A nano-hydroxyapatite composite hydrogel, its preparation method and application

Abstract

The present invention discloses a nano-hydroxyapatite composite hydrogel, its preparation method and application, belonging to the field of advanced biomaterials. The present invention designs a dynamic Schiff base reaction to combine carboxymethyl chitosan, oxidized dextran and nano-hydroxyapatite to prepare a multifunctional hydrogel that takes into account the advantages of all three. This composite hydrogel has excellent biocompatibility, injectability, self-healing property, adhesiveness and antibacterial property, can regulate the cell microenvironment, stop bleeding locally, and then repair damaged periodontal tissues, showing great potential in the treatment of periodontitis and the preparation of hemostatic materials.

Description

Technical Field

[0001] The present invention belongs to the field of advanced biomaterials, and particularly relates to a nano-hydroxyapatite composite hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Hydroxyapatite has good biocompatibility and osteoconductivity, and is the main inorganic component of human and animal bones and teeth. It can tightly bind to human soft and hard tissues in a short time, release harmless ions, participate in human metabolism, stimulate or induce bone hyperplasia, promote the repair of defective tissues, and has been widely used in the repair of human bone tissues. However, as a sintered material, hydroxyapatite has poor brittleness and fatigue resistance, which seriously hinders its clinical application. Selecting a suitable organic matrix material to tightly combine with hydroxyapatite to prepare a composite biomimetic material with good bioactivity, mechanical properties and degradation properties has gradually become the focus of current research.

[0003] However, in the preparation process of traditional biomimetic bone repair materials, it is very difficult to select a suitable organic matrix material due to the defects in the performance of the organic matrix material. For example, high-density polyethylene does not have biodegradability; collagen fibers have poor mechanical properties, fast degradation rate and low mechanical strength; the composite process of starch-based composites is relatively difficult; polylactic acid (PLA) degrades rapidly in the human body, resulting in rapid loss of mechanical properties. At the same time, many organic matrix materials may show aseptic inflammatory reactions and osteolytic changes at the implantation site. Therefore, it is necessary to find a suitable organic matrix material to combine with hydroxyapatite.

[0004] Hydrogel materials have a high water content, good biocompatibility and appropriate physical properties, and can provide the best biological environment for cell adhesion, proliferation and differentiation. Natural polysaccharide hydrogels, with good biocompatibility, biodegradability, biosafety, antibacterial activity, anti-inflammatory activity and other properties, are widely used as matrix materials for bone tissue regeneration scaffolds. However, these hydrogels often show obvious deficiencies in osteogenesis, angiogenesis and mechanical properties, seriously limiting their practical clinical applications. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a nano-hydroxyapatite composite hydrogel, a preparation method thereof, and an application thereof. The present invention designs a dynamic Schiff base reaction to combine carboxymethyl chitosan, oxidized dextran and nano-hydroxyapatite to prepare a multifunctional hydrogel that takes into account the advantages of the three. The composite hydrogel has excellent biocompatibility, injectability, self-repairability, adhesiveness and antibacterial properties, can regulate the cell microenvironment, stop bleeding locally, and then repair damaged periodontal tissues, showing great potential in the treatment of periodontitis and other aspects.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] On the one hand, a nano-hydroxyapatite composite hydrogel is formed by crosslinking a polysaccharide and nanoparticles in an aqueous medium;

[0008] The nanoparticles include nano-hydroxyapatite or particles containing nano-hydroxyapatite;

[0009] The polysaccharide includes two of dextran, carboxymethyl chitosan, chitosan, hyaluronic acid, and hydroxyethyl chitosan.

[0010] On the other hand, a method for preparing the above nano-hydroxyapatite composite hydrogel includes the following steps:

[0011] Dissolve a polysaccharide in an aqueous medium to obtain solution A;

[0012] Add the nanoparticles to solution A to obtain solution B;

[0013] Dissolve another polysaccharide in an aqueous medium to obtain solution C;

[0014] Mix solution B and solution C to obtain the nano-hydroxyapatite composite hydrogel.

