Composite gel for alveolar bone defect repair as well as preparation method and application of composite gel

Abstract

The invention discloses composite gel for alveolar bone defect repair as well as a preparation method and application of the composite gel. Main raw materials of the composite gel are regenerated silk fibroin (RSF), collagen (Col) and hydroxyapatite (HAP) (or the composite gel further comprises poly-deoxyribonucleotide (PDRN) as a main raw material), and an auxiliary raw material of the composite gel is sodium hyaluronate (HA). The preparation method comprises the following steps: forming hydrogel from RSF, Col and HAP (or further comprising PDRN crosslinking) through 1, 4-butanediol glycidyl ether (BDDE), performing alcohol washing and freeze-drying, mechanically crushing the material to obtain a particle material, and dispersing the particles in an HA solution to obtain the composite gel. The prepared composite gel has injectability and can completely fill an alveolar bone defect area. The composite gel has excellent biocompatibility, and can promote migration of stem cells, induce osteogenic differentiation of cells and promote bone tissue regeneration.

Description

Technical Field

[0001] The present invention relates to a composite gel for repairing alveolar bone defects, a preparation method thereof, and an application thereof, belonging to the technical field of biomedicine. Background Art

[0002] The alveolar bone is the part of the upper and lower jaws that surrounds and supports the root tissues of teeth, mainly acting on the generation of teeth and daily chewing functions. Alveolar bone defects are mainly caused by periodontal diseases, trauma, tumor resection, systemic diseases, etc. The main clinical symptoms are tooth defects, exposed tooth roots, loose teeth, etc. At present, the main methods for treating alveolar bone tissue defects are autologous bone transplantation and allogeneic bone transplantation, but these methods have certain limitations and may lead to disease transmission and immune rejection. Hydrogels have a porous three-dimensional network structure, soft texture, variable shape, strong inclusiveness, physical properties similar to biological tissues, and excellent biocompatibility, and are excellent biomaterials.

[0003] Regenerated silk fibroin is a biopolymer material extracted from natural silk and reprocessed, with excellent biocompatibility, degradability, and strong chemical modifiability. However, regenerated silk fibroin lacks bioactive signals and has low cell adhesion, and needs to be used in combination with other biomaterials. Recombinant humanized collagen is obtained by optimizing and recombinantly expressing the original gene sequence of human skin collagen using genetic engineering technology and through biotechnological fermentation. Recombinant humanized type III collagen (rhCol-III) is highly similar to human type III collagen, has high biological activity and low immunogenicity, and is suitable for in vivo implantation. However, recombinant humanized type III collagen may be rapidly degraded due to enzymatic hydrolysis or physicochemical changes in the in vivo environment, and its stability needs to be improved by crosslinking or compounding with other materials. Animal-derived type I collagen (Col-I) has higher biocompatibility with the human body, higher stability against thermal degradation, is beneficial to the progress of blood coagulation activities, is beneficial to cell proliferation, serves as a growth matrix for fibroblasts and vascular endothelial cells, and promotes tissue repair. Hydroxyapatite is the main inorganic component of bones and teeth, has excellent biocompatibility and osteoconductivity, has no immune rejection reaction when implanted in the body, can promote cell adhesion and differentiation, and supports bone tissue regeneration. Polydeoxyribonucleotide is a class of bioactive substances extracted from fish sperm or mammalian cells, mainly composed of deoxyribonucleotides (DNA fragments). Polydeoxyribonucleotide has the functions of promoting cell proliferation, anti-inflammation, and promoting angiogenesis, and can be applied to orthopedic repair materials.

[0004] However, for the repair after alveolar bone defects, although there are already some hydrogel products, their effects are still relatively limited. How to fully exert the maximum advantages of this product type in the repair of alveolar bone defects is still a technical problem to be solved urgently. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a composite gel for repairing alveolar bone defects, its preparation method and application, which can solve the problem of repairing alveolar bone defects.

[0006] The present invention provides a composite gel for repairing alveolar bone defects, which is characterized in that the composite gel is made of composite microparticles and sodium hyaluronate, wherein the raw materials of the composite microparticles include regenerated silk fibroin (RSF), collagen (Col), and hydroxyapatite (HAP).

[0007] In some embodiments, the raw materials of the composite microparticles further include polydeoxyribonucleotide (PDRN).

[0008] In some embodiments, the raw materials of the composite microparticles are made of regenerated silk fibroin (RSF), collagen (Col), and hydroxyapatite (HAP).

[0009] In some embodiments, the raw materials of the composite microparticles are made of regenerated silk fibroin (RSF), collagen (Col), hydroxyapatite (HAP), and polydeoxyribonucleotide (PDRN).

