Composite sponge based on calcium silicate biological ceramic as well as preparation method and application of composite sponge

Abstract

The invention relates to a composite sponge based on calcium silicate biological ceramic and a preparation method and application thereof, St-Nor and HS-PEG-SH are prepared into a polymer precursor solution, a proper amount of modified anion polysaccharide-sodium alginate is introduced, then the prepared CS is added into the polymer solution, and after foaming treatment, cross-linking curing and freeze drying, the composite sponge based on calcium silicate biological ceramic is obtained. A series of composite sponges based on calcium silicate biological ceramics are obtained, the composite sponges not only have good water absorption capacity and rapid shape recovery capacity, but also have relatively strong mechanical properties, have the potential of expandable hemostatic sponges in incompressible and massive hemorrhage applications, have relatively strong water absorption capacity and macroporous structures, and are suitable for large-scale popularization and application. When the sponge is in contact with blood, blood components such as blood platelets and red blood cells can be effectively enriched, and meanwhile, CS in the sponge plays an important role in accelerating the blood coagulation process. The negative charges in the sponge activate the blood coagulation factor XII and thrombin to trigger the blood coagulation cascade rapid reaction, and further enhance the thrombus strength through the interaction with fibrinogen, so that the sponge has an enhanced hemostatic effect in a massive hemorrhage model and a massive hemorrhage model in a blood dilution state.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a composite sponge based on calcium silicate bioceramics, its preparation method, and its application. Background Technology

[0002] The inventive team of this application proposed online a rapid shape-recovery hemostatic sponge based on modified starch (St-Nor) and a contacting agent (HS-PEG-SH) for controlling non-compressible bleeding. However, subsequent studies found that the hemostatic efficiency of polymer-based synthetic hemostatic sponges is limited by their insufficient procoagulant activity. Furthermore, some silicate biomaterials have been found to possess high procoagulant activity; using them as a procoagulant component in the construction of organic / inorganic composite hemostatic sponges holds promise for achieving more efficient treatment of acute bleeding wounds. Summary of the Invention

[0003] To address the technical deficiencies of existing technologies, this invention provides a composite sponge based on calcium silicate bioceramics, its preparation method, and its application.

[0004] The technical solution adopted in this invention is: a composite sponge based on calcium silicate bioceramics. The composite sponge is obtained by foaming modified starch, modified anionic polysaccharide-sodium alginate, crosslinking agent HS-PEG-SH, and calcium silicate bioceramic particles CS, followed by crosslinking curing and freeze drying.

[0005] The modified starch mentioned is norbornene-modified starch St-Nor.

[0006] The modified anionic polysaccharide-sodium alginate is norbornene-modified sodium alginate SA-Nor.

[0007] The cross-linking curing is performed using ultraviolet light cross-linking.

[0008] The concentration of calcium silicate bioceramic particles (CS) in the composite sponge is 25% w / v.

[0009] The calcium silicate bioceramic particles CS have a particle size of 1 μm.

[0010] A method for preparing a composite sponge based on calcium silicate bioceramics includes the following steps: (1) Synthesis of norbornene-modified starch (St-Nor): Starch was dispersed in anhydrous DMSO and heated to complete dissolution under an inert atmosphere. The reaction mixture was then cooled to 25 °C. Norbornene anhydride, triethylamine and N,N-dimethylaminopyridine were dissolved in anhydrous DMSO and added dropwise with stirring. The product was then purified by alternating dialysis with NaHCO3 solution and deionized water. After freeze-drying, St-Nor was obtained. (2) Synthesis of norbornene-modified sodium alginate (SA-Nor): Sodium alginate was dissolved in MES buffer and stirred overnight. N-hydroxysuccinimide (NHS) was added to activate the carboxyl group. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to the sodium alginate solution, followed by the slow addition of 5-norbornene-2-methylamine. The coupling reaction was completed by stirring at room temperature. Finally, the solution was dialyzed with deionized water and then freeze-dried to obtain modified sodium alginate. (3) Synthesis of calcium silicate nanoparticles: Hexadecyltrimethylammonium bromide (CTAB) was dissolved in deionized water and stirred continuously until completely dissolved. Ethyl acetate was then added and stirred. Then, ammonia water (NH3·H2O) was added and stirred. Subsequently, tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] were added sequentially and reacted. The resulting product was washed with ethanol and deionized water multiple times, centrifuged, and dried. The dried powder was heated to 700°C at a heating rate of 2°C / min and held to obtain calcium silicate ceramic particles. (4) Preparation of composite sponge: Norbornene modified starch (St-Nor) and norbornene modified sodium alginate (SA-Nor) were dissolved in phosphate buffered saline (PBS) containing 0.1 wt% photoinitiator PI 2959. Then, calcium silicate nanoparticles were added to the solution. After ensuring complete dissolution, the pH of the mixture was adjusted to neutral. HS-PEG-SH was dissolved in PBS at room temperature and then added to the solution. SDS solution was added. The resulting mixture was stirred at 1500 rpm to form a foam solution. The foam solution was transferred to a mold, irradiated with ultraviolet light, and then freeze-dried to obtain a composite sponge based on calcium silicate bioceramics.

