue regeneration; 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.

[0020] Preferably, the hemostatic component comprises at least one of thrombin and fibrin.

[0021] Preferably, the initiator comprises a photoinitiator.

[0022] Preferably, the photoinitiator comprises lithium phenyl-2,4,6-trimethylbenzenesulfonate.

[0023] Preferably, in the preparation raw materials of the composite hydrogel, the proportion of the thermosensitive positive gel material is 2-10wt%, the proportion of the thermosensitive reverse gel material is 2-15wt%, the proportion of the biomaterial is 1-10wt%, the proportion of the initiator is 0.05-0.5wt%, and the concentration of the hemostatic component is 10-500U / mL.

[0024] Preferably, the preparation raw materials of the composite hydrogel further comprise a solvent.

[0025] Preferably, in the preparation raw materials of the composite hydrogel, the proportion of the thermosensitive positive gel material is 2-10wt%, the proportion of the thermosensitive reverse gel material is 2-15wt%, the proportion of the biomaterial is 1-10wt%, the proportion of the initiator is 0.05-0.5wt%, and the concentration of the hemostatic component is 10-500U / mL, and the balance is a solvent.

[0026] Preferably, the solvent comprises water.

[0027] Preferably, the gel temperature of the composite hydrogel is less than 18.6℃, or the gel temperature of the composite hydrogel is greater than 24.3℃.

[0028] Preferably, the gel temperature of the composite hydrogel is less than 18.56℃, or the gel temperature of the composite hydrogel is greater than 24.31℃.

[0029] Further preferably, the gel temperature of the composite hydrogel is greater than or equal to 4℃ and less than 18.56℃, or the gel temperature of the composite hydrogel is greater than 24.31℃ and less than or equal to 45℃.

[0030] Still further preferably, the gel temperature of the composite hydrogel is 10-17℃, or the gel temperature of the composite hydrogel is 25-40℃.

[0031] Preferably, the gel time of the composite hydrogel is 18-45s; further preferably, the gel time of the composite hydrogel is 20-40s.

[0032] The second aspect of the present application provides a preparation method of the composite hydrogel according to the first aspect of the present application.

[0033] Specifically, the preparation method of the composite hydrogel comprises the following steps:

[0034] The raw materials of the composite hydrogel are mixed, incubated at a temperature lower than 18.6℃ or higher than 24.3℃, and then photo-cured to obtain the composite hydrogel.

[0035] Specifically, the raw materials of the composite hydrogel are first incubated at a temperature lower than 18.6℃ or higher than 24.3℃ to form a preliminary gel, and then further cross-linked and cured by photo-curing to obtain the composite hydrogel.

[0036] Preferably, the photo-curing mode comprises ultraviolet light irradiation.

[0037] Specifically, after the preliminary gel is formed, further cross-linking and curing by photo-curing can enhance the mechanical strength of the composite hydrogel and stabilize the three-dimensional network structure.

[0038] Preferably, the preparation method of the hydroxybutyl chitosan comprises the following steps:

[0039] After the chitosan is fully alkalized, it is fully stirred in a dispersion medium, and an epoxy alkane reactant is slowly added dropwise under heating conditions, and then the pH value is adjusted to neutral, followed by dialysis, freeze-drying to obtain the hydroxybutyl chitosan.

[0040] Preferably, the process of alkalization is to mix the alkali solution and the chitosan, and then complete the reaction.

[0041] Preferably, the alkali solution comprises a sodium hydroxide solution.

[0042] Preferably, the mass concentration of the alkali solution is 9-33%; further preferably, the mass concentration of the alkali solution is 10-30%.

[0043] Preferably, the temperature of the reaction during the alkalization is 25-55℃, and the time of the reaction is 1.8-6.5h; further preferably, the temperature of the reaction during the alkalization is 25-50℃, and the time of the reaction is 2-6h.

[0044] Preferably, the dispersion medium comprises isopropyl alcohol.