[0010] In some embodiments, the collagen is selected from type I collagen (Col-I), type II collagen, and type III collagen; preferably type I collagen or type III collagen; more preferably animal-derived type I collagen or recombinant humanized type III collagen (rhCol-III).

[0011] The present invention also provides a preparation method of a composite gel for repairing alveolar bone defects. The specific steps of the preparation method of the composite gel for repairing alveolar bone defects are as follows:

[0012] Step 1: Prepare the composite material: Dissolve RSF and Col in lithium bromide solution respectively. After mixing the two solutions of RSF and Col evenly, add HAP, stir for 20 - 40 min, then add BDDE in sequence, stir for 20 - 40 min, and then let it stand for 24 - 72 h to obtain a hydrogel.

[0013] Step 2: Wash the composite material: Extrude the prepared hydrogel through a sieve and wash it 6 - 10 times with 40 - 60% ethanol, and then freeze-dry the hydrogel particles.

[0014] Step 3: Prepare the composite microparticles: Obtain the composite microparticles by crushing the freeze-dried hydrogel particles.

[0015] Step 4: Prepare the composite gel: Dissolve HA in PBS solution. After complete dissolution, uniformly disperse and mix the prepared composite microparticles in the HA solution to obtain the composite gel.

[0016] In some embodiments, in step 1, the following operations are further included: dissolving PDRN in ultrapure water to obtain a PDRN solution, and adding the PDRN solution immediately after adding BDDE.

[0017] In some embodiments, the mass ratio of RSF to Col in the composite microparticles is 2:1 to 5:1, preferably 2:1.

[0018] In some embodiments, the mass ratio of RSF to HAP in the composite microparticles is 1:3 to 1:1, preferably 1:2.57.

[0019] In some embodiments, the mass ratio of RSF to PDRN in the composite microparticles is 25:1 to 30:1, preferably 26.67:1.

[0020] In some embodiments, the mass ratio of the composite microparticles to sodium hyaluronate is 5:1 to 10:1, preferably 7.5:1.

[0021] In some embodiments, in step 1, the final concentrations of RSF and Col are 80 mg / mL and 40 mg / mL respectively, the final concentration of HAP is 205.9 mg / mL, and the final concentration of PDRN is 3 mg / mL.

[0022] In some embodiments, in step 1, the concentration of lithium bromide is 12 M.

[0023] In some embodiments, in step 1, the stirring time is 25 - 35 min, and the standing time is 36 - 60 h. Preferably, the stirring time is 30 min and the standing time is 48 h.

[0024] In some embodiments, in step 2, the mesh aperture of the sieve is 6 - 50 mesh.

[0025] In some embodiments, in step 2, the concentration of the ethanol solution is 50%, the number of cleaning times is 8 times, and the cleaning solution is replaced every 30 min.

[0026] In some embodiments, in step 3, the pulverization is mechanical pulverization by an electric mill.

[0027] In some embodiments, in step 4, the concentration of the sodium hyaluronate solution is 10 - 50 mg / mL, preferably 20 mg / mL.

[0028] In some embodiments, in step 4, the concentration of the composite microparticles is 100 - 500 mg / mL, preferably 150 mg / mL.

[0029] The regenerated silk fibroin protein of the present invention is a biological macromolecule material with excellent biocompatibility, degradability, and multifunctionality of structural adjustment. Collagen has high biological activity and low immunogenicity, can promote cell adhesion and migration, and create a good repair environment. Hydroxyapatite has excellent biocompatibility and osteoconductivity, can promote cell adhesion and osteogenic differentiation, and support bone tissue regeneration. Polydeoxyribonucleotide has the functions of promoting cell proliferation, anti-inflammation, and promoting angiogenesis, and can accelerate tissue repair.

[0030] Advantages of the present invention:

[0031] 1. The present invention combines regenerated silk fibroin protein, collagen, and hydroxyapatite through the cross-linking action of BDDE, or further adds polydeoxyribonucleotide for cross-linking and binding, giving full play to the specific advantages of various biomaterials and providing a good repair environment for alveolar bone defects.

[0032] 2. The composite gel prepared by dispersing composite microparticles in HA has injectability, can adapt to the defect location, does not require open surgery, and avoids the risk of postoperative infection.

[0033] 3. The hydrogel material can be in direct contact with human tissues, prevent external microbial infection, effectively prevent body fluid loss, and can transmit oxygen and nutrients, providing a good defect repair environment.

[0034] 4. In vitro and in vivo studies show that the composite gel prepared by the present invention can promote cell migration and osteogenic regeneration of cells, promote bone tissue regeneration, and has obvious therapeutic effects on alveolar bone defects. Description of the drawings

[0035] Figure 1 It is a schematic flow chart of the preparation method of RSF-Col-HAP-PDRN composite gel.