[0011] In step (4), the ultraviolet light intensity is 15 mW / cm² and the irradiation time is 180 seconds.

[0012] Application of a composite sponge based on calcium silicate bioceramics in the preparation of hemostatic and coagulant materials.

[0013] The beneficial effects of this invention are as follows: This invention provides a composite sponge based on calcium silicate bioceramics, its preparation method, and its application, with the following beneficial effects: (1) In order to fully utilize the procoagulant activity of calcium silicate bioceramics during the construction of composite sponges, the present invention adopts the following strategy: First, based on the characteristics of calcium silicate bioceramics, modified anionic polysaccharide-sodium alginate (SA-Nor) is introduced into the St-Nor and HS-PEG-SH solution system, and the bioceramics are assembled into the cross-linked network of the sponge through hydrogen bonds and ionic bonds, thereby increasing the effective loading of calcium silicate bioceramics. Second, the size of calcium silicate bioceramics and its proportion in the composite are controlled, and the relationship between the procoagulant properties of the composite material and its composition and structure is clarified through a series of chemical, materials science and biological characterizations. (2) A series of composite sponges (CS / StSA) based on calcium silicate bioceramics were obtained. These sponges not only have good water absorption and rapid shape recovery, but also strong mechanical properties. They have the potential to be expandable hemostatic sponges in incompressible and massive hemorrhage applications. The composite sponges of this invention have strong water absorption and macroporous structure. When in contact with blood, they can effectively enrich blood components such as platelets and red blood cells. At the same time, CS in the sponge plays an important role in accelerating the coagulation process. The negative charge in the sponge activates coagulation factor XII and thrombin, triggering a rapid coagulation cascade reaction. Furthermore, through interaction with fibrinogen, it further enhances thrombus strength. In massive hemorrhage and massive hemorrhage models under blood dilution conditions, it has an enhanced hemostatic effect. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the preparation of composite sponges.

[0015] Figure 2 Performance characterization of CS with different particle sizes.

[0016] Figure 3 The residual amount of CS in the composite sponge at different particle sizes.

[0017] Figure 4 For polymers and composite sponges 1 1H NMR and FT-IR spectra.

[0018] Figure 5 These are SEM and EDS images of the composite sponge.

[0019] Figure 6 The results show the water contact angle of the composite sponge.

[0020] Figure 7 This refers to the liquid absorption properties of the composite sponge.

[0021] Figure 8 This refers to the expandability of the composite sponge.

[0022] Figure 9 The mechanical properties of composite sponges.

[0023] Figure 10 The hemolysis rate and cytotoxicity of the composite sponge.

[0024] Figure 11 The in vitro coagulation time and coagulation index of the composite sponge are given.

[0025] Figure 12 This describes the adhesion of the composite sponge to red blood cells and platelets.

[0026] Figure 13 To investigate the platelet activation effect of composite sponges by flow cytometry.

[0027] Figure 14 The effect of composite sponge on XII and thrombin activity.

[0028] Figure 15 The hemostatic effect of the composite sponge in a liver volume defect model of SD rats.

[0029] Figure 16 The hemostatic effect of the composite sponge in the femoral artery transection model of SD rats.

[0030] Figure 17 The hemostatic effect of composite sponge in a hemodiluted liver volume defect model in SD rats.

[0031] Figure 18 The hemostatic effect of the composite sponge in a rabbit blood-diluted liver volume defect model. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Synthesis of norbornene-modified starch (St-Nor) In a reaction flask, potato starch (4 g) was dispersed in anhydrous DMSO (150 ml) and heated to 125 °C under an argon atmosphere until completely dissolved. The reaction mixture was then cooled to 25 °C. Specific amounts of norbornene, triethylamine, and N,N-dimethylaminopyridine were dissolved in anhydrous DMSO (10 ml) and added dropwise to the reaction flask over 1 h using a constant-pressure dropping funnel. The reaction was stirred at 25 °C for 24 h. The product was then poured into a dialysis bag (MWC = 3500) and purified by alternating dialyzing with NaHCO3 solution and deionized water for 72 h. After freeze-drying, St-Nor was obtained.

[0034] Example 2: Synthesis of norbornene-modified sodium alginate (SA-Nor) 0.5 g of sodium alginate (molecular weight 20,000–50,000) was dissolved in 20 mL of 0.1 M MES buffer (pH 6.0) and stirred overnight. Then, 0.21 g of N-hydroxysuccinimide (NHS) was added and the reaction was allowed to proceed for 1 hour to activate the carboxyl groups. Next, 0.32 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to the sodium alginate solution. Following this, 0.15 g of 5-norbornene-2-methylamine was slowly added, and the reaction was stirred at room temperature for 24 hours to complete the coupling reaction. Finally, the solution was transferred to a dialysis bag (molecular weight cutoff = 3500 Da), dialyzed against deionized water for 3 days, and then freeze-dried to obtain modified sodium alginate.