[0045] Specifically, isopropyl alcohol as the dispersion medium can not only provide a solvent environment, but also effectively regulate the reaction rate and inhibit the occurrence of side reactions.

[0046] Preferably, the alkylene oxide reaction agent comprises at least one of 1,2-epoxyhexane, 1,2-epoxybutane, 1,2-epoxypentane, and propylene oxide.

[0047] Specifically, the alkylene oxide reaction agent comprises but is not limited to the above-mentioned types, such as other alkylene oxides capable of undergoing ring-opening addition reaction with the amino or hydroxyl groups of chitosan.

[0048] Preferably, the time of the stirring is 22-52h; further preferably, the time of the stirring is 24-48h.

[0049] Preferably, the temperature of the heating is 45-65℃; further preferably, the temperature of the heating is 50-60℃.

[0050] Preferably, the time of the reaction during the addition of the alkylene oxide reaction agent is 11-26h; further preferably, the time of the reaction is 12-24h.

[0051] Preferably, the preparation method of the methacrylated gelatin comprises the following steps:

[0052] The gelatin is dissolved in a PBS buffer, the methacrylic anhydride is added dropwise, the reaction is carried out, the PBS buffer is added to terminate the reaction, and then the dialysis and freeze-drying are carried out to obtain the methacrylated gelatin.

[0053] Preferably, the temperature of the reaction during the preparation of the methacrylated gelatin is 35-55℃, and the time of the reaction is 2-4h; further preferably, the temperature of the reaction is 40-50℃, and the time of the reaction is 2.5-3.5h; more preferably, the temperature of the reaction is 45℃, and the time of the reaction is 3h.

[0054] The third aspect of the present application provides a biomaterial.

[0055] Specifically, the biomaterial comprises the composite hydrogel according to the first aspect of the present application.

[0056] With respect to the prior art, the technical scheme provided by the present application has the following beneficial effects:

[0057] (1) The heat-sensitive positive gel material and the heat-sensitive reverse gel material of the present application are used in combination, so that the composite hydrogel has a double-temperature threshold heat response, breaking through the limitation of traditional single-temperature point, and can quickly form a gel under the condition of less than 18.6℃ or more than 24.3℃, realizing physical plugging of the bleeding site, constructing a physical hemostatic barrier, and being suitable for in-situ rapid hemostasis and repair under the condition of less than 18.6℃ or more than 24.3℃. Meanwhile, it maintains a flow state between 18.6-24.3℃, which is an operation window, facilitating intraoperative operation, injection and positioning, and having good injectability and controllability. At the same time, combined with the biological material and the hemostatic component, they jointly act to make the composite hydrogel have good hemostatic and bone repair effects.

[0058] (2) After the gel of the present application quickly forms, it seals the wound and constructs a physical hemostatic barrier; at the same time, the heat-sensitive positive gel material, such as hydroxybutyl chitosan, can also synergistically act with the hemostatic component to promote blood coagulation and quickly form a gel barrier to effectively stop bleeding.

[0059] (3) The heat-sensitive reverse gel material, such as methacrylated gelatin, of the present application can provide a cell attachment environment; the heat-sensitive positive gel material, such as hydroxybutyl chitosan, has osteogenic activity; the biological material, such as hydroxyapatite, can promote bone induction; the three jointly act to make the composite hydrogel have good bone repair ability, and be suitable for bone defect repair of the skull, limbs and other parts.

[0060] (4) The present application integrates the hemostatic and repair functions in a single material system, reduces the operation steps, and avoids secondary damage; and the composite hydrogel of the present application can provide a shaped support matrix in minimally invasive or precise injection surgery.

[0061] (5) The present application can still be applied in scenarios without constant temperature or device support, such as high-altitude extreme cold, high-temperature fire scene, battlefield, remote field medical emergency, and other complex and harsh application scenarios.