[0036] Figure 2 It is a schematic structural flow chart of the preparation method of RSF-Col-HAP-PDRN composite gel.

[0037] Figure 3 It is a scanning electron microscope image of RSF-Col-HAP-PDRN composite microparticles.

[0038] Figure 4 It is a distribution diagram of Col and PDRN in RSF-Col-HAP-PDRN.

[0039] Figure 5 It is a Fourier transform infrared spectrum diagram of RSF-Col-HAP-PDRN composite microparticles.

[0040] Figure 6This is the appearance of RSF-Col-HAP-PDRN composite gel.

[0041] Figure 7 This is a scanning electron micrograph of the RSF-Col-HAP-PDRN composite gel.

[0042] Figure 8 This is the rheological diagram of RSF-Col-HAP-PDRN composite gel.

[0043] Figure 9 This is the CCK-8 cytotoxicity data of RSF-Col-HAP-PDRN composite gel on bone marrow mesenchymal stem cells.

[0044] Figure 10 This is a comparison chart of the cytotoxicity of the RSF-Col-HAP-PDRN composite gels prepared in Example 9 and Example 10.

[0045] Figure 11 This is a statistical chart showing the effect of RSF-Col-HAP-PDRN composite gel on the migration of bone marrow mesenchymal stem cells.

[0046] Figure 12 This is a diagram showing the effect of RSF-Col-HAP-PDRN composite gel on the expression of osteogenic genes in bone marrow mesenchymal stem cells.

[0047] Figure 13 These are photos of the alveolar bone defects in rats treated with RSF-Col-HAP-PDRN composite gel.

[0048] Figure 14 This is a micro-CT scan of the alveolar bone defect in rats treated with RSF-Col-HAP-PDRN composite gel. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0052] Regenerated silk fibroin is abbreviated as RSF and is purchased from Nanjing Siyuan Medical Technology Co., Ltd.; Recombinant humanized type III collagen is abbreviated as Col-III and is purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.; Animal-derived type I collagen is written as Col-I and is purchased from Desheng Biology; Hydroxyapatite is written as HAP and is purchased from Linkete Medical Technology (Wuxi) Co., Ltd.; Polydeoxyribonucleotide is written as PDRN and is purchased from Ruijiming Biotechnology Co., Ltd.; Sodium hyaluronate is abbreviated as HA and is purchased from Bloomage Biotechnology Co., Ltd.

[0053] As Figure 1 and Figure 2 shown, the preparation method of the composite gel of the present invention includes the following steps:

[0054] Step 1: Prepare the composite material: Dissolve RSF and Col in lithium bromide solution respectively, dissolve PDRN in ultrapure water. After mixing the two solutions of RSF and Col evenly, add HAP, stir for 30 min, then add BDDE and PDRN solution in sequence, stir for 30 min, and then let it stand for 48 h to obtain RSF-Col-HAP-PDRN hydrogel.

[0055] Step 2: Wash the composite material: Extrude the prepared hydrogel through a sieve and wash it 8 times with 50% ethanol, and then freeze-dry the hydrogel particles.

[0056] Step 3: Prepare the composite particles: Mechanically crush the freeze-dried hydrogel particles through an electric grinder to obtain RSF-Col-HAP-PDRN composite particles.

[0057] Step 4: Prepare the composite gel: Dissolve HA in PBS solution. After complete dissolution, uniformly disperse and mix the prepared composite particles in the HA solution to obtain RSF-Col-HAP-PDRN composite gel.

[0058] In the present invention, in order to obtain a sterile RSF-Col-HAP-PDRN composite gel for in vitro and in vivo experiments, the prepared RSF-Col-HAP-PDRN gel needs to be sterilized by moist heat, and the sterilization conditions are 121 °C for 12 min.

[0059] The following will be described in detail with specific examples:

[0060] Example 1

[0061] A kind of RSF composite particle is prepared by the following method:

[0062] Step S1: Dissolve 720 mg of RSF in 6 mL of 12 M lithium bromide solution, stir for 1 h at room temperature. After dissolution, an RSF solution with a concentration of 120 mg / mL is obtained. Add 170 μL of BDDE crosslinking agent dropwise, and continue stirring for 30 min after the addition. Let the solution stand at 30 °C for 48 h to obtain an RSF hydrogel.

[0063] Step S2: Extrude the crosslinked RSF hydrogel through a 6-mesh sieve, wash it in 50% ethanol for 30 min, then extrude the hydrogel through a 50-mesh sieve. Stir and wash the hydrogel particles in 50% ethanol solution 7 times, changing the washing solution every 30 min. After washing, place the hydrogel particles in a freeze dryer for freeze-drying.

[0064] Step S3: After freeze-drying, mechanically crush the RSF material using an electric grinder, and the crushed microparticles are RSF microparticles.

