Multifunctional COFs (covalent organic frameworks) basal bone filler with efficient anti-inflammatory and osteogenesis-promoting effects and preparation method of multifunctional COFs basal bone filler

Abstract

The invention discloses a multifunctional COFs base bone filler with efficient anti-inflammatory and osteogenesis promoting effects and a preparation method thereof, and relates to the technical field of orthopedic implants. The preparation method comprises the following steps: (1) synthesizing a COFs nano material through a simple Schiff base reaction; (2) introducing polydopamine to obtain COFs (PCOFs) with the surface modified by PDA (Poly (Covalent Organic Frameworks)); and (3) loading traditional Chinese medicine active ingredients onto the PCOFs nano-carrier by utilizing the adhesion capability of PDA to prepare the multifunctional COFs basal bone filler. According to the invention, COFs and PDA are combined to obtain PDA modified COFs (PCOFs), and traditional Chinese medicine active ingredients with anti-inflammatory, osteogenic and vascularization effects are loaded on a PCOFs nano-carrier by utilizing the adhesion capacity of PDA, so that the problems of inflammatory response, functional vascularization, bone regeneration and the like in a bone defect area in the prior art are effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of orthopedic implants, and in particular to a multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects and a preparation method thereof. Background Art

[0002] Bone defects caused by trauma, infection, tumor resection, osteoporosis and congenital malformations seriously affect the physical and mental health of patients, and bring heavy burdens and pressures to patients, medical systems and public resources. Relevant studies show that about 900 million bone reconstruction surgeries are performed worldwide each year to treat bone defect diseases. Bone transplantation and related expenses reached US$2.4 billion in 2016 and are expected to reach US$11.5 billion in 2025. It can be seen that the problem of bone defect repair has become an important global public health issue.

[0003] Traditionally, autologous bone transplantation is regarded as the gold standard for bone defect repair. However, its disadvantages are also obvious: autologous bone is usually taken from the ilium or fibula, which is limited in source; it will increase the surgical trauma of the patient; there are risks of bleeding and wound infection in the donor site. In recent years, by combining new knowledge and technologies in materials science, biology and medicine, bone tissue engineering has made great progress and is one of the most promising methods for repairing bone defects. However, bone defect reconstruction is a complex biological process involving interactions between different complex biological events, including anti-inflammation, osteogenic differentiation of stem cells, angiogenesis, collagen formation, etc. Therefore, the ideal bone defect regeneration tissue engineering material should simultaneously achieve the biological functions required for bone regeneration in terms of both efficient anti-inflammatory and osteogenic efficacy.