[0015] In the third aspect, the above nano-hydroxyapatite composite hydrogel is used in the preparation of anti-inflammatory drugs and hemostatic materials. Preferably, the drugs include those for treating periodontitis, gingivitis, and oral ulcers.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention designs a dynamic Schiff base reaction to combine carboxymethyl chitosan, oxidized dextran, and nano-hydroxyapatite to prepare a multifunctional hydrogel that takes into account the advantages of all three. This composite hydrogel has excellent biocompatibility, injectability, self-repairability, adhesiveness, and antibacterial properties, can regulate the cell microenvironment, stop local bleeding, and then repair damaged periodontal tissues, showing great potential in the treatment of periodontitis and other aspects.

[0018] In the present invention, a certain amount of nano-hydroxyapatite is added to a hydrogel composed of oxidized dextran and carboxymethyl chitosan to synthesize a novel composite hydrogel that exhibits more excellent adhesion, hemostatic, and antibacterial properties. Through SEM detection, the hydrogel has a porous structure. Even under the action of rapid water flow impact in different directions, the nano-hydroxyapatite composite hydrogel still adheres strongly to pigskin. Through the self-assembly test of the nano-hydroxyapatite composite hydrogel, at 37 °C, two halves of the composite hydrogel of different colors can be firmly combined together. The blood compatibility of the composite hydrogel is also good and does not cause damage to red blood cells. A hemostatic test was carried out on the livers of mice with the composite hydrogel. The composite hydrogel was injected into the wound with a syringe, and rapid hemostasis could be achieved after 10 seconds, indicating its excellent hemostatic performance. The composite hydrogel has good antibacterial effects against Staphylococcus aureus and Escherichia coli, especially the antibacterial effect against Escherichia coli reaches more than 90%. Therefore, the nano-hydroxyapatite composite hydrogel has good application prospects in hemostasis and antibacterial aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 It is a process diagram of the nano-hydroxyapatite composite hydrogel prepared in Example 1 of the present invention;

[0021] Figure 2 It is the SEM image and pore size distribution diagram of the composite hydrogel without nano-hydroxyapatite prepared in Comparative Example 1 of the present invention;

[0022] Figure 3 It is the SEM image and pore size distribution diagram of the nano-hydroxyapatite composite hydrogel prepared in Example 1 of the present invention;

[0023] Figure 4 It is a data graph of the swelling ratio of the hydrogels obtained in Example 1 and Comparative Example 1 in PBS (pH = 7) measured by the swelling test;

[0024] Figure 5 It is the infrared absorption spectra of nano-hydroxyapatite, the composite hydrogel without nano-hydroxyapatite, and the nano-hydroxyapatite composite hydrogel;

[0025] Figure 6This is the adhesion detection diagram of the nano-hydroxyapatite composite hydrogel in Experimental Example 2 of the present invention. Among them, A - C are pictures of the stained nano-hydroxyapatite composite hydrogel on the surface of pigskin at different twisting angles, and D - F are photos of the stained nano-hydroxyapatite composite hydrogel adhering to the surface of pigskin under the impact of rapid water flow at different angles;

[0026] Figure 7 This is the self-healing performance detection diagram of the nano-hydroxyapatite composite hydrogel in Experimental Example 3 of the present invention. Among them, A is the nano-hydroxyapatite composite hydrogel stained with green dye, B is the nano-hydroxyapatite composite hydrogel stained with red dye, and (C) is the self-healing diagram of the nano-hydroxyapatite composite hydrogel with the green and red halves combined;

[0027] Figure 8 This is the picture of the hemolysis situation of each group in the hemolysis performance test of the nano-hydroxyapatite composite hydrogel in Experimental Example 3 of the present invention;

[0028] Figure 9 This is the bar chart of the hemolysis rate of each group in the hemolysis performance test of the nano-hydroxyapatite composite hydrogel in Experimental Example 3 of the present invention;