[0065] Example 2

[0066] A kind of RSF-Col composite microparticle is prepared by the following steps:

[0067] Step S1: Dissolve 480 mg of RSF in 4 mL of 12 M lithium bromide solution, stir for 1 h at room temperature. After dissolution, an RSF solution with a concentration of 120 mg / mL is obtained; dissolve 240 mg of rhCol-Ⅲ in 2 mL of 12 M lithium bromide solution, and ultrasonically dissolve to obtain an rhCol-Ⅲ solution with a concentration of 120 mg / mL. Use a dropper to suck the Col-Ⅲ solution and add it dropwise into the RSF solution, continuously stir during the addition. After the addition of the rhCol-Ⅲ solution, continue stirring for 20 min to make the two solutions mix evenly. Add 170 μL of BDDE crosslinking agent dropwise, and continue stirring for 30 min after the addition. Let the solution stand at 30 °C for 48 h to obtain an RSF-Col hydrogel.

[0068] Step S2: Extrude the crosslinked RSF-Col hydrogel through a 6-mesh sieve, wash it in 50% ethanol for 30 min, then extrude the hydrogel through a 50-mesh sieve. Stir and wash the hydrogel particles in 50% ethanol solution 7 times, changing the washing solution every 30 min. After washing, place the hydrogel particles in a freeze dryer for freeze-drying.

[0069] Step S3: After freeze-drying, mechanically crush the RSF-Col material using an electric grinder, and the crushed microparticles are RSF-Col microparticles.

[0070] Example 3

[0071] An RSF-Col-HAP composite particle is prepared by the following steps:

[0072] Step S1: Dissolve 480 mg of RSF in 4 mL of 12 M lithium bromide solution and stir at room temperature for 1 h. After dissolution, an RSF solution with a concentration of 120 mg / mL is obtained. Dissolve 240 mg of rhCol-Ⅲ in 2 mL of 12 M lithium bromide solution and dissolve it ultrasonically to obtain a Col-Ⅲ solution with a concentration of 120 mg / mL. Use a dropper to suck the rhCol-Ⅲ solution and drop it into the RSF solution. Keep stirring during the dropping process. After the dropping of the rhCol-Ⅲ solution is completed, continue to stir for 20 min to mix the two solutions evenly. Weigh 1235.4 mg of HAP powder and add it to the above solution, and stir for 30 min. Drop 170 μL of BDDE cross-linking agent, and continue to stir for 30 min after the dropping is completed. Let the solution stand in an environment of 30 °C for 48 h to obtain an RSF-Col-HAP hydrogel.

[0073] Step S2: Extrude the cross-linked RSF-Col-HAP hydrogel through a 6-mesh sieve, wash it in 50% ethanol for 30 min, then extrude the hydrogel through a 50-mesh sieve, and continue to stir and wash the hydrogel particles 7 times in 50% ethanol solution, changing the washing solution every 30 min. After the washing is completed, put the hydrogel particles into a freeze dryer for freeze-drying.

[0074] Step S3: After freeze-drying, use an electric grinder to mechanically crush the RSF-Col-HAP material, and the crushed particles are RSF-Col-HAP particles.

[0075] Example 4

[0076] An RSF-Col-HAP-PDRN composite particle is prepared by the following steps:

[0077] Step S1: Dissolve 480 mg of RSF in 4 mL of 12 M lithium bromide solution, stir at room temperature for 1 h, and obtain an RSF solution with a concentration of 120 mg / mL after dissolution; dissolve 240 mg of rhCol-Ⅲ in 2 mL of 12 M lithium bromide solution, and ultrasonically dissolve to obtain a rhCol-Ⅲ solution with a concentration of 120 mg / mL. Weigh 18 mg of PDRN and dissolve it in 0.36 mL of UP, and ultrasonically treat until completely dissolved. Use a dropper to suck the rhCol-Ⅲ solution and drop it into the RSF solution, continuously stir during the dropping process. After the dropping of the rhCol-Ⅲ solution is completed, continue to stir for 20 min to make the two solutions mix evenly. Weigh 1235.4 mg of HAP powder and add it to the above solution, and stir for 30 min. Drop 170 μL of BDDE cross-linking agent, then add the PDRN solution, and continue to stir for 30 min. Let the solution stand in an environment of 30 °C for 48 h to obtain the RSF-Col-HAP-PDRN hydrogel.

[0078] Step S2: Extrude the cross-linked RSF-Col-HAP-PDRN hydrogel through a 6-mesh sieve, wash it in 50% ethanol for 30 min, then extrude the hydrogel through a 50-mesh sieve, and continue to stir and wash the hydrogel particles 7 times in a 50% ethanol solution, replacing the washing solution every 30 min. After the washing is completed, put the hydrogel particles into a freeze dryer for freeze-drying.