[0062] (6) The preparation process of the present application is simple, and is convenient for large-scale popularization and application, and the photo-crosslinking solidification in the preparation process can further enhance the mechanical properties of the gel. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 is the thermal response rheological graph of the composite hydrogel of Example 1 of the present application;

[0064] Figure 2 is the thermal response rheological graph of the composite hydrogel of Comparative Example 3 of the present application;

[0065] Figure 3 is the thermal response rheological graph of the composite hydrogel of Comparative Example 4 of the present application;

[0066] Figure 4 Cell culture live and dead staining chart of the leaching solution of the composite hydrogel of Example 1 and Comparative Example 1-2 of the present application;

[0067] Figure 5 Cell toxicity test result chart of the composite hydrogel of Example 1 and Comparative Example 1-2 of the present application;

[0068] Figure 6 Chart of the expression influence of the composite hydrogel of Example 1 and Comparative Example 1-2 of the present application on bone-related genes;

[0069] Figure 7 Micro-computed tomography chart of the rat skull defect model after 12 weeks;

[0070] Figure 8 Chart of the bone repair volume and bone volume fraction in the modeling area of the rat skull defect model after 12 weeks;

[0071] Figure 9 Chart of the mouse liver hemostasis time and total blood loss. DETAILED DESCRIPTION

[0072] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0073] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0074] Example 1

[0075] A preparation method of a composite hydrogel, comprising the following steps:

[0076] (1) Preparation of hydroxybutyl chitosan (HBC): Chitosan and 50% sodium hydroxide solution were mixed and reacted at 25°C for 6h to fully alkalinize the chitosan, then fully stirred in isopropyl alcohol for 24h, 1,2-epoxyhexane was slowly added under heating conditions at 60°C, and the reaction was carried out for 12h, then the pH value was adjusted to pH=7, and after sufficient dialysis, freeze-drying was carried out to obtain HBC;

[0077] Preparation of methacrylated gelatin (GelMA): 5g of gelatin was dissolved in 75mL of PBS buffer, and methacrylic anhydride was added dropwise, and the reaction was carried out at 45°C for 3h, then 375mL of PBS was added to terminate the reaction, and after sufficient dialysis, freeze-drying was carried out to obtain GelMA;

[0078] (2) Preparation of composite hydrogel: 0.5g HBC is dissolved in 10mL ultrapure water, 0.5g GelMA is added, stirred until completely dissolved, 0.01g photo initiator lithium acyl phenyl-2, 4, 6-trimethyl benzene sulfonate (LAP) is added, after mixing, 0.3g nano hydroxyapatite (nHA) is added, 1000U thrombin (concentration is 100U / mL) is added, after mixing, incubation at 37℃, a preliminary gel is formed, then irradiated by 365nm ultraviolet light for 10s, a stable gel is formed, which is the composite hydrogel.

[0079] Example 2

[0080] A preparation method of a composite hydrogel, comprising the following steps:

[0081] (1) Preparation of HBC: chitosan and sodium hydroxide solution with a mass concentration of 50% are mixed, reacted at 25℃ for 6h, so that the chitosan is fully alkali, then fully stirred in isopropyl alcohol for 24h, 1, 2-epoxyhexane is slowly added under the condition of heating at 60℃, after reaction for 12h, the pH value is adjusted to pH=7, after sufficient dialysis, freeze-drying is performed, and HBC is obtained;

[0082] Preparation of GelMA: 5g gelatin is dissolved in 75mL PBS buffer, methacrylic anhydride is added dropwise, reacted at 45℃ for 3h, then 375mL PBS is added to terminate the reaction, after sufficient dialysis, freeze-drying is performed, and GelMA is obtained;

[0083] (2) Preparation of composite hydrogel: 0.3g HBC is dissolved in 10mL ultrapure water, 0.3g GelMA is added, stirred until completely dissolved, 0.005g photo initiator LAP is added, after mixing, 0.2g nHA is added, 500U thrombin (concentration is 50U / mL) is added, after mixing, incubation at 37℃, a preliminary gel is formed, then irradiated by 365nm ultraviolet light for 10s, a stable gel is formed, which is the composite hydrogel.