[0029] Figure 10 This is the SEM diagram of red blood cells on the surface of the composite hydrogel in the adhesion detection of red blood cells by the nano-hydroxyapatite composite hydrogel in Experimental Example 4 of the present invention. Among them, A is the adhesion of red blood cells on the surface of the composite hydrogel without nano-hydroxyapatite at a magnification of 2000 times; B is the adhesion of red blood cells on the surface of the nano-hydroxyapatite composite hydrogel at a magnification of 2000 times; C is the adhesion of red blood cells on the surface of the composite hydrogel without nano-hydroxyapatite at a magnification of 5000 times; D is the adhesion of red blood cells on the surface of the nano-hydroxyapatite composite hydrogel at a magnification of 5000 times;

[0030] Figure 11 This is the picture taken at a specific time of the hemostasis of different composite hydrogels and the blank control group in the liver of mice in the hemostasis performance detection of the nano-hydroxyapatite composite hydrogel in Experimental Example 5 of the present invention;

[0031] Figure 12 This is the hemostasis time diagram of the liver of mice with different composite hydrogels and the blank control group in the hemostasis performance detection of the nano-hydroxyapatite composite hydrogel in Experimental Example 5 of the present invention;

[0032] Figure 13 This is the blood loss mass diagram of the liver of mice with different composite hydrogels and the blank control group in the hemostasis performance detection of the nano-hydroxyapatite composite hydrogel in Experimental Example 5 of the present invention;

[0033] Figure 14This is the evaluation diagram of the antibacterial activity in the in vitro antibacterial test of the nano-hydroxyapatite composite hydrogel in Experimental Example 6 of the present invention. Among them, A is the diagram of the living bacteria colonies of Escherichia coli on the agar plate after co-culturing with the hydrogel; B is the diagram of the living bacteria colonies of Staphylococcus aureus on the agar plate after co-culturing with the hydrogel.

[0034] Figure 15 This is the average number of colonies on the agar plate after the composite hydrogels in each group are co-cultured with Staphylococcus aureus and Escherichia coli in the in vitro antibacterial test of the nano-hydroxyapatite composite hydrogel in Experimental Example 6 of the present invention. Detailed implementation mode

[0035] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing the specific implementation mode and are not intended to limit the exemplary implementation mode according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] In view of the fact that the brittleness and poor fatigue resistance of existing hydroxyapatite hinder its application in clinical practice, it is very necessary to find a suitable organic matrix material to combine with it to improve its performance and expand its application; the deficiencies of hydrogels in osteogenesis, angiogenesis and mechanical properties also seriously limit their practical application in clinical practice. The present invention proposes a nano-hydroxyapatite composite hydrogel and its preparation method and application.

[0038] A typical implementation mode of the present invention provides a nano-hydroxyapatite composite hydrogel, which is cross-linked by polysaccharides and nanoparticles in an aqueous medium;

[0039] The nanoparticles include nano-hydroxyapatite or particles containing nano-hydroxyapatite;

[0040] The polysaccharides include two of dextran, carboxymethyl chitosan, chitosan, hyaluronic acid and hydroxyethyl chitosan.

[0041] In some embodiments of this implementation mode, the polysaccharides are dextran and carboxymethyl chitosan.

[0042] In some embodiments of this implementation mode, the molecular weight of the dextran is 10 - 200 kDa, preferably 20 - 150 kDa.

[0043] In some embodiments of this embodiment, the mass concentration of the nanoparticles is 5-15 wt%, preferably 10 wt%.

[0044] Another typical embodiment of the present invention provides a method for preparing the above-mentioned nano-hydroxyapatite composite hydrogel, comprising the following steps:

[0045] Dissolve a polysaccharide in an aqueous medium to obtain solution A;

[0046] Add the nanoparticles to solution A to obtain solution B;

[0047] Dissolve another polysaccharide in an aqueous medium to obtain solution C;

[0048] Mix solution B and solution C to obtain the nano-hydroxyapatite composite hydrogel.

[0049] In some embodiments of this embodiment, the specific preparation method of solution A is: dissolve dextran and sodium periodate in water and stir to synthesize an oxidized dextran solution.