[0079] Step S3: After freeze-drying is completed, use an electric grinder to mechanically crush the RSF-Col-HAP-PDRN material, and the crushed fine particles are RSF-Col-HAP-PDRN fine particles.

[0080] Example 5

[0081] An RSF-Col-HAP-PDRN composite fine particle is prepared by the following steps:

[0082] Step S1: Dissolve 480 mg of RSF in 4 mL of 12 M lithium bromide solution and stir at room temperature for 1 h. After dissolution, an RSF solution with a concentration of 120 mg / mL is obtained. Dissolve 240 mg of Col-I in 2 mL of 12 M lithium bromide solution and dissolve it ultrasonically to obtain a Col-I solution with a concentration of 120 mg / mL. Weigh 18 mg of PDRN and dissolve it in 0.36 mL of UP, and ultrasonicate until completely dissolved. Use a dropper to suck the Col-I solution and drop it into the RSF solution, and continuously stir during the dropping process. After the dropping of the Col-I solution is completed, continue to stir for 20 min to make the two solutions mix evenly. Weigh 1235.4 mg of HAP powder and add it to the above solution, and stir for 30 min. Drop 170 μL of BDDE cross-linking agent, then add the PDRN solution, and continue to stir for 30 min. Let the solution stand in an environment of 30 °C for 48 h to obtain the RSF-Col-HAP-PDRN hydrogel.

[0083] Step S2: Extrude the cross-linked RSF-Col-HAP-PDRN hydrogel through a 6-mesh sieve, wash it in 50% ethanol for 30 min, then extrude the hydrogel through a 50-mesh sieve, and continue to stir and wash the hydrogel particles 7 times in 50% ethanol solution, changing the washing solution every 30 min. After washing, put the hydrogel particles into a freeze dryer for freeze-drying.

[0084] Step S3: After freeze-drying, use an electric grinder to mechanically crush the RSF-Col-HAP-PDRN material, and the crushed fine particles are RSF-Col-HAP-PDRN fine particles.

[0085] Example 6

[0086] A composite gel for alveolar bone defect repair is prepared by the following steps:

[0087] Slowly add 20 mg of HA to 1 mL of PBS and stir at room temperature until HA is completely dissolved. Add 150 mg of the RSF fine particles prepared in Example 1 to 1 mL of HA solution, and use magnetic stirring to make the fine particles completely and evenly dispersed in HA to obtain the RSF gel. Then, sterilize the RSF gel by moist heat, and the sterilization conditions are 121 °C for 12 min. After sterilization, dispense it into 1 mL syringes on a sterile operating table and store it at 4 °C.

[0088] Example 7

[0089] A composite gel for alveolar bone defect repair is prepared by the following steps:

[0090] Slowly add 20 mg of HA to 1 mL of PBS and stir until the HA is completely dissolved. Add 150 mg of the RSF-Col microparticles prepared in Example 2 to 1 mL of the HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain an RSF-Col gel. Then, subject the RSF-Col gel to moist heat sterilization under the conditions of 121 °C for 12 min. After sterilization, dispense it into 1 mL syringes in a sterile operating table and store it at 4 °C.

[0091] Example 8

[0092] A composite gel for alveolar bone defect repair is prepared by the following steps:

[0093] Slowly add 20 mg of HA to 1 mL of PBS and stir until the HA is completely dissolved. Add 150 mg of the RSF-Col-HAP microparticles prepared in Example 3 to 1 mL of the HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain an RSF-Col-HAP gel. Then, subject the RSF-Col-HAP gel to moist heat sterilization under the conditions of 121 °C for 12 min. After sterilization, dispense it into 1 mL syringes in a sterile operating table and store it at 4 °C.

[0094] Example 9

[0095] A composite gel for alveolar bone defect repair is prepared by the following steps:

[0096] Slowly add 20 mg of HA to 1 mL of PBS and stir until the HA is completely dissolved. Add 150 mg of the RSF-Col-HAP-PDRN microparticles prepared in Example 4 to 1 mL of the HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain an RSF-Col-HAP-PDRN gel. Then, subject the RSF-Col-HAP-PDRN gel to moist heat sterilization under the conditions of 121 °C for 12 min. After sterilization, dispense it into 1 mL syringes in a sterile operating table and store it at 4 °C.

[0097] Example 10

[0098] A composite gel for alveolar bone defect repair is prepared by the following steps:

[0099] Slowly add 20 mg of HA to 1 mL of PBS and stir until the HA is completely dissolved. Add 150 mg of the RSF-Col-HAP-PDRN microparticles prepared in Example 5 to 1 mL of the HA solution and, with the aid of magnetic stirring, completely and uniformly disperse the microparticles in the HA to obtain the RSF-Col-HAP-PDRN gel. Then, subject the RSF-Col-HAP-PDRN gel to moist heat sterilization under the conditions of 121 °C for 12 min. After sterilization is completed, aliquot it into 1 mL syringes on a sterile workbench and store at 4 °C.