[0084] Example 3

[0085] A preparation method of a composite hydrogel, comprising the following steps:

[0086] (1) Preparation of HBC: chitosan and sodium hydroxide solution with a mass concentration of 50% are mixed, reacted at 25℃ for 6h, so that the chitosan is fully alkali, then fully stirred in isopropyl alcohol for 24h, 1, 2-epoxyhexane is slowly added under the condition of heating at 60℃, after reaction for 12h, the pH value is adjusted to pH=7, after sufficient dialysis, freeze-drying is performed, and HBC is obtained;

[0087] Preparation of GelMA: 5 g gelatin was dissolved in 75 mL PBS buffer, and methacrylic anhydride was added dropwise, and reacted at 45℃ for 3 h, then 375 mL PBS was added to terminate the reaction, and after sufficient dialysis, it was freeze-dried to obtain GelMA;

[0088] (2) Preparation of composite hydrogel: 0.6 g HBC was dissolved in 10 mL ultrapure water, 0.5 g GelMA was added, stirred until completely dissolved, 0.02 g photoinitiator LAP was added, mixed, then 0.5 g nHA was added, 2000 U thrombin (concentration 200 U / mL) was added, mixed, and incubated at 37℃ to form a preliminary gel, then irradiated with 365 nm ultraviolet light for 10 s to form a stable gel, which was the composite hydrogel.

[0089] Comparative Example 1

[0090] The difference between Comparative Example 1 and Example 1 is only that Comparative Example 1 does not contain nHA and thrombin, and the mass of the missing components is supplemented with an equal mass of deionized water to maintain the total mass of the formula consistent, and the others are the same as Example 1.

[0091] Comparative Example 2

[0092] The difference between Comparative Example 2 and Example 1 is only that Comparative Example 2 does not contain thrombin, and the mass of the missing components is supplemented with an equal mass of deionized water to maintain the total mass of the formula consistent, and the others are the same as Example 1.

[0093] Comparative Example 3

[0094] The difference between Comparative Example 3 and Example 1 is only that Comparative Example 3 does not contain GelMA, and the mass of the missing components is supplemented with an equal mass of deionized water to maintain the total mass of the formula consistent, and the others are the same as Example 1.

[0095] Since Comparative Example 3 does not contain a light-crosslinkable component, further crosslinking does not occur after ultraviolet light irradiation, and the stability of the gel depends on the temperature-responsive physical gel structure.

[0096] Comparative Example 4

[0097] The difference between Comparative Example 4 and Example 1 is only that Comparative Example 4 does not contain HBC, and the mass of the missing components is supplemented with an equal mass of deionized water to maintain the total mass of the formula consistent, and the others are the same as Example 1.

[0098] Since Comparative Example 4 does not contain HBC with positive temperature responsiveness, no preliminary gel is formed during incubation at 37℃, and by irradiating with 365 nm ultraviolet light for 10 s, the photocrosslinking of GelMA is achieved, forming a stable gel, which is the composite hydrogel.

[0099] Performance Test

[0100] 1. Thermal-responsive rheological behavior test

[0101] To evaluate the thermal-responsive rheological behavior of the composite hydrogel, rheological tests under temperature gradient were carried out on an Anton Paar MCR 302 rheometer (Anton Paar, Austria).

[0102] During the test, the C25 concentric cylinder measuring system was used, and the shear frequency was set to 1 Hz. During the test, the composite hydrogel precursor solution (solution without gelation after mixing the raw materials) prepared in advance in Example 1 and Comparative Examples 3-4 was placed in the measuring fixture, and a layer of anhydrous mineral oil was covered on the surface of the sample to prevent water evaporation caused by temperature change during the test, which would interfere with the data. The temperature was set to rise from 10°C to 40°C at a rate of 1°C / min. Within this temperature gradient range, the oscillation mode was used to continuously monitor the storage modulus (G') and loss modulus (G'') of the sample, which was used to judge the elastic-viscous transition behavior and gelation characteristics. Each sample was tested at least three times, and the average value was taken to draw the modulus-temperature response curve.

