Biomedical material based on egg shells as well as preparation method and application of biomedical material

Abstract

The invention relates to the technical field of biomedical materials, in particular to a biomedical material based on an egg shell and a preparation method and application of the biomedical material based on the egg shell, the biomedical material is crosslinked with genipin through in-situ compounding, a stable chemical bonding network is constructed among chitosan, eggshell membrane protein and crude eggshell powder, and a three-dimensional porous bionic scaffold is formed. According to the invention, efficient synergistic hemostasis is realized, and double mechanisms of physical adsorption (porous structure) and chemical activation (continuous release of Ca < 2 + >) are realized. The scaffold can actively promote healing, the scaffold provides three-dimensional support for cell migration and proliferation, calcium ions released by the scaffold and natural bioactive factors can synergistically regulate and control cell behaviors, granulation growth, angiogenesis and epithelization are accelerated, and the healing period is remarkably shortened. The material has built-in antibiosis and biological safety, the chitosan component endows the material with inherent antibiosis capability, and infection is effectively prevented. Meanwhile, the material has good biocompatibility and controllable degradability and does not need to be taken out for the second time.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a biomedical material based on eggshells, its preparation method, and its application. Background Technology

[0002] Uncontrolled bleeding is a major cause of death from clinical trauma, and developing novel materials that can rapidly stop bleeding and also possess antibacterial properties is crucial to reducing mortality. Existing wound repair materials mainly include passive dressings such as gauze, hydrocolloids, and films that absorb exudate by covering the wound; natural polymers such as collagen, gelatin, and fibrin glue; inorganic materials such as zeolite, montmorillonite, and kaolin; and synthetic polymers such as polyhydroxyalkanoates (PHAs) and polycaprolactone (PCL). However, existing materials still have significant shortcomings in the three core performance aspects: hemostasis speed, healing promotion effect, and antibacterial properties. Most materials rely solely on physical absorption (such as gauze) or chemical activation (such as calcium-based materials), resulting in low hemostatic efficiency. In complex scenarios such as arterial pulsating bleeding or organ wounds, existing materials (such as Arista™) struggle to form stable clots, leading to significant fluctuations in hemostasis time.

[0003] The healing effect is passive and inefficient, lacking the three-dimensional structure of the biomimetic extracellular matrix (ECM), making cell ingrowth difficult; the release of active factors (such as EGF and VEGF) is too rapid and cannot match the healing cycle; in chronic wound models such as diabetes, collagen deposition and angiogenesis are insufficient.

[0004] It is difficult to achieve both antibacterial properties and bioactivity simultaneously, and bacterial biofilms can easily form on the surface of implanted materials, leading to chronic infections. In addition, there are also issues with biocompatibility and biodegradability. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method that offers rapid hemostasis, good healing effects, and excellent antibacterial properties.

[0006] The objective of this invention is achieved through the following technical solution: a method for preparing a biomedical material based on eggshells, comprising the following process steps: Step 1: Pretreatment of raw materials: Collect fresh eggshells, peel off and collect the inner membrane of the eggshells, then grind the eggshells into powder and pass them through a 90-150 mesh sieve to obtain coarse eggshell powder; Step 2: Extraction of eggshell membrane protein SEP: The eggshell inner membrane collected in step 1 was extracted under weakly acidic conditions to obtain eggshell membrane protein powder (SEP). Step 3: Preparation of chitosan-egg shell membrane protein CS-SEP mixed solution: Dissolve 1-3g of chitosan CS in 100 mL of 0.5%-2% acetic acid aqueous solution and stir magnetically until completely clear; add 1g of eggshell membrane protein powder SEP extracted in step 2 and continue stirring for 2-6 hours to ensure thorough mixing and obtain a chitosan-eggshell membrane protein CS-SEP mixed solution; at this point, the positively charged CS and the negatively charged SEP are initially combined through electrostatic interaction. Step 4: Introduce the inorganic phase and form a mold: Slowly add 4-6g of the crude eggshell powder obtained in step 1 to the chitosan-eggshell membrane protein CS-SEP mixed solution obtained in step 3, emulsify and disperse to form a uniform suspension, and inject the suspension into a polytetrafluoroethylene mold. Step 5: Freeze-drying and cross-linking: The polytetrafluoroethylene mold is frozen at -60℃ to -100℃ for 2-4 hours to set its shape, and then freeze-dried to form a three-dimensional porous sponge scaffold. Three-dimensional porous sponge scaffolds were cross-linked in ethanol vapor of genipin for 24-72 hours to obtain biomedical materials based on eggshells. At this time, strong chemical bonds were formed between CS, SEP and coarse eggshell powder, and the network structure was more stable.