[0035] Example 3 Synthesis of calcium silicate nanoparticles Calcium silicate nanoparticles were synthesized using a microemulsion-sol-gel method. First, 0.56 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in an appropriate amount of deionized water and stirred continuously until completely dissolved. After complete dissolution, 8 mL of ethyl acetate was added, and the mixture was stirred for 30 minutes. Then, 5.6 mL of ammonia water (NH3·H2O) was added, and the mixture was stirred for another 30 minutes. Subsequently, tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] were added sequentially, and the reaction proceeded for 4 hours. The resulting product was washed repeatedly with ethanol and deionized water, centrifuged, and then dried at a constant temperature of 60°C for 12 hours. Finally, the dried powder was heated to 700°C in a muffle furnace at a heating rate of 2°C / min and held for 4 hours. After drying, the resulting calcium silicate ceramic particles of different particle sizes were obtained by sieving for subsequent testing.

[0036] Example 4: Preparation of Composite Sponge 0.5 g of modified starch (St-Nor) and 0.5 g of modified sodium alginate (SA-Nor) were dissolved in phosphate-buffered saline (PBS, pH=7.4, 0.01 M) containing 0.1 wt% photoinitiator PI 2959. Then, calcium silicate powder with particle sizes of 5 μm, 1 μm, and 300 nm was added to the solution at concentrations of 0%, 10%, 25%, and 50% (w / v), respectively. After screening for the desired particle size of calcium silicate powder, solutions with concentrations of 0%, 10%, 25%, and 50% (w / v) were prepared. After ensuring complete dissolution, the pH of the mixture was adjusted to neutral. Subsequently, 0.5 g of HS-PEG-SH was dissolved in 2 mL of PBS at room temperature and added to each solution after pH adjustment. Then, 50 μL of SDS solution (10 mg / mL) was added to each solution, and the resulting mixture was stirred at high speed (1500 rpm) to form a foam solution. It is worth noting that the foaming heights differed among the different groups. The foam solution was transferred to a cylindrical mold using a syringe, irradiated with ultraviolet light (15 mW / cm²) for 180 seconds, and then freeze-dried. The resulting composite ceramic sponges were named CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA, respectively. Similarly, additional composite ceramic sponge groups were prepared using calcium silicate powders with particle sizes of 1 μm and 300 nm at the same concentrations (0%, 10%, 25%, and 50% w / v), named St-SA-1μmCS0, St-SA-1μmCS2, St-SA-1μmCS5, St-SA-1μmCS10, St-SA-300 nmCS0, St-SA-300 nmCS2, St-SA-300 nmCS5, and St-SA-300 nmCS10.

[0037] Experimental Analysis: This invention prepared CS particles of different sizes (5 μm, 1 μm, 300 nm) and analyzed them by scanning electron microscopy, particle size, potential, and coagulation time. Figure 2To control the size of the coagulant sac (CS) and its proportion in the composite, a series of chemical, materials science, and biological characterizations were used to clarify the relationship between the procoagulant properties of the composite material and its composition and structure. As shown in the figure, the sizes of the three CS at different magnifications are approximately 5 μm, 1 μm, and 300 nm, respectively. The corresponding particle sizes of each group are also shown in the figure, confirming this. As is well known, in the intrinsic coagulation pathway, when blood comes into contact with a negatively charged surface, coagulation factor XII is activated, leading to the proteolytic activation of other downstream coagulation factors. Therefore, this invention analyzed the potentials of the three CS sizes. The potentials of 1 μm CS and 300 nm CS reached approximately -20 mV, while that of 5 μm CS reached approximately -35 mV. Furthermore, the in vitro coagulation time of the three CS sizes was tested. The experimental results showed that the coagulation time of all CS powders was shorter than that of the blank group. Among them, the coagulation time of the 1 μm group CS and the 300 nm group CS in whole blood was shorter than that of the 5 μm CS powder, but there was no significant difference between the two.

[0038] The efficient loading of CS in composite sponges is a key research focus of this project. Besides the aforementioned studies, another criterion for selecting CS particle sizes is the binding effect of CS in the sponge at different particle sizes. Therefore, this invention investigated the loading efficiency using thermogravimetric analysis. As shown in the figure, the mass percentage of each composite sponge group decreased slightly between 30℃ and 200℃, possibly due to moisture loss. Furthermore, when the temperature rose to 800℃, the mass percentage of each composite sponge group decreased significantly. Ultimately, the mass percentage of sponges prepared with 1μm CS (CS25 / StSA and CS50 / StSA) was significantly higher than that prepared with 5μm and 300nm CS (CS25 / StSA and CS50 / StSA). Figure 3 ).

[0039] Based on the above research, this invention decided to load 1 μm CS into a polymer sponge. Specifically, this invention fixed the contents of St-Nor and SA-Nor, and further optimized the CS content from 0% to 50% (w / v). Four types of sponges were prepared, named CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA, respectively. The sponge prepared without ceramic addition (CS0 / StSA) served as a negative control material. Firstly, this invention utilizes... 1 H NMR and FT-IR confirmed the chemical structures of St-Nor and SA-Nor. Figure 4 The chemical structures of the four composite sponges were determined by Fourier transform infrared spectroscopy (FT-IR). In the spectra of all samples, the signal ranged from 3300 to 3290 cm⁻¹. -1 and 1738 to 1725cm -1 These are the absorption bands O The H···O and C=O tensile vibrations are likely due to hydrogen bonds formed between the silanol groups on the CS surface of the composite sponge and the hydroxyl or carboxyl groups on the polymer segments, as well as the C=O composition of the chemically modified starch and sodium alginate. Notably, in the spectrum of the composite sponge, these vibrations are observed at 3624 and 3695 cm⁻¹. -1 The peaks at that location belong to the stretching vibrations of interlayer and surface hydroxyl groups. The CS50 / StSA sample also clearly exhibits the same stretching vibrations, suggesting that excess CS may be exposed on the sponge surface rather than bound internally.