[0103] The thermal-responsive rheological diagrams of the composite hydrogels of Example 1 and Comparative Examples 3-4 are shown in FIGS. 1-4, respectively. Figures 1-3

[0104] As can be seen from the rheological test results of Figures 1-3 The composite hydrogel system of the present application has a significant double-temperature response characteristic. Within the two temperature ranges 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 an elastic-dominant gel state, which is conducive to achieving physical plugging of the bleeding site at low temperature or body temperature environment and constructing 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 good sol state of fluidity, which is suitable for injection operation.

[0105] As a comparison, the HBC-nHA-thrombin system of Comparative Example 3 only has a storage modulus higher than a loss modulus at high temperature (>23.7°C), forming a gel structure, while at a lower temperature, it is in a solution state and is difficult to maintain a stable gel state. The GelMA-nHA-thrombin system of Comparative Example 4 only exhibits a storage modulus higher than a loss modulus at a lower temperature (<11.98°C), entering a gel state, and turns into a fluid after the temperature rises, lacking structural support. Comparative Examples 3 and 4 cannot achieve a double-temperature threshold response.

[0106] ​It can be seen that the present application realizes good fluidity of the composite hydrogel material in the intraoperative temperature range (18.56-24.31°C) by synergistic design of the cold gel properties of GelMA (low temperature gel, high temperature solution) and the heat-sensitive gel properties of HBC (low temperature solution, high temperature gel), and the composite hydrogel material can be stored at low temperature before operation or quickly gels at body temperature after operation, showing a typical double-temperature threshold response behavior, which is not possessed by the single-component GelMA or HBC system, and has higher application adaptability and clinical operability.

[0107] 2. Cell compatibility test

[0108] The composite hydrogels of Example 1, Comparative Examples 1-2 were sterilized by 75% (volume fraction) ethanol, and were cut into small pieces. The small pieces were added into Gibco DMEM medium containing double antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL) at a mass-volume ratio of 0.1 g / mL, and were incubated at 37°C on a 60 r / min shaking table for 24 h. Then, the obtained solution was sterilely 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, which was ready for use. The obtained extract can be used for subsequent cell proliferation activity observation, morphological evaluation, cytotoxicity detection, and osteogenesis-related gene expression analysis.

[0109] (a) Cell viability evaluation of bone marrow mesenchymal stem cells: Live / Dead double staining experiment

[0110] The influence of the composite hydrogels of Example 1, Comparative Examples 1-2 on the cell viability of bone marrow mesenchymal stem cells (BMSCs) was evaluated by Live / Dead double staining method. Specifically, 1 mL of BMSC cell suspension with a density of 1×10 5 cells / mL was uniformly inoculated into a 24-well plate, and the extract of the composite hydrogel of Example 1, Comparative Examples 1-2 and the complete DMEM culture medium of the control group were added, respectively. Then, the plate was incubated in a constant temperature incubator at 37°C in 5% (volume fraction) carbon dioxide for 1, 3, and 5 days. After incubation, the plate was gently washed twice with pre-cooled PBS, and Live / Dead double staining working solution (Calcein-AM / PI staining working solution) was added for double staining of green fluorescence of living cells (Calcein-AM) and red fluorescence of dead cells (PI), and the plate was incubated in the dark for 20 minutes. After staining, the plate was observed and photographed under a fluorescence microscope (FITC / TRITC channel), and the cell survival and morphological distribution were analyzed.

[0111] The preparation process of the BMSC cell suspension is as follows:

[0112] 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.

[0113] Example 1, Comparative Example 1-2 composite hydrogel extract cell culture live-dead staining images as shown Figure 4 Group 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.

[0114] 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.