[0007] Preferably, the pretreatment of raw materials in step 1 specifically includes: Collect fresh eggshells, rinse them with purified water, and then place them in a vacuum drying oven at 35-39℃ and 4-6KPa for 2-4 hours to dry. After drying, remove the eggshells, peel off and collect the inner membrane of the eggshells. After soaking the eggshells in a 1.5M NaOH solution for 80-100 minutes, wash them with purified water until neutral, dry them at 100-110℃, grind them into powder using a ball mill, and pass them through a 90-150 mesh sieve to obtain coarse eggshell powder. Preferably, the extraction of eggshell membrane protein SEP in step 2 specifically involves: placing the eggshell inner membrane collected in step 1 into a 2-4 mol / L dilute acetic acid solution, stirring at 2-8°C for 24-72 h, centrifuging, collecting the supernatant, adjusting the pH to neutral, and then freeze-drying to obtain eggshell membrane protein powder (SEP). The preparation method of this invention employs a low-temperature, weakly acidic, flexible extraction technique to separate active proteins from the eggshell membrane, avoiding the destruction of biological activity by high temperature, high pressure, or strong chemical solvents.

[0008] Preferably, in step 2, the solution is adjusted to neutral using a 0.5 mol / L NaOH solution.

[0009] Preferably, in step 5, the freeze-drying is carried out by a staged heating method.

[0010] Preferably, the 0.5%-2% acetic acid aqueous solution in step 3 refers to the mass-volume ratio (m / V), meaning that each 100 mL of water contains 0.5-2 g of acetic acid.

[0011] Preferably, in step 6, rinsing is performed using 0.01 M phosphate-buffered saline (PBS) at pH 7.4.

[0012] Preferably, the freeze-drying temperature in steps 2 and 6 is the same as the temperature in step 5.

[0013] Preferably, the preparation method of this eggshell-based biomedical material further includes step 6: post-processing and sterilization: the obtained eggshell-based biomedical material is washed with sterile PBS buffer, freeze-dried again, and finally terminally sterilized by Co-60 γ-ray irradiation.

[0014] A biomedical material based on eggshells is prepared from the following raw materials: chitosan, eggshell membrane protein extract, and crude eggshell powder.

[0015] Application of an eggshell-based biomedical material in wound dressings or tissue engineering scaffolds.

[0016] This invention utilizes not only the inorganic components (calcium carbonate) in eggshells, but also the organic components of the eggshell membrane (rich in collagen, elastin, glycosaminoglycans, growth factors, etc.), thus realizing the utilization of all components of eggshells and greatly enhancing resource value.

[0017] The basic principle of the raw materials of this invention: Chemical composition: The main component of eggshell is calcium carbonate (CaCO3, accounting for about 94%), and it contains about 4% organic matrix (mainly including proteins, such as collagen X, osteopontin, growth factors, etc.). Calcium carbonate is a good hemostatic agent and can activate the coagulation pathway; while the proteins and growth factors in the organic matrix have excellent biological activity and the ability to promote cell growth.

[0018] Microstructure: Eggshells have a natural porous micro / nano structure, which gives them a huge specific surface area, enabling them to efficiently absorb water from the blood, concentrate platelets and clotting factors, and thus physically accelerate the clotting process.