[0004] More and more studies have found that many active ingredients of traditional Chinese medicine, such as icariin (ICA), ginsenoside (Rg1), psoralen (Psl), naringin (Nrg), salidroside (Sal), etc., have excellent anti-inflammatory, osteogenic, and angiogenic effects. Among them, ICA is a major active ingredient in the genus Epimedium of the Berberidaceae family, and has a variety of biological activities such as promoting osteogenesis, inhibiting bacteria and inflammation, and regulating immunity. Studies have shown that ICA can not only reduce the mRNA expression of classic pro-inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) induced by lipopolysaccharide (LPS) by inhibiting the JNK / NF-κB pathway, thereby achieving its anti-inflammatory and immunomodulatory effects; it can also promote angiogenesis by activating the autophagy pathway and upregulating the expression of transforming growth factor-β1 (TGFβ1). In addition, ICA can promote bone formation by inducing osteogenic genes such as alkaline phosphatase (ALP), Runx2, Osx, and type I collagen, and can also inhibit bone resorption by regulating osteoprotegerin / receptor activator of nuclear factor-κb ligand (OPG / RANKL). Psl is a furanocoumarin compound derived from psoralea corylifolia, and is also widely found in traditional Chinese medicines such as Adenophora acuta, Saposhnikovia divaricata, and Angelica dahurica. It has pharmacological activities such as anti-tumor, neuroprotective, anti-inflammatory, and antioxidant. Psl can regulate bone homeostasis by affecting bone metabolism-related pathways, promoting osteoblast proliferation and differentiation, and inhibiting osteoclast proliferation. Among them, the signaling pathways related to bone formation include BMP-Smads, Wnt / β-catenin, NF-κB, MAPK, Hedgehog, and estrogen signaling pathways; the signaling pathways related to inhibiting bone resorption include AKT, AP-1, OPG / RANKL / RANK, and estrogen pathways. In addition to direct effects, psoralen can also inhibit bone lesions caused by inflammatory responses by acting on the interleukin-1β (IL-1β) signaling pathway. Nrg, as a natural flavonoid compound, is the main active ingredient of traditional Chinese medicines such as Drynaria fortunei, Citrus aurantium, Citrus aurantium and Citrus aurantium. Modern pharmacological studies have found that naringin has anti-osteoporosis, antioxidant, anti-inflammatory, and antibacterial effects. Studies have shown that Nrg can promote fracture healing and improve osteoporotic fractures by upregulating bone morphogenetic protein-7 (BMP-7) and basic fibroblast growth factor (BFGF) proteins and increasing femoral bone mineral density (BMD). At the same time, it was also found that Nrg can reduce the bone mass coefficient, bone length, bone ash, calcium and phosphorus content in the model group rats, and can significantly improve the symptoms of osteoporosis. Sal is a chemical extracted from Rhodiola rosea, which has multiple pharmacological effects such as anti-hypoxia, anti-inflammatory, anti-fatigue, cold resistance, cardiotonic, and anti-arrhythmic. A large number of studies have shown that Sal can regulate [Ca 2+]_i, cAMP, NF-κB, ERK and p38MAPKs pathways to inhibit the secretion of cytokines TNF-α, IL-6, IL-1β and the synthesis of inflammatory mediators NO and PGE_2, effectively reducing the damage caused by ROS and promoting DNA damage repair. In addition, Sal also has a significant effect of upregulating BMP-2, which can promote osteogenic bone formation and has a good application prospect in anti-osteoporosis. Therefore, the above traditional Chinese medicine active ingredients with anti-inflammatory, osteogenic and angiogenic effects as bioactive factors of bone tissue engineering materials have broad application prospects.

[0005] Therefore, in order to solve both problems at the same time, the present invention provides a multifunctional COFs-based bone filler with high efficiency in anti-inflammatory and osteogenic effects and a preparation method thereof. The method constructs a multifunctional COFs-based bone filler (such as ICA@PCOF) with high efficiency in anti-inflammatory and osteogenic effects, providing a new idea for the field of bone defect repair. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects and a preparation method thereof, COFs and polydopamine (PDA) are combined to obtain PDA-modified COFs (PCOFs), and then the adhesion ability of PDA is utilized to load the active ingredients of traditional Chinese medicine with anti-inflammatory, osteogenesis and angiogenesis effects onto the PCOFs nanocarrier, which effectively solves the problems of inflammatory response, functional vascularization and bone regeneration in the bone defect area in the prior art.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing a multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects is provided, comprising the following steps:

[0008] (1) COFs synthesis: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde are ultrasonically dissolved in a solvent, and a Schiff base reaction is performed under the action of a catalyst, followed by elution, centrifugation and vacuum drying to obtain COFs nanomaterials;

[0009] (2) adding the COFs nanomaterial obtained in step (1) to a Tris buffer solution to obtain a COFs-Tris suspension, then dropping a dopamine hydrochloride solution into the COFs-Tris suspension under stirring conditions for reaction, dialyzing to remove unreacted dopamine hydrochloride, and then centrifuging and vacuum drying to obtain a PDA-modified COFs powder, i.e., PCOFs;

[0010] (3) The PCOFs obtained in step (2) and the active ingredients of traditional Chinese medicine are mixed and reacted in an organic solvent, and then eluted with the organic solvent, centrifuged and vacuum dried to obtain a multifunctional COF-based bone filler with high anti-inflammatory and osteogenic effects.

[0011] Furthermore, in step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxybenzene-1,4-dicarbaldehyde is 1:1-3.

[0012] Furthermore, in step (1), the molar volume ratio of 1,3,5-tris(4-aminophenyl)benzene, 2,5-dimethoxybenzene-1,4-dicarbaldehyde, solvent and catalyst is 0.1 mmol: 0.15 mmol: 16 mL: 1 mL.

[0013] Furthermore, in step (1), the solvent is acetonitrile solution and the catalyst is glacial acetic acid.