[0100] Performance analysis of the RSF-Col-HAP-PDRN composite gel:

[0101] Figure 3 Scanning electron micrographs of the four composite microparticles of RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN prepared in Examples 6-9 are shown, and the structures of the composite microparticles are observed microscopically. The size distributions of the composite microparticles prepared by mechanical pulverization through the same procedure are uniform.

[0102] Figure 4 The fluorescence image of the RSF-Col-HAP-PDRN composite microparticles prepared in Example 9 is shown. Fluorescein isothiocyanate-labeled rhCol-III is used to crosslink with RSF, HAP, and PDRN to prepare the composite microparticles. The composite microparticles are soaked and washed in a DAPI solution, freeze-dried, and then observed under a laser confocal microscope. The results show that the labeled rhCol-III exhibits green fluorescence and PDRN exhibits blue fluorescence, and the fluorescence is uniformly dispersed in the microparticles, indicating the successful crosslinking of rhCol-III and PDRN in the composite microparticles.

[0103] Figure 5 Infrared spectra of the RSF-Col-HAP-PDRN, RSF-Col-HAP, RSF-Col, and RSF composite microparticles prepared in Examples 6-9 are shown successively from top to bottom. The peak at 1651 cm -1 in RSF-Col comes from the C=O segment of the amide I bond of rhCol-III, and the peak at 1550 cm -1 is the N-H segment of the amide II bond. The peak at 1035 cm -1 in the RSF-Col-HAP and RSF-Col-HAP-PDRN composite microparticles is the asymmetric stretching vibration of P-O in the phosphate group, confirming the presence of HAP in the composite microparticles.

[0104] Figure 6The preparation appearance diagram of the RSF-Col-HAP-PDRN composite gel prepared in Example 9 is shown. The composite gel can be plastically formed by a mold to produce different shapes and can adapt to various environments, indicating that the gel can adapt to the defect location.

[0105] Figure 7 The scanning electron microscope images of the composite gels prepared in Examples 6-9 are shown. It can be observed from the figures that the gels have a porous network structure, and nutrients and metabolic wastes can be exchanged and transported in vivo, creating a good environment for the regeneration of alveolar bone defects.

[0106] Figure 8 The rheological diagrams of the composite gels prepared in Examples 6-9 are shown. The figure shows that under a constant stress of 0.01%, within the scanning frequency range of 0.1-100 Hz, the storage modulus (G') of the four groups of gels is always greater than the loss modulus (G"), indicating that the prepared RSF-Col-HAP-PDRN gel is in a gel state, with a stable structure and good mechanical properties.

[0107] Figure 9 The CCK-8 cytotoxicity data of the control group and the RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN composite gels prepared in Examples 6-9 on bone marrow mesenchymal stem cells (BMSCs) are shown from left to right. A certain weight of RSF, RSF-Col-HAP-PDRN, RSF-Col-HAP-PDRN, and RSF-Col-HAP-PDRN composite gels with a concentration of 0.2 g / mL was placed in the cell complete medium and extracted in an environment at 37°C for 24 h. BMSC cells in good growth state were evenly seeded in a 96-well plate at a density of 6×10 4 / mL. After adding 100 μL of cell suspension to each well and culturing for 24 h, the cells were completely adherent. The original culture medium was removed, and the extraction solutions of different groups of hydrogels were added. The control group was added with cell complete medium. After continuing to culture in the incubator for 24 h, 100 μL of complete medium containing 10% CCK-8 was added to each well. After incubating for 1 h, the cell absorbance was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. The results showed that the composite gels prepared in the present invention did not affect cell growth, and compared with the control group, the cell survival rate of the material group increased and the cell number increased, indicating that the prepared hydrogel had good cell compatibility and could promote cell proliferation.

[0108] Figure 10Cytotoxicity data of the RSF-Col-HAP-PDRN composite gels prepared for Example 9 and Example 10. It can be seen from the result graph that the cell survival rates of the composite gels prepared in Example 9 and Example 10 are much higher than those of the control group, indicating good cell compatibility. The addition of the composite gel increases cell viability and promotes cell proliferation, and the composite gel prepared in Example 9 has a better effect.