[0019] The hemostatic principle of this invention: Physical adsorption and concentration effect: The porous structure of the material rapidly absorbs water from the blood, causing a sharp increase in the concentration of platelets and coagulation factors, thus accelerating the coagulation cascade reaction.

[0020] Chemical activation: Calcium ions (CaO) provided by the surface of calcium carbonate (CaCO3) 2+Factor IV is an indispensable cofactor in both intrinsic and common coagulation pathways. It can significantly activate prothrombin, thereby efficiently catalyzing the formation of fibrinogen into a fibrin network for strong hemostasis.

[0021] The principle of promoting healing in this invention: Provides a healing microenvironment: The material acts as a temporary scaffold, providing three-dimensional support for the migration and proliferation of fibroblasts, vascular endothelial cells, and other cells.

[0022] Release of bioactive factors: Bioactive molecules such as proteins and polysaccharides retained in the material or released from the organic matrix of eggshells can regulate cell behavior and promote cell adhesion, proliferation and differentiation.

[0023] Release of beneficial ions: The material slowly degrades in the wound microenvironment (weakly acidic), continuously releasing calcium ions (Ca). 2 + Calcium ions are not only clotting factors, but also key messengers in cell signaling. The bioactive factors they carry can actively regulate cell behavior, accelerate granulation tissue growth, angiogenesis and epithelialization, and play an important role in multiple stages of wound healing (such as cell migration, proliferation and skin barrier reconstruction).

[0024] The antibacterial principle of this invention is that by loading antibacterial drugs or utilizing the inherent properties of the material (such as chitosan components), it has a certain ability to prevent wound infection.

[0025] The beneficial effects of this invention: This invention constructs a stable chemically bonded network between chitosan (CS), eggshell membrane protein (SEP), and crude eggshell powder through in-situ composite and genipin crosslinking, forming a three-dimensional porous biomimetic scaffold. This structure not only simulates the physical characteristics of the extracellular matrix, but also effectively enhances the interfacial bonding force through chemical crosslinking between components, overcoming the defects of easy phase separation in simple blending, thereby improving the structural stability and mechanical properties of the material. This invention provides highly efficient and synergistic hemostasis, combining physical adsorption (porous structure) and chemical activation (continuous release of Ca). 2+ This invention employs a dual mechanism, resulting in significantly faster hemostasis compared to traditional materials. It actively promotes healing; the scaffold not only provides three-dimensional support for cell migration and proliferation, but its released calcium ions and natural bioactive factors synergistically regulate cell behavior, accelerating granulation tissue growth, angiogenesis, and epithelialization, thus significantly shortening the healing cycle. Furthermore, this invention possesses built-in antibacterial and biosafety properties; the chitosan component endows the material with inherent antibacterial capabilities, effectively preventing infection. Simultaneously, the material exhibits excellent biocompatibility and controllable degradation, eliminating the need for secondary removal. Attached Figure Description

[0026] Figure 1The following is a diagram showing the changes in the bleeding site and surrounding tissues when the biomedical material based on eggshells and the Arista™ hemostatic material were used in a liver bleeding model. Figure 2 The following is a diagram showing the changes in the bleeding site and surrounding tissues when the biomedical material based on eggshells and the Arista™ hemostatic material were used in a kidney bleeding model. Detailed Implementation

[0027] The present invention will be further described in conjunction with the following embodiments.