[0040] Next, the physical properties of the sponge were studied. When used as a hemostatic material and wound dressing, the pore structure of the sponge is one of the most important parameters. Large pores in the sponge facilitate the absorption of blood and excess exudate. During the sponge synthesis process, the pore structure varies due to differences in the degree of cross-linking reaction and CS content. To demonstrate the porous structure of the sponge, the morphology of CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA was characterized using scanning electron microscopy. For example... Figure 5 As shown, all sponges exhibit a connected macroporous structure, possessing high water absorption and rapid water absorption capacity. With increasing CS content, the microstructure of the sponges becomes loose and disordered. This is because foam solutions containing high CS have excessively high viscosity, which may lead to uneven bubble distribution during foaming. Figure 5 As shown, the pore distribution in the CS25 / StSA foam solution is significantly more uniform compared to the CS50 / StSA foam solution. This uniform porous structure may also play an important role in the mechanical properties and elasticity of the StCA sponge. Furthermore, at 1.0 kx magnification, it is clearly observed that the CS loading increases accordingly with the increasing proportion of CS in the sponge. In addition, the present invention also recorded the EDS spectra of the sponges and identified the elemental composition of the four sponges, including C, O, Ca, and Si. As can be seen from the figure, with the increasing proportion of CS, the proportions of Ca and Si in the composite sponge increase accordingly, with the highest proportions of Ca and Si in CS50 / StSA reaching 12.07% and 8.93%, respectively. This also confirms the effective loading of CS.

[0041] To evaluate the change in hydrophilicity of the composite sponge surface properties after CS assembly into the sponge crosslinking network, this invention conducted contact angle tests on each group of composite sponges, such as... Figure 6 The higher the proportion of CS in the sponge, the smaller the contact angle of the composite sponge, and the greater its hydrophilicity. This may be because the silanol groups on the CS surface form hydrogen bonds with the hydroxyl and carboxyl groups on the polymer chain segments, and the formation of non-covalent bonds makes it more hydrophilic.

[0042] Cross-linked macroporous sponges possess the ability to rapidly absorb liquids, enabling the fixed-shape sponge to quickly absorb blood and tissue exudate from wounds, maintaining a moist environment and promoting wound healing. Furthermore, its rapid absorption capacity allows the sponge to quickly accumulate clotting factors at the bleeding site, promoting coagulation and wound repair. To evaluate the sponge's liquid absorption performance, experiments were conducted to determine the absorption rates of water and blood, as the sponge's rapid liquid absorption capacity is beneficial for uncontrolled bleeding. The figure shows the water absorption ratio of the sponge within 60 seconds. However, as... Figure 7 As shown, all the sponges could absorb the vast majority of water within 10 seconds. Compared to CS50 / StSA, CS0 / StSA sponges exhibited higher water absorption rates, while CS10 / StSA and CS25 / StSA showed comparable absorption capacities. This is due to the large-pore, cross-linked structure within CS0 / StSA, which results in a higher water absorption ratio; after 60 seconds of contact with water, CS0 / StSA can absorb approximately 30 times its weight in water. Furthermore, CS10 / StSA also demonstrated similar performance, while CS50 / StSA absorbed only about 20 times its weight in water. These results indicate that the excellent liquid absorption properties of the composite ceramic sponges are attributed to their porous structure and hydrophilic surface characteristics.

[0043] It has been reported that because blood has a higher viscosity than water, it prolongs the expansion time of sponges. A slight decrease in the blood absorption rate of the composite sponges was observed in the blood. Figure 7 Compared to CS0 / StSA, CS25 / StSA sponge exhibits a higher blood absorption rate. After 60 seconds of contact with blood, CS25 / StSA can absorb approximately 25 times its weight in blood. Furthermore, CS10 / StSA shows similar performance, while CS0 / StSA and CS50 / StSA absorb only about 18 times their weight in blood. This is likely because CS25 / StSA has an appropriate CS loading and a better porosity, resulting in a higher blood absorption rate and absorption ratio compared to the other three sponge groups.