[0115] (b) BMSC cell proliferation activity detection: Cell counting kit-8 (CCK-8) experiment

[0116] 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.

[0117] The preparation process of BMSC cell suspension is the same as above, except that the cell concentration is different.

[0118] Figure 5In 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).

[0119] 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.

[0120] 3. Analysis of Osteogenesis-related Gene Expression

[0121] 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.

[0122] 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.

[0123] 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;

[0124] 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).

[0125] Depend on Figure 6 It 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.

[0126] 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.

[0127] 4. Bone defect repair test

[0128] 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,

[0129] Twenty adult male SD rats (190-210 g) were randomly divided into four groups. After anesthesia, a 6 mm diameter defect model was established on the skull of each rat, and then the composite hydrogel precursor solution (i.e. the solution before gelation) of Example 1, Comparative Examples 1-2 was injected into the defect site of three groups of rats respectively, and then ultraviolet light was cured for 10 s. The rats without any treatment were used as a blank control group. Finally, the rat head skin was sutured and disinfected, and the rat skull was taken for Micro-CT 12 weeks after the operation, and the bone repair volume and volume fraction of the modeling area were calculated.

[0130] The micro-computed tomography graph of the rat skull defect model 12 weeks after modeling is shown in Figure 7 . Among them, Figure 7 the (a) graph, the (b) graph, the (c) graph and the (d) graph in

[0131] It can be seen from Figure 7 that the composite hydrogels of Example 1, Comparative Example 1 and Comparative Example 2 all promote the formation of new bone tissue to some extent, but there are obvious differences in the size of the repair area. After using the composite hydrogel of Example 1 of the application for repair, the bone defect residual area is significantly smaller than that of Comparative Examples 1 and 2, the bone bridge formation is more continuous, and the bone density is higher, indicating that it has a more significant repair effect in promoting new bone formation in the skull defect area. The repair area of Comparative Examples 1 and 2 is smaller, and there is still a large non-healing area at the edge of the bone defect, and the blank control group has almost no obvious bone regeneration.

[0132] The results show that the composite hydrogel system of the application has excellent effect in promoting bone defect repair. The system combines the physical gelation of methacrylated gelatin at low temperature and the enhanced three-dimensional scaffold after photo-crosslinking, the temperature-responsive physical support structure of hydroxybutyl chitosan at body temperature, and the introduction of nano-hydroxyapatite to release osteogenic active calcium and phosphorus ions, and supplemented with thrombin to promote hemostasis and cell migration, forming a functional platform with physical support, biological activity and osteogenic microenvironment. The Micro-CT evaluation results further verify the application potential of the system in bone tissue regeneration.

[0133] The bone repair volume and bone volume fraction results of the modeling area 12 weeks after the rat skull defect modeling are shown in Figure 8 . Among them, Figure 8 the (a) graph in Figure 8Figure (b) in the results of the bone volume fraction (bone volume and tissue volume ratio, BV / TV) of the modeling area after 12 weeks of rat skull defect modeling. Hydroxybutyl chitosan+methylacrylated gelatin, hydroxybutyl chitosan+methylacrylated gelatin+nanoscale hydroxyapatite, hydroxybutyl chitosan+methylacrylated gelatin+nanoscale hydroxyapatite+thrombin represent Comparative Example 1-2, the composite hydrogel of Example 1, respectively.

[0134] From 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 site are significantly higher than those of Comparative Example 1, Comparative Example 2 and the blank control group. Example 1 forms a larger volume of new bone tissue in the defect area, and the proportion of bone tissue in the entire area of interest is higher, indicating 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 show lower new bone volume and bone volume fraction, with limited repair effect; Comparative Example 2 contains nanoscale hydroxyapatite, making its effect on osteogenesis better than that of Comparative Example 1, but its bone formation and bone volume fraction are still significantly lower than those of Example 1.