[0050] Preferably, the molar ratio of dextran to sodium periodate is 1-3:1, and further preferably 2:1.

[0051] In some embodiments of this embodiment, the specific preparation method of solution B is: add the nanoparticles to 10 wt% solution A and stir evenly.

[0052] Preferably, the ratio of the nanoparticles to 10 wt% solution A is 1:5-20, and further preferably 1:5.

[0053] In some embodiments of this embodiment, the specific preparation method of solution C is: dissolve carboxymethyl chitosan in double-distilled water to obtain an aqueous carboxymethyl chitosan solution. Preferably, the concentration of the aqueous carboxymethyl chitosan solution is 10 wt%.

[0054] In some embodiments of this embodiment, the volume ratio of solution B to solution C is 1-2:1-2, preferably 1:1.

[0055] The third typical embodiment of the present invention provides the application of the above-mentioned nano-hydroxyapatite composite hydrogel in the preparation of anti-inflammatory drugs and hemostatic materials. Preferably, the drugs include those for treating periodontitis, gingivitis, and oral ulcers.

[0056] The main manifestations of periodontitis are local inflammatory responses caused by the colonization of pathogenic microorganisms, accompanied by gingival bleeding and alveolar bone resorption, which often lead to premature loss of tooth function and even tooth loss. Gingivitis refers to acute and chronic inflammation occurring in the gingival tissue, manifested as gingival bleeding, swelling, and pain. If it continues to develop and invades hard tissues, periodontitis will form. The occurrence of oral ulcers is the result of the combined action of multiple factors, but oral microorganisms such as Streptococcus sanguinis may play an important role in the occurrence and development of oral ulcers. The present invention designs a dynamic Schiff base reaction, combines carboxymethyl chitosan, oxidized dextran, and nano-hydroxyapatite to prepare a multifunctional hydrogel that takes into account the advantages of all three. This composite hydrogel has excellent biocompatibility, injectability, self-repairability, adhesiveness, and antibacterial properties, can regulate the cell microenvironment, stop local bleeding, and thus repair damaged periodontal tissues, showing great potential in the treatment of periodontitis and other aspects.

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] The materials required for synthesis in the following examples: carboxymethyl chitosan, dextran, and sodium periodate (provided by Shanghai YuanYe Biotechnology Co., Ltd., China). Nano-hydroxyapatite (provided by Shanghai Aladdin Biotechnology Co., Ltd.).

[0059] Example 1

[0060] A preparation method of a nano-hydroxyapatite composite hydrogel is as follows:

[0061] (1) Dissolve dextran (20 - 150 kDa) and sodium periodate in water at a molar ratio of 2:1 and stir for 24 h to synthesize an oxidized dextran solution.

[0062] (2) Weigh 2 grams of nano-hydroxyapatite and add it to 10 milliliters of 10 wt% oxidized dextran aqueous solution and stir evenly.

[0063] (3) Weigh 1 gram of carboxymethyl chitosan and dissolve it in 10 milliliters of double-distilled water to obtain a 10 wt% carboxymethyl chitosan aqueous solution.

[0064] (4) Mix the two solutions obtained in (2) and (3) at a volume ratio of 1:1 to obtain a nano-hydroxyapatite composite hydrogel containing 10%.

[0065] As Figure 1 shown, in the synthesis process of the nano-hydroxyapatite composite hydrogel, when the oxidized dextran aqueous solution containing nano-hydroxyapatite and the carboxymethyl chitosan solution are mixed at a ratio of 1:1, the nano-hydroxyapatite composite hydrogel is formed under the action of Schiff base.

[0066] Comparative Example 1

[0067] A composite hydrogel, the specific steps are as follows:

[0068] (1) Dextran (20 - 150 kDa) and sodium periodate were dissolved in water at a molar ratio of 2:1 and stirred for 24 h to synthesize an oxidized dextran solution.

[0069] (2) Weigh 1 g of carboxymethyl chitosan and dissolve it in 10 mL of double-distilled water to obtain a 10 wt% aqueous carboxymethyl chitosan solution.