[0044] Hemostatic sponges with shape-restoring or expandability have previously been shown to treat incompressible bleeding because they expand upon contact with blood and block narrow penetrating wounds. Therefore, this invention qualitatively and quantitatively evaluates the water / blood-triggered shape-restoring behavior of all sponges. The expandability of composite ceramic sponges, such as… Figure 8 As shown, the initial state, compressed state, water-absorbed and swelled state, free water squeezed out, and the recovery state after water reabsorption are illustrated. The shape recovery mechanism of the sponge has been described in detail in previous work. When the sponge comes into contact with liquid, due to its good resilience, it absorbs moisture through the residual pores and recovers its shape. This reflects the excellent mechanical and expansion properties of the composite ceramic sponge. To obtain quantitative data, the expansion properties of four types of sponges were tested in this invention. Figure 8As shown, when a fixed-shape sponge comes into contact with water within 1 minute, CS0 / StSA expands to 490%, while CS10 / StSA, CS25 / StSA, and CS50 / StSA expand to approximately 400%. This phenomenon may be because the ionic cross-linking network formed by Ca2+ released from CS and SA-Nor, and the hydrogen bonds formed between the silanol groups on the CS surface and the two polymer chains, can inhibit the excessive expansion behavior of the sponge. Therefore, inhibiting the expansion of the sponge helps to maintain its initial shape and mechanical properties. Hence, within the limited time of 1 minute, the expansion rates of composite sponges with different CS ratios also differ. The four calcium silicate composite sponges also exhibited a trend in blood that corresponds to their expansion rates in water.

[0045] Stable mechanical properties of the sponge are crucial for effective sealing and filling of wounds. Next, to compare the mechanical strength of four sponges, compressive stress-strain tests were conducted on CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA under different conditions. Figure 9 As shown, in the dry state, the compressive strength of all four sponges significantly increased with increasing CS ratio. In the fully expanded state, at 60% strain, the compressive strengths of CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA were 9.61 ± 0.99 KPa, 19.70 ± 9.87 KPa, 38.65 ± 13.03 KPa, and 22.46 ± 5.47 KPa, respectively. This is attributed to the enhanced mechanical strength and strong mechanical elasticity resulting from the covalently cross-linked porous network and the macroporous structure formed by additional hydrogen bond interactions. These results indicate that CS-loaded sponges possess enhanced mechanical properties and can withstand sufficient arterial pressure to block the wound in the oral cavity and prevent blood outflow. To further evaluate the mechanical properties of all sponges, modulus tests were performed on plasma clots and plasma clot / sponge mixtures. Figure 9 Plasma clots are protein gels based on cross-linked fibrin, providing a physical barrier for physiological hemostasis. Sponges can absorb plasma, forming a complex gel after clotting. As shown in the figure, the plasma clots formed by sponges have a significantly higher elastic modulus. The elastic modulus of the plasma clot is 62.95 Pa, which increases to 6748.84 Pa and 26299.7 Pa in the CS0 / StSA and CS25 / StSA groups, respectively. Therefore, the blood clots formed by CS25 / StSA provide a stronger barrier than physiological blood clots and CS0 / StSA blood clots, resulting in better hemostasis. Figure 9The time-scan linear rheological properties of three groups at a fixed frequency of 1 Hz are shown. In the shortest time, plasma clots and plasma clot / sponge mixtures exhibit gel behavior. The storage modulus (G') of each group increases with time. The CS25 / StSA group has the highest storage modulus, reaching 10751.1 Pa. An important characteristic of an ideal hemostatic sponge for controlling bleeding is its ability to rapidly absorb fluid and apply sufficient force to the bleeding site. Reported sponges and cryogels can absorb large amounts of water from the blood and concentrate fibrinogen, coagulation factors, and platelets. However, their mechanical strength is weak, and they cannot apply sufficient pressure to incompressible large bleeding wounds. Therefore, this greatly limits their therapeutic effect. To solve this problem, this invention incorporates CS into the sponge, giving it a certain degree of swelling inhibition after water absorption while retaining sufficient mechanical strength. Furthermore, the fully expanded sponge is elastic and does not generate rigid compressive pressure after expansion, thus avoiding damage to surrounding tissues or nerves.

[0046] In summary, the prepared composite ceramic sponge not only has good water absorption and rapid shape recovery, but also strong mechanical properties, and has the potential to become an expandable hemostatic sponge in applications involving incompressibility and massive bleeding.

[0047] Biocompatibility of hemostatic materials is crucial. This invention employs an in vitro hemolysis method, a widely used approach, to evaluate the blood compatibility of sponges. A saline group served as the negative control, and a deionized water group as the positive control. The hemolysis rates of CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA sponges were measured. Images of the centrifuged supernatant and final hemolysis rates for all sponge groups and control groups are shown below. Figure 10 As shown. The ratios of CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA were found to be only 3.7±0.1%, 2.2±0.07%, 1.5±0.08%, and 1.6±0.13%, respectively, all within the acceptable range (5%) for biomaterials. Furthermore, the cell compatibility of the sponge was assessed using CCK-8 assays, and the live / dead state of L929 fibroblasts was determined. Figure 10 As shown, the results indicated that after 24 hours of culture, the cell viability values ​​in both the low-concentration and high-concentration experimental groups reached as high as 75%. The composite ceramic sponge exhibited low toxicity to L929 fibroblasts. Fluorescent staining results further confirmed this conclusion. Figure 10 There were no significant differences in cell number and morphology between the CS10 / StSA and CS25 / StSA sponge groups. The hemolysis rate and CCK8 results together demonstrate that the hemostatic sponge exhibits good blood and cell compatibility.