[0014] Furthermore, in step (1), the reaction is carried out at a temperature of 0-120° C. for 3 days.

[0015] Furthermore, in step (1), tetrahydrofuran is used for elution 2-4 times, centrifuged at 5000-10000 r / min for 5-10 min, and vacuum dried at 40-60° C. and minus 0.02-0.08 MPa for 6-12 h.

[0016] Furthermore, in step (2), the Tris buffer solution has a concentration of 10 mM and a pH of 8.5.

[0017] Furthermore, in step (2), the mass ratio of COFs nanomaterial to dopamine hydrochloride is 5:1-5.

[0018] Furthermore, in step (2), the reaction is carried out under magnetic stirring at 300-500 r / min and at room temperature for 24-48 h.

[0019] Furthermore, in step (2), dialyze with a 8-15 kDa dialysis bag for 24-48 hours, the volume ratio of the reaction solution to deionized water is 1:100-400, centrifuge at 5000-10000 r / min for 10-30 minutes, and vacuum dry at 40-60° C. and minus 0.02-0.08 MPa for 6-12 hours.

[0020] Furthermore, in step (3), the mass ratio of PCOFs to active ingredients of traditional Chinese medicine is 1-1000:1.

[0021] Furthermore, in step (3), the reaction is carried out at 300-500 r / min and room temperature for 24-48 hours.

[0022] Furthermore, in step (3), dialyze with a 8-15 kDa dialysis bag for 24-48 hours, the volume ratio of the reaction solution to deionized water is 1:100-400, centrifuge at 5000-10000 r / min for 10-30 minutes, and vacuum dry at 40-60° C. and minus 0.02-0.08 MPa for 6-12 hours.

[0023] Furthermore, in step (3), the organic solvent is at least one of methanol, ethanol, isopropanol, n-butanol and 2-methoxyethanol.

[0024] Furthermore, in step (3), the active ingredient of the traditional Chinese medicine is at least one of icariin (ICA), ginsenoside (Rg1), psoralen (Psl), naringin (Nrg) and salidroside (Sal).

[0025] Furthermore, in step (3), the drug loading rate of the multifunctional COFs-based bone filler is 5-30%.

[0026] The present invention also provides a multifunctional COFs-based bone filler with high efficiency anti-inflammatory and osteogenic effects obtained by the preparation method of the multifunctional COFs-based bone filler with high efficiency anti-inflammatory and osteogenic effects.

[0027] The present invention also provides the use of the multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenic effects in preparing stem cell-derived reagents with anti-inflammatory and osteogenic effects.

[0028] Furthermore, the stem cells are derived from humans, mice or rabbits.

[0029] The present invention has the following beneficial effects:

[0030] 1. The multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects of the present invention introduces PDA into the nanocarrier. The high adhesion and anti-inflammatory ability of PDA make the material more widely used in the biomedical field. The filler can simultaneously solve the problems of immune regulation, functional vascularization and bone regeneration in the bone defect area, providing a new idea for the field of bone defect repair and treatment.

[0031] 2. The multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects of the present invention can serve as an integrated nano-drug delivery system, which can improve the bioavailability of drugs, reduce drug degradation in vivo and in vitro, increase drug stability, achieve precise drug release, and improve therapeutic effects, providing new ideas for the field of bone defect repair and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a scanning electron microscope image of the product obtained in Example 5;

[0033] Figure 2 This is a scanning electron microscope image of the product obtained in Comparative Example 1;

[0034] Figure 3 The CCK8 test results of the ICA@PCOFs extract obtained from Example 5 with different concentrations;

[0035] Figure 4 This is an immunofluorescence comparison of the OCN protein expression after the extracts of the products obtained in Example 5 and Comparative Example 1 induced human bone marrow mesenchymal stem cells (HBMSCs);

[0036] Figure 5 Statistical results of semi-quantitative analysis of immunofluorescence comparison of OCN protein expression. DETAILED DESCRIPTION

[0037] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0038] Example 1

[0039] A multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects, the preparation method of which comprises the following steps:

[0040] (1) COFs synthesis: At room temperature, 18 mg of 1,3,5-tris(4-aminophenyl)benzene and 30 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL of acetonitrile solution, 1 mL of glacial acetic acid catalyst was added and ultrasonication was continued for 10 min. The reaction was carried out at room temperature for 3 days, and tetrahydrofuran was used for elution 3 times. The mixture was centrifuged at 7000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 6 h to obtain COFs nanomaterials.