[0028] Example 1 A method for preparing a biomedical material based on eggshells, comprising the following process steps: Step 1: Pretreatment of raw materials: Collect fresh eggshells, peel off and collect the inner membrane of the eggshells, then grind the eggshells into powder and pass them through a 90-mesh sieve to obtain coarse eggshell powder. Step 2: Extraction of eggshell membrane protein SEP: The eggshell inner membrane collected in step 1 was extracted under weakly acidic conditions to obtain eggshell membrane protein powder (SEP). Step 3: Preparation of chitosan-egg shell membrane protein CS-SEP mixed solution: Dissolve 1g of chitosan CS in 100 mL of 0.5% acetic acid aqueous solution and stir magnetically until completely clear; add 1g of eggshell membrane protein powder SEP extracted in step 2 and continue stirring for 2 hours to mix thoroughly and evenly to obtain a chitosan-eggshell membrane protein CS-SEP mixed solution. Step 4: Introduce the inorganic phase and form a mold: 4g of the coarse eggshell powder obtained in step 1 is slowly added to the chitosan-eggshell membrane protein CS-SEP mixed solution obtained in step 3, and the mixture is emulsified and dispersed to form a uniform suspension. The suspension is then injected into a polytetrafluoroethylene mold. Step 5: Freeze-drying and cross-linking: The polytetrafluoroethylene mold was frozen at -60℃ for 4 hours to set its shape, and then freeze-dried to form a three-dimensional porous sponge scaffold. A three-dimensional porous sponge scaffold was placed in a sealed container containing a 1% (w / v) genipin ethanol solution and crosslinked at 38°C for 24 hours to obtain a biomedical material based on eggshell.

[0029] Specifically, the pretreatment of raw materials in step 1 includes: Collect fresh eggshells, rinse them with purified water, and then place them in a vacuum drying oven at 35°C and 4 kPa for 4 hours to dry. After drying, remove the eggshells, peel off and collect the inner membrane of the eggshells. After soaking the eggshells in a 1M NaOH solution for 80 minutes, they were washed with purified water until neutral, dried at 100°C, ground and pulverized using a ball mill, and passed through a 90-mesh sieve to obtain coarse eggshell powder. Specifically, the extraction of eggshell membrane protein SEP in step 2 is as follows: the eggshell inner membrane collected in step 1 is placed in a 2 mol / L dilute acetic acid solution, stirred at 2℃ for 72 h, centrifuged, the supernatant is collected, the pH is adjusted to neutral, and then freeze-dried to obtain eggshell membrane protein powder SEP.

[0030] In step 2, the solution is adjusted to neutral using a 0.5 mol / L NaOH solution.

[0031] The specific freeze-drying temperature in step 5 is as follows: Sublimation / desorption stage 1: Temperature -20℃, Time 1200min; Sublimation / desorption stage 2: temperature 0℃, time 600min; Sublimation / desorption stage 3: temperature 10℃, time 300min; Sublimation / desorption stage 4: temperature 20℃, time 300min; Sublimation / desorption stage 5: temperature 25℃, time 990min.

[0032] In step 3, the 0.5% acetic acid aqueous solution refers to the mass-to-volume ratio (m / V), which means that 0.5g of acetic acid is contained in every 100mL of water.

[0033] In step 6, 0.01 M phosphate-buffered saline (PBS) at pH 7.4 is used for rinsing.

[0034] The freeze-drying temperature in step 6 is the same as that in step 5.

[0035] The preparation method of this eggshell-based biomedical material also includes step 6: post-processing and sterilization: the obtained eggshell-based biomedical material is washed with sterile PBS buffer, freeze-dried again, and finally terminally sterilized by Co-60 γ-ray irradiation.

[0036] A biomedical material based on eggshells is prepared from the following raw materials: chitosan, eggshell membrane protein extract, and crude eggshell powder.

[0037] Application of an eggshell-based biomedical material in wound dressings or tissue engineering scaffolds.