[0070] (3) The two solutions obtained in (1) and (2) were mixed at a volume ratio of 1:1 to obtain a composite hydrogel without nano-hydroxyapatite.

[0071] In the following experimental examples, the nano-hydroxyapatite composite hydrogels and the composite hydrogels without nano-hydroxyapatite obtained in Example 1 and Comparative Example 1 were used as different samples, and a control group was set up for detection.

[0072] Experimental Example 1

[0073] (1) Characterization analysis of nano-hydroxyapatite composite hydrogel

[0074] The composite hydrogel materials obtained in Example 1 and Comparative Example 1 were observed for their characteristics using Fourier transform infrared spectroscopy (FTIR, Thermo Fisher Scientific, Waltham, Ma, USA) and scanning electron microscopy (SEM, TESCAN Co., Ltd., VEGA3, China).

[0075] (2) Detection of the swelling behavior of nano-hydroxyapatite hydrogel

[0076] Take different masses of nano-hydroxyapatite composite hydrogel and the composite hydrogel of Comparative Example 1, add a certain volume of phosphate buffered saline (PBS, PH = 7) solution, and soak at 37 °C until swelling equilibrium is reached. When measuring the mass of the composite hydrogel, use tweezers to take out the hydrogel, dry it on absorbent paper, and weigh the moisture of the hydrogel. (The swelling rate of the hydrogel was determined by the gravimetric method)

[0077]

[0078] Analysis and detection results:

[0079] Through Figure 2 It can be seen that the composite hydrogel obtained in Comparative Example 1 has a porous structure, and the pores are evenly distributed. After quantifying the pore size, the average pore size is 216.99 microns. As Figure 3As shown, after adding nano-hydroxyapatite, the pore size in the composite hydrogel becomes uneven because the gelation rate is relatively fast after adding nano-hydroxyapatite and the mixing becomes uneven. After adding nano-hydroxyapatite, the pore size of the composite hydrogel decreases, and the average pore size is 170.75 μm.

[0080] As Figure 4 shown, from the hydrogel swelling experiment, both composite hydrogels can absorb sufficient water in a short time, reach saturation within 1 hour, and the swelling rate of the hydrogel decreases after 1 hour. The solution in the hydrogel is in dynamic water absorption equilibrium in the PBS solution. In the PBS (pH = 7) solution, the shapes of both hydrogels do not change anymore after 120 minutes, maintaining a stable structure.

[0081] Fourier transform infrared spectroscopy was used to detect the infrared absorption spectra of pure nano-hydroxyapatite and nano-hydroxyapatite composite hydrogels. As Figure 5 shown, pure nano-hydroxyapatite has absorption peaks at 1020 cm -1 and 564 cm -1 for PO 3 4- , while the absorption peak of HPO 2 4- is at 605 cm -1 . The composite hydrogel without adding nano-hydroxyapatite shows a vibration peak of C=O in oxidized dextran at 1590 cm -1 . After adding nano-hydroxyapatite, the absorption peaks at 564 cm -1 , 603 cm -1 and 1030 cm -1 in the composite hydrogel are obvious. Therefore, nano-hydroxyapatite is successfully loaded into the composite hydrogel, and at the same time, the wave peak of C=O in the gel is significantly enhanced, proving that the composite hydrogel can still be successfully gelated.

[0082] Experimental Example 2

[0083] Adhesion detection of nano-hydroxyapatite composite hydrogel

[0084] Porcine skin was selected for testing to simulate the adhesion of the hydrogel material on the human skin surface. Solution B and C of Example 1 were injected onto the surface of porcine skin through a dual mixing syringe to form a nano-hydroxyapatite composite hydrogel on the surface of porcine skin. The adhesion force of the composite hydrogel on the tissue surface was detected by changing the shape of the porcine skin. Specifically: a certain amount of red or green dye was added to solution B. 1 ml of solution B and solution C were taken with two sterile medical needles respectively, added to the mixing syringe, and injected onto the surface of porcine skin. After gelation, photos of different groups of porcine skin were taken.