[0048] Based on coagulation mechanisms, hemostatic materials are classified into active and passive hemostatic materials. The prepared sponge possesses morphological recovery capabilities and can apply sustained pressure to blood vessels. The composite hemostatic sponge prepared in this invention exhibits high procoagulant activity. Therefore, the in vitro coagulation time of the composite sponge is assessed based on blood flow. Figure 11 As shown, the spontaneous recalcification clotting time in the blank control group was 286±2 s, while the clotting time in the gelatin group (251±2 s) was slightly shorter than that in the blank control group. CS0 / StSA slightly promoted the clotting process (246±7 s). After the addition of CS, the whole blood clotting time of the C10 / StSA, CS25 / StSA, and CS50 / StSA groups decreased to 228±2 s, 205±8 s, and 238±2 s, respectively. The results indicate that the composite sponge assembled with sufficient CS has significant procoagulant activity, which may be related to the concentration of blood components and platelet activation. In the CS-loaded sponge, the negatively charged silanol groups interact with the positively charged amino acids on the coagulation factor XII chain, activating the intrinsic coagulation pathway and accelerating the coagulation cascade.

[0049] Following the coagulation cascade, plasma transforms from unstable platelet plugs into stable, insoluble fibrin, gradually trapping red blood cells. Therefore, in vitro coagulation can be evaluated by the release of red blood cells from the clot in water. Lower hemoglobin concentrations in water and a more mature clot result in faster coagulation. Therefore, the coagulation index (BCI) of all samples was measured after 3 minutes. Figure 11 As shown, the BCI values ​​of CS0 / StSA, CS10 / StSA, CS25 / StSA, and CS50 / StSA were 48.2±0.5%, 36.7±0.5%, 39.3±1.0%, and 51.7±1.8%, respectively. The addition of CS improved the coagulation ability of the sponge. With a significant increase in CS dosage (CS50 / StSA), the coagulation ability of the sponge decreased, which may be related to the porous structure and blood absorption capacity of the sponge. Even so, compared with commercial gelatin sponge (87.5±2.2%), the composite ceramic sponge still exhibited better coagulation ability.

[0050] Study on the procoagulant mechanism of composite sponge Previous reports have shown that sponges, due to their three-dimensional structure, can rapidly enrich blood components through their liquid absorption properties, thereby promoting the coagulation process. During coagulation, enriched red blood cells promote platelet activation by releasing adenosine diphosphate (ADP). Subsequently, activated platelets can promote thrombin formation, activate coagulation factors XI and XII, and accelerate the intrinsic coagulation process. This invention focuses on the adhesion behavior of sponges to platelets and red blood cells.

[0051] Figure 12This illustrates the localized situation of erythrocyte adhesion. Scanning electron microscopy images show that erythrocytes effectively adhere to all sponge surfaces, exhibiting irregular aggregation and activated deformation. Notably, the sponges with added CS (CS10 / StSA, CS25 / StSA, CS50 / StSA) showed a higher level of erythrocyte enrichment than the sponges without added CS (CS0 / StSA). To further quantify the data, this invention quantitatively detected the erythrocytes adhering to the sponges. Figure 12 The results showed that the adsorption of erythrocytes by the composite ceramic sponge increased, while the CSO / StSA sponge exhibited the lowest erythrocyte adhesion. This difference in adhesion may be due to the polymer network structure of the composite sponge, which helps restrict blood flow and capture erythrocytes. Furthermore, scanning electron microscopy images revealed platelet aggregation and activation on all sponges; the platelets deformed and extended tentacles after activation. As shown in the figures, sponges containing CS adsorbed more platelets than those without CS, and the adhesion was stronger with a higher proportion of CS. Regarding the platelet deformation and activation, this invention hypothesizes that the addition of CS during the synthesis of the composite sponge altered the surface properties of the sponge, affecting the specific binding of the sponge to clotting proteins, thereby activating the free amino groups on the proteins and activating the platelets. This platelet activation enhanced their adhesion to the sponge.

[0052] Furthermore, this invention investigated the effect of the composite sponge on platelet activation using flow cytometry. This invention selected FITC and PE to label CD41 (typically expressed in inactive platelets) and CD62P (expressed in activated platelets), respectively. This invention screened platelet populations by FITC expression and identified activated platelets expressing PE. The results are as follows: Figure 13 As shown, the platelet activation rate in the untreated platelet group was only 0.38%, while the activation rate of the bioceramic particles CS was the highest, at 61.6%. The platelet activation rates of CS0 / StSA and CS25 / StSA sponges were 3.93% and 25.7%, respectively, which may be because the CS component was added during the synthesis of the composite ceramic sponge, thereby stimulating platelet activation. Therefore, these findings indicate that the composite ceramic sponge is effective in activating platelets and can promote the extrinsic coagulation process.