[0038] Example 2 A method for preparing a biomedical material based on eggshells, comprising the following process steps: Step 1: Pretreatment of raw materials: Collect fresh eggshells, peel off and collect the inner membrane of the eggshells, then grind the eggshells into powder and pass them through a 100-mesh sieve to obtain coarse eggshell powder; Step 2: Extraction of eggshell membrane protein SEP: The eggshell inner membrane collected in step 1 was extracted under weakly acidic conditions to obtain eggshell membrane protein powder (SEP). Step 3: Preparation of chitosan-egg shell membrane protein CS-SEP mixed solution: Dissolve 2g of chitosan CS in 100 mL of 1% acetic acid aqueous solution and stir magnetically until completely clear; add 1g of eggshell membrane protein powder SEP extracted in step 2 and continue stirring for 4 hours to mix thoroughly and evenly to obtain a chitosan-eggshell membrane protein CS-SEP mixed solution. Step 4: Introduce the inorganic phase and form a mold: Slowly add 5g of the coarse eggshell powder obtained in step 1 to the chitosan-eggshell membrane protein CS-SEP mixed solution obtained in step 3, emulsify and disperse to form a uniform suspension, and inject the suspension into a polytetrafluoroethylene mold. Step 5: Freeze-drying and cross-linking: The polytetrafluoroethylene mold was frozen at -80℃ for 3 hours to set its shape, and then freeze-dried to form a three-dimensional porous sponge scaffold. A three-dimensional porous sponge scaffold was placed in a sealed container containing a 1% (w / v) genipin ethanol solution and crosslinked at 37°C for 48 hours to obtain a biomedical material based on eggshell.

[0039] Specifically, the pretreatment of raw materials in step 1 includes: Collect fresh eggshells, rinse them with purified water, and then place them in a vacuum drying oven at 36°C and 5 kPa for 3 hours to dry. After drying, remove the eggshells, peel off and collect the inner membrane of the eggshells. After soaking the eggshells in a 3M NaOH solution for 90 minutes, they were washed with purified water until neutral, dried at 105°C, ground and pulverized using a ball mill, and passed through a 100-mesh sieve to obtain coarse eggshell powder. Specifically, the extraction of eggshell membrane protein SEP in step 2 is as follows: the eggshell inner membrane collected in step 1 is placed in a 3 mol / L dilute acetic acid solution, stirred at 4℃ for 48 h, centrifuged, the supernatant is collected, the pH is adjusted to neutral, and then freeze-dried to obtain eggshell membrane protein powder SEP.

[0040] In step 2, the solution is adjusted to neutral using a 0.5 mol / L NaOH solution.

[0041] The specific freeze-drying temperature in step 5 is as follows: Sublimation / desorption stage 1: Temperature -20℃, Time 1200min; Sublimation / desorption stage 2: temperature 0℃, time 600min; Sublimation / desorption stage 3: temperature 10℃, time 300min; Sublimation / desorption stage 4: temperature 20℃, time 300min; Sublimation / desorption stage 5: temperature 25℃, time 990min.

[0042] In step 6, 0.01 M phosphate-buffered saline (PBS) at pH 7.4 is used for rinsing.

[0043] The freeze-drying temperature in step 6 is the same as that in step 5.

[0044] The preparation method of this eggshell-based biomedical material also includes step 6: post-processing and sterilization: the obtained eggshell-based biomedical material is washed with sterile PBS buffer, freeze-dried again, and finally terminally sterilized by Co-60 γ-ray irradiation.

[0045] A biomedical material based on eggshells is prepared from the following raw materials: chitosan, eggshell membrane protein extract, and crude eggshell powder.

[0046] Application of an eggshell-based biomedical material in wound dressings or tissue engineering scaffolds.

[0047] Example 3 A method for preparing a biomedical material based on eggshells, comprising the following process steps: Step 1: Pretreatment of raw materials: Collect fresh eggshells, peel off and collect the inner membrane of the eggshells, then grind the eggshells into powder and pass them through a 150-mesh sieve to obtain coarse eggshell powder. Step 2: Extraction of eggshell membrane protein SEP: The eggshell inner membrane collected in step 1 was extracted under weakly acidic conditions to obtain eggshell membrane protein powder (SEP). Step 3: Preparation of chitosan-egg shell membrane protein CS-SEP mixed solution: Dissolve 1-3g of chitosan CS in 100 mL of 2% acetic acid aqueous solution and stir magnetically until completely clear; add 1g of eggshell membrane protein powder SEP extracted in step 2 and continue stirring for 6 hours to mix thoroughly and evenly to obtain a chitosan-eggshell membrane protein CS-SEP mixed solution. Step 4: Introduce the inorganic phase and form a mold: Slowly add 6g of the crude eggshell powder obtained in step 1 to the chitosan-eggshell membrane protein CS-SEP mixed solution obtained in step 3, emulsify and disperse to form a uniform suspension, and inject the suspension into a polytetrafluoroethylene mold. Step 5: Freeze-drying and cross-linking: The polytetrafluoroethylene mold was frozen at -100℃ for 2 hours to set its shape, and then freeze-dried to form a three-dimensional porous sponge scaffold. A three-dimensional porous sponge scaffold was placed in a sealed container containing a 1% (w / v) genipin ethanol solution and crosslinked at 36°C for 72 h to obtain a biomedical material based on eggshell.