[0085] Detection results:

[0086] As Figure 6 shown, the hydrogel was injected onto the surface of porcine skin through a dual - mixing needle, and the nano - hydroxyapatite composite hydrogel could firmly adhere to the surface of porcine skin at different twisting angles ( Figure 6 A - C). The hydrogel on the porcine skin surface was vigorously impacted by rapid water flows at different angles, and the nano - hydroxyapatite composite hydrogel could firmly adhere to the porcine skin ( Figure 6 D - F).

[0087] This indicates that the nano - hydroxyapatite composite hydrogel can still firmly seal the wound under a huge impact force, avoiding secondary injury caused by the cracking of the hydrogel.

[0088] Experimental Example 3

[0089] Self - healing property detection and hemolysis property test of nano - hydroxyapatite composite hydrogel

[0090] (1) Self - repair ability of nano - hydroxyapatite composite hydrogel

[0091] Red and green dyes were respectively added to two equal - volume solutions B. Different groups of solutions were injected into a circular mold using a dual - mixing syringe. After gelation for 5 minutes, the nano - hydroxyapatite composite hydrogels of two different colors were cut in half with a blade. The two semi - circular nano - hydroxyapatite composite hydrogels of different colors were placed in the same circular mold. After a period of time, the self - repair situation of the nano - hydroxyapatite composite hydrogel was observed and recorded.

[0092] Test results:

[0093] The self - healing property of the hydrogel can maintain the structural integrity and restore the hemostatic performance. Green and red dyes were added to the nano - hydroxyapatite composite hydrogel to prepare two different - colored hydrogels, as shown in Figure 7 A and Figure 7 B. The composite hydrogels of different colors were cut in half, and half of the green nano - hydroxyapatite composite hydrogel and half of the red nano - hydroxyapatite composite hydrogel were placed together respectively. After 10 minutes, under the action of gravity, the nano - hydroxyapatite composite hydrogels with different colors joined together could be lifted intact with tweezers, and the other half of the hydrogel did not shift, ensuring the self - healing property of the nano - hydroxyapatite composite hydrogel ( Figure 7 C).

[0094] (2) Biocompatibility of nano - hydroxyapatite composite hydrogel

[0095] The nano - hydroxyapatite composite hydrogel will come into contact with blood during the hemostasis process. The blood compatibility of the material was evaluated by examining the hemolysis characteristics of the composite hydrogel. The purpose of the blood compatibility test is to observe whether the material can cause red blood cell rupture.

[0096] According to the steps of Example 1 and Comparative Example 1, 200 μL of composite hydrogel without nano-hydroxyapatite and nano-hydroxyapatite composite hydrogel were prepared respectively. After washing three times with PBS solution (pH = 7), they were put into 1 mL of PBS (pH = 7) solution. According to relevant laws and institutional guidelines, whole blood of healthy volunteers was collected. 1 mL of whole blood was added to 5 mL of PBS (pH = 7) solution, mixed evenly, and 50 μL was taken from the mixed blood and then added to the PBS solution (pH = 7) containing the hydrogel. The above four groups of mixed solutions (i.e., double-distilled water treatment group control group, blank group, composite hydrogel group, nano-hydroxyapatite composite hydrogel group) were placed in an incubator at 37 °C for 1 hour.

[0097] Test results:

[0098] As Figure 8 shown, after centrifugation, a large amount of bright red blood was contained in the upper layer solution of the control group. While the red blood cells of the other three groups were deposited at the bottom of the centrifuge tube, and the upper layer solution was colorless and transparent, similar to the blank group. This indicates that the composite hydrogel without nano-hydroxyapatite water and the nano-hydroxyapatite composite hydrogel have no hemolytic activity on red blood cells. From the bar chart Figure 9 it can be obtained that the hemolysis rates of the composite hydrogel without nano-hydroxyapatite and the nano-hydroxyapatite composite hydrogel are both lower than 5%, and the hemolysis rate of the nano-hydroxyapatite composite hydrogel group is lower than that of the composite hydrogel group, which indicates that the addition of nano-hydroxyapatite to the gel further improves its blood compatibility.