[0109] Figure 11 (a) and (b) respectively show, from left to right in sequence, the crystal violet staining photographs and the statistical graphs of cell migration rates of the RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN composite gels prepared in the control group and Examples 6-9 that affect the migration of BMSC cells. The BMSC cells were dispersed in serum-free medium at a density of 3×10 4 cells / mL. 200 μL of the cell suspension was added above the Transwell chamber. The hydrogel material was evenly spread in a 24-well plate, and 500 μL of serum-containing medium was added. The Transwell chamber was placed in the 24-well plate, and after culturing in an incubator for 24 h, crystal violet staining was performed, photographs were taken, and the cell migration rate was calculated. The results showed that, compared with the control group (without adding materials), the number of cells migrating in the material group increased significantly, and with the addition of Col-III, HAP, and PDRN materials, the cell migration rate increased, indicating that RSF-Col-HAP-PDRN can promote cell migration. Among them, the effects of RSF-Col-HAP and RSF-Col-HAP-PDRN in promoting cell migration are significantly better than those of the control group, RSF, and RSF-Col groups; RSF-Col-HAP-PDRN is the best (P<0.0001). Excellent cell migration ability helps bone marrow mesenchymal stem cells to aggregate at the defect site, creating a good environment for cell osteogenic differentiation and bone tissue regeneration.

[0110] Figure 12 (a), (b), (c), and (d) respectively show, from left to right in sequence, the diagrams of the effects of the RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN composite gels prepared in the control group and Examples 6-9 on the osteogenic differentiation of BMSC cells. 500 μL of the cell suspension was evenly spread in a 24-well plate, and the cell concentration was 4×10 4 / mL. When the cells in the well plate grew to 80%, Transwell chambers were placed in, and an equal amount of RSF, RSF-Col-HAP-PDRN, RSF-Col-HAP-PDRN, and RSF-Col-HAP-PDRN composite gels were added to the chambers for co-incubation. In the control group, no material was added, and osteogenic induction medium was used for osteogenic induction instead. The osteogenic induction medium was changed every 2 days. After 14 days of induction, cell RNA was extracted using the RNAeasyTM Animal RNA Extraction Kit (spin column type), and the BeyoFastTM SYBR Green One-Step qRT-PCR Kit was used to detect the expression of related genes in the RNA. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and Runt-related transcription factor 2 (Runx2) are osteogenic-related matrix proteins and transcription factors. The gene expression of these proteins can be detected by qRT-PCR experiments to judge the osteogenic differentiation of cells. The results showed that compared with the control group, the expression of osteogenic genes in the stem cells co-cultured with the four groups of composite gels increased, indicating that this material has the ability to promote the osteogenic differentiation of cells. Among them, the RSF-Col-HAP-PDRN composite gel could significantly increase the expression of osteogenic genes related to ALP, BSP, OCN, and RUNX2, indicating that the RSF-Col-HAP-PDRN composite gel could promote the osteogenic differentiation of BMSC cells, which was significantly better than the control group, RSF, RSF-Col, and RSF-Col-HAP groups (P<0.001).

[0111] Figure 13Pictures showing the treatment of alveolar bone defect repair with the RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN composite gels prepared in Examples 6 to 9 are presented. In the animal experiment, a total of four groups were set up, namely the control group (Control), the RSF group, the RSF-Col group, the RSF-Col-HAP group, and the RSF-Col-HAP-PDRN group. Male Sprague-Dawley rats at 8 weeks of age were selected. First, the rats were raised for one week. After the rats were fully adapted to the environment, the animal experiment was carried out. The rats were anesthetized, placed supine on the operating table, the hair in the mandibular region of the rats was removed, the surgical area was gently wiped with iodophor, an incision was made in the vertical alveolar bone area of the edentulous area of the rat mandible, the skin was bluntly dissected and the gingiva was peeled off, and a defect with a diameter of 3 mm and a depth of 1.5 mm was drilled with a low-speed dental drill. During the operation of the dental drill, ice-cold normal saline was continuously dropped to cool down. After the defect was formed, the RSF, RSF-Col, RSF-Col-HAP, and RSF-Col-HAP-PDRN composite gels were filled in the defect position. In the control group, no material was filled. The tissue was repositioned to cover the bone, and the tissue and skin were sutured layer by layer. After the operation, the state of the rats was observed to prevent death due to wound infection. The rats were sacrificed at four weeks and six weeks after the operation respectively. The repair situation of the defect position of the rats was photographed and counted, and the alveolar bone area of the rats was collected and fixed in 4% paraformaldehyde. The direct-view photos of the defect repair showed that after four weeks of repair, obvious defect positions could be observed in each group. The defect position in the control group was more obvious, while the repair effects of the RSF-Col-HAP and RSF-Col-HAP-PDRN groups were better, and the defect positions were repaired to a certain extent. After six weeks, the bone regeneration of the defect positions in the material groups was good. The defect position in the RSF-Col-HAP-PDRN material group was basically filled with newly formed bone tissue, and it was well integrated with the surrounding bone tissue, and the defect repair effect was good.