[0041] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (50 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension magnetically stirred at 500 r / min, reacted at room temperature for 24 h, dialyzed with an 8-12 kDa dialysis bag for 24 h, the volume ratio of the reaction solution to deionized water was 1:400, centrifuged at 10000 r / min for 10 min, and vacuum dried at 60°C and -0.06 MPa for 6 h to obtain PDA-modified COFs powder, i.e., PCOFs;

[0042] (3) 50 mg of the PCOFs obtained in step (2) was ultrasonically dispersed in 20 mL of isopropanol to obtain a PCOFs-isopropanol suspension, and then 20 mg of icariin (ICA) was ultrasonically dissolved in 20 mL of isopropanol, and then slowly added dropwise to the PCOFs-isopropanol suspension magnetically stirred at 500 r / min, reacted at room temperature for 24 h, and then eluted with 20 mL of isopropanol three times, centrifuged at 10000 r / min for 10 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects, ICA@PCOFs.

[0043] Example 2

[0044] A multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects, the preparation method of which comprises the following steps:

[0045] (1) COFs synthesis: 54 mg 1,3,5-tris(4-aminophenyl)benzene and 30 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL acetonitrile solution at room temperature, 1 mL glacial acetic acid catalyst was added and ultrasonication was continued for 10 min, the reaction was carried out at room temperature for 3 days, tetrahydrofuran was used for elution 3 times, the mixture was centrifuged at 6000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 8 h to obtain COFs nanomaterials;

[0046] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (10 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension under magnetic stirring at 400 r / min, and the mixture was reacted at room temperature for 30 h, and dialyzed with an 8-12 kDa dialysis bag for 24 h. The volume ratio of the reaction solution to deionized water was 1:200. The mixture was centrifuged at 7000 r / min for 20 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain PDA-modified COFs powder, i.e., PCOFs.

[0047] (3) 50 mg of the PCOFs obtained in step (2) was ultrasonically dispersed in 20 mL of ethanol to obtain a PCOFs-ethanol suspension, and then 1 mg of psoralen (Psl) was ultrasonically dissolved in 1 mL of methanol, and then slowly added dropwise to the PCOFs-ethanol suspension magnetically stirred at 500 r / min, reacted at room temperature for 30 h, and then eluted three times with 20 mL of ethanol, centrifuged at 7000 r / min for 20 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects, Psl@PCOFs.

[0048] Example 3

[0049] A multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects, the preparation method of which comprises the following steps:

[0050] (1) COFs synthesis: At room temperature, 18 mg of 1,3,5-tris(4-aminophenyl)benzene and 30 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL of acetonitrile solution, 1 mL of glacial acetic acid catalyst was added and ultrasonication was continued for 10 min. The reaction was carried out at room temperature for 3 days, and tetrahydrofuran was used for elution 3 times. The mixture was centrifuged at 8000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 10 h to obtain COFs nanomaterials.

[0051] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (30 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension under magnetic stirring at 300 r / min, and the mixture was reacted at room temperature for 36 h, and dialyzed with an 8-12 kDa dialysis bag for 36 h. The volume ratio of the reaction solution to deionized water was 1:200, and the mixture was centrifuged at 5000 r / min for 10 min. The mixture was vacuum dried at 40°C and -0.02 MPa for 12 h to obtain PDA-modified COFs powder, i.e., PCOFs.

[0052] (3) 50 mg of the PCOFs obtained in step (2) was ultrasonically dispersed in 20 mL of n-butanol to obtain a PCOFs-n-butanol suspension, and then 5 mg of naringin (Nrg) was ultrasonically dissolved in 5 mL of n-butanol, and then slowly added dropwise to the PCOFs-n-butanol suspension magnetically stirred at 500 r / min, reacted at room temperature for 36 h, and then eluted three times with 20 mL of n-butanol, centrifuged at 8000 r / min for 20 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects, Nrg@PCOFs.