[0099] Experimental Example 4

[0100] Detection of the adhesion of nano-hydroxyapatite composite hydrogel to red blood cells

[0101] Red blood cells can adhere and aggregate on its surface through the Schiff base reaction of nano-hydroxyapatite composite hydrogel, and accelerate its own coagulation process. To verify this, the present invention verified the coagulation effect of composite hydrogel with and without nano-hydroxyapatite on red blood cells with whole blood.

[0102] According to the preparation methods of Example 1 and Comparative Example 1, 100 μL of composite hydrogel without nano-hydroxyapatite and nano-hydroxyapatite composite hydrogel were prepared respectively, placed in a 12-well plate, 200 μL of anticoagulated whole blood was taken out and dropped on the surface of the hydrogel. The non-adherent red blood cells on the surface of the hydrogel were removed, and after diluting 2.5% glutaraldehyde solution with PBS solution, it was fixed at 4 °C for 24 hours. After drying, the adhesion morphology on the surface of the composite hydrogel of different groups was observed under SEM.

[0103] Detection results:

[0104] As shown Figure 10 in the figure, nano-hydroxyapatite composite hydrogel (Gel-nHA) can be adhered to by red blood cells and protect their integrity. After magnifying 5000 times, observing the morphology of red blood cells, it is found that the morphology of red blood cells adhered to the composite hydrogel without nano-hydroxyapatite is good, and there is no rupture of the cell membrane( Figure 10 C). Similarly, the morphology of red blood cells on the surface of nano-hydroxyapatite composite hydrogel also does not show rupture( Figure 10 D), and the number of adsorbed red blood cells is more. Therefore, nano-hydroxyapatite composite hydrogel can adhere red blood cells on its surface without damaging red blood cells, which is more conducive to the formation of blood clots and creates good conditions for hemostasis.

[0105] Experimental Example 5

[0106] Detection of Hemostatic Performance of Nano-Hydroxyapatite Composite Hydrogel

[0107] Nano-hydroxyapatite composite hydrogel shows strong adhesiveness and excellent external hemostatic performance. Therefore, the hemostatic activities of different groups of hydrogels were studied. Specifically: A mouse hemostasis model was established to stop bleeding in the mouse liver. A blank group (i.e., the group without any treatment on the mouse liver wound) and a composite hydrogel without nano-hydroxyapatite were set as control groups. The mice were injected with 200 μl of 4% chloral hydrate. After anesthesia, the liver was taken out, and the liver was placed on a pre-weighed filter paper. At the same time, a wound with a length of 3 mm and a depth of 1 mm was made on the liver with a scalpel. The prepared different groups of hydrogels were injected into the wound with a syringe to stop bleeding. At this time, the timing started. Photos were taken of the hemostatic site of the mouse liver at 0, 15, 30, 60, 120, and 240 s to record the changes in the blood on the filter paper. After 5 minutes, the filter paper was taken out and the mass of the filter paper was weighed. According to the difference in mass before and after the filter paper, the blood loss of the mouse liver was calculated.

[0108] Detection Results:

[0109] As can be seen Figure 11 , in the blank group, the blood of the mice basically coagulated at 240 s, while in the nano-hydroxyapatite composite hydrogel, there was basically no bleeding at the mouse liver site within 15 s after injection.

[0110] Through the observation of the bleeding site of the mouse liver, the self-coagulation time of the blood in the mouse liver in the blank group was 290 s( Figure 12 ), while for the wound treated with hydrogel, the bleeding time of the liver was shortened to 10 s( Figure 12 ), showing a good hemostatic effect. When the nano-hydroxyapatite composite hydrogel was injected into the bleeding wound of the mouse liver, the hemostasis time was within 10 s( Figure 12 ). Therefore, after adding nano-hydroxyapatite, the hemostatic effect of the composite hydrogel is not affected.