[0048] Specifically, the pretreatment of raw materials in step 1 includes: Collect fresh eggshells, rinse them with purified water, and then place them in a vacuum drying oven at 39°C and 6 kPa for 4 hours to dry. After drying, remove the eggshells, peel off and collect the inner membrane of the eggshells. After soaking the eggshells in a 1.5M NaOH solution for 100 minutes, they were washed with purified water until neutral, dried at 110°C, ground and pulverized using a ball mill, and passed through a 150-mesh sieve to obtain coarse eggshell powder. Specifically, the extraction of eggshell membrane protein SEP in step 2 is as follows: the eggshell inner membrane collected in step 1 is placed in a 4 mol / L dilute acetic acid solution, stirred at 8°C for 24 h, centrifuged, the supernatant is collected, the pH is adjusted to neutral, and then freeze-dried to obtain eggshell membrane protein powder SEP.

[0049] In step 2, the solution is adjusted to neutral using a 0.5 mol / L NaOH solution.

[0050] The specific freeze-drying temperature in step 5 is as follows: Sublimation / desorption stage 1: Temperature -20℃, Time 1200min; Sublimation / desorption stage 2: temperature 0℃, time 600min; Sublimation / desorption stage 3: temperature 10℃, time 300min; Sublimation / desorption stage 4: temperature 20℃, time 300min; Sublimation / desorption stage 5: temperature 25℃, time 990min.

[0051] In step 6, 0.01 M phosphate-buffered saline (PBS) at pH 7.4 is used for rinsing.

[0052] The freeze-drying temperature in step 6 is the same as that in step 5.

[0053] The preparation method of this eggshell-based biomedical material also includes step 6: post-processing and sterilization: the obtained eggshell-based biomedical material is washed with sterile PBS buffer, freeze-dried again, and finally terminally sterilized by Co-60 γ-ray irradiation.

[0054] A biomedical material based on eggshells is prepared from the following raw materials: chitosan, eggshell membrane protein extract, and crude eggshell powder.

[0055] Application of an eggshell-based biomedical material in wound dressings or tissue engineering scaffolds.

[0056] Experimental data

[0057] 2.1 Experimental bleeding model 2.1.1 Liver hemorrhage model Rabbits were anesthetized via the marginal ear vein with 3% sodium pentobarbital injection (30-40 mg / kg). After fixation on the experimental table, the abdominal hair was shaving, and the abdomen was disinfected with 75% ethanol and draped. A 5 cm incision was made along the midline of the abdomen below the xiphoid process, and the abdomen was opened layer by layer to expose the liver. A 1 cm long and 0.5 cm deep incision was made in the middle lobe of the liver with a scalpel. Rapid blood seepage from the wound surface and active bleeding were considered a successful model. The blood from the incision site was quickly absorbed with pre-weighed dry gauze. According to the group, the most obvious bleeding site was immediately covered with eggshell-based biomedical material for hemostasis, and the timing was started. The gauze was applied to the wound for 30 seconds, and the coagulation process of the hemostatic material was observed. If no bright red blood seeped out within 2 minutes, hemostasis was considered successful. Otherwise, the blood clot and hemostatic material were removed from the wound, the medication was reapplied, and gauze was applied for 30 seconds. If hemostasis still could not be achieved, it was considered unsuccessful. The amount of bleeding and the time of hemostasis were recorded, and the adhesion between the hemostatic material and the wound was observed.