[0053] Example 4

[0054] A multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects, the preparation method of which comprises the following steps:

[0055] (1) COFs synthesis: 54 mg 1,3,5-tris(4-aminophenyl)benzene and 30 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL acetonitrile solution at room temperature, 1 mL glacial acetic acid catalyst was added and ultrasonication was continued for 10 min, the reaction was carried out at room temperature for 3 days, tetrahydrofuran was used for elution 3 times, the solution was centrifuged at 8000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 10 h to obtain COFs nanomaterials;

[0056] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (40 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension under magnetic stirring at 500 r / min, reacted at room temperature for 48 h, dialyzed with an 8-12 kDa dialysis bag for 36 h, the volume ratio of the reaction solution to deionized water was 1:400, centrifuged at 8000 r / min for 30 min, and vacuum dried at 60°C and -0.06 MPa for 6 h to obtain PDA-modified COFs powder, i.e., PCOFs;

[0057] (3) 50 mg of the PCOFs obtained in step (2) was ultrasonically dispersed in 20 mL of 2-methoxyethanol to obtain a PCOFs-2-methoxyethanol suspension, and then 10 mg of salidroside (Sal) was ultrasonically dissolved in 10 mL of 2-methoxyethanol, and then slowly added dropwise to the PCOFs-2-methoxyethanol suspension magnetically stirred at 500 r / min, reacted at room temperature for 48 h, and then eluted 3 times with 20 mL of 2-methoxyethanol, centrifuged at 8000 r / min for 30 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects, Sal@PCOFs.

[0058] Example 5

[0059] A multifunctional COFs-based bone filler with high-efficiency anti-inflammatory and osteogenesis effects, the preparation method of which comprises the following steps:

[0060] (1) COFs synthesis: 36 mg 1,3,5-tris(4-aminophenyl)benzene and 30 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL acetonitrile solution at room temperature, 1 mL glacial acetic acid catalyst was added and ultrasonication was continued for 10 min, the reaction was carried out at room temperature for 3 days, tetrahydrofuran was used for elution 3 times, the mixture was centrifuged at 6000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 10 h to obtain COFs nanomaterials;

[0061] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (20 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension magnetically stirred at 400 r / min, reacted at room temperature for 24 h, dialyzed with an 8-12 kDa dialysis bag for 24 h, the volume ratio of the reaction solution to deionized water was 1:200, centrifuged at 8000 r / min for 30 min, and vacuum dried at 50°C and -0.06 MPa for 8 h to obtain PDA-modified COFs powder, i.e., PCOFs;

[0062] (3) 50 mg of the PCOFs obtained in step (2) was ultrasonically dispersed in 20 mL of methanol to obtain a PCOFs-methanol suspension, and then 50 mg of icariin (ICA) was ultrasonically dissolved in 5 mL of methanol, and then slowly added dropwise to the PCOFs-methanol suspension magnetically stirred at 500 r / min, reacted at room temperature for 24 h, and then eluted three times with 20 mL of methanol, centrifuged at 8000 r / min for 10 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects, ICA@PCOFs.

[0063] Comparative Example 1

[0064] A bone filler, the preparation method of which comprises the following steps:

[0065] (1) COFs synthesis: 36 mg 1,3,5-tris(4-aminophenyl)benzene and 30 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 16 mL acetonitrile solution at room temperature, 1 mL glacial acetic acid catalyst was added and ultrasonication was continued for 10 min, the reaction was carried out at room temperature for 3 days, tetrahydrofuran was used for elution 3 times, the mixture was centrifuged at 6000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 10 h to obtain COFs nanomaterials;

[0066] (2) 50 mg of the COFs nanomaterial obtained in step (1) was added to 20 mL of Tris buffer solution (10 mM, pH 8.5) to obtain a COFs-Tris suspension, and then a dopamine hydrochloride solution (20 mg / 5 mL) was slowly added dropwise to the COFs-Tris suspension under magnetic stirring at 400 r / min, and the mixture was reacted at room temperature for 24 h. The mixture was dialyzed with an 8-12 kDa dialysis bag for 24 h. The volume ratio of the reaction solution to deionized water was 1:200. The mixture was centrifuged at 8000 r / min for 30 min, and vacuum dried at 50°C and -0.06 MPa for 8 h to obtain a bone filler.

[0067] Test example

[0068] The scanning electron microscope images of the products obtained in Example 5 and Comparative Example 1 were obtained, and the results were as follows: Figure 1-2 shown.