[0111] The blood loss of mice was weighed and quantified. For the blank group, the blood loss of mice reached 160 mg, while for the gel group and the nano-hydroxyapatite composite gel group, the average blood loss of the mouse liver was 23.3 mg and 20 mg respectively( Figure 13 ). Therefore, the nano-hydroxyapatite composite hydrogel has a more obvious hemostatic effect.

[0112] Experimental Example 6

[0113] In vitro antibacterial test of nano-hydroxyapatite composite hydrogel

[0114] The skin is a barrier to protect the human body. After the skin is damaged, bacteria enter the body through the wound, causing the wound to be infected by bacteria. Therefore, it is necessary to avoid the invasion of bacteria while the hydrogel stops bleeding. The antibacterial ability of the nano-hydroxyapatite composite hydrogel was verified through the in vitro antibacterial experiment of the hydrogel in this invention.

[0115] First, according to the preparation methods of Example 1 and Comparative Example 1, 200 μL of the composite hydrogel without nano-hydroxyapatite and the nano-hydroxyapatite composite hydrogel were respectively prepared. After being irradiated with ultraviolet light for 30 min in a super-clean workbench, they were placed in a bacterial broth with the same concentration and co-cultured in a bacterial incubator at 37 °C for 12 h. Then, the co-cultured bacterial broth was diluted 10-6 times with PBS (pH = 7), 20 μL of the diluted PBS bacterial broth was sampled and evenly spread on an agar plate, and then the plate was placed in a bacterial incubator at 37 °C for 24 h and the number of colonies was observed. Three plates were coated for each sample. Calculate the number of bacterial colonies in the agar plate.

[0116] Detection results:

[0117] Figure 14 It shows the antibacterial effects of the agar plates coated with the hydrogel after co-culturing with Escherichia coli and Staphylococcus aureus and diluted 10-6 times. The antibacterial effects of each group were determined by CFU counting. The average number of colonies of Staphylococcus aureus in the blank group was 5.38×10 9 CFU / ml, while the number of colonies in the composite hydrogel group without nano-hydroxyapatite and the nano-hydroxyapatite composite hydrogel group were 1.2×10 9 CFU / ml and 1.65×10 9 CFU / ml respectively. The average number of colonies of Escherichia coli in the blank group was 3.18×10 9 CFU / ml, while the number of colonies in the composite hydrogel group without nano-hydroxyapatite and the nano-hydroxyapatite composite hydrogel group were 0.15×10 9 CFU / ml and 0.07×10 9 CFU / ml(Figure 15 )。

[0118] From the above results, it can be concluded that the gel group has a good antibacterial effect on bacteria. When nano-hydroxyapatite is added to the gel, it does not affect the antibacterial rate of the hydrogel, but instead further inhibits the growth of bacteria. In particular, the antibacterial effect against Escherichia coli reaches more than 90%.

[0119] Example 7

[0120] Statistical analysis

[0121] One-way analysis of variance (ANOVA) was performed using Origin 8.0 to determine the statistical significance of the data. P < 0.05 was considered to be statistically significant.

[0122] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nano-hydroxyapatite composite hydrogel, characterized in that, the preparation method of the nano-hydroxyapatite composite hydrogel comprises the following steps: (1) Dissolve dextran and sodium periodate in water at a molar ratio of 2:1 and stir for 24 h to synthesize an oxidized dextran solution; the molecular weight of the dextran is 20 - 150 kDa; (2) Weigh 2 g of nano-hydroxyapatite and add it to 10 mL of a 10 wt% oxidized dextran aqueous solution and stir evenly; (3) Weigh 1 g of carboxymethyl chitosan and dissolve it in 10 mL of double-distilled water to obtain a 10 wt% carboxymethyl chitosan aqueous solution; (4) Mix the two solutions obtained in (2) and (3) at a volume ratio of 1:1 to obtain the nano-hydroxyapatite composite hydrogel.

2. Use of the nano-hydroxyapatite composite hydrogel according to claim 1 in the preparation of a medicament for treating periodontitis, gingivitis, and oral ulcers.

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