[0135] The Micro-CT quantitative analysis results show that the composite hydrogel material of the application has good bone repair ability in the process of bone tissue regeneration, can effectively promote new bone formation and filling in the defect area, and provides important experimental basis for its application in the field of skull defect repair and bone tissue engineering.

[0136] 5. Hemostatic effect test

[0137] In the mouse liver puncture injury model, the precursor solutions of the composite hydrogels of Example 1, Comparative Example 1 and Comparative Example 2 (i.e. the solutions before gelation after mixing the raw materials) were injected onto the surface of the liver and cured by light, and the hemostatic time and total blood loss were recorded. Mice with no treatment after liver puncture injury were used as a blank control group.

[0138] The hemostatic time and total blood loss of mouse liver are shown in Figure 9 , wherein Figure 9 Figure (A) in the results of the hemostatic time of mouse liver, *** indicates a very significant difference (P<0.001); Figure 9 Figure (B) in the results of the total blood loss of mouse liver; hydroxybutyl chitosan+methylacrylated gelatin, hydroxybutyl chitosan+methylacrylated gelatin+nanoscale hydroxyapatite, hydroxybutyl chitosan+methylacrylated gelatin+nanoscale hydroxyapatite+thrombin represent the composite hydrogels of Comparative Example 1, Comparative Example 2 and Example 1, respectively.

[0139] From Figure 9It can be seen that the composite hydrogel of Example 1 has the most stable performance in terms of bleeding control, and the average hemostatic time and bleeding volume are significantly lower than those of the blank control group and Comparative Examples 1 and 2, and the difference is statistically significant (p<0.05). The composite hydrogel of Example 1 can achieve effective hemostasis in a short time after application, and the bleeding volume is significantly reduced, indicating that the composite hydrogel has good hemostatic efficiency in in vivo application. In contrast, Comparative Examples 1 and 2 have certain hemostatic effect, but their hemostatic time and bleeding volume are higher than that of Example 1, especially in the early stage of wound bleeding, the blood exudation control is poorer than that of the application. The blank control group has the most bleeding volume and the longest hemostatic time without any treatment. It shows that the selection of materials plays a key role in bleeding control.

[0140] The results of the liver bleeding model experiment show that the composite hydrogel of the application has good in vivo hemostatic ability under the synergistic action of coagulation factor and other functional components, can quickly form a barrier and reduce bleeding in a short time, and lays a reliable foundation for its promotion in practical wound treatment, intraoperative hemostasis and other clinical application scenarios.

[0141] In summary, the composite use of the heat-sensitive positive gel material and the heat-sensitive reverse gel material can make the composite hydrogel have a double-temperature threshold heat response, break through the limitation of traditional single-temperature point, and quickly form a gel under the condition of less than 18.6℃ or more than 24.3℃, realize physical plugging of the bleeding site, construct a physical hemostatic barrier, and be suitable for in situ rapid hemostasis and repair in low temperature environment (<18.6℃) or body temperature (>24.3℃). In the interval of 18.6-24.3℃, it maintains a flow state, which is an operation window, facilitating 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 hemostatic and bone defect repair effect.

[0142] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application 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 application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A composite hydrogel, characterized in that The raw materials for preparing the composite hydrogel are composed of a thermosensitive forward gel material, a thermosensitive reverse gel material, a biomaterial, an initiator, a hemostatic component and a solvent; The thermosensitive positive gel material is hydroxybutyl chitosan; The thermosensitive reverse gel material is methacrylated gelatin; The biomaterial is hydroxyapatite; 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.; The initiator is a photoinitiator.

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: 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.

4. The composite hydrogel according to any one of claims 1 to 3, characterized in that: The gel time of the composite hydrogel is 18-45s.

5. The method for preparing the composite hydrogel according to any one of claims 1 to 4, 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.

6. The preparation method according to claim 5, characterized in that: The light curing method includes ultraviolet light irradiation.

7. A biomedical material, characterized in that: The composite hydrogel comprises the composite hydrogel according to any one of claims 1 to 4.

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