[0069] Depend on Figure 1-2 It can be seen that the product of Comparative Example 1 is spherical particles with a diameter of about 300-600nm, and most of the particles have irregular pits and cracks on the surface; while the spherical particles in Example 5 are also about 300-600nm in diameter, but different from Comparative Example 1, the surface morphology of the COFs particles has changed significantly, showing that ICA is evenly covered on the surface of the PCOFs spherical particles, causing the pits and cracks on the original surface to become significantly shallower, thus confirming that the ICA loading is successful.

[0070] The sample extracts of the product obtained in Example 6 (ICA@PCOFs and α-basal medium were shaken at a concentration of 1 mg / mL at 37°C for 24 hours) were prepared into complete medium (10% FBS + 1% double antibody) for CCK-8 test; human bone marrow mesenchymal stem cells were induced with appropriate ICA@PCOFs concentrations, and the protein expression of OCN was detected by immunofluorescence; the above results were as follows: Figure 3-5 shown.

[0071] Depend on Figure 3 It can be seen that the ICA@PCOFs of the present invention has an inhibitory effect on cell proliferation at high concentrations, but has a significant promoting effect at low concentrations, among which 1 μg / mL has the best promoting effect.

[0072] Depend on Figure 4-5 It can be seen that compared with the control group, the fluorescence intensity of the PCOFs group did not change significantly, while the fluorescence intensity of the ICA@PCOFs group was significantly enhanced, which indicates that the nanomaterials loaded with ICA have a significant effect in promoting the osteogenic differentiation of stem cells.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects, characterized in that: The following steps are involved: (1) COFs synthesis: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde are ultrasonically dissolved in a solvent, and a Schiff base reaction is performed under the action of a catalyst, followed by elution, centrifugation and vacuum drying to obtain COFs nanomaterials; (2) adding the COFs nanomaterial obtained in step (1) to a Tris buffer solution to obtain a COFs-Tris suspension, then dropping a dopamine hydrochloride solution into the COFs-Tris suspension under stirring conditions for reaction, dialyzing to remove unreacted dopamine hydrochloride, and then centrifuging and vacuum drying to obtain a PDA-modified COFs powder, i.e., PCOFs; (3) The PCOFs obtained in step (2) and the active ingredients of traditional Chinese medicine are mixed and reacted in an organic solvent, and then eluted with the organic solvent, centrifuged and vacuum dried to obtain a multifunctional COF-based bone filler with high anti-inflammatory and osteogenic effects.

2. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxybenzene-1,4-dicarbaldehyde is 1:1-3.

3. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (1), the molar volume ratio of 2,5-dimethoxybenzene-1,4-dicarbaldehyde, solvent and catalyst is 0.1 mmol: 0.15 mmol: 16 mL: 1 mL.

4. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (2), the weak alkaline solution is a Tris buffer solution with a concentration of 10 mM and a pH of 8.5; the mass ratio of COFs nanomaterials to dopamine hydrochloride is 5:1-5.

5. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (2), dialyze with a 8-15 kDa dialysis bag for 24-48 hours, the volume ratio of the reaction solution to deionized water is 1:100-400, centrifuge at 5000-10000 r / min for 10-30 minutes, and vacuum dry at 40-60° C. and minus 0.02-0.08 MPa for 6-12 hours.

6. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (3), the organic solvent is at least one of methanol, ethanol, isopropanol, n-butanol and 2-methoxyethanol.

7. The method for preparing the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenesis effects according to claim 1, characterized in that: In step (3), the active ingredient of the traditional Chinese medicine is at least one of icariin, ginsenoside, psoralen, naringin and salidroside.

8. The multifunctional COFs-based bone filler with high efficiency in anti-inflammatory and osteogenic effects obtained by the preparation method of the multifunctional COFs-based bone filler with high efficiency in anti-inflammatory and osteogenic effects according to any one of claims 1 to 7.

9. Use of the multifunctional COFs-based bone filler with high anti-inflammatory and osteogenic effects as claimed in claim 8 in the preparation of stem cell-derived agents with anti-inflammatory and osteogenic effects.

10. The use according to claim 9, characterized in that The stem cells are derived from humans, mice or rabbits.

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