[0112] Figure 14 Micro-CT images showing the bone repair of alveolar bone defects are presented. The results showed that at four weeks, the defect positions in each group were clearly visible. The defect positions in the control group, the RSF group, and the RSF-Col group were the most obvious, while the defect positions in the RSF-Col-HAP group and the RSF-Col-HAP-PDRN group were repaired better. After six weeks, the alveolar bone defects in each group were repaired to a certain extent. In the control group, an obvious defect area could still be seen. The defect recovery in the RSF group and the RSF-Col group was good, but the defect area was still visible. The defect areas in the RSF-Col-HAP group and the RSF-Col-HAP-PDRN group almost completely disappeared, and complete bone tissue formation could be seen. The results of new bone formation were continuous and the morphology was complete. It indicates that the RSF-Col-HAP-PDRN gel has a good therapeutic effect on the repair of alveolar bone defects.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A composite gel for repairing alveolar bone defects, characterized in that: The composite gel is made of composite particles and sodium hyaluronate (HA), wherein the raw materials of the composite particles include regenerated silk fibroin (RSF), collagen (Col) and hydroxyapatite (HAP).

2. A composite gel for repairing alveolar bone defects as claimed in claim 1, characterized in that: The raw materials of the composite particles also include polydeoxyribonucleotide (PDRN).

3. A method for preparing a composite gel for repairing alveolar bone defects according to claim 1 or 2, wherein the preparation method of the composite gel is as follows: Step 1: preparing a composite material: dissolving regenerated silk fibroin (RSF) and collagen (Col) in lithium bromide solution respectively, adding hydroxyapatite (HAP) after the two solutions of regenerated silk fibroin (RSF) and collagen (Col) are evenly mixed, adding BDDE after stirring for 20-40 minutes, continuing to stir for 20-40 minutes and standing for 24-72 hours to obtain a hydrogel; Step 2: Cleaning the composite material: The prepared hydrogel was squeezed through a mesh and washed with 40-60% ethanol for 6-10 times, and the hydrogel particles were freeze-dried; Step 3: preparing composite microparticles: grinding the freeze-dried hydrogel particles to obtain composite microparticles; Step 4: preparing a composite gel: dissolving hyaluronic acid (HA) in a PBS solution, and after the hyaluronic acid (HA) is completely dissolved, uniformly dispersing and mixing the prepared composite microparticles in the hyaluronic acid (HA) solution to prepare a composite gel.

4. A method for preparing the composite gel for repairing alveolar bone defects as claimed in claim 3, characterized in that: The step 1 also includes the following operations: dissolving polydeoxyribonucleotide (PDRN) in ultrapure water to prepare a polydeoxyribonucleotide (PDRN) solution, and adding the polydeoxyribonucleotide (PDRN) solution immediately after adding BDDE.

5. A method for preparing a composite gel for repairing alveolar bone defects as claimed in claim 3 or 4, characterized in that: The mass ratio of regenerated silk fibroin to collagen in the composite particles in the composite gel is 2:1 to 5:1, preferably 2:1; The mass ratio of regenerated silk fibroin to hydroxyapatite in the composite particles in the composite gel is 1:3 to 1:1, preferably 1:2.

57.

6. A method for preparing a composite gel for repairing alveolar bone defects as claimed in claim 4, characterized in that: The mass ratio of the regenerated silk fibroin to the polydeoxyribonucleic acid in the composite particles in the composite gel is 25:1 to 30:1, preferably 26.67:

1.

7. A method for preparing a composite gel for repairing alveolar bone defects as claimed in claim 3 or 4, characterized in that: The mass ratio of the composite microparticles to sodium hyaluronate in the composite gel is 5:1 to 10:1, preferably 7.5:

1.

8. A method for preparing a composite gel for repairing alveolar bone defects as claimed in claim 3 or 4, characterized in that: In step 1, the stirring time is 25-35 minutes, and the standing time is 36-60 hours. Preferably, the stirring time is 30 minutes, and the standing time is 48 hours. In step 2, the sieve mesh size is 6 to 50 meshes; In step 2, the concentration of the ethanol solution is 50%, the number of cleanings is 8, and the cleaning solution is replaced every 30 minutes; In step 3, the pulverization is mechanical pulverization by an electric grinding mill; In step 4, the concentration of the sodium hyaluronate solution is 10 to 50 mg / mL, preferably 20 mg / mL; In step 4, the concentration of the composite microparticles is 100-500 mg / mL, preferably 150 mg / mL.

9. The composite gel prepared by the preparation method according to any one of claims 3 to 8.

10. Use of the composite gel according to claim 1 or 2 or the composite gel prepared by the preparation method according to any one of claims 3 to 8 in preparing a drug for repairing alveolar bone defects.

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