[0058] 2.1.2 Renal hemorrhage model The anesthesia method was the same as above. After the anesthetized animal was fixed on the experimental table, the abdomen was opened layer by layer to expose one kidney. A longitudinal incision of about 1 cm long and 0.5 cm deep was made on the kidney with a scalpel. Blood quickly seeped out of the wound. If the bleeding was active, it was considered a successful model. After quickly absorbing the blood from the incision site with a pre-weighed dry gauze, the application of the eggshell-based biomedical material was the same as in the liver hemorrhage model. The amount of bleeding and the time to hemostasis were recorded in the same way as above, and the adhesion between the eggshell-based biomedical material and the wound was observed.

[0059] 2.1.3 Results In liver and kidney bleeding models, after surgical incisions were made on the surface of normal organs, blood gushed out rapidly. After the blood was absorbed with dry gauze, a biomedical material based on eggshells was quickly covered on the incision. The dry gauze was applied for 30 seconds and then removed. The rapidly accumulated blood formed a gel-like protective film on the wound surface, with no oozing, and the bleeding stopped. In the control group, after applying Arista™ hemostatic material, no gel-like protective film was formed, but there was also no obvious oozing, and the bleeding stopped. In the femoral artery bleeding model, after the femoral artery was freed and incised, blood gushed out rapidly. After wiping with dry gauze, eggshell-based biomedical material was quickly sprinkled onto the incision. The eggshell-based biomedical material in the bleeding area immediately turned from white to red. After the dry gauze was applied for 30 seconds, it was removed, and there was obvious blood seepage. After removing the blood clot and the eggshell-based biomedical material from the wound, the eggshell-based biomedical material was reapplied and gauze was applied for 30 seconds. There was no obvious blood seepage, and hemostasis was successful. In the control group, hemostasis using Arista™ hemostatic material failed.

[0060] 2.2 Observation of bleeding indicators

[0061] 2.2.1 Bleeding volume: The gauze was weighed precisely before and after hemostasis using an electronic balance, and the bleeding volume was calculated (bleeding volume = gauze mass after hemostasis - gauze mass before hemostasis).

[0062] 2.2.2 Hemostasis time was recorded using an electronic precision stopwatch timer, from the time the eggshell-based biomedical material was first applied to the wound until the bleeding from the wound stopped or the area of ​​red staining by the eggshell-based biomedical material no longer expanded.

[0063] 2.2.3 Results - Bleeding volume and time: The bleeding volume and hemostasis time of the liver and kidney bleeding models are shown in Table 1. The experimental group based on eggshell biomedical materials and Arista... TM There was no statistically significant difference between the hemostatic powder and control groups (P>0.05), which suggests that the hemostatic powder-based biomedical material and Arista... TM Hemostatic powder has the same effect on stopping bleeding from kidney and liver wounds.

[0064]

[0065] Note: n=5, data is based on... (mean ± standard deviation) is expressed as a denoted value.

[0066] 2.3 Histological Observation: Tissue surrounding the wound in successfully hemostatic bleeding models was stained with Hematoxylin and eosin (H&E) to observe structural changes. Normal organ tissue from each bleeding model served as a control. All specimens were prepared into histological sections using standard procedures, stained with H&E, and then observed by a single-blind pathologist to assess histological changes.

[0067] Tissue section examination of liver and kidney tissues from the eggshell-based biomedical material experimental group and the Arista™ hemostatic material control group, performed using H-E staining, revealed a large accumulation of erythrocytes in the wound and surrounding tissues, forming an effective hemostatic plug. Simultaneously, the wound and surrounding tissues remained morphologically intact, without obvious burns, necrosis, or abnormal inflammatory cell infiltration. Figure 1 and Figure 2 This indicates that the material of the present invention achieves rapid hemostasis without irritating or causing thermal damage to surrounding tissues, and has excellent biocompatibility.