[0053] Platelet activation and coagulation depend on the activation of coagulation factors in the coagulation cascade. In the intrinsic coagulation pathway, activation of coagulation factor XII leads to activation of coagulation factor X, which in turn induces thrombin production. To further investigate the effect of composite sponges on the intrinsic coagulation pathway, this invention examined the interaction between composite sponges and coagulation factor XII. Platelet-rich plasma was incubated with composite sponges for 2, 5, and 10 min. After incubation, the production of coagulation factor XII was assessed. Interestingly, after 2 min of incubation, the content of coagulation factor XII in the CS50 / StSA sponge was significantly higher than that in other sponge groups, reaching a peak at 2 min. After 5 min, the coagulation factor content decreased slightly, but the overall content in the CS-loaded sponge group was still higher than that in the blank group and the CS0 / StSA group. Figure 14 Furthermore, the same method was used to test the production of thrombin after incubation with the material for 2, 5, 10, and 20 minutes. Specifically, compared to the control group and CS0 / StSA, the thrombin levels produced by CS10 / StSA, CS25 / StSA, and CS50 / StSA were significantly higher, peaking after 5 minutes and then beginning to decline. Similarly, in the control group, the thrombin content gradually increased within the first 20 minutes, reaching its highest value. Figure 14 Research has found that after reaching its peak, the animal's coagulation system does release substances to lower the levels of coagulation factors and thrombin, creating a negative feedback regulation.

[0054] Therefore, the blood coagulation process involving composite ceramic sponges was summarized. Sponges possess strong water absorption capacity and a macroporous structure, effectively accumulating blood components such as platelets and erythrocytes upon contact with blood. Simultaneously, the CS (calcium carbonate) in the sponge plays a crucial role in accelerating the coagulation process. The negative charge in the sponge activates coagulation factor XII, triggering the intrinsic coagulation pathway within the activation pathway, and the released Ca... 2+ It is a key component in the coagulation process, and it can regulate thrombin activity to form fibrin clots.

[0055] Study on the in vivo hemostatic effect of composite sponge Uncontrollable bleeding due to lack of internal hemostasis is a cause of many trauma-related deaths. In particular, deep and incompressible bleeding from explosions, shootings, traffic accidents, and other incidents is nearly impossible to treat quickly. To treat deep, lethal, massive, and incompressible hemorrhages, researchers have developed various sponges or hydrogels with rapid shape recovery or expansion capabilities. Some studies focus on the material's water absorption and shape recovery capabilities, while others attempt to enhance the material's mechanical properties or coagulant properties through chemical structural design or the addition of active ingredients. In this work, the invention introduces CS (chemically cross-linked components) into a chemically cross-linked framework, which enhances the sponge's mechanical properties while also endowing it with active coagulation capabilities.

[0056] This invention uses an in vivo trauma model in SD rats to evaluate the hemostatic performance of CS0 / StSA and CS25 / StSA compared to commercially available gelatin hemostatic sponges (CS25 / StSA was chosen in this invention considering its physical properties and in vitro coagulation effect). Due to the rich blood supply in the liver, compression should not be used to stop massive hemorrhage, as strong pressure can lead to liver rupture. To simulate incompressible hemorrhage in the liver, this invention creates a standard circular penetrating wound on the liver (…). Figure 15 ).

[0057] like Figure 15 As shown, the hemostasis time and blood loss in a liver hemorrhage model of SD rats were recorded. The results showed that the hemostasis time in the blank control group was 836±35 s, and the blood loss was 4.63±0.3 g. The commercial gelatin group achieved complete hemostasis in 740±17 s, with a blood loss of 3.35±0.37 g. In contrast, the prepared composite sponge exhibited better hemostatic performance. When CS0 / StSA was injected into the penetrating wound, it immediately absorbed blood and restored its original shape to fill the wound due to its superabsorbent and swelling capacity. CS0 / StSA successfully stopped bleeding after 630±30 s of treatment, with a total blood loss of 2.91±0.6 g. Compared with the control group, the blood loss at the bleeding site treated with CS25 / StSA (1.55±0.14 g) was significantly reduced, and the hemostasis time was shortened (485±8.6 s). This result may be due to its active coagulation ability; the sponge can rapidly activate coagulation FXII and initiate the coagulation cascade locally.

[0058] Further evaluation of the various sponges in the femoral artery hemorrhage experiment in SD rats. Surgical procedures and statistical results are as follows: Figure 16 As shown, massive bleeding occurred after arterial transection. In the blank control group, the total blood loss was 11.27 ± 0.98 g. All hemostatic sponges, including the commercial and experimental groups, reduced bleeding time and blood loss. The blood loss for Gel-Sp and CS0 / StSA was 9.2 ± 0.98 g and 6.9 ± 0.84 g, respectively. The prepared sponge CS25 / StSA exhibited strong mechanical properties and improved procoagulant activity, resulting in the shortest bleeding time and the lowest blood loss (5.97 ± 0.20 g), but there was no significant difference between CS0 / StSA and CS25 / StSA sponges.

[0059] Severe trauma often involves a significant risk of bleeding, and timely control of the bleeding source is crucial for a successful outcome. Approximately half of post-traumatic deaths are due to bleeding and coagulation disorders. Trauma-related coagulation disorder (TAC) is an early and major complication in patients with severe trauma. Coagulation disorders can be caused by systemic diseases that lead to a reduction in clotting factors.

[0060] Therefore, this invention establishes hemodiluted SD rat liver volume defect models and hemodiluted rabbit liver volume defect models to simulate coagulation disorders caused by trauma, leading to a decrease in coagulation factors and resulting in bleeding. Figure 17 As shown, in the hemodiluted SD rat liver volume defect model, the hemostasis time in the blank control group was 873±25 s, and the total blood loss was 2.51±0.37 g. All hemostatic sponges, including the commercially available group and the experimental group, reduced bleeding time and blood loss. The bleeding time for the gelatin group, SSt-CS0, and SSt-CS5 was 691±30 s, 595±13 s, and 501±33 s, respectively, and the blood loss was 1.62±0.17 g, 1.42±0.048 g, and 0.71±0.09 g, respectively.