[0068] 2.4 Statistical Analysis: SPSS 13.0 statistical software was used for analysis and processing. Experimental data are presented as follows: The t-test was used for comparisons between groups, with a significance level (α) of 0.05.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a biomaterial based on chicken eggshells, characterized by: It comprises the following process steps: Step 1: Pretreatment of raw materials: Collect fresh eggshells, peel and collect the inner membrane of the eggshell, and then grind the eggshell into powder, pass through a 90-150 mesh sieve to obtain coarse eggshell powder; Step 2: Extraction of eggshell membrane protein SEP: The inner membrane of the eggshell collected in step 1 is extracted under weak acid conditions to obtain eggshell membrane protein powder SEP; Step 3: Preparation of chitosan-eggshell membrane protein CS-SEP mixed solution: Dissolve 1-3 g of chitosan CS in 100 mL of 0.5%-2% acetic acid aqueous solution, and magnetically stir until completely clear; add 1 g of eggshell membrane protein powder SEP extracted in step 2, continue to stir for 2-6 hours to make it fully mixed and uniform, to obtain a chitosan-eggshell membrane protein CS-SEP mixed solution; Step 4: Introduction of inorganic phase and molding: Slowly add 4-6 g of coarse eggshell powder obtained in step 1 to the chitosan-eggshell membrane protein CS-SEP mixed solution obtained in step 3 to form a uniform suspension, and inject the suspension into a polytetrafluoroethylene mold; Step 5: Freeze-drying and crosslinking: Place the polytetrafluoroethylene mold in a freezer at -60℃ to -100℃ for 2-4 hours to shape, and then perform freeze-drying to form a three-dimensional porous sponge scaffold; Place the three-dimensional porous sponge scaffold in a ginpin ethanol vapor for crosslinking for 24-72 hours to obtain a chicken eggshell-based biomedical material.

2. The method for preparing a biomedical material based on eggshells according to claim 1, characterized in that: The pretreatment of raw materials in step 1 is specifically as follows: Collect fresh eggshells, rinse with purified water, and then place them in a vacuum drying oven at 35-39℃ under a vacuum environment of 4-6 KPa for 2-4 hours; after drying, remove them, peel and collect the inner membrane of the eggshell; Soak the eggshell in a 1-3 M NaOH solution for 80-100 minutes, then wash it with purified water until it is neutral, dry it at 100-110℃, and then grind it with a ball mill, pass it through a 90-150 mesh sieve to obtain coarse eggshell powder.

3. The method for preparing a biomedical material based on eggshells according to claim 1, characterized in that: The extraction of eggshell membrane protein SEP in step 2 is specifically as follows: Place the inner membrane of the eggshell collected in step 1 in a 2-4 mol / L dilute acetic acid solution, stir at 2-8℃ for 24-72 hours, centrifuge, take the supernatant, adjust the pH to neutral, and then freeze-dry to obtain eggshell membrane protein powder SEP.

4. The method for preparing a biomedical material based on eggshells according to claim 3, characterized in that: In step 2, adjust to neutral with 0.5 mol / L NaOH solution.

5. The method for preparing a biomedical material based on eggshells according to claim 1, characterized in that: The freeze-drying in step 5 is performed in a staged heating manner.

6. The method for preparing a biomedical material based on eggshells according to claim 1, characterized in that: The preparation method of the chicken eggshell-based biomedical material further comprises step 6: post-treatment and sterilization: rinse the obtained chicken eggshell-based biomedical material with sterile PBS buffer, freeze-dry again, and finally perform terminal sterilization using Co-60 γ-ray irradiation.

7. A biomaterial based on chicken eggshell characterized in that, It is prepared from the following raw materials: chitosan, eggshell membrane protein extract, and coarse eggshell powder.

8. Application of a chicken eggshell-based biomedical material in wound dressings or tissue engineering scaffolds.

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