[0061] Then, a blood-dilution rabbit liver volume defect model was used to verify the hemostatic performance of the composite sponge under coagulation disorders. Figure 18 Without any treatment, the injured liver in the control group experienced a blood loss of 4.17±0.27g within 723.33±25s, with continuous bleeding. After treatment of the wound with gelatin, hemostasis was achieved in 626.66±30s, with a blood loss of 2.75±0.12g. SSt-CS0, which has excellent fluid absorption capacity, achieved hemostasis within 506.66±30s, but could not form a stable physical barrier, resulting in 2.3±0.12g of bleeding. However, SSt-CS5, prepared by loading CS in this invention, has strong mechanical properties and improved procoagulant activity, which can significantly shorten the hemostasis time of the wound (380±20s) and reduce the blood loss (1.50±0.43g). The key to SSt-CS5 hemostasis lies in the negatively charged silanol groups on the CS surface, which can interact with the positively charged amino acids on the coagulation factor XII chain, activating the intrinsic coagulation pathway and accelerating the coagulation cascade, thereby enhancing the hemostatic effect. This also explains that under blood dilution, the CS loaded in this invention can achieve better active coagulation ability, which is significantly different from commercially available gelatin sponges and SSt-CS0.

[0062] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0063] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A composite sponge based on calcium silicate bioceramics, characterized in that, The composite sponge is obtained by foaming modified starch, modified anionic polysaccharide-sodium alginate, crosslinking agent HS-PEG-SH, and calcium silicate bioceramic particles CS, followed by crosslinking curing and freeze drying.

2. The composite sponge based on calcium silicate bioceramics according to claim 1, characterized in that, The modified starch mentioned is norbornene-modified starch St-Nor.

3. The composite sponge based on calcium silicate bioceramics according to claim 1, characterized in that, The modified anionic polysaccharide-sodium alginate is norbornene-modified sodium alginate SA-Nor.

4. The composite sponge based on calcium silicate bioceramics according to claim 1, characterized in that, The cross-linking curing is performed using ultraviolet light cross-linking.

5. The composite sponge based on calcium silicate bioceramics according to claim 1, characterized in that, The concentration of calcium silicate bioceramic particles (CS) in the composite sponge is 25% w / v.

6. The composite sponge based on calcium silicate bioceramics according to claim 1, characterized in that, The calcium silicate bioceramic particles CS have a particle size of 1 μm.

7. A method for preparing a composite sponge based on calcium silicate bioceramics as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of norbornene-modified starch (St-Nor): Starch was dispersed in anhydrous DMSO and heated to complete dissolution under an inert atmosphere. The reaction mixture was then cooled to 25 °C. Norbornene anhydride, triethylamine and N,N-dimethylaminopyridine were dissolved in anhydrous DMSO and added dropwise with stirring. The product was then purified by alternating dialysis with NaHCO3 solution and deionized water. After freeze-drying, St-Nor was obtained. (2) Synthesis of norbornene-modified sodium alginate (SA-Nor): Sodium alginate was dissolved in MES buffer and stirred overnight. N-hydroxysuccinimide (NHS) was added to activate the carboxyl group. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to the sodium alginate solution, followed by the slow addition of 5-norbornene-2-methylamine. The coupling reaction was completed by stirring at room temperature. Finally, the solution was dialyzed with deionized water and then freeze-dried to obtain modified sodium alginate. (3) Synthesis of calcium silicate nanoparticles: Hexadecyltrimethylammonium bromide (CTAB) was dissolved in deionized water and stirred continuously until completely dissolved. Ethyl acetate was then added and stirred. Then, ammonia water (NH3·H2O) was added and stirred. Subsequently, tetraethyl orthosilicate (TEOS) and calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] were added sequentially and reacted. The resulting product was washed with ethanol and deionized water multiple times, centrifuged, and dried. The dried powder was heated to 700°C at a heating rate of 2°C / min and held to obtain calcium silicate ceramic particles. (4) Preparation of composite sponge: norbornene-modified starch (St-Nor) and norbornene-modified sodium alginate (SA-Nor) are dissolved in a solution containing... 0.1 wt% of photoinitiator PI 2959 was added to phosphate-buffered saline (PBS), and then calcium silicate nanoparticles were added to the solution. After ensuring complete dissolution, the pH of the mixture was adjusted to neutral. HS-PEG-SH was dissolved in PBS at room temperature and then added to the solution. SDS solution was then added, and the resulting mixture was stirred at 1500 rpm to form a foam solution. The foam solution was transferred to a mold, irradiated with ultraviolet light, and then freeze-dried to obtain a composite sponge based on calcium silicate bioceramics.

8. The preparation method according to claim 7, characterized in that, In step (4), the ultraviolet light intensity is 15 mW / cm² and the irradiation time is 180 seconds.

9. The application of the composite sponge based on calcium silicate bioceramics according to claim 1 in the preparation of hemostatic and coagulant